Study of the Influence in osteoblastic Behavior of compressive residual Stress in smooth and rough Surfaces of titanio dental Implants

STUDY OF THE INFLUENCE IN OSTEOBLASTIC BEHAVIOR OF COMPRESSIVE RESIDUAL STRESS IN SMOOTH AND ROUGH SURFACES OF TITANIO DENTAL IMPLANTS Thesis presented by: Ana Rita Costa e Curto de Carvalho Pereira To qualify for the degree of Doctor in Dentistry Department of Stomatology Stomatology College of Universidad de Sevilla 2024 Directors: Doctor Mariano Herrero Climent. Professor Doctor Vicent Ríos Santos. Co-director: Doctor Paulo Maia. Recomeça…se puderes sem angustia e sem pressa e os passos que deres, nesse caminho duro do futuro, dá-os em liberdade, enquanto não alcances não descanses, de nenhum fruto queiras só metade Miguel Torga To my parents, Elsa e Vitor: Once I heard that children choose their parents. I will choose you for the eternity. To my brothers and sisters: Pedro, Elsa, Ricardo e Mafalda. My best friends in all world. I will also choose you all for the eternity. To my brother and sister in law, Paulo and Margarida: a brother and a sister, that came just a little after. To all my nieces and nephews: Catarina, João, Bernardo, Miguel, Martim, Tomás, Simão, Guilherme and Matilde: I will always do all your wishes. To Jorge: if I could imagine my life without you… probably, I would just survive. Acknowledgments To my Directors PROFESSOR DOCTOR JOSÉ VICENTE RíOS SANTOS and DOCTOR MARIANO CLIMENT HERRERO, for the opportunity to carry out this work with me, for all your patient, guidance and availibility. I’m very fortunate to have both in this project with me. To PROFESSOR DOCTOR JAVIER GIL MUR, a ligthhouse most of the time. Thank you so much for your encorage words, kindness, guidance, patient, and opportunity. To Doctor PAULO MAIA, my co-Director, my professor and, above all, a friend. This work possibly wouldn’t exist without him. To all the team that was involved in the realization of this investigation, from the laboratory part to the processing of data. To SOADCO/Klockner that kindly provided the necessary material to the investigation. To all my friends and collegues that daily work with me and had to be more patient in recent times.You all made my days lighter. To my family for the love, unconditional support and cofidence in this moment and in all moments in my live. To Jorge, who always take my hand in all our journeys and walk side by side with me until the end. Abstract In oral rehabilitation, the necessity of excellent results in long term, translated in a physiologic fully adapted masticatory function and irrepressible aesthetics, totally satisfactory to individual requirements, become the ultimately and principal objective to the surgical and dental clinician. Nowadays, the use of dental implants in oral rehabilitation have become an usual process with predictable results. All the efforts to improve the biological relationship of these materials with the host, have allowed the rehabilitation of cases with increasing complexity. All the process of conceptualization and execution of the rehabilitation treatment is based in an well adapted individual plan. The treatment clinical decision must be supported on broad and enlightened knowledge of the available instruments. The evolution of dental implants, and changes resulting from this, has followed a line towards optimizing the relationship of these devices with neighboring tissues, namely with the surrounding bone, through osseointegration process, and with soft tissues maintaining their integrity. In this sense, implant surface treatments concerning the topography, chemical composition and surface energy have made possible to enhance these results making the osseointegration process faster, more effective and predictable. Surface treatments can be categorized as additive, subtractive or bioactive. Many of these treatments are carried out in a combined way, enhancing the added value of each one. It is generally agreed that the topographical change of an implant through these treatments will consequently bring a chemical change to the surface of the implants. The surface energy and wettability inherent to the material also change, which, in terms of osteointegration, becomes an added value in being able to enhance the relationship between the implant and the biomolecular environment, since these energetic properties may increase adhesion, proliferation and cell differentiation. It is known that subtraction treatments change the residual energy of materials. The effect that this energetic alteration promotes at the molecular, cellular and bacteriological level is still an open field whose knowledge and comprehension may enhance the results to obtain. The aim of this work is to establish a relationship between two titanium surfaces, rough and smooth, and the residual energy on each one of these surfaces with the adhesion, proliferation and differentiation of osteoblastic cells, as well as understanding whether there is a relationship between this residual energy and the development of bacterial species present in the oral cavity, using two colonizing species with different characteristics. Doctoral Thesis Ana Rita Pereira - 17 - Introduction In 1960s Per-Ingvar Bränemark and co-workers gave rise to the concept of osseointegration. Such concept defined this biological response as a “direct structural and functional connection between ordered living bone and the surface of a load-carrying endoosseous implant”(1). Shortly after, André Schroeder and his coworkers histologically demonstrated this osteointegration referring it as “functional ankylosis”(2). Nowadays, and even with the success rates in implantology treatments, efforts continue to be made to achieve more efficient relation between implant and the host(3). The implant surface is one of the key factors for integration in bone. An important discovery was that rough surfaces implants possess a higher bone-to-implant contact and favour biomechanical stability. Surface characteristics, such as topography and chemical modifications of the titanium implant surface, were recognized as factors that significantly affect the speed of osseointegration(4, 5). The series of events leading to osseointegration encompass coagulum formation, granulation tissue formation, development of provisional matrix, woven bone formation, parallel-fibered bone formation and eventually lamellar bone formation. This way, implant’s osseointegration initiates when blood cells meet the implant surface. In a first moment, in the presence of a blood clot and consequently, blood proteins and other biomolecules, there is a formation of an amorphous layer of unmineralized collagen, proteoglycans and fibrin net(6). To this amorphous layer, and immediately after, will occur platelet adhesion and activation. Platelets are very rich in growth factors that will enrol mesenquimal cells. These cells will be responsible for the formation and mineralization of the bone tissue(7). The main reason in choosing metallic implants as a biomaterial lies in their stable oxide film. It is desirable that this oxide surface undergo electrochemical changes in the physiologic environment. This can be enhanced by incorporating calcium, phosphorus and sulphur to the composition of the oxide film. The host will respond through a series of cell and matrix events which should culminate in bone healing and apposition of bone in the implant surface: osseointegration. Doctoral Thesis Ana Rita Pereira - 18 - 1 Osseointegration Osseointegration of implants is dependent on successful wound healing around the implant. 1.1 Stages of osseointegration 1.1.1 Haemostasis It begins with the surgical trauma created by the drilling and can last minutes or hours. With the trauma, bone matrix release proteins, growth factors and differentiation factors that are stored. Also, blood vessels are affected and, in this case, polymerization of fibrinogen takes place, performed by thrombin and initiated by platelets (extrinsic system) and intrinsic clotting cascade (Hageman Factor). Immediately after the insertion, the implant surface interacts with water molecules and ions like calcium ions. These ions are followed by plasma proteins (albumin, globulins, fibrin) – protein adsorption process(8). The first proteins biding to the surface are those in a high concentration in the blood (albumin); slowly they are replaced by proteins in a low concentration but with a higher affinity to the surface (vitronectin and fibronectin) – Vroman effect(9). The adsorption of the proteins depends on their properties (charge, size, stability of the structure, amino acid composition, steric conformation) but also on the surface characteristics such as topography, surface energy and hydrophilicity. Titanium hydrophilic surface preserves better proteins conformation and function(9). Through this initial coating of the titanium with blood proteins, cells can attach to the titanium surface. Cellular adhesion is achieved through cell adhesion proteins - integrins - that interact with cell biding sites in proteins (ex. fibronectin) with amino acid specific sequences like the sequence RGD (arginine, glycine, aspartate). The integrin receptors act as an interface between the intracellular and extracellular compartments(9-12). Doctoral Thesis Ana Rita Pereira - 19 - In vascular site, platelets aggregate and form a white thrombus closing the continuity solution. Various biomolecules are freed (thrombin, collagen, fibrinogen, thrombospondin, ADP) in this process stimulating and amplify all this mechanism. Platelets also bind to vitronectin and fibrin present in the surface of the titanium. These bindings result in activation and degranulation of the platelets. Released serotonin and thromboxane participate in vasoconstriction. Cytokines resultant from platelets degranulation begin the inflammatory phase(11). 1.1.2 Inflammatory phase Starts around 10 minutes after injury, with the degranulation of the platelets, and can last for the first days. The degranulation brings to the wound several molecules, for instance: growth factors, bradykinin, histamine. These molecules last to promote an increase of the vascular permeability, increase blood flow and decrease blood stream velocity which in turn brings a state of hyperaemia to the wound. This way, the initial vasoconstriction in the haemostatic phase turns into vasodilatation with consequent swelling and warming of the local. Also, in this phase the host defence is activated. The innate immune system is activated by unspecific molecules of bacterial origin and is not adaptable. The first cellular elements arriving to the wound are polymorphonuclear leucocytes (PMN), also called neutrophil granulocytes and macrophages. These white cells invade the blood clot passing through little gaps in the vessel’s walls (ameboid migration) in a process called diapedesis. Also involved in this defence process are molecular elements such like complement system. These elements have the capacity for perforate and damage bacterial wall cells; also labels it - opsonization - to future phagocytosis by the immune cells. This defence process is promoted by chemotactic substances originating from the host and from the bacteria. The abundance of bacteria my prolong and amplifie all this cellular immune response, which can, in turn, be harmful to the surrounding tissues. A clean environment and antibacterial measures are of summit importance to overtake this inflammatory phase. Advancing to the end of this phase, PMN are replaced for lymphocytes and macrophages. The roll of these cells is not well stablished. Although, they appear to clean the wound of old neutrophils and secret cytokines that are mitogens and Doctoral Thesis Ana Rita Pereira - 20 - chemoattractants for fibroblast. They can act as a switch for the end of the inflammatory phase and the beginning of the proliferative phase(11). 1.1.3 Proliferative phase This phase is characterized by the formation of granulation tissue which components are new extracellular matrix and blood vessels (angiogenesis). It may take a few days or weeks leading the 4 to 6 weeks after surgery. Fibroblast from the surrounding tissue invades the blood clot, binding through integrins to the RGD peptides of fibronectin and start replacing the provisional clot matrix with insoluble cellular fibronectin, collagens, vitronectin, decorin and other proteoglycans. In parallel, angiogenesis is stimulated by hypoxia through a mechanism mediate by macrophages and vascular endothelial growth factor (VEGF). Angiogenesis is the prerequisite for osteogenesis. New bone forms only in close connection to blood vessels – angiogenetic osteogenesis(11). The process of osteogenesis starts with an osteoprogenitor cell that binds through cellular integrins to the extracellular matrix/protein layer in the implant surface. After firm attachment, the osteoprogenitor cell became an osteoblast, a secretory cell that starts to express osteocalcin and alkaline phosphatase. Bone morphogenetic proteins (BMP) bind to receptors on the cell surface of the bone precursor cells and this will lead to a nuclear cell transcription and activation of specific genes for regulation of phosphatase alkaline, collagen type I and osteopontin. The insertion of the implant and its friction in the bone during this process promotes a primary stability. This mechanical stability cannot be subject to excessive external load. Micromovements of the implant at this point may compromise the implant stability and consequent osseointegration. Under normal conditions, the first weeks are a vulnerable phase because primary stability can decrease to critical levels before secondary stability has developed(11). After one week of the implantation, the primary stability will be reinforced by the formation of a woven bone. In histological terms, woven bone is characterized by collagen fibres not Doctoral Thesis Ana Rita Pereira - 21 - parallel and randomly orientated, numerous and irregular shaped osteocytes. Woven bone grows along the existing bone and along the dental implant surface towards the groves of the threads. It usually starts growing from the surrounding bone towards the implant, except in narrow gaps, where it is simultaneously deposit upon the implant surface. Remaining bone debris from the drilling preparation can be incorporated in woven bone(13). The secreted collagen is type III, which is characteristic of intramembranous ossification. Later is replaced for collagen type I. In this phase, mineralization of the matrix will occur through an unorganized, rapid and not very closed to collagen deposition of hydroxyapatite. Figure 1: Line drawings to illustrate distance (A e B) and contact (C and D) osteogenesis. In A, osteogenic cells line the old bone surface, while in C, osteogenic cells have first been recruited to the implant surface. The blood supply to these cells is between the cells and the implant in A, but between the cells and the old bone in C. In terms of bone matrix elaboration, A results in B, in which bone is laid down on the old bone surface. This is in sharp contrast to D, where new (de novo) bone is laid down on the implant surface. In each case (A+B; C+D), the secretory bone cells recede towards their bone supply(14). Doctoral Thesis Ana Rita Pereira - 22 - 1.1.4 Remodelling phase This phase involves the removal of woven bone by the osteoclast and replacement by lamellar bone. Also, mineralization will incorporate hydroxyapatite within the collagen fibres (interfibrillar) which will provide a unique mechanical and biologic properties. It starts around the third month and, after several weeks of increasingly high activity, slowdowns again, but continues for the rest of life(13). In contrast to woven bone, in lamellar bone the collagen fibres have a parallel orientation. Also, lamellar bone attaches to the tips of macro threads of the implant forming a particular trabecular design. These features promote the distribution of occlusal loads to the surrounding bone and makes the bone structure as light as possible - Wolfs Law(11). The action of osteoclast is dependent of osteoblast control as well as the reverse. An imbalance in this mechanism promotes pathologic entities such as osteopenia and osteopetrosis(9, 13). Osteoblasts secret RANKL (receptor activator of nuclear factor kappa beta ligand) and osteoprotegerin. RANKL stimulates osteoclast activity establishing contact with receptor RANK in these cells. Osteoprotegerin prevents RANKL to achieve RANK receptor. Thus, osteoprotegerin prevents bone by inhibition of osteoclastogenesis. Osteoclast are called to act after osteocytes digest remnants of osteoid by collagenases and leave exposed RGD peptide ends from non-collagenous bone matrix proteins. Osteoclast are attracted for this surface and form a structure comparable to a suction cup, selling the margin in contact with the bone surface through integrins. An isolate space is created under the osteoclast – resorption lacuna or Howship’s lacuna(9). Neighbourhood cells are kept safe from digestive enzymes and ion pumps that produce hydrochloric acid and demineralizes bone matrix and liberates bone collagen. Later, osteoclast activate osteoblast to fill the void after resorption. The concrete mechanism by which osteoclast interact with osteoblast in this purpose is not well stablished(13). However, osteoblastic precursor cells can sense the surface topography in the resorption lacuna by creating pseudopodia and thus obtain information about how much bone is needed to fill the lacuna. In a parallel way, osteoblast perceive the micro Doctoral Thesis Ana Rita Pereira - 23 - and nanostructured of treated surface on dental implants in the same way they recognized the gaps left by osteoclasts giving the surface an osteoconductive property(9, 11). The result of osteoclast/osteoblast interaction promote the formation of a new osteon with a central tunnel with blood vessel – Haversian system. Figure 2: Bone healing on an implant surface. (A) initial adhesion on surface is by plasma proteins in higher concentration, such as albumin. Later, higher affinitive proteins (fibronectin, vitronectin, and so forth) replace them. (B) Inflammatory phase. Neutrophils and mononuclear cells are found in this stage. (C) Stimulation by foreign-body giant cells, fibrogenesis, and various cytokines differentiate mesenchymal stem cells into osteoblasts that form new bone around the implant surface. Alb, albumin; Fb, fibroblast; FBGC, foreign-body giant cell; FN, fibronectin; MC, mononuclear cell; MsC, mesenchymal stem cell; NP neutrophil; Ob, osteoblast; VN, vitronectin(9). This temporal sequence of osseointegration have been well elucidated in the results of an animal study(15). The objective of such study was to describe a novel model to investigate these different phases of wound healing involved in osseointegration. The installation of an implant implies a series of events, as previously described, that among others includes necrosis and subsequent resorption of traumatized bone around the titanium body concomitant with new bone formation. For that animal study, solid-screw implants with a sand-blasted and acid etch Doctoral Thesis Ana Rita Pereira - 24 - surface were installed in the mandible of dogs and, wound healing and osseointegration were studied over a period of 2 hours to 12 weeks after implant installation. More recently, and with the necessity of expand these knowledges to human field, a combination of histological, histomorphometrical and gene analysis techniques, from human biopsy material of retrieved dental implants, allowed to compare results with the previews animals’ results. Donos, N. and colleagues (4) summarized the temporal events of osseointegration in animal models and humans. Figure 3: Experimental implant device. (A): screw-shaped titanium implant. (B): Cross-section of the wound chamber: A, pitches engaging the bone tissue walls; B, the inner U-shaped wound chamber proper. The dotted line indicates the lateral wall of the chamber (i.e. the position of the cut bone surface)(15). Doctoral Thesis Ana Rita Pereira - 25 - 1.2 Osseointegration in animal models - Early events Two hours after implant installation, the threads were in contact with pristine bone and the pitches of the threads provided mechanical anchorage in the pristine bone. This provided initial or primary mechanical stability. The inner U shaped chamber was filled with a clot that was replaced with a primitive granulation tissue in 4 days. This tissue contains numerous mesenchymal cells, matrix components and newly formed vascular structures (angiogenesis). A provisional connective tissue matrix had been established. Figure 4: Upper part of the wound chamber containing coagulum, including fibrin and large number of erythrocytes. Decalcified section. Magnification ´200(16). Doctoral Thesis Ana Rita Pereira - 26 - - Bone modeling One week after installation. The provisional connective tissue in the wound chambers was rich in vascular structures and contain numerous mesenchymal cells. Some inflammatory cells were present. Woven bone was seen in the provisional connective tissue that surrounded the blood vessels. Woven bone formation occurs in the centre of the chamber as well apparently in direct contact with the surface of the implant – contact osteogenesis. After 14 days, woven bone formation was more pronounced in all compartments. Portions of newly bone apparently extended from the old bone into the provisional connective tissue – distance osteogenesis. In many regions woven bone was bridging to the surface of the titanium implant. A more osseointegration had been established. Osteoclast formation was notice in the pristine bone resulting in bone resorption adjacent to the implant surface, especially in areas of pressure of the implant to the body bed. Thus, areas of bone located lateral to the device and in direct contact immediately after implant insertion (primary stability) had undergone resorption and were involved in new bone formation: mechanical stability of the implant was replaced by biological bonding and stability. Four weeks after implant installation, new formed mineralized bone extended from the implant bed (drilling preparation) to the walls/chambers of the implant. The central part of the chamber was filled with a primary sponjosa, which was rich in vascular structures and contains a multitude of mesenchymal cells. Doctoral Thesis Ana Rita Pereira - 33 - The chemical composition and surface energy depends on bulk composition and surface treatments(27). The surface topography is dependent on surface grooves orientation and roughness(28). Different machining procedures will produce different orientations and different roughness. The modification of topography increases the macro, micro and nanoroughness of the surface. In a biomechanical approach and comparing to a smooth surface, as far as to micrometer level, a rough surface develops a more extended area, increasing the interlocking of the bone with the implant. At the nanometer level, the biologic processes are prevalent and improve matrix protein adsorption, bone cell migration and proliferation(8). In topographic terms, a surface with a clear orientation is called anisotropic surface. This kind of surface is achieved in a turned (or milled or polished) surface. A surface with no orientation at all is called isotropic surface and can be achieved in blasting or etching treatments(26). A systematic review from Wennerberg A. and Alberktsson T.(28), on surface topography and importance in bone integration, analyzed one hundred (100) articles. The authors start highlighting some flaws concerning the poorly described surfaces analyses and the lack of consensus on “smooth” and “rough” surfaces. Only a few studies have had characterized the surface topography more than in the height direction, and this height-descriptive twodimensional was the most used parameter. Many studies used imprecise techniques in the evaluation of the topography of the surface and many of them had an incomplete evaluation because they don’t consider the importance of the nanotopography resulting from surface treatments. The authors split the analyze concerning surface orientation and surface treatments. In the former, they conclude that whether the surface was isotropic or anisotropic no differences had in implant incorporation on bone. Relatively to surface treatments, the authors found better bone integration in blasted, etched and blasted-etch surfaces than in machined ones. Also, in oxidized treatment the implants demonstrated stronger bone anchorage than in machined ones. From additive treatments such as titanium plasma spray (TPS) the results were very ambiguous since some authors point different conclusions. Several advocate TPS surfaces to be better integrated in bone compared with smooth implants, whereas others found no correlation. The Doctoral Thesis Ana Rita Pereira - 34 - few studies that had investigated the importance of nanometer structures on implant integration, indicate that these had an impact on the early bone healing. What concerns to surface roughness, evaluation can be made by several parameters. These parameters are used to numerically describe the appearance of the surface topography. Normally, these parameters are categorized into three groups according to its functionality: amplitude or height parameters, spacing parameters and hybrid parameters(29). Height parameters are solely descriptive of height. Spatial or texture parameters describe the horizontal distance between the irregularities. Hybrid parameters include spatial and height information. Roughness parameters can also be calculated in either two dimensional (2D) or three dimensional (3D) forms. Amplitude parameters are the most important parameters to characterize surface topography. They are used to measure the vertical characteristics of the surface deviation. The arithmetic average height parameter, also known as the center line average (CLA) is the most universally used and gives a good general description of the height variation. It is the most used roughness parameter in the biomaterials field and is defined as Ra parameter(29). Albrektsson and Wennerberg (30) proposed a classification of implant surfaces based on their topographical features. Most of the dental implants available have a Ra ranging from 1 to 2 µm. The implant surface topography can be classified as smooth (Ra < 0,5 µm), minimally rough Ra 0,5-1,0 µm) moderately rough (Ra 1,0-2,0 µm) and highly rough (Ra > 2,0 µm)(31). The optimal values of amplitude parameters are controversial. However, beneficial effects have been correlated between surfaces with Ra in the range 0,5-8,5 µm (32) and optimal results are achieved for intermediately rough implants, in a range of 1 to 1,5 µm(33). Also controversial are the results from most studies. This is because different topographic measure parameters, different instruments and techniques strongly influence the outcome of a topographic characterization. One of the first questions are the “control” machined implants used as comparison in many studies. Some authors do not mention that machined surfaces span a wide range of surface textures. Most of the times inappropriate instruments are used to Doctoral Thesis Ana Rita Pereira - 35 - measure the surface of “screw type implant”. Many times, there is no consensus on which parameters – 2 or 3 dimensional – and resolution should be evaluated, and finally, the concepts of form, waviness, and roughness are not well defined. 3.1 Roughness analyzes Wennerberg, A and colleagues (33) published some measuring guidelines to standardize the investigations. The authors divide in 3 major groups the measure instruments: 3.1.1 Mechanical contact profilometers (contact stylus instruments) A pickup with a stylus is traversed over the surface at a constant velocity. The tip it’s coupled to a cantilever and, as passing through the relief, it is registered in an analog or digital signal. The tip has a radius of 2 or 10 µm and an angle of 60º or 90º. Naturally this will determine the smallest tips the tip can enter and the steepest slopes that may be measured. The tip returns to its starting point, scanning again the surface. This can give a 3D image. The tip is always in contact with the sample and is exposed to wear and contamination. The authors recommend inspection in a light microscope before measurement. Contact profilometers have been used almost exclusively for cylinder and flat samples investigations in dental research. In a screw design only parts of the screw can be analyzed with this method and, generally no information is given about precisely which regions of the screw were really measured. However, the measurements must refer to some areas without threads, since a mechanical contact profilometer cannot evaluate threaded regions, which is a limitation on this method. Another disadvantage is the possibility of damage to the surface of the implant from the load applied on the tip. Still, these instruments have a large horizontal measuring range (typical 100×100 mm) and a vertical range up to 8 mm. Doctoral Thesis Ana Rita Pereira - 36 - Figure 8: Diagram of the influence of tip radius on the measurement profile. A radius that is to large will result in a loss of information(33). 3.1.2 Optical Profilling Instruments They are non-contacting which make them attractive for biomaterials with soft and vulnerable surfaces. They are generally faster and have better resolution than mechanical contact instruments. There are a wide variety of these kind of instruments, ex.: focus detection system, confocal laser scanning microscopy, white light interferometer. 3.1.3 Scanning Probe Microscopes (SPM) Measure the interaction between a sharp tip and the sample surface. The most common technics are atomic force microscopy (AFM) and scanning tunnel microscopy. Doctoral Thesis Ana Rita Pereira - 37 - All these techniques have advantages and disadvantages. Mechanical contact profilometers have been used extensively in industry, whereas optical instruments and 3-D techniques are much newer and related to the development of computers. Optical measuring techniques and 3-D measures have attracted increased interest and standards are expected soon for calibration of the instruments and evaluation of the results. The different measuring equipment’s influence the results, and these results are even more influenced by how roughness measurement is separated from errors of form and waviness. For 2-D measurements, a cut-off length is used to remove errors forms. The standard filter for digital 3-D measurements is a Gaussian filter. For 3-D standards, the authors recommend the work by Stout and colleagues (34) that functions as a guideline and has gained international acceptance in surface metrology. Some other details are also pointed by the authors: - the possible importance of surface nanoscale measurements - at least 9 measurements on each screw (3 tops, 3 valleys and 3 flanks). Surface topography cannot be characterized well with only one parameter, since one surface may have the same height deviation but differ in spatial distribution when compared to other. Thus, different parameters should be included in evaluation. Nevertheless, there is still uncertainty about which set of parameters is the most suitable for implants evaluations, but at least some representative parameters from the 3 major groups must be included. The authors conclude the study, stating that confocal laser scanning profilometers and interferometers are the only acceptable methods available at present, able to evaluate densely threaded oral implant designs. For falt or cylindric implants, mechanical contact profilometers may additional be used. Preferably, 3-D measurements should be performed. In exceptional cases, 2-D measurements may be acceptable, but at least 25 scans should be performed no more than 20 µm apart. 2-D measurements must always be measured perpendicular to the main direction of the irregularities. On screw type implants, tops, valleys and flanks should be evaluated, in 3 different measuring areas. The measuring areas should be as large as possible to allow for different wavelengths inherent in the topography. Doctoral Thesis Ana Rita Pereira - 38 - 4 Surface Treatments Implant surface characteristics is a factor affecting the rate and extent of the implant bone response as well the quality of bone/interface(27, 35). A variety of different modified surfaces have been developed in the last years to improve the clinical performance of the titanium implants. Different methods are used for this purpose. Implants with smooth surfaces are not used mainly because their poor interaction with tissues, both hard and soft tissues. Smooth, polished surfaces show poor mechanical integration with bone. Without surfaces irregularities, such surfaces provide no resistance to mechanical forces at the bone implant interface. Furthermore, very smooth surfaces can allow epithelial downgrowth and are associated with deeper peri-implant pockets(36). Implant surface characteristics, such topography and chemical modifications of the titanium surface can be altered to accomplish rapid osseointegration and establish strong implant-bone contact (BIC)(37). Rough surfaced implants possess a higher bone to implant contact, favour biomechanical stability and enhance the production of cytokines and growth factors(4, 5). Different implant surface modification techniques are employed to enhance implant surface roughness and, at same time, surface energy and wettability, improving cell progression and osteoblastic differentiation. These surface treatments have evolved from simple modification of the oxide surface to precise nanoscale modification that involve the formation of a uniform and consistent surface that leads to altered cellular response(19). The main objective of the various techniques used for surface modification is to create irregularities on the surface. Some techniques add material to give positive irregularities; other techniques remove particles from the surface, creating negative pits on the surface(7, 19, 20, 37, 38). Doctoral Thesis Ana Rita Pereira - 39 - Figure 9: Implant surface technics(39) . 4.1 Additive treatments Additive treatments include titanium plasma sprayed (TPS), hydroxyapatite (HA) and other calcium phosphate coatings, ion deposition and oxidation. Plasma spraying is a kind of thermal spray which coats molten material on a shell. Partially molten particles of titanium powder are projected on the implant surface resulting in a rough surface containing round or irregular pore, pit and depression which helps in better wettability. Some disadvantages pointed out are the possibility of crack of the coating on implant insertion and bacterial infiltration(37). Calcium phosphate coatings, mainly composed of hydroxyapatite have osteoinductive and osteoconductive properties and good biointegration. The two main advantages of calcium phosphate materials include acceleration of early healing and its ability to bond to bone. It is suggested that the effect of calcium phosphate on accelerating early healing lies on its capacity to readily adsorb proteins to the surface. This could increase the binding of fibrinogen that in turn can result in an increase in platelet adhesion and activation. In addition, fibrin binding to the implant surface can serve as a bridge between the osteogenic cells and the implant surface. Doctoral Thesis Ana Rita Pereira - 40 - Some authors reported that bone adjacent to those implants is better organized and has a higher degree of mineralization(19, 39). Hydroxyapatite has some advantages over the plasma titanium coating: it shows faster healing of bone interface, increased healing between hydroxyapatite and bone, stronger interface, less corrosion. It also, after implantation, releases calcium phosphate into the periimplant region increasing the saturation of body fluids and precipitate a biological apatite in the surface of the implant. This layer of biological apatite might contain endogenous proteins and serve as matrix for osteogenic cell attachment and growth. The bone healing process around the implant is therefore enhanced by this biological apatite layer. Some disadvantages are pointed to plasma-spraying hydroxyapatite: - It is more susceptible to fatigue - is subject to drastic changes in the composition and crystallinity of the initial calcium phosphate powder - the strength of the bond between hydroxyapatite and substrate is questionable, being considered a weak point. Indeed, the most common problem with this coating surface is the separation from the metallic substrate, a phenomenon referred to as delamination(19, 36). A retrospective study (27, 40) put forward the hypothesis that delamination occurs due to the discrepancy in dissolution behavior between amorphous and crystalline calcium phosphate phases. The technique is also not very effective for coating tiny dental implants with a complex shape(35). Hydroxyapatite promotes bacterial adhesion(6). For the previous reasons, several studies have observed failures with hydroxyapatite implants and there for, several other techniques have been introduced in which calcium phosphate coatings are thin and there is an increase in bond strength between calcium phosphate and the implant surface(41). Some of these techniques are sol-gel deposition, pulsed laser deposition, sputtering coating techniques, electrophoretic deposition, ion-beam-assisted deposition and biomimetic precipitation. However, only the plasma-spraying coating method has been used for titanium dental implants in clinical practice(35). Doctoral Thesis Ana Rita Pereira - 41 - Figure 10: SEM micrographs of a titanium plasma-sprayed (TPS) surface(35). Figure 11: SEM micrographs of a titanium plasma-sprayed (TPS) surface(35). 4.2 Subtractive treatments Remove material from the surface. These modifications can be categorized at macro, micro and nano levels and these features are related to distinct effects during wound healing and osseointegration. Macro-level modifications are defined by visible geometry ranging on a millimeter scale, ex. implant body shape and thread patterns. It corresponds to a surface roughness of more than 10 µm. It gives the assessable implant surface area and the mechanical interlocking between the implant and the bone, achieving primary stability at implant placement. However, a major risk of a high roughness is an increase in periimplantitis as well as an increase in ionic leakage. A moderate roughness of 1-2 µm may limit these two parameters(35). Doctoral Thesis Ana Rita Pereira - 42 - Micro-level modifications increase implant’s surface area on a micrometer scale (range from 1 to 10 µm), reinforcing the mechanical interlocking and enhancing fibrin matrix formation as an osteoconductive scaffold for osteogenic cells and bone matrix deposition. It improves secondary integration including bone growth, turnover, remodeling and overall interlocking of bone at the implant interface. Bone growth is encouraged at cellular level as microroughness attracts differentiating osteogenic cells. It also encourages platelets to secret various mediators that help stabilize the blood clot and induce the formation of a fibrin matrix on the implant. This fibrin matrix plays the role of an osteoconductive scaffold for osteogenic cells to migrate and leading to bone formation at the surface of the implant. The microscale can also have an impact on biofilm formation since they have a similar topographical size as the colonizing microorganisms (~1µm). Roughness and wettability are affected. The most common micro-level modifications are machining, grit blasting and acid etching. Nano-level modification affects both the morphology and the chemistry of the implant surface(35). It can increase the implant’s surface roughness, wettability, and surface energy, enhancing cell growth and osteoblastic differentiation. Nanometer roughness size ranges between 1 and 100 nm and has an important role in the adsorption of proteins, adhesion of osteoblastic cells and thus the rate of osseointegration. Examples of nanometer modifications are laser ablation. These kind of treatments results in absorption of proteins and adhesion of osteoblast on implant’s surface, enhancing osseointegration at both cellular and protein levels(38). Doctoral Thesis Ana Rita Pereira - 49 - Bioactive drugs such like antibiotic can be bind to and released from calcium-based implants coatings(37). Tetracycline, for instance, in addiction to kill microorganisms, can also remove the smear layer, increase cell proliferation and inhibit collagenase activity hence it promotes enhanced cell attachment and bone healing(6). Bisphosphonates are agents that have strong chemical similarities to calcium phosphate particles. They can be combined with RGD peptides to produce synergistic osteogenic effects(37). However, chemical structure of bisphosphonates is attribute to bacterial attraction since it displays a direct electrostatic interaction with bacteria(38). Simvastatin is a drug used to decrease serum cholesterol concentration. In animal studies, simvastatin increased cancellous bone intensity as well as its compressive intensity. For this, it has been suggested simvastatin could promote bone formation by inducing expression of the bone morphogenetic protein BMP-2(6). Among the various questions that these approaches come up with are the sustained released of the molecules, since the active product as to be released progressively and not in a single burst(35). In literature, various available studies are very promising in this field of search. However, a systematic review (27) that includes several of the most representative implants, with in vitro, in vivo and animal studies, conclude that, despite efforts, there is no definitive proof of an clear advantage on thin-film calcium phosphate coatings neither in the use of extracellular matrix peptides sequences or proteins (growth factors) in coated titanium implant surfaces. 4.4 Surface energy, wettability and nanoroughness Although later in the case of topography and roughness, surface chemical composition and surface energy have been reported to influence interfacial reactions of a biomaterial, more specifically, the bone formation in vivo. Surface roughness is a determinant of surface wettability. Roughness and wettability both interfere with protein adsorption processes during the formation of macromolecular films, which in turn condition further cellular reactions(47). These became particularly evident from Doctoral Thesis Ana Rita Pereira - 50 - the moment that osseointegration starts to be seen as a healing process with all the necessity of interaction between the implant surface proteins and bone cells. Surface topography and roughness have already proved the superiority in the healing process. Combined micro and nano-scale modifications of titanium implants has shown beneficial conditions for the osteogenic cell growth, since it provides a random dimensional structure by mimicking the hierarchical structure of bone(43). Nevertheless, highly hydrophobic surface characteristic has been seen in these rough surfaces and it can be attribute to surface treatments, namely typical combined treatment grit blasted and acid etch in dental titanium implants. In this way, the continuous optimization of the topography has generated microrough and nanorough surfaces that induced very poor wetting. A possible reason given for this fact is the entrapped air below the wetting liquid contributing to an inhomogeneous and hydrophobic material/air-liquid interface(48). Likewise, titanium surfaces underly a reduction of bioactivity in ambient atmosphere due to carbon contamination accompanied by changes in surface energy and wettability. Researchers have been trying to develop strategies to clean, decontaminate and storage implants. One example is the technique of radio frequency glow-discharge (RFGD) treatments which allows to obtain decontaminated surfaces with high energy. Hydrocarbon layers, the most common residual contamination on solid surfaces, can be efficiently stripped by this cleaning process. By the previous, wettability and surface energy of implants is influenced by material, manufacturing process, cleaning, sterilization, and management of the implant throughout surgical process. Generally, positively charged surface is hydrophilic which is essential for the initial osteogenic interactions(49). A higher surface energy has been hypothesized to be desirable for bone implants because increased wettability enhances interactions between the implant and the biologic environment(50). Commercial brands have developed and launched to the market several technics to retain hydrophilicity of implant surfaces during longer storage times or to improve wetting by a direct treatment for immediate use. Several approaches have been created in the sense of changing Doctoral Thesis Ana Rita Pereira - 51 - these surfaces from hydrophobic to hydrophilic ones with very low water contact angles(5158). Figure 18: Different approaches enabling hydrophilization of dental implants and biomaterial surfaces to increase wettability with aqueous host bioliquids(51). 4.4.1 Wettability measurement In the same way that there is not a total consensus on the evaluation of microroughness, also, no guidelines exist concerning proper analysis in atomic force microscopy for nanoroughness measurement or even for the measurement of wettability. In relation to this last surface characteristic, measurement of wettability can be made by several methods(51, 59, 60): Doctoral Thesis Ana Rita Pereira - 52 - 4.4.1.1 Sessile drop method The most common approach. It is appliable on flat surfaces. In this technique a drop of a desired wetting liquid is placed on the surface of the specimen, and the angle between the tangent of the drop at the solid/liquid/gas three-phase boundary and the horizontal baseline of the solid is measured. This angle, the so-called contact angle (CA or θ), quantifies the wetting of the surface by the specific liquid used. The well-known Young Equation describes the balance at the three-phase contact of solid/ liquid/gas (the interfacial tension between solid and liquid): γSV = γSl + γlv cosθY Figure 19: Three-phase contact point where solid, fluid and gas meet. The interfacial tensions, γSV (Surface tension of the liquid), γSl (The interfacial tension between solid and liquid) and γlv (Surface tension of the solid, ex. surface free energy). This method only provides a static and approximate evaluation for microrough surfaces. The minimal possible drop size and volume of conventional drop shape analytic systems restrains measurements on very small areas of dental implants as between or on top of threads. Other factors that complicate the sessile-drop technique include variability in the chemistry of the wetting liquid as well changes in the vapor pressure and evaporating with temperature and time(59). Some trials have used small sessile drops condensed in the surface and analyzed in Doctoral Thesis Ana Rita Pereira - 53 - scanning electron microscope. Recently available droplet generation systems allowed drop volumes in the lower picoliter range with corresponding small evaluation of areas of wettability. The contact angle (CA) can range from 0º to 180º, indicating that the wetting liquid is being drawn towards the surface (ex. spreading of the top) or is being repelled by the surface (ex. beading of the drop), respectively. CA lower than 90º designate surfaces as hydrophilic, while CA close to 0º ascribe surfaces as superhydrophilic. Surface with CA above 90º are considered hydrophobic and above 150º superhydrophobic. Figure 20: Interrelations between wetting tension and the wetting of a solid. To improve wetting, the surface tension of the wetting liquid should be lower than the surface tension of the solid substrate(60). Doctoral Thesis Ana Rita Pereira - 54 - 4.4.1.2 Environmental scanning electron microscope (ESEM) It can give a better representation of the true nature of the wettability of surfaces with complex topography. ESEM provides enhanced spatial resolution and an environment with control pressure, temperature and humidity for microscale assessment of contact angle during nucleation, growth and coalescence of condensed droplets. The visualization of water condensation can help minimize confounding results by avoiding air entrapment between microscale surface roughness features, which may make the surface appear more hydrophobic. 4.4.1.3 Tensiometry In this technique, contact angles in a geometrically defined sample surface, are calculated based on force measurement during immersion in a wetting liquid with known surface tension. Tensiometry enables the direct access to dynamic advancing and receding contact angles during immersion/emersion loops – hysteresis loops. Wetting of dental implants screws was successfully tensiometrically analyzed by means of the Wilhelmy balance method. If a sample is repeatedly immersed during multiloop measurements, hysteresis can be distinguished in thermodynamic and time-dependent kinetic hysteresis, the latter becoming apparent by differences between the successive force-loops. Among several factors which are responsible for kinectics hysteresis, time-dependent changes of surface heterogeneity are a main reason, ex. caused by adsorption of proteins(47). To get a general idea of the contribution of the interfacial tensions acting at the three-phase line it should be referred the role of surface tension. Generally, surface tension is caused by the asymmetry of the cohesive forces of molecules at a surface compared to molecules in the bulk where each molecule has surrounding partners resulting in a net force of zero. Correspondingly, the surface energy is minimized in the bulk, whereas at the surface the energy is increased due to the missing surrounding molecules. Therefore, to reduce the surface energy, the surface area Doctoral Thesis Ana Rita Pereira - 55 - must be minimized, thus resulting in a phenomena like spherical water drops. A higher energetic surface presents the spreading of aqueous liquids. Wetting is favored by combining high energetic solid surfaces and wetting liquids with low surface tension. During wetting, the exposed area of a high energetic surface is reduced in favor of a solid/liquid interface, thus lowering the energy of the complete system as much as possible to reach a stable equilibrium state. A plausible way to enhance wetting, which has been associated with improved implant success, is there for to increase the surface tension of biomaterials. Besides Young’s equation, refered above in the sessil drop method, an additional equation is necessary to acquire information about the surface free energy of a solid material. Several empirical and semi-empirical approaches have been proposed to estimate surface energy. The geometric mean calculation of this surface parameter divides the total surface energy of a solid or a liquid in hidrogen bond (polar) and nonpolar/dispersive components. Owens and Wendt proposed a geometric mean to combine the polar and dispersion components of the solid and liquid surface interfacial energies. This new equation can be combined with Young’s equation. Thus, CA measurments with several test liquids can provide estimates of the polar and dispersion components of the surface energy. Other proposals have been being suggested to calculate these values (60) and to interpret the results of CA and wettability. Worth mentioning E. A. Vogler inference about values of hydrophilicity and hidrophobicity. In contrast to the earlier described, which technical division of hydrophilicity and hydrophobicity was at CA 90º, Vogler defined hydrophobic surfaces as those exhibiting water CA > 65º, corresponding to an adhesion tension < 30mN cm-1. Should also be also mentioned in a succinct way, that not always the Young’s equation is a consensual approach, even because real surfaces are characterized by a certain roughness. For instance, in dynamic evaluation like in the tensiometry technique, and with the analyze by Wilhelmy balance method, another wetting phenomenon, already mention previously, can be observed during dynamic measurements: contact angle hysteresis. The definition of contact angle hysteresis is the difference between the advanced contact angle and the receding contact angle for a contact line moving in an opposite direction at the same velocity. When the contact Doctoral Thesis Ana Rita Pereira - 56 - angle hysteresis is large, the surface has probably some chemical or roughness inhomogeneities. In other words, hysteresis is proportional to the force required to start moving a droplet on the surface. For instance, lower hysteresis contact angle is the result of a higher surface hydrophobicity. An increase in hysteresis can be attribute to the surface heterogeneity of an inconsistent overlayer adsorbed protein, whereas a decrease in hysteresis is caused by surface homogenization due to further protein adsorption. The general idea that must be retained is that contact angle measure and wetting analysis is of essential importance. From a theoretical point of consideration, the valid surface free energy of a solid material based on Young’s equation can be only performed using contact angle drops that are resting in thermodynamic equilibrium on an ideal solid surface. Although, surface energy calculations are generally based on the Young equation, using the most stable contact angle that can be achieved. From all the above, recently it has been seen in all fields of investigation, an attempted to improve the implant’s surfaces characteristics managing the morphology, wettability and bioactivity combining micro and nano-scale modifications and even functionalization with protein, peptides and bioactive compounds. Such modifications determine the early bone formation around the implants by inducing the migration and differentiation of osteogenic cells followed by enhancement of the mineral matrix formation that accelerate the osseointegration process. The wettability is believed to have a preponderant role in the initial process of osseointegration since it influences the adsorption of molecules from the physiologic environment and then cells receptors bind to functional groups of the adsorbed proteins(61). For instance, the impact of hydrophilicity in blood proteins is seen by the different processes that can be triggered depended on the adherent proteins to the surface: activation of the complement system, adhesion and activation of thrombocytes, adhesion and expression of polymorphonuclear leucocytes and/or maturation of dendritic cells. This influence is notorious on the behavior of fibronectin, a fundamental element of extra cellular matrix. Fibronectin adsorbed to hydrophobic surfaces shows a marked reduction in cell-adhesive function. On hydrophilic surfaces fibronectin improves cells response. The kind of protein or/and its orientation leads to different cells response such as adhesion, spreading, proliferation and several aspects of differentiation, and these may be seen on the behavior of osteoblast, fibroblast or keratinocytes(62). Doctoral Thesis Ana Rita Pereira - 57 - Osteoblast grown in modified surfaces presenting hydrophilicity characteristics exhibit a more differentiated phenotype, characterized by increased alkaline phosphatase and osteocalcin activity and generate an osteogenic microenvironment through higher production of PGE2 and TGF-b1(50, 61). Titanium implants with high surface energy and low water contact angle values reveal a high blood wettability and affinity for protein adsorption that enhance the osseointegration process(43). Surface nano-scale features can increase the adsorption of proteins and stimulate the osteogenic cell migration leading to the osseointegration speeding up. Topographies in the nanometer range, differently from micrometre-roughned surfaces, are not able to have a mechanical impact on implant retention in the bone, whereas they mainly act at molecular and cellular level(26). Procedures like grit blasting increases the titanium roughness at micro-scale while etching procedures can modify the roughness at nano-scale. The combination of these two procedures results in a nanostructure surface. Nano-structured implant surfaces have been studied to shorten the time of osseointegration. Another examples of surface nanoroughness treatments, some of them already implemented and other in study and development, are anodization, that depending on the specifications may create nanotubes in implant surface, and laser treatments. This kind of technology is an emerging field not only for the created nanoroughness but also for the possibility to delivery certain drugs and coat the surface with several peptides, polymers and other bioactive molecules. Nanoroughness mimics the natural structure of bone, and therefore enhance the bone healing process(43). Doctoral Thesis Ana Rita Pereira - 58 - Figure 21: Schematic illustration of interrelations between roughness and wettability and the biological response at biomaterial-biosystem interfaces after implantation(47). From the cited above some ideas should be retained: - Roughness and wettability of implants may influence initial macromolecular biological responses such as the adsorption of plasma proteins - Implant surfaces with high surface energy and low contact angle presents higher wettability - Nanoroughness increase protein adsorption, cell adhesion leading to a faster initial osseointegration - Protein bind itself changes the wettability of a surface - Static methods such as static sessile drop method lack an important part of information about the time dependencies of initial reactions of implants in aqueous systems - Dynamic wettability studies should be used to investigate structured rough implants to reveal most possible information about time-depended changes in the wetting behavior. Doctoral Thesis Ana Rita Pereira - 65 - Figure 25: Number of MG63 osteoblast-like cells released at 24h(66). Figure 26: [3H]-thymidine incorporation by MG63 osteoblast-like cells during culture on plastic or Ti disks(66). Doctoral Thesis Ana Rita Pereira - 66 - Figure 27: Alkaline phosphatase specific activity of cell layers produced by MG63 osteoblast-like cells after culture on Ti disks(66). Figure 28: Alkaline phosphatase specific activity of MG63 osteoblast-like cells after culture on Ti disks(66). Doctoral Thesis Ana Rita Pereira - 67 - Figure 29: Osteoclacin production by MG63 osteoblast-like cells during culture on Ti disks(66). Figure 30: Percentage collagen production by MG63 osteoblast-life cells during culture on Ti disks(66). Doctoral Thesis Ana Rita Pereira - 68 - Figure 31: [35S]-sulfate incorporation by MG63 osteoblast-like cells during culture on Ti disks(66). Figure 32: Latent transforming growth factor b (LTGFb) production by MG63 osteoblast-like cells during culture on Ti disks(66). Doctoral Thesis Ana Rita Pereira - 69 - Figure 33: Prostaglandin E2 (PGE2) production by MG63 osteoblast-like cells during culture on Ti disks(66). 6 Genetic response It has been shown that the insertion of a dental implant changes the nature of bone healing, with an alteration of osteogenesis-associate mechanisms being observed. For this process, not only contribute the implant characteristics, namely the surface properties, as also the molecular biology of the host. Only recently, the molecular basis of osseointegration has been considered. However, recent activities have begun to highlight both the fundamental processes that contribute to interfacial bone formation and how alterations in the implant surface may alter the biologic mechanisms, which lead to bone formation at the alloplastic/tissue interface. It is possible to investigate the molecular processes through in vitro and different in vivo models of animals (mouse, rat, minipig, rabbit) and, more recently, through human studies. It has been seen a broad consensus in most of the existing studies relating to this issue. One possible explanation for this alignment is that the early question asked, regarding the molecular process underlying osseointegration, was directed toward known aspects of bone formation and repair in general. A systematic review by Thalji, G and Cooper, L. F (1) considered about 30 articles of in vivo studies about this subject. The spectrum of molecular events target in all Doctoral Thesis Ana Rita Pereira - 70 - the studies was narrow. Osteoinduction (the process by which primitive, undifferentiated cells are stimulated to develop into osteoprogenitor cells) was represented by the expression or abundance of Runx2 and Osx genes, which are key transcriptional regulators of stem cell commitment to osteoblastogenesis. Osteogenesis was reflected by the expression of bone specific or enriched protein-encoding mRNAs, including type I collagen (col 1), osteopontin (OPN), oasteonectin (ON), bone sialoprotein (BSP) and decorin. Greater expression of bonespecific proteins was suggestive of greater osteogenesis and more bone formation. All the reported studies included in this systematic review pointed out an increased expression of mRNAs with time and with the “enhanced” on surface topography. One general conclusion of the authors was that, regardless of possible modulation of mRNA abundance quantitatively or over time, osseointegration involves osseoinduction from progenitor cells and the subsequent elaboration of a bone matrix composed of the basic components involved in bone repair. Also was seen a clear progression of cellular events indicated by gene expression. The majority of the included studies focused on the effect of implant surface on the molecular process of osseointegration and the general observation was that surface topography influenced the pattern of expression of bone related proteins. Doctoral Thesis Ana Rita Pereira - 71 - Figure 34: The process(es) that lead to this result are dependent on the differentiation of mesenchymal stem cells into osteoblasts by the process of osteoinduction. The key transcriptional regulators that control osteoblastogenesis are Runx2 and Osterix. One important question regarding implant surface influence on osseointegration is: “what is the effect of surface on the expression of these transcriptional regulators in implantadherent or adjacent cells?” Committed osteoblasts elaborate a collagen-rich matrix that is embellished with bonespecific extracellular matrix (ECM) proteins that control tissue formation and mineralization. Many studies have revealed that the nature of the implant surface alters the expression of bone-specific ECM proteins. These fundamental relationships between the implant and cells that produce bone matrix are accompanied by many other cell type-implant relationships that have not yet been elucidate(1). Another systematic review (71) on this theme, focused on in vitro studies using only osteoblast-like osteosarcoma MG63 cells. The authors conclude that osteoblasts were induced to differentiate and produce a mineralized matrix in response to titanium surfaces, which was associated with an osteogenesis-associated gene expression profile(4, 71). The described in vitro studies supported the hypothesis that the presence of a titanium surface, especially surface roughness, may have direct effects on osteoblast migration, attachment, proliferation and differentiation, which is associated with an osteogenesis-associated gene-expression profile. Another study, using a human model(67), evidence a temporal gene transcriptional change during osseointegration. An earlier stage of proliferation and immune-inflammatory response Doctoral Thesis Ana Rita Pereira - 72 - involves genes expression which are ultimately replaced by genes associated with the process of expression of extracellular matrix, as well morphogenesis and differentiation processes, which is consistent with a maturing wound. In the early phase (day 4) exists an over-expression of a variety of cytokines (TNF-a, IL-2, IL-6), as well as the proliferation and/or activation of different immune-inflammatory system-related cells. The major signalling pathway that was seen positively regulated at day 4 was the I-kB kinase/NF-kB cascade. This pathway is associated with inflammation, play a key role in inflammation-induced bone loss and the regulation of this pathway is particularly important considering the apparent critical role of bone debris to the osseointegration. The TGF-b/BMP and Notch signalling pathways were predominantly represented at day 14, as was Ras and Rho protein signal transduction. At day 14 there was an over-expression of genes associated to the regeneration-related mechanisms of skeletogenesis, angiogenesis and neurogenesis. Most of the genes were up-regulated at day 14, with very few genes being up-regulated at day 4. Among the extracellular matrix-related genes that were up-regulated at day 14 compared with the day 4 were a few collagens (Type V, XI, XIII) as well as the broadly recognized osteogenesis-associated genes like osteocalcin, osteonectin and alkaline phosphatase. This original study, despite some limitations, provided a broad overview of biological processes and signalling pathways that are involved in the process of osteointegration, leaving pointed some potential targets mechanisms for modulating the osseointegration process. Also highlight several mechanisms like neurogenesis, which have not received much attention. It probably plays a relevant role in osseointegration, suggesting that neurogenic tissues are regenerated during this process; it has been postulated that neuropeptide Y may also modulate osseointegration and some literature point the eventual participation of the nervous system in bone function. In this line of human models and regarding gene expression profile of osseointegration, another study (69) following observations at 4, 7 and 14 days, compared two kinds of surfaces: moderately rough and a chemical modified hydrophilic moderately rough surface. At initial period of 4 days there were not see significantly differences. However, by day 7, osteogenesis and angiogenesis-associated gene expression were up regulated in the chemical modified surface, being regulated apparently by BMP and VEGF signalling pathways, respectively. At the final period of 14 days, the chemical modified surface maintained an up regulation of VEGF pathway, but BMP pathway increased in moderately rough surface as, probably, a Doctoral Thesis Ana Rita Pereira - 73 - compensatory response. Very interestingly was to notice, again, a prominent neurogenic process in both surfaces. In general: - the study of gene profile during the process of osseointegration have confirmed the important role of surface topography and/or hydrophobicity associated with this feature. The influence has a large range that includes the initial process of inflammation until the wound healing, osteogenesis and bone mineralization. 7 Hormonal response The process of bone formation and its regulation is complex. Osteoblasts respond to local factors such growth factors and cytokines through autocrine and paracrine mechanisms. In addition, these local regulatory events occur in a background of endocrine regulation involving systemic hormones such as vitamin D3 and other estrogens. In vitro MG63 cells respond to 1a,25-(OH)2D3, an active metabolite of vitamin D3, in several ways. 1a,25-(OH)2D3 inhibits cell proliferation, increases alkaline phosphatase expression and activity, and increases collagen Type I and osteocalcin production. This indicates that 1a,25-(OH)2D3 stimulates MG63 cells to differentiate into a more osteoblastic phenotype. A pioneer study from Boyan, B.D and colleagues (72) demonstrates that surface roughnessdependent changes in MG63 cells can modify how the cells respond to extrinsic regulatory agents, such as 1a,25-(OH)2D3. The authors corroborated the previous statements. Further add that roughest surfaces, in their in vitro study, had a markedly enhanced response to vitamin D3; indeed, it was synergistic with the effect of surface roughness alone. The authors advanced the hypothesis that cellular growth on the rougher surfaces result in a more differentiated cellular phenotype, with a corresponding change in the response to 1a,25-(OH)2D3. It also was possible that 1a,25-(OH)2D3 altered the response of MG63 to surface roughness by promoting the differentiation of the cells into a more mature osteoblastic phenotype. Treatment with the vitamin D metabolite increased osteocalcin production demonstrating that osteoblastic Doctoral Thesis Ana Rita Pereira - 74 - differentiation occurs. The authors made their initial point with respect to growth factors and cytokines production getting production of both PGE2 and LTGFb increased in rough surfaces. They conclude that surface roughness and not the 1a,25-(OH)2D3 was the primary factor sensitizing the cell. Lohmann, C.H (73) and colleagues had based is some studies that examined the response of bone cells to the surface roughness to show that cell maturation play a role in the results. The authors used three distinctly different stages of osteoblastic differentiation: undifferentiated osteoblasts, well differentiated osteoblasts and osteocytes. All originated in cell lineage from rat/mouse (animal study). The authors also analyzed the effect of surface roughness on the response of those cells to 1a,25-(OH)2D3. In accordance with the previous authors, this work confirmed that osteoblasts are sensitive to surface roughness with cell proliferation being inhibited and the amount of PGE2, TGF-b1 and osteocalcin being increased as a function of roughness. The results led to believe that cell attachment to the surface also may be dependent on cell maturation state. It was confirmed the idea that surface roughness and not 1a,25-(OH)2D3 was the primary trigger for phenotypic expression. 1a,25-(OH)2D3 had market stimulatory effects in more immature cells. The study showed that surface roughness is not only the overriding variable in early wound healing, but also in latter osteoblastic differentiation, and affects even terminally differentiated osteocytes that are involved in maintenance of the bone-implant interface. Another observation of this study was that exists cells specific differences in response to surface roughness. In this case, and contrary to what other authors stated, phosphatase alkaline had diminished in rough surfaces, especially when compared with MG63 cells. Supposelly, it could be related to an alteration of the chemical surface between the studies and a small change in Ti hydride particles that could be sufficiently important to modulate cell response. Doctoral Thesis Ana Rita Pereira - 81 - Material and Methods Material To determine the effect of compressive residual energy on osteoblasts cells behavior in rough and smooth titanium surfaces, eighty (80) discs of commercially pure titanium (grade 3) were tested. The eighty disks were the result of analyze tests in which was considered the necessity of sixty (60) disks for the study. Although, another twenty disks were added in case of any occurrence. The Cp-Ti grade 3 discs used in this study measured 5 mm diameter and 2.5 mm thickness. The microstructure of Cp-Ti disks was composed by a particles equiaxiales of commercial pure titanium, as can be seen in figure 36. Figure 36: Titanium grade 3 microstructure, composed by equiaxiales a particles. Doctoral Thesis Ana Rita Pereira - 82 - Disks preparation All the discs were washed with 70% ethanol, acetone, distilled water, dried at room temperature and sterilized in an autoclave. The disks were divided equitably into four groups and each group was composed by twenty (20) disks: - Group 1: smooth titanium surface with no residual stress (S) - Group 2: smooth titanium surface with residual stress (S+RS) - Group 3: rough titanium surface with residual stress (R+RS) - Group 4: rough titanium surface without residual stress (R) Shot blasting treatment Smooth disks were obtained from regular manufacturing processes and no additional treatment for roughness has been made posteriorly (group 1 and group 2). Rough disks (group 3 and group 4) were shot-blasted with 600 micrometer-sized alumina at a distance of 100 mm with a pressure of 2.5 bar for 120 s, in order to obtain such treatment as Klockner Dental Implants, Escaldes-Engordany, Andorra. The discs obtained showed roughness and had a residual surface tension due to the projection of the abrasive particles. Roughness can be changed by altering the size of the shooting particles, pressure, velocity of the shoot and the distance from the device to the object. Figure 37 shows a detail of shot blasting machined. Doctoral Thesis Ana Rita Pereira - 83 - Figure 37: Shot blasting device used to prepare rough surfaces. Determination of Residual Stress on titanium surfaces To determine the compressive stress to be applied to the smooth discs, the residual stress of these discs was first determined by X-ray diffraction (Bragg-Bentano). Residual stresses were measured with a diffractometer incorporating a Bragg– Bentano configuration (D500, Siemens, Munich, Germany). The measurements were performed for the family of planes (213), which diffracts at 2θ = 139.5°. The elastic constants of Ti at the direction of this family of planes are EC = (E/1 + ν)(213) = 90.3 (1.4) GPa. Eleven Ψ angles, 0°, five positive, and five negative angles, were evaluated. The positions of the peaks were adjusted with a pseudo-Voigt function using appropriate software (WinplotR, free access on-line), and then converted to interplanar distances (dΨ) using Bragg’s equation. The d Ψ vs. sen2Ψ graphs and the calculation of the slope of the linear regression (A) were performed with appropriate software (Origin, Microcal, Piscataway, NJ, USA). The residual stress is σ = EC(1/d0)A; where d0 is the interplanar distance for Ψ = 0°(75). Doctoral Thesis Ana Rita Pereira - 84 - Figure 38: Rx to determine residual stress. Application of mechanical stress to obtain residual stress on smooth the surface Once the residual stress value was known, mechanical compression tests were carried out on the smooth discs of group 2 (S+SR). The residual stress was obtained through a compression test, performed with a Bionix MTS electromechanical machine (MTS, Minneapolis, MN, USA). The crosshead speed was 1 mm/min. In order to obtain a residual stress very similar to that produced by grit blasting, in groups 3 and 4, the stress applied for this residual stress was 678 ± 35 MPa within the plastic field of the disc, reaching a compressive stress of 978 MPa. Doctoral Thesis Ana Rita Pereira - 85 - Figure 39: Mechanical testing machine to produce surface tension. Elimination of compressive surface tension on the treated surface by shot blasting After the same blasted treatment of group 3 with 600 micrometer-sized alumina at a distance of 100 mm with a pressure of 2.5 bar for 120 s, the group 4 disks were heat treated at a temperature of 800 °C for 30 min under vacuum with a vacuum furnace (Hobersal 6300XB, Caldes de Montbui, Spain) in order to remove the residual stress. These thermal treatments are called annealing treatments. The temperature is used to eliminate the residual stress and any sort of defects. The treatment is performed in vacuo to avoid titanium surface oxidation, which could alter the results of the study. Doctoral Thesis Ana Rita Pereira - 86 - Figure 40: Heat treatment oven to diminish the residual stress of implants treated with grit-blasting. Figure 41: inside the oven. Doctoral Thesis Ana Rita Pereira - 87 - Roughness avaliation Three different specimens of each of the four groups were measured to determine the amplitude parameter (Sa), the maximum peak value (Sz), and the hybrid parameter (index area). White light interferometry (Wyko NT1100 Interferometer, Veeco Instruments, USA), in the vertical scanning interferometry mode, was used to produce, evaluate and quantify the topographical features of the tested surfaces. The interferometric technique is ideal for imaging these surfaces as a large area of the surface can be imaged with a high vertical resolution (≈2 nm). The analysis area was 124.4×94.6 µm. Data analysis was performed with Wyko 32 (Veeco Instruments, USA), which allows the application of a Gaussian filter to separate waviness and form from roughness. In Figure 42, the scheme of the different roughness parameters studied can be observed. Doctoral Thesis Ana Rita Pereira - 88 - Figure 42: Roughness parameters scheme. Sa expresses, as an absolute value, the difference in height of each point compared to the arithmetical mean of the surface. This parameter is generally used to evaluate surface roughness. The maximum height Sz is equivalent to the sum of the maximum peak height Sp and maximum valley depth Sv. Wettability/Contact angle measurment The contact angle analysis, and consequently the wettability of surface, was performed on n = 3 samples of which of four groups, with ultrapure distilled water (Millipore Milli-Q, Merck Millipore Corporation, Darmstadt, Germany) and formamide (Contact Angle System OCA15plus, Dataphysics, Filderstadt, Germany), and the corresponding data were analyzed with an SCA20 goniometer (Dataphysics, Filderstadt, Germany). Contact angle measurements were made using the sessile drop method. Drops were generated with a micrometric syringe and were deposited over discs. A total of 3 μL of distilled water and 1 μL of formamide were deposited on each sample at 200 μL/min. Doctoral Thesis Ana Rita Pereira - 89 - Figure 43: Contact angle equipment. Surface free energy measurment The surface free energy was determined by applying the Owens, Wendt, Rabel, and Kaelble (OWRK) equation, with wettability values obtained with distilled water and formamide, and the Wenzel equation for the correction of contact angles with the roughness. Total surface energy and the Lifshit-van der Waals (LW) and the acid and basic components of surface energy for all the series were determined from contact angle measurements using three liquids on each material: ultrapure distilled water (MilliQ), formamide, which are polar liquids, and di-iodomethane, which is a non-polar liquid. A series of four drops (3 l/drop) were placed on the samples, and the contact angle, θ, was measured using the Sessile Drop method. Doctoral Thesis Ana Rita Pereira - 90 - A series of at least four drops were used in three trials, for a total of 12 contact angle measurements per liquid on a particular materials surface. Contact angles (CA) were determined semi manually from the image of the drop with an accuracy of ±1º. The measurements were obtained at room temperature (T=25ºC) in an environmental chamber previously saturated with the liquid of study. To determine the components of acid, base and LW surface tensions of a material surface A and liquid B the Young equation was used: γSV = γSL+ γLV•cosθ (Equation 1) Where θ, γSV , γSL , γLV , represent the contact angle, solid (S)/vapour(V), solid/liquid (L) and liquid/vapour surface energies respectively, is combined with the interfacial energy (γSL) equation between two surfaces. In the case of a solid surface and a liquid surface, (denoted S and L respectively) this relation is given by, γL (1+ cos θ) = 2(γL LWγSLW)1/2 + 2(γL+ γS - )1/2 + 2(γL - γS+)1/2 (Equation 2) γS = γS LW+ γS AB (Equation 3) γS AB=2(γS¯ · γS+)1/2 (Equation 4) where γLW is due to the interactions arising from induced dipoles (Lifshit-van der Waals), γ+ is the Lewis acid or electron acceptor and γ¯ is the Lewis base or electron donor component(76-78). From a practical point of view, the Lifshit-van der Waals and the electron donor and electron acceptor parameters of solid surfaces can be calculated by contact angle measurement with liquids of known parameters, through a system of three equations and three unknowns. Thus, the contact angle measurement with, at least three liquids, is required. The electron donor-electron acceptor approach is, as of today, the most advanced theory and the one that uses the presently accepted physical knowledge to account for interfacial interactions(79). Doctoral Thesis Ana Rita Pereira - 97 - Results Table 1 show the roughness parameters in the four different study surfaces. The index area is also stated. Sa expresses, as an absolute value, the difference in height of each point compared to the arithmetical mean of the surface. Sz, is an average measure, equivalent to the sum of the maximum peak height (Sp) and maximum valley depth (Sv), of the surface. These rough values (vertical and horizontal measures) are altered according to the dimension, velocity and pressure of the applied grit particles. Sa μm Sz (μm) Index Area S 0.21 ± 0.02 * 0.34 ± 0.02 * 1.09 ± 0.01 * S+RS 0.24 ± 0.10 * 0.41 ± 0.11 * 1.08 ± 0.06 * R 2.04 ± 0.15 ** 4.67 ± 1.07 ** 1.66 ± 0.04 ** R+RS 1.99 ± 0.18 ** 4.67 ± 1.07 ** 1.76 ± 0.04 ** Table 1: Roughness parameters Sa, Sz, and index area. The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks. The difference present at p < 0.001. Although denominated smooth surfaces, the previous results evidence the inherent roughness that these surfaces present, and this is the result of the fabrication process. As expected, the disks with blasted treatment show a higher degree of roughness. Doctoral Thesis Ana Rita Pereira - 98 - The difference in roughness between smooth and rough surfaces is statistically different, and the R and R+RS surfaces have a roughness statistically higher that both S and S+Rs surfaces. The index area represents the raise in the surface that will be in contact with the biological environment, in this case, with bone cells. In S and S+RS surfaces, the index area values are very similar, showing no differences after applying the compressive residual treatment. The same observation can be made relatively to both rough surfaces, with and without compressive residual treatment. Although, as would be expected, the index area value from rough surfaces (R and R+RS) show a raise of about 166-176% in comparison to both smooth surfaces (S and S+RS). In table 1, we can also understand that whatever is the surface, the compressive residual treatments do not alter the roughness property. Contact angle CA (◦) S 77 ± 5 * S+RS 58 ± 3 ** R 69 ± 4 * R+RS 53 ± 2 ** Table 2: Contact angle (CA). The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks. The difference present at p < 0.001. The contact angle, in accordance with the results found in literature, is higher in smooth disks, which can be translate in a smaller wettability of these surfaces. On the contrary, and as expected, the rough surfaces presented statistically evidence of diminished contact angle, inferring a higher wettability of these surfaces. Doctoral Thesis Ana Rita Pereira - 99 - Surprising, and in accordance with our hypothesis, both rough and smooth surfaces, that presents residual stress, had the lower contact angle values. Even the smooth surface with residual stress presented a lower value comparing with the rough surface without residual stress. This highlights the importance of the value of the residual value, possible overlapping the rough parameter, and the influence that it may have in the hydrophobic and hydrophilic behavior of the surfaces. This value represents the quantity of proteins that will be adsorbed in the surface. The lowest value of contact angle represents a higher value in wettability and a more hydrophilic surface. The more hydrophilic surface the more adsorbed proteins in the surface. DC (mJ/m2) PC (mJ/m2) Total SFE (mJ/m2) S 24.8 ± 1.2 * 10.2 ± 2.0 * 35.0 ± 3.2 * S+RS 27.2 ± 1.2 ** 18.3 ± 1.8 ** 45.5 ± 2.2 ** R 27.7 ± 1.3 ** 12.5 ± 2.1 * 40.2 ± 1.2 *** R+RS 29.0 ± 2.2 ** 20.4 ± 1.9 ** 49.4 ± 1.8 ** Table 3: Dispersive component of the surface energy (DC), polar component (PC), total surface energy (SFE). The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks and those marked with three asterisks show statistically significant differences with respect to those marked with two asterisks. The difference present at p < 0.001. Surface energy is a measure of the energy at the surface of the material and results from an incomplete bound of the surface atoms. The surface energy comprises a nonpolar component and a polar component. The nonpolar or dispersive component implies weak interactions like Van-der-Waals forces. The polar component comprises all the other forces, like hydrogen forces, and include forces between permanent dipoles and between permanent and induced dipoles. The Owens and Wendt Doctoral Thesis Ana Rita Pereira - 100 - analyze/equation express the total surface free energy as a sum of dispersion and polar intermolecular forces. A surface will always try to minimize its surface energy, and this can be done through the adsorption process. Raising the total SFE makes the surface to become more reactive and, from the table 3, we can observe that S+RS and R+RS surfaces have the highest values of SFE. Though, these surfaces show the most reactive surfaces comparing to S and R, respectively. It is of major importance to note that the polar component is the one that suffers a highest augmentation with the residual force treatment: smooth surfaces show values of 10.2 ± 2.0 mJ/m2 and after RS suffer a rise to 18.3 ± 1.8 mJ/m2. Rough surfaces present 12.5 ± 2.1 mJ/m2 that alter after RS treatment to 20.4 ± 1.9 mJ/m2. The polar component promotes a negative energetic cloud in the surface. In biologic fluids this will be translate in a preferred surface adsorption of positive charges, like fibronectin. It has been suggested that Arg-Gly-Asp (RGD) sequence, in fibronectin, is in an active state on hydrophilic surfaces, which will increase the attachment of osteoblastic cells and consequently the osseointegration process(82). The total surface energy showed higher results in disks with residual stress, as expected and in accordance with our expectations. Doctoral Thesis Ana Rita Pereira - 101 - σresidual (MPa) S −10 ± 2 * S+RS −189 ± 20 ** R −8 ± 3 * R+RS −201 ± 12 ** Table 4: Residual stress (σresidual). The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks. The difference present at p < 0.001. Residual stress is an elastic response to non-uniform plastic deformation. This residual stress can be achieved by surface finish, like machining or grit blasting or by material phase changes during a heat treatment(83). The x-ray diffraction method is widely used for residual stress measurement(84). In our study, the residual stress was applied by high pressure of aluminum oxide particles and mechanical compression. Although, as can be seen in table 4, the smooth surfaces that passed through only for machining process, also present some residual stress. In the case of S and R surfaces the σresidual value is nearly 0 (zero). The compressive nature of the residual compressive stress gives to the value presentation a negative sign. The residual stress value presente significant higher values in smooth and rough disks in which this component was raised. Although, outside the scope of our study, it can be remembered that fatigue behavior of implants submitted to grit blasting treatment is better due to the compressive effect of the residual stresses on the surface that difficult the crack nucleation. In this case, the grit blasting treatment improves not only the osseointegration of the implants due to the increase in the metal surface roughness but also their fatigue life thanks to the layer of compressive residual stresses that is formed(84). Doctoral Thesis Ana Rita Pereira - 102 - Table 5: Proliferation of Saos-2 cells on surfaces after 3 days of incubation. The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks and those marked with three asteriscs show statistically significant differences with respect to those marked with two asterisks. The differences present at p < 0.001 . Table 5 and 6 shows the number of osteoblastic cells on the different surfaces, demonstrating a greater number on both smooth and rough surfaces where there is compressive residual stress, with statistically significant differences p < 0.001 for each of them. However, the rough surfaces R and R+RS presents the highest values even in comparison with S+SR, demonstrating that increased surface area of rough surfaces create the ideal conditions to adhesion and proliferation of osteoblastic cells. Although, it can be seen the synergic effect that residual stress produces with roughness surfaces to the osteoblastic behavior. As referred before, the more reactive titanium surface with residual compressive stress is due to the raise in wettability values and the alteration in surface energy trough the polar component. In the smooth surface, the residual compressive stress also increased the osteoblastic proliferation. Doctoral Thesis Ana Rita Pereira - 103 - Table 6: Proliferation of Saos-2 cells on surfaces after 14 days of incubation. The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks and those marked with three asterisks show statistically significant differences with respect to those marked with two asterisks. The differences present at p < 0.001. The same trend is also observed in the levels of ALP, showing a better osteoblast differentiation on surfaces with residual stress (Table 7). The results clearly demonstrate that roughness favors cellular responses with respect to smooth surfaces. In fact, the topography of titanium surfaces affects the osteoblastic response towards bone regeneration the most. Doctoral Thesis Ana Rita Pereira - 104 - Table 7: Production of ALP by Saos-2 cells on different surfaces. The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks and those marked with three asterisks show statistically significant differences with respect to those marked with two asterisks. The differences present at p < 0.001. The mineralization activity of osteoblastic cells can be seen through the values of phosphatase alkaline (ALP). This value gives the capacity of the cells to produce new bone tissue. Surfaces with residual compressive stress (S+RS and R+RS) demonstrate higher values of ALP comparing to S surfaces. The table also demonstrates that roughness favors the mineralization activity, even because S+RS and R do not present significant different values of ALP activity. Data demonstrates the value of residual compressive stress in ALP activity and, once again, the synergistic behavior that it has with roughness surfaces given that R+RS presents the higher value of ALP. Doctoral Thesis Ana Rita Pereira - 105 - Table 8: Streptococcus Sanguinis colonies (gram positive) adhered on the different surfaces studied. Samples with the same symbol show no statistically significant differences between them. The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks. The differences present at p < 0.001. Doctoral Thesis Ana Rita Pereira - 106 - Table 9: Lactobacillus Salivarius colonies (gran negative) adhered on the different surfaces studied. Samples with the same symbol show no statistically significant differences between them. The results marked with one asterisk show statistically significant differences with respect to those marked with two asterisks. The differences present at p < 0.001. The quantification of colony forming units (CFUs) per square millimeter (p < 0.001) can be observed in Table 8 and 9. Both strains showed a low trend in attaching to smooth (L. salivarius~1×102/mm2, S. sanguinis~8.22×103/mm2) compared to rougher surfaces. The smooth samples with residual stress show values of 1.53 × 102 for Lactobacillus salivarius and 8.70 ×103 for Streptococcus sanguinis with respect to the values of 1.00 ×102 and 8.22×103, respectively. These results do not show statistically significant differences. When we studied the influence of residual stress on the rough samples, a slight increase in colony formation by both bacteria was observed: from 2.20×102 to 2.35×102 for Lactobacillus salivarius and from 9.80×103 to 10.32×103 for Streptococcus sanguinis. However, the influence of residual stress does not present statistically significant differences with respect to the samples with no residual stress. Doctoral Thesis Ana Rita Pereira - 113 - Osteocalcin, a late osteoblast differentiation marker, was also increased by surface energy. The modSLA surfaces values were 40% higher than in SLA surfaces. With these results, the authors managed to prove that prevention of rough Ti surfaces from contact with air reduced hydrocarbon contamination, increases surface energy and hydrophilicity extensively on rough Ti surfaces without changing surface topography. Osteoblast cells cultures on these cleaner, higher surface energy and hydrophilic surfaces produced more differentiation markers represented by increased cell layer alkaline phosphatase specific activity and osteocalcin. Osteocalcin have not been subject of our study, although osteocalcin is among the most abundant proteins in bone and is produced primarily by osteoblasts and, in smaller quantities, also produced by odontoblasts of the teeth and hypertrophic chondrocytes. It has been used as a marker of osteoblastic bone formation and believed to act in the bone matrix to regulate mineralization, but new genetic and pharmacological evidence points to a hormonal role for the protein. It is also referred as γ-carboxyglutamic acid (Gla) protein or BGP(50). The idea that bone is an endocrine organ secreting a growing number of hormones, and the concept that osteocalcin secreted by osteoblasts is responsible for several physiological processes such like energy metabolism, brain development, cognition and gonadal functions had gain emphasis through several works, namely with the work and experiments of Karsenty, G and colleagues(50, 96). The investigations carried out by this author have been based in mouse genetics. Other authors like Manolagas (97) refute those ideas claiming, between other justifications, that osteocalcin genetics and function differ between humans and mice; for instance, humans have a single osteocalcin gene, whereas in mice, there are two adjacent osteocalcin genes. Besides that, other discrepancies between osteocalcin and obesity, insulin resistance, testosterone synthesis or muscle mass have not been found. Anyway, osteocalcin has a mechanical role in the bone matrix leading to alignment and tightly bind of biological apatite crystallites parallel to collagen fibers influencing bone quality and resistance to fracture. Some other works had been more compatible with the notion that osteocalcin functions as an inhibitor of bone mineralization since it inhibits the precipitation of calcium salts from saturated solutions. Therefore, the precise role of osteocalcin within the bone matrix remains unclear(98). Doctoral Thesis Ana Rita Pereira - 114 - Bone is built by a process of self-assembly under control of a gene regulatory network (GNR). Chekroun and colleagues (70) studied the behaviour of bone gene regulation as a control of mineralization using mathematical equations. The author estimated direct interactions between genes encoding transcription factors and those encoding bone proteins, as well as performing mathematical modelling of the bone GNR using a system of nonlinear differential equations modelling the interactions. The author showed negative indirect interactions from negative feedback loops or micro-RNAs. The study showed theoretical evidence of osteoblast self-inhibition following activation of the genetic regulatory network controlling mineralization. Bone metabolism is very complicated and further research is still needed to improve understanding. With respect to the bacterial colonies study, in our work, the quantification of bacteria attachment was performed with two normal inhabitants of the mouth: S. sanguinis and L. salivarius. Streptococcus presents a membrane with a hydrophobic character and Lactobacillus a hydrophilic membrane. Both strains presented a real significant statistically difference which favors attachment to rough surfaces. Regarding surfaces with residual stress, it has been seen a slight increase in both strains in these surfaces; the raise had been more notorious on roughness surfaces but even there the difference between values was not statistically relevant. It could be though that the variation in wettability caused by residual stress would favor bacterial adhesion to hydrophobic or hydrophilic surfaces, depending on the character of the bacterial membrane. The results may be explained by the fact that the residual stress may be not sufficient to observe behavior changes in bacteria, but it was sufficient for osteoblastic cells. Contribution of bacteria to implant failure starts from their attachment to the implant. Surface properties such as material composition, roughness and wettability can affect bacterial adhesion to the surfaces. Bacterial growth on the surface can show different behaviour depending on the bacterial species even under the same surface conditions(99). Several studies have made efforts to understand the contribution of the bacterial infection to the survival of dental implants. In this context, Choi, S (99) and collaborates carried out a study with the purpose to investigate the effect of surface properties on biofilm formation on the implant surface. For that, they used one early colonizer, Streptococcus gordonii (S. Doctoral Thesis Ana Rita Pereira - 115 - gordonii) and two late colonizers: Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis). Titanium and zirconia were tested with two types of surfaces: roughness and machined, both in 24 and 72 hours each. The surface roughness of the smooth surfaces was less than 0,2 µm and for rough surfaces the value range between 1 and 2 µm (Sa value) which was recommended for effective osseointegration. Due to the different physical properties of titanium and zirconia, different processes of surface treatment were required to have the same level of roughness. The three bacteria exhibit the highest growth on the rough titanium surface and the lowest growth on the smooth zirconia surface for 72h of proliferation. The authors noted that the bacterial adhesion and proliferation seems more influenced by the bacteria species rather for the material or surface roughness. The contact angle, which is related to the surface energy, was also analysed in this study. All surfaces presented different values of contact angles; however, all of them were less than 90º, indicating hydrophilic surfaces. Even this factor did not evidence consistent correlation with the extent of bacterial film. These results can be correlated with the ones in our study, in which we could not determinately find a consistent relationship with values of residual energy. With the objective of investigate the properties of different implant materials and the affinity of each one to bacteria adhesion, an in vitro study was accomplished by Al-Rada and colleagues(100). They used zirconia and titanium discs in different versions: polished partially stabilized zirconia, titanium blasted with zirconia, titanium blasted with zirconia and acid etching and polished titanium. Contact angle, surface free energy, surface microhardness and chemical composition were analysed. Streptococcus mitis and Prevotella nigrescens have been the strains used. Also, the discs have been or not precoated with saliva. In general, the results demonstrated zirconia material and titanium blasted with zirconia with lower surface energy and lesser surface wettability. Also, these surfaces had the lowest bacterial adhesion especially after coating with saliva. The authors highlight as cause of these results the strong effect of material type on surface wettability and contact angle since zirconia and titanium blasted with zirconia did not have significant differences in results. The zirconia discs had the same roughness has the polished titanium, so the effects could not be related with roughness. The authors claim that the ability of zirconia to decrease bacterial adhesion could be attributed to its lower surface energy. The significant reduction of bacterial adhesion to the surfaces coated with saliva was hypothesized by a direct effect of saliva in transferring the Doctoral Thesis Ana Rita Pereira - 116 - lower surface energy, “shine through theory”, or indirectly by affecting the type of proteins adsorbed to the substrate, as the bacteria have different affinities to different proteins. Wassman, T and colleagues (101) used zirconia and titanium surfaces, in vitro, with different roughness and wettability to study bacterial adhesion and define the predominant factor for bacterial adhesion for each material. Selected strains were Streptococcus sanguinis and Staphylococcus epidermidis, both hydrophobic. They start from the assumption that the type of implant material and its specific texture and physico-chemical surface properties influence the quantity and quality of microbial colonization. Not only implant surfaces are, nowadays, modified to increase osseous integration, but also to reduce biofilm formation after exposure to the oral cavity. In this way, zirconia has been introduced as an alternative to titanium implants, mainly because of their supposedly reduced potential to adhere microorganisms. For a better classification of the surface roughness and trying to eliminate possible discrepancies, in this study the authors applied atomic force microscopy (AFM) for a three-dimensional assessment of the surface topography of the tested materials. The titanium and zirconia specimens were subject to the same treatments to modify surface roughness and surface free energy. The results indicated no statistically differences on S. epidermis for the different rough surfaces (titanium and zirconia), except for smooth titanium on which significantly more adhering bacteria were found. In general, significantly more S. sanguinis adhere to ceramic surfaces than to titanium, except for smooth ceramic compared to rough titanium, where a higher Ra led to an increased adhesion. Relatively to the wettability, S. epidermis showed higher bacterial adhesion on hydrophobic than on hydrophilic surfaces, but these differences were not statistically significant. In general, the potential to adhere S. sanguinis was significantly higher for all ceramics surfaces – hydrophobic and hydrophilic – than for titanium specimens, with no significant statistically differences in the wettability changes. No preferences could be found to S. epidermis. An explanation advanced by the authors to these results advocate that titanium is coated by a layer of surface oxide, which physical and mechanical characteristics are more closely related to ceramic than to metal. This phenomenon leads to a similar protein bind properties on both surfaces. This could explain why zirconia did not show any reduced bacterial adhesion in the study. S. epidermis showed higher adhesion to hydrophobic surfaces probably because of the hydrophobic properties of this strain and because the thermodynamic model of microbial adhesion. Doctoral Thesis Ana Rita Pereira - 117 - On the contrary of the previous authors and attending to the idea that specific ranges of roughness favour not only osteoblast proliferation but also adhesion and proliferation of bacteria, Robles, D and colleagues (102) proposed a researched with the objective of physicochemical characterization of surfaces in hybrid implants and comprehension of osteoblastic and microbiological behaviour in those. The authors used titanium grade 3 discs with different surfaces: smooth, smooth-rough and completely rough. Human osteoblasts (SaOS-2) were used and two common bacterial strains: Enterococcus faecalis and Streptococcus gordonii. Roughness was determined as also wettability and surface energy. Hybrid implants have two types of surface finishes. The coronal part, up to the third turn, has machined titanium (smooth) and the apical part, from the third turn, has a rough surface. The machined coronal part with very small roughness values hinders bacterial adhesion and therefore reduces biofilm formation. This effect is probably due to several reasons, including the decreased wettability. This smooth surface also causes a decrease in cell adhesion. The rough side favors bacterial and osteoblast adhesion. Hybrid implants have showed good behavior, marginal bone stability and fewer biologic complications, preventing periimplantitis. Figure 47: Microstructures of smooth and rough zones of the hybrid implant and their 3D topographic maps(102) . In this study, the abrasive projection with alumina in the rough discs, decreased the wettability of the surface and increased the contact angle. This surface became more Doctoral Thesis Ana Rita Pereira - 118 - hydrophobic with respect to the smooth surface. Comparing the contributions of the dispersive and polar components of the surface energy, the polar component decreased in a significant manner in the rough surfaces with residual alumina. Cell morphology, obtained by fluorescent microscopy, revealed polygonal shape in smooth surface and irregular shape on rough surface. The behavior of cells denoted statistically differences: the cells had a large area in the smooth samples compared with the shot blasted ones. These last samples presented cells scattered over the entire surface, while in the smooth samples, cells formed clusters in specific areas. The authors referred that an explanation for the difference in shape and adhesion of osteoblast may reside in the limiting effect of the rough topography on osteoblast extension and, osteoblastic cells on rough surfaces present a greater number of focal points and filopodia than those on smooth surfaces. The results lead to a higher mineralization in rough surfaces that increased progressively from the first day to day 14, and then decreased until day 21. This behavior is well described in literature and indicates an onset on cell differentiation after a certain time. The authors observed, in a clear way, a greater adhesion of human osteoblasts on rough surfaces referring that the values of roughness and surface energy had a greater influence than the contact angle. Furthermore, in the study, although the rough surface had a greater contact angle than the smooth surface, the difference was around 10º, which not produced a greater difference in the hydrophilicity of the surface and do not play a decisive role in osteoblastic adhesion. Bacteria metabolism presented significant differences between the surfaces, and, in this case, smooth discs had a lower bacterial adhesion. The authors refer that the residual tension caused by the projection of particles at high pressure to form roughness facilitates colonization. The compressive stress allows the bacteria to establish a better adhesion with the titanium. The study concludes that hybrid implants have a smooth coronal surface that does not have optimal properties for osteoblast but hinders the bacterial colonization, what taking in account the coronal area close to the oral cavity may prevent bacterial infection biofilm formation. In alignment with this last work, Rodriguez-González, R and colleagues (103) launched the question that rough surfaces presented a greater capacity for osteoblastic differentiation and a greater adhesion of bacterial plaque on its surface. For this study have been used grade 4 Doctoral Thesis Ana Rita Pereira - 119 - titanium discs with different treatments: machined titanium, acid-etch titanium, titanium sprayed with abrasive alumina particles under pressure and titanium sprayed with abrasive alumina particles and then subject to acid-etching. The strains used were Streptococcus gordonii, Streptococcus oralis, Actinomyces viscosus and Enterococcus faecalis. As expected, the samples treated with blasting and blasting acidetches presented the roughness surface with changes in the wettability, making them slightly more hydrophobic and with lower surface energy. These surfaces influenced both cell proliferation as well as the osteogenic differentiation of SAOs-2 cells. In principle, the degree of hydrophilicity should increase the degree of osseointegration, but the rough topography favored adhesion, proliferation and osteoblastic differentiation as well as lower internal energy. Regarding the initial bacteria adhesion on the different surfaces, no significant differences were found between the different treatments, with a tendency for the machined samples to have a lower number of adhered bacteria, but without being significant. Thus, not statistically difference was explained due to the smaller size of the bacteria with respect to the cells, which makes them insensitive to the topography obtained on the four surfaces studied. Doctoral Thesis Ana Rita Pereira - 120 - Conclusion From the results of our investigation and from consequent discussion of these, we reached the following conclusions. - Compressive residual stress promotes an increase in wettability and in the polar component of surface energy, which raises the osteoblastic behavior of Saos-2. Moreover, we can conclude that exist a synergic behavior between roughness and compressive residual stress that favors biologic activity. - Residual compressive stress promotes an increase in smooth and rough surfaces wettability through the diminished contact angle from 77º ± 5º to 58º ± 3º and 69º ± 4º to 53º ± 2º, respectively. - Compressive residual stress promotes an augmentation of the total value of surface free energy, indicating a higher reactivity aspect of these surfaces. The polar component gives the higher contribution for this augmentation, promoting an electrostatic environment which favors protein adsorption and consequent cell adhesion. - Hydrophilic behavior increases through the increase in compressive residual stress. - Statistically higher difference exists when compressive residual stress is increased, in both smooth and rough surfaces, in osteoblastic adhesion and proliferation by cell/cm2. Also, an increase in ALP activity is observed in this case, showing that compressive residual stress favors mineralization and, consequently, osseointegration. - From microbiologic study, we can conclude that roughness favors bacterial colonization of both study strains. Although no significant changes in these bacteria behavior were seen in compressive residual stress alteration. This kind of studies are necessary to improve implants surface treatments to enhance biological response achieving faster and more effective osseointegration. Doctoral Thesis Ana Rita Pereira - 121 - For further research, it would be interest to do in vivo observations, use different values of compressive residual stress stablishing an energy limit value for which biologic behavior is positively influenced. Also determine if compressive residual stress may have any influence in corrosion behavior of titanium surface. We can also suggest new studies using titanium alloys instead cp titanium. It can also be interest the use of different oral cavity bacterial strains colonizers, looking for the interaction inside colonies environment, with the residual stress tension alteration. Doctoral Thesis Ana Rita Pereira - 122 - Bibliography 1. Thalji G, Cooper LF. Molecular assessment of osseointegration in vivo: a review of the current literature. Int J Oral Maxillofac Implants. 2013;28(6):52134. 2. Schroeder A, van der Zypen E, Stich H, Sutter F. The reactions of bone, connective tissue, and epithelium to endosteal implants with titanium-sprayed surfaces. J Maxillofac Surg. 1981;9(1):15-25. 3. Stanford CM. Application of oral implants to the general dental practice. J Am Dent Assoc. 2005;136(8):1092-100;165-6. 4. Salvi GE, Bosshardt DD, Lang NP, Abrahamsson I, Berglundh T, Lindhe J, et al. Temporal sequence of hard and soft tissue healing around titanium dental implants. Periodontol 2000. 2015;68(1):135-52. 5. Elias CN, Oshida Y, Lima JH, Muller CA. Relationship between surface properties (roughness, wettability and morphology) of titanium and dental implant removal torque. J Mech Behav Biomed Mater. 2008;1(3):234-42. 6. Barfeie A, Wilson J, Rees J. Implant surface characteristics and their effect on osseointegration. Br Dent J. 2015;218(5). Doctoral Thesis Ana Rita Pereira - 129 - 52. Lang NP, Salvi GE, Huynh-Ba G, Ivanovski S, Donos N, Bosshardt DD. Early osseointegration to hydrophilic and hydrophobic implant surfaces in humans. Clin Oral Implants Res. 2011;22(4):349-56. 53. Orsini G, Assenza B, Scarano A, Piattelli M, Piattelli A. Surface analysis of machined versus sandblasted and acid-etched titanium implants. Int J Oral Maxillofac Implants. 2000;15(6):779-84. 54. Buser D, Broggini N, Wieland M, Schenk RK, Denzer AJ, Cochran DL, et al. Enhanced bone apposition to a chemically modified SLA titanium surface. J Dent Res. 2004;83(7):529-33. 55. Sarinnaphakorn L, Di Silvio L. Surface-modified titanium to enhance osseointegration in dental implants. In Cellular Response to Biomaterials. 2009;572-588. 56. Wall I, Donos N, Carlqvist K, Jones F, Brett P. Modified titanium surfaces promote accelerated osteogenic differentiation of mesenchymal stromal cells in vitro. Bone. 2009;45(1):17-26. 57. Vlacic-Zischke J, Hamlet SM, Friis T, Tonetti MS, Ivanovski S. The influence of surface microroughness and hydrophilicity of titanium on the upregulation of TGFβ/BMP signalling in osteoblasts. Biomaterials. 2011;32(3):665-71. 58. Wennerberg A, Svanborg LM, Berner S, Andersson M. Spontaneously formed nanostructures on titanium surfaces. Clin Oral Implants Res. 2013;24(2):203-9. Doctoral Thesis Ana Rita Pereira - 130 - 59. Gittens RA, Olivares-Navarrete R, Cheng A, Anderson DM, McLachlan T, Stephan I, et al. The roles of titanium surface micro/nanotopography and wettability on the differential response of human osteoblast lineage cells. Acta Biomater. 2013;9(4):6268-77. 60. Rupp F, Gittens RA, Scheideler L, Marmur A, Boyan BD, Schwartz Z, et al. A review on the wettability of dental implant surfaces I: theoretical and experimental aspects. Acta Biomater. 2014;10(7):2894-906. 61. Kilpadi DV, Lemons JE. Surface energy characterization of unalloyed titanium implants. J Biomed Mater Res. 1994;28(12):1419-25. 62. Gittens RA, Scheideler L, Rupp F, Hyzy SL, Geis-Gerstorfer J, Schwartz Z, et al. A review on the wettability of dental implant surfaces II: Biological and clinical aspects. Acta Biomater. 2014;10(7):2907-18. 63. Sculean A, Stavropoulos A, Bosshardt DD. Self-regenerative capacity of intra-oral bone defects. J Clin Periodontol. 2019;46 Suppl 21:70-81. 64. Blumer MJF. Bone tissue and histological and molecular events during development of the long bones. Ann Anat. 2021;235:151704. 65. Sakka S, Coulthard P. Bone quality: a reality for the process of osseointegration. Implant Dent. 2009;18(6):480-5. 66. Lincks J, Boyan BD, Blanchard CR, Lohmann CH, Liu Y, Cochran DL, et al. Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition. Biomaterials. 1998;19(23):2219-32. Doctoral Thesis Ana Rita Pereira - 131 - 67. Ivanovski S, Hamlet S, Salvi GE, Huynh-Ba G, Bosshardt DD, Lang NP, et al. Transcriptional profiling of osseointegration in humans. Clin Oral Implants Res. 2011;22(4):373-81. 68. Velasco-Ortega E, Fos-Parra I, Cabanillas-Balsera D, Gil J, Ortiz-García I, Giner M, et al. Osteoblastic Cell Behavior and Gene Expression Related to Bone Metabolism on Different Titanium Surfaces. Int J Mol Sci. 2023;24(4). 69. Donos N, Hamlet S, Lang NP, Salvi GE, Huynh-Ba G, Bosshardt DD, et al. Gene expression profile of osseointegration of a hydrophilic compared with a hydrophobic microrough implant surface. Clin Oral Implants Res. 2011;22(4):365-72. 70. Chekroun A, Pujo-Menjouet L, Falcoz S, Tsuen K, Yueh-Hsun Yang K, Berteau JP. Theoretical evidence of osteoblast self-inhibition after activation of the genetic regulatory network controlling mineralization. J Theor Biol. 2022;537:1-12. 71. Bächle M, Kohal RJ. A systematic review of the influence of different titanium surfaces on proliferation, differentiation and protein synthesis of osteoblast-like MG63 cells. Clin Oral Implants Res. 2004;15(6):683-92. 72. Boyan BD, Batzer R, Kieswetter K, Liu Y, Cochran DL, SzmucklerMoncler S, et al. Titanium surface roughness alters responsiveness of MG63 osteoblast-like cells to 1 alpha,25-(OH)2D3. J Biomed Mater Res. 1998;39(1):7785. Doctoral Thesis Ana Rita Pereira - 132 - 73. Lohmann CH, Bonewald LF, Sisk MA, Sylvia VL, Cochran DL, Dean DD, et al. Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D3. J Bone Miner Res. 2000;15(6):116980. 74. Lohmann CH, Tandy EM, Sylvia VL, Hell-Vocke AK, Cochran DL, Dean DD, et al. Response of normal female human osteoblasts (NHOst) to 17betaestradiol is modulated by implant surface morphology. J Biomed Mater Res. 2002;62(2):204-13. 75. Velasco E, Monsalve-Guil L, Jimenez A, Ortiz I, Moreno-Muñoz J, Nuñez-Marquez E, et al. Importance of the Roughness and Residual Stresses of Dental Implants on Fatigue and Osseointegration Behavior. In Vivo Study in Rabbits. J Oral Implantol. 2016;42(6):469-76. 76. Vogler EA. Structure and reactivity of water at biomaterial surfaces. Adv Colloid Interface Sci. 1998;74:69-117. 77. J D. Static contact angles for liquids at heterogeneous rigid solid surfaces. Polish J Chem 1997;71:525-549. 78. Good R. Contact Angle, Wetting, and Adhesion: A Critical Review. Journal of Adhesion Science and Technology - J Adhes Sci Technol. 1992;6(12):1269-1302. 79. Morra M, Cassinelli C. Bacterial adhesion to polymer surfaces: a critical review of surface thermodynamic approaches. J Biomater Sci Polym Ed. 1997;9(1):55-74. Doctoral Thesis Ana Rita Pereira - 133 - 80. Buser D, Mericske-Stern R, Bernard JP, Behneke A, Behneke N, Hirt HP, et al. Long-term evaluation of non-submerged ITI implants. Part 1: 8-year life table analysis of a prospective multi-center study with 2359 implants. Clin Oral Implants Res. 1997;8(3):161-72. 81. Klinge B, Flemming T, Cosyn J, De Bruyn H, Eisner BM, Hultin M, et al. The patient undergoing implant therapy. Summary and consensus statements. The 4th EAO Consensus Conference 2015. Clin Oral Implants Res. 2015;26 Suppl 11:64-7. 82. Wei J, Igarashi T, Okumori N, Maetani T, Liu B, Yoshinari M. Influence of surface wettability on competitive protein adsorption and initial attachment of osteoblasts. Biomed Mater. 2009;4(4):1-7. 83. Dlhý P, Poduška J, Pokorný P, Jambor M, Náhlík L, Hutař P. Residual stress determination by the layer removal and X-ray diffraction measurement - correction method. MethodsX. 2022;9:1-9. 84. Gil FJ, Espinar E, Llamas JM, Sevilla P. Fatigue life of bioactive titanium dental implants treated by means of grit-blasting and thermo-chemical treatment. Clin Implant Dent Relat Res. 2014;16(2):273-81. 85. Godoy-Gallardo M, Manzanares-Céspedes MC, Sevilla P, Nart J, Manzanares N, Manero JM, et al. Evaluation of bone loss in antibacterial coated dental implants: An experimental study in dogs. Mater Sci Eng C Mater Biol Appl. 2016;69:538-45. Doctoral Thesis Ana Rita Pereira - 134 - 86. Rakic M, Galindo-Moreno P, Monje A, Radovanovic S, Wang HL, Cochran D, et al. How frequent does peri-implantitis occur? A systematic review and meta-analysis. Clin Oral Investig. 2018;22(4):1805-16. 87. Chan HL, Lin GH, Suarez F, MacEachern M, Wang HL. Surgical management of peri-implantitis: a systematic review and meta-analysis of treatment outcomes. J Periodontol. 2014;85(8):1027-41. 88. Monje A, Pons R, Amerio E, Wang HL, Nart J. Resolution of periimplantitis by means of implantoplasty as adjunct to surgical therapy: A retrospective study. J Periodontol. 2022;93(1):110-22. 89. Katsikogianni M, Missirlis YF. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions. Eur Cell Mater. 2004;8:37-57. 90. Boyan BD, Lohmann CH, Dean DD, Sylvia VL, Cochran DL, Schwartz Z. Mechanisms involved in osteoblast response to implant surface morphology. Annu. Rev. 2001;31:357-71. 91. Pieuchot L, Marteau J, Guignandon A, Dos Santos T, Brigaud I, Chauvy PF, et al. Curvotaxis directs cell migration through cell-scale curvature landscapes. Nat Commun. 2018;9(1):1-13. 92. Degasne I, Baslé MF, Demais V, Huré G, Lesourd M, Grolleau B, et al. Effects of roughness, fibronectin and vitronectin on attachment, spreading, and proliferation of human osteoblast-like cells (Saos-2) on titanium surfaces. Calcif Tissue Int. 1999;64(6):499-507. Doctoral Thesis Ana Rita Pereira - 135 - 93. Aragoneses J, Valverde NL, Fernandez-Dominguez M, Mena-Alvarez J, Rodriguez C, Gil J, et al. Relevant Aspects of Titanium and Zirconia Dental Implants for Their Fatigue and Osseointegration Behaviors. Materials (Basel). 2022;15(11):1-15. 94. Orimo H. The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J Nippon Med Sch. 2010;77(1):4-12. 95. Vimalraj S. Alkaline phosphatase: Structure, expression and its function in bone mineralization. Gene. 2020;754:1-8. 96. Karsenty G, Ferron M. The contribution of bone to whole-organism physiology. Nature. 2012;481(7381):314-20. 97. Manolagas SC. Osteocalcin promotes bone mineralization but is not a hormone. PLoS Genet. 2020;16(6):e1008714. 98. Zoch ML, Clemens TL, Riddle RC. New insights into the biology of osteocalcin. Bone. 2016;82:42-9. 99. Choi S, Jo YH, Luke Yeo IS, Yoon HI, Lee JH, Han JS. The effect of surface material, roughness and wettability on the adhesion and proliferation of. J Dent Sci. 2023;18(2):517-25. 100. Al-Radha AS, Dymock D, Younes C, O'Sullivan D. Surface properties of titanium and zirconia dental implant materials and their effect on bacterial adhesion. J Dent. 2012;40(2):146-53. Doctoral Thesis Ana Rita Pereira - 136 - 101. Wassmann T, Kreis S, Behr M, Buergers R. The influence of surface texture and wettability on initial bacterial adhesion on titanium and zirconium oxide dental implants. Int J Implant Dent. 2017;3(1):321. 102. Robles D, Brizuela A, Fernández-Domínguez M, Gil J. Osteoblastic and Bacterial Response of Hybrid Dental Implants. J Funct Biomater. 2023;14(6):321. 103. Rodriguez-González R, Monsalve-Guil L, Jimenez-Guerra A, VelascoOrtega E, Moreno-Muñoz J, Nuñez-Marquez E, et al. Relevant Aspects of Titanium Topography for Osteoblastic Adhesion and Inhibition of Bacterial Colonization. Materials (Basel). 2023;16(9):3553. Doctoral Thesis Ana Rita Pereira - 137 - Appendix Citation: Pereira, R.; Maia, P.; Rios-Santos, J.V.; Herrero-Climent, M.; Rios-Carrasco, B.; Aparicio, C.; Gil, J. Influence of Titanium Surface Residual Stresses on Osteoblastic Response and Bacteria Colonization. Materials 2024,17, 1626. https://doi.org/10.3390/ ma17071626 Academic Editors: Gilberto Sammartino, Marco Tatullo and Giuseppina Ambrogio Received: 3 March 2024 Revised: 27 March 2024 Accepted: 28 March 2024 Published: 2 April 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). materials Article Influence of Titanium Surface Residual Stresses on Osteoblastic Response and Bacteria Colonization Rita Pereira 1,PauloMaia2,JoseVicenteRios-Santos1,MarianoHerrero-Climent3,BlancaRios-Carrasco1, Conrado Aparicio 4and Javier Gil 5,* 1Facultad de Odontología, Universidad de Sevilla, Calle Avicena s/n, 41009 Sevilla, Spain; [email protected] (R.P.); [email protected] (J.V.R.-S.); [email protected] (B.R.-C.) 2Facultade Ciências da Saúde, Universidad Europeia de Lisboa,1500-210 Lisboa, Portugal; [email protected] 3Porto Dental Institute, 4150-518 Porto, Portugal; [email protected] 4Facultad de Odontología, Universitat Internacional de Catalunya, c/ Josep Trueta s/n, 08195 Sant Cugat del Vallés, Spain; [email protected] 5Bioengineering Institute of Technology, Universidad Internacional de Catalunya, c/ Josep Trueta s/n, 08195 Sant Cugat del Vallés, Spain *Correspondence: xavier[email protected] Abstract: Grit basting is the most common process applied to titanium dental implants to give them a roughness that favors bone colonization. There are numerous studies on the influence of roughness on osseointegration, but the influence of the compressive residual stress associated with this treatment on biological behavior has not been determined. For this purpose, four types of surfaces have been studied using 60 titanium discs: smooth, smooth with residual stress, rough without stress, and rough with residual stress. Roughness was studied by optic interferometry; wettability and surface energy (polar and dispersive components) by contact angle equipment using three solvents; and residual stresses by Bragg–Bentano X-ray diffraction. The adhesion and alkaline phosphatase (ALP) levels on the different surfaces were studied using Saos-2 osteoblastic cultures. The bacterial strains Streptococcus sanguinis and Lactobacillus salivarius were cultured on different surfaces, determining the adhesion. The results showed that residual stresses lead to increased hydrophilicity on the surfaces, as well as an increase in surface energy, especially on the polar component. From the culture results, higher adhesion and higher ALP levels were observed in the discs with residual stresses when compared between smooth and roughened discs. It was also found that roughness was the property that mostly influenced osteoblasts’ response. Bacteria colonize rough surfaces better than smooth surfaces, but no changes are observed due to residual surface tension. Keywords: titanium; grit blasting; dental implants; residual stress; osteoblasts 1. Introduction Dental implants represent a valid therapeutic option for the replacement of missing teeth [ 1 ]. The development of implantology has made it possible to broaden the scope of dental treatment in that, through the placement of an implant, it is possible to provide longterm stable support for a dental prosthesis subjected to masticatory load [ 1 , 2 ]. The biological principles underlying the functioning of implants have been described by several authors and are summarized in the concept of osseointegration. Osseointegration is defined as the direct and structural connection of living and ordered bone with the surface of an implant subjected to a functional load [ 3 ]. The first studies on this phenomenon were carried out by Branemark in the 50s, 60s, and 70s [ 4 , 5 ], and in parallel by Schröeder [ 6 – 8 ], who were able to demonstrate that the alveolar bone is able to form a direct connection with an alloplastic material such as titanium in the form of a screw, once placed in a surgically created bed [ 9 – 11 ]. Implant surface characteristics have been shown to influence bone healing of the surrounding bone [ 2 ]andit Materials 2024,17, 1626. https://doi.org/10.3390/ma17071626 https://www.mdpi.com/journal/materials Doctoral Thesis Ana Rita Pereira - 138 - Materials 2024,17, 1626 2 of 13 has been histologically demonstrated that osseointegration can be achieved in a time range of six weeks under normal conditions with rough surfaces [11]. The morphology of these surfaces is involved in a number of biological events that occur after implant placement, ranging from protein adhesion to peri-implant bone remodeling. These phenomena are favored by a certain surface roughness allowing a faster osseointegration which, from the clinical point of view, leaves room for the possibility of placing the prosthesis in shorter times. Usually, dental implants have roughened surfaces to optimize the processes of adhesion, proliferation, and osteoblastic differentiation, with the aim of generating bone around the dental implant and obtaining a mechanical and biological fixation [ 12 , 13 ]. The most common method of obtaining this topography is by grit blasting, in which abrasive particles, usually aluminum oxide, are projected to create a surface with a specific roughness. As is well known, osteoblastic cells are sensitive to roughness between 1 and 2 µ m of Sa, and these values can be achieved by optimizing factors such as the particle projection pressure, distance from the projection gun to the surface, and size of the abrasive particles, among others [ 14 , 15 ]. Roughness optimization studies should also consider the affinity of bacteria for roughness. It is well known that increases in roughness favor bacterial adhesion and favor the creation of biofilms that can lead to peri-implantitis disease [ 16 ]. For this reason, roughness values have been adjusted to Sa between 0.7 to 1.2 µ m, so that they provide a balanced roughness topography that favors osteoblastic response without notably increasing bacterial colonization [17,18]. Many studies have studied the influence of surface roughness on the biological response in vitro and in vivo, but the influence of surface compressive residual stresses as a result of grit particle projection has not been determined yet. For this purpose, smooth and rough titanium without and with residual stresses have been studied here. The hypothesis of this research is that the residual surface tension generated by the grit blasting treatment used to roughen the surface enhances osteoblastic cellular and microbiological activity. This hypothesis is based on the fact that the residual stress causes an increased hydrophilicity on the surface of the dental implant, favoring protein adsorption and facilitating osteoblastic cell adhesion as well as bacterial colonization. 2. Materials and Methods 2.1. Materials Eighty discs of commercially pure titanium (grade 3) (Klockner Dental Implants, Escaldes-Engordany, Andorra) were tested and divided into four groups: •Smooth titanium with no residual stress (S); •Smooth titanium with residual stress (S + RS); •Rough titanium without residual stress (R); •Rough titanium with residual stress (R + RS). Twenty original smooth discs formed the first studio group (S). Another twenty smooth discs were subjected to a compression test, reaching a compressive stress. The mechanical tests were performed with a Bionix MTS electromechanical machine (MTS, Minneapolis, MN, USA) (S + RS). The crosshead speed was 1 mm/min. The other 40 discs were shot-blasted with 600 micrometer-sized alumina at a distance of 100 mm with a pressure of 2.5 bar for 120 s. The discs obtained showed roughness and had a residual surface tension due to the projection of the abrasives (R + RS). Twenty of these discs were heat treated at a temperature of 800  C for 30 min under vacuum with a vacuum furnace (Hobersal 6300XB, Caldes de Montbui, Spain) in order to remove the residual stress (R). To determine the compressive stress to be applied to the smooth discs, the residual stress of these discs was first determined by X-ray diffraction (Bragg-Bentano) (Siemens, Munich, Germany), as will be explained in the following sections. Once this was known, mechanical compression tests were carried out on the smooth discs in order to obtain a residual stress very similar to that produced by grit blasting. The stress applied for this residual stress was 678 ±35 MPa within the plastic field of the disc. Doctoral Thesis Ana Rita Pereira - 145 - Materials 2024,17, 1626 9 of 13 Materials 2024, 17, x FOR PEER REVIEW 9 of 13 producing an increase in fractures, as can be seen in Figure 5, where the dental implants fracture at the connection necks with the screw that joins the implant and abutment. Studies have been carried out with conventional implants and it has been determined that on many occasions conventional implants made of commercially pure titanium cannot withstand physiological stresses. Therefore, there are two ways to solve this situation: the manufacture of dental implants with an alloy that increases the maximum resistance of the implant, as is the case with the Roxolid® implants of Straumann; or the manufacture of implants with Optimum® titanium that are cold worked at 30%, such as those from Klockner Dental Implants. Bacteria colonize rough surfaces better than smooth surfaces, but no changes are observed due to residual surface tension. Currently, this last type of implant obtains better results since it produces a better osseointegration due to the compressive residual stress caused by the cold working. However, alloyed titanium increases the mechanical properties of the implant, but does not favor osteoblastic activity, as in the case of cold-worked implants [38–40]. Figure 5. Fractures on the neck of the implant produced by fatigue in dental implants made of commercially pure titanium. Osteoblast cell culture studies show that cell proliferation values are higher in both smooth and rough osteoblast samples. This fact can be justified by the higher hydrophilicity values generated by surface tension [34–36]. Likewise, the surface energy values, and especially their polar component, causes the increases in the degree of proliferation and also in the levels of mineralization of the cells, as demonstrated by the higher levels of alkaline phosphatase [36]. The favoring effect of residual stress is seen in both types of surfaces. However, when comparing the results between smooth and rough surfaces it can be seen that the main role for cell activity is topography. Biomineralization is the process by which hydroxyapatite is deposited in the extracellular matrix. The first step of mineralization is the formation of hydroxyapatite crystals within matrix vesicles that bud from the surface membrane of hypertrophic chondrocytes, osteoblasts, and odontoblasts. The hydroxyapatite then propagates into the extracellular matrix and is deposited between collagen fibrils. Extracellular inorganic pyrophosphate, provided by NPP1 and ANKH, inhibits hydroxyapatite formation [41,42]. ALP increases local inorganic phosphate rates and facilitates mineralization, in addition to reducing the extracellular pyrophosphate concentration. Among several isoforms, the nontissue-specific isoenzyme of ALP (TNAP) is strongly expressed in bone, liver, and kidney and plays a key role in bone calcification. TNAP hydrolyzes pyrophosphate and supplies inorganic phosphate to enhance mineralization. The biochemical substrates of TNAP are believed to be inorganic pyrophosphate and pyridoxal phosphate. These substrates are concentrated under conditions of NAPT deficiency, resulting in hypophosphatasia. Increasing the level of Figure 5. Fractures on the neck of the implant produced by fatigue in dental implants made of commercially pure titanium. Osteoblast cell culture studies show that cell proliferation values are higher in both smooth and rough osteoblast samples. This fact can be justified by the higher hydrophilicity values generated by surface tension [ 34 – 36 ]. Likewise, the surface energy values, and especially their polar component, causes the increases in the degree of proliferation and also in the levels of mineralization of the cells, as demonstrated by the higher levels of alkaline phosphatase [ 36 ]. The favoring effect of residual stress is seen in both types of surfaces. However, when comparing the results between smooth and rough surfaces it can be seen that the main role for cell activity is topography. Biomineralization is the process by which hydroxyapatite is deposited in the extracellular matrix. The first step of mineralization is the formation of hydroxyapatite crystals within matrix vesicles that bud from the surface membrane of hypertrophic chondrocytes, osteoblasts, and odontoblasts. The hydroxyapatite then propagates into the extracellular matrix and is deposited between collagen fibrils. Extracellular inorganic pyrophosphate, provided by NPP1 and ANKH, inhibits hydroxyapatite formation [41,42]. ALP increases local inorganic phosphate rates and facilitates mineralization, in addition to reducing the extracellular pyrophosphate concentration. Among several isoforms, the non-tissue-specific isoenzyme of ALP (TNAP) is strongly expressed in bone, liver, and kidney and plays a key role in bone calcification. TNAP hydrolyzes pyrophosphate and supplies inorganic phosphate to enhance mineralization. The biochemical substrates of TNAP are believed to be inorganic pyrophosphate and pyridoxal phosphate. These substrates are concentrated under conditions of NAPT deficiency, resulting in hypophosphatasia. Increasing the level of ALP expression and development in this setting would undoubtedly provide new and essential information on the fundamental molecular mechanisms of bone formation and offer therapeutic possibilities for the treatment of bone-related diseases [ 43 ]. Alkaline phosphatase has sometimes been confused with osteocalcin. Alkaline phosphatase is an immediate indicator of mineralization processes, whereas the role of osteocalcin is more complicated but effects on osteoblastic differentiation have been attributed to it. Over the past few years, a great deal of research has been devoted to some sometimes controversial theories about osteocalcin (OCN). OCN is a 46-amino acid protein produced and secreted almost exclusively by osteoblasts, terminally differentiated cells responsible for the synthesis and mineralization of bone matrix during skeletal development and its periodic regeneration throughout life. Osteoblasts originate from mesenchymal progenitors and are short-lived cells that are constantly replaced, depending on the need for bone formation [ 44 , 45 ]. OCN, secreted by osteoblasts, contains three  -carboxyglutamic acid residues that confer a high affinity for the hydroxyapatite bone matrix. However, when bone is resorbed by osteoclasts, a cell type derived from macrophages, the acidic pH of the Doctoral Thesis Ana Rita Pereira - 146 - Materials 2024,17, 1626 10 of 13 resorption compartment causes the removal of the carboxyl groups of OCN, and the decarboxylated OCN is released into the circulation [ 46 – 50 ]. Circulating levels of decarboxylated OCN, thus, depend on the rate of bone turnover, also known as remodeling [51–53]. Initially thought to be an inhibitor of bone mineralization, recent studies suggest a broader role for osteocalcin that extends to the regulation of whole-body metabolism, reproduction, and cognition. Osteoblasts are specialized mesenchymal cells primarily responsible for the synthesis and deposition of the collagen-rich mineralized matrix that makes up bone tissue. In the last decade, studies have elaborated an expanded biological role for the osteoblast that focuses on the actions of bone-derived osteocalcin [ 49 , 50 ]. Osteocalcin has been commonly used as a serum marker of osteoblast bone formation and is thought to act in the bone matrix to regulate mineralization [54–57]. Bone is built by a process of self-assembly under the control of a gene regulatory network (GRN). Chekroun et al. [ 58 ] studied the behavior of bone gene regulation as a control of mineralization using mathematical equations. These authors estimated direct interactions between genes encoding transcription factors and those encoding bone proteins, as well as performing mathematical modeling of the bone GRN using a system of nonlinear differential equations modeling the interactions. The authors showed negative indirect interactions from negative feedback loops or micro-RNAs. These authors showed theoretical evidence of osteoblast self-inhibition following activation of the genetic regulatory network controlling mineralization. Bone metabolism is very complicated and further research is still needed to improve understanding. With respect to the bacterial colonies studied, no significant differences were observed when comparing the presence or absence of residual stress on both surfaces. Although it could be thought that the variation in wettability would favor bacterial adhesion to hydrophilic or hydrophobic surfaces, depending on the character of the bacterial membrane, this could not be observed in this case, where Streptococcus has a more hydrophobic membrane and Lactobacillus more hydrophobic. It is possible that the difference in hydrophilic/hydrophobic character caused by residual stress is not sufficient to observe behavioral changes at the bacterial level but is sufficient for osteoblastic cells. The influence of surface energy and its two components, dispersive and polar, on bacterial adhesion should be further studied. It would be necessary to know the physicochemical properties of bacterial membranes in order to determine the mechanisms of adsorption and microbiological colonization. What has been observed is that rough surfaces cause an increase in bacterial colony formation in both types of strains. This fact is common and has been observed by several authors and is a problem in dental implants since a rough surface is necessary for the improvement in osteoblastic activity and mechanical fixation of implant–bone tissue, but it also presents worse behavior towards bacteria and facilitates the formation of peri-implantitis. With these results we can confirm the fulfillment of a part of the hypothesis, since we have been able to verify how residual stress favors osteoblast activity with the number of cells as well as osteocalcin levels in mineralization. However, the part of the hypothesis in which residual stress favors bacterial proliferation is not fulfilled. At this point it was only possible to verify that roughness favors the adhesion of the bacterial strains studied, but no significant differences were observed with the residual surface stress. This manuscript is of clinical importance because in the placement of dental implants by clinicians, especially in narrow dental implants, the good properties, not only mechanical but also cellular activity, of dental implants obtained by cold working can be verified. The results of this study also give clinicians confidence that there is no increase in bacterial colonization caused by residual surface stresses. 5. Conclusions It was determined that the compressive residual stress on the surface of both smooth and rough titanium improves the hydrophilicity and increases the surface energy of titanium, especially in its polar component. This fact causes osteoblastic proliferation to Doctoral Thesis Ana Rita Pereira - 147 - Materials 2024,17, 1626 11 of 13 improve at 7 and 14 days of culture. It was observed that the main factor was roughness, since rough implants without residual stress showed a higher proliferation and degree of mineralization than smooth surfaces with residual stress. However, residual stress on smooth and rough surfaces caused proliferation and osteocalcin levels to be higher, with statistically significant differences. An increase in bacterial colonization was observed on rough surfaces compared to smooth surfaces, but the influence of residual stress on bacterial behavior could not be demonstrated. Author Contributions: Conceptualization, P.M., J.G., C.A. and M.H.-C.; methodology, J.G., B.R.-C. and J.V.R.-S.; software, R.P.; validation, M.H.-C., J.V.R.-S., B.R.-C. and P.M.; formal analysis, R.P.; investigation, R.P., P.M., J.G., B.R.-C. and J.V.R.-S.; resources, M.H.-C. and J.V.R.-S.; data curation, P.M. and R.P.; writing—original draft preparation, R.P., J.G. and C.A.; writing—review and editing, J.G.; visualization, P.M.; supervision, J.V.R.-S. and M.H.-C.; project administration, P.M.; funding acquisition, P.M., R.P., J.V.R.-S. and B.R.-C. All authors have read and agreed to the published version of the manuscript. Funding: The authors thank the MINECO (PID2022-137496OB-I00) and Klockner dental system (Esclades Engordany, Andorra). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The authors can provide details of the research requirements by letter and comments if needed. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Blanes, R.J.; Bernard, J.P.; Blanes, Z.M.; Belser, U.C. A 10-year prospective study of ITI dental implants placed in the posterior region. I: Clinical and radiographic results. Clin. Oral Implant. Res. 2007,18, 699–706. [CrossRef][PubMed] 2. Buser, D.; Schenk, R.K.; Steinemann, S.; Fiorellini, J.P.; Fox, C.H.; Stich, H. Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs. J. Biomed. Mater. Res. 1991,25, 889–902. [CrossRef][PubMed] 3. Brânemark, P.I.; Hansson, B.O.; Adell, R.; Breine, U.; Lindstrom, J.; Hallen, O.; Ohman, A. Osseointegrated implants in the treatment of edentulous jaw. Experience from a 10-year period. Scand. J. Plast Reconstr. Surg. 1977,16, 1–132. 4. Branemark, P.I. Tissue-Integrated Prostheses. Osseointegration in Clinical Dentistry; Quintessence Publishing Co., Inc.: Berlin, Germany, 1985. 5. Brånemark, P.I.; Breine, U.; Adell, R.; Hansson, B.O.; Lindström, J.; Ohlsson, Å. Intra-osseous anchorage of dental prostheses I. Experimental studies. Scand. J. Plast Reconstr. Surg. 1969,3, 81–100. [CrossRef][PubMed] 6. Schroeder, A.; Pohler, O.; Sutter, F. Gewebsreaktion auf ein Titan-Hohlzylinder Implantatmit Titan-Spritzschicht oberflächlich. Schweiz. Monatsschrift Zahnheilkd. 1976,86, 713–727. 7. Schroeder, A.; Stich, H.; Straumann, F.; Sutter, F. Leber die Anlagerung von Osteo Zement an einen belasteten Implantatkörper. Schweiz. Monatsschrift Zahnheilkd. 1978,88, 1051–1058. 8. Schroeder, A.; van der Zypery, E.; Stitch, H.; Sutter, F. The reactions of bone, connective tissue, and epithelium to endosteal implants with titanium-sprayed surfaces. J. Maxillofac. Surg. 1981,9, 15–25. [CrossRef][PubMed] 9. Johansson, C.; Albrektsson, T. Integration of screw implants in the rabbit. A 1-year follow-up of removal of titanium implants. Int. J. Oral Maxillofac. Implant. 1987,2, 69–75. 10. Buser, D.; Mericske-Stern, R.; Bernard, J.P.; Behneke, A.; Behneke, N.; Hirt, H.P.; Belser, U.; Lang, N.P. Long-term evaluation of non-submerged ITI implants. Part 1: 8-year life table analysis of a prospective multicenter study with 2359 implants. Clin. Oral Implant. Res. 1997,8, 161–172. [CrossRef][PubMed] 11. Berglundh, T.; Abrahamsson, I.; Lang, N.P.; Lindhe, J. De novo alveolar bone formation adjacent to endosseous implants. Clin. Oral Implant. Res. 2003,14, 251–262. [CrossRef][PubMed] 12. Aparicio, C.; Gil, F.J.; Planell, J.A.; Engel, E. Human-osteoblast proliferation and differentiation on grit-blasted and bioactive titanium for dental applications. J. Mater. Sci. Mater. Med. 2002,13, 1105–1111. [CrossRef][PubMed] 13. Herrero-Climent, M.; Lázaro, P.; Rios, J.V.; Lluch, S.; Marqués, M.; Guillem-Martí, J.; Gil, F.J. Influence of acid-etching after gritblasted on osseointegration of titanium dental implants: In Vitro and in vivo studies. J. Mater. Sci. Mater. Med. 2013,24, 2047–2055. [CrossRef][PubMed] 14. Aparicioa, C.; Padrósb, A.; Gil, F.-J. In Vivo evaluation of micro-rough and bioactive titanium dental implants using histometry and pull-out tests. J. Mech. Behav. Biomed. Mater. 2011,4, 1672–1682. [CrossRef][PubMed] Doctoral Thesis Ana Rita Pereira - 148 - Materials 2024,17, 1626 12 of 13 15. Esposito, M. Titanium for Dental Implants (I). En “Titanium in Medicine: Material Science, Surface Science, Engineering, Biological Responses and Medical Applications”; Brunette, D.M., Tengvall, P., Textor, M., Thomsen, P., Eds.; Springer: Berlin/Heidelberg, Germany, 2001. 16. Berglundh, T.; Armitage, G.; Araujo, M.G.; Avila-Ortiz, G.; Blanco, J.; Camargo, P.M.; Chen, S.; Cochran, D.; Derks, J.; Figuero, E.; et al. Peri-implant diseases and conditions: Consensus report of Workgroup 4 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J. Clin. Periodontol. 2018,45,S286–S291.[CrossRef][PubMed] 17. Pjetursson, B.E.; Thoma, D.; Jung, R.; Zwahlen, M.; Zembic, A. A systematic review of the survival and complication rates of implant-supported fixed dental prostheses (FDPs) after a mean observation period of at least 5 years. Clin. Oral Implant. Res. 2012,23, 22–38. [CrossRef][PubMed] 18. Gallego, L.; Sicilia, A.; Sicilia, P.; Mallo, C.; Cuesta, S.; Sanz, M. A retrospective study on the crestal bone loss associated with different implant surfaces in chronic periodontitis patients. Clin. Oral Implant. Res. 2018,29, 557–567. [CrossRef][PubMed] 19. Pegueroles, M.; Aparicio, C.; Bosio, M.; Engel, E.; Gil, F.J.; Planell, J.A.; Altankov, G. Spatial Organization of Osteoblast Fibronectin-Matrix on Titanium Surface—Effects of Roughness, Chemical Heterogeneity, and Surface Free Energy. Acta Biomater. 2010,6, 291–301. [CrossRef][PubMed] 20. Velasco-Ortega, E.; Monsalve-Guil, L.; Jiménez-Guerra, A.; Ortiz, I.; Moreno-Muñoz, J.; Nuñez-Marquez, E.; Pequeroles, M.; Perez, R.A.; Gil, F.J. Importance of the roughness and residual stresses of dental implants on fatigue and osseointegration be-havior. In Vivo study in rabbits. J. Oral Implantol. 2016,42, 469–476. [CrossRef][PubMed] 21. Klinge, B.; Flemming, T.; Cosyn, J.; De Bruyn, H.; Eisner, B.M.; Hultin, M.; Isidor, F.; Lang, N.P.; Lund, B.; Meyle, J.; et al. The patient undergoing implant therapy. Summary and consensus statements. The 4th EAO Consensus Conference 2015. Clin. Oral Implant. Res. 2015,26, 64–67. [CrossRef][PubMed] 22. Godoy-Gallardo, M.; Manzanares-Céspedes, M.C.; Sevilla, P.; Nart, J.; Manzanares, N.; Manero, J.M.; Gil, F.J.; Boyd, S.K.; Rodríguez, D. Evaluation of bone loss in antibacterial coated dental implants: An experimental study in dogs. Mater. Sci. Eng. C Mater. Biol. Appl. 2016,69,538–545.[CrossRef][PubMed] 23. Rakic, M.; Galindo-Moreno, P.; Monje, A.; Radovanovic, S.; Wang, H.-L.; Cochran, D.; Sculean, A.; Canullo, L. How frequent does peri-implantitis occur? A systematic review and meta-analysis. Clin. Oral Investig. 2018,22, 1805–1816. [CrossRef][PubMed] 24. Chan, H.L.; Lin, G.H.; Suarez, F.; MacEachern, M.; Wang, H.L. Surgical management of peri-implantitis: A systematic review and meta-analysis of treatment outcomes. J. Periodontol. 2014,85, 1027–1041. [CrossRef][PubMed] 25. Monje, A.; Pons, R.; Amerio, E.; Wang, H.L.; Nart, J. Resolution of peri-implantitis by means of implantoplasty as adjunct to surgical therapy: A retrospective study. J. Periodontol. 2021,93, 110–122. [CrossRef][PubMed] 26. Pieuchot, L.; Marteau, J.; Guignandon, A.; Dos Santos, T.; Brigaud, I.; Chauvy, P.-F.; Cloatre, T.; Ponche, A.; Petithory, T.; Rougerie, P.; et al. Curvotaxis directs cell migration through cell-scale curvature landscapes. Nat. Commun. 2018,9,3995.[CrossRef][PubMed] 27. Song, K.H.; Park, S.J.; Kim, D.S.; Doh, J. Sinusoidal wavy surfaces for curvature-guided migration of T lymphocytes. Biomaterials 2015,51, 151–160. [CrossRef][PubMed] 28. Charest, J.L.; Eliason, M.T.; García, A.J.; King, W.P. Combined microscale mechanical topography and chemical patterns on polymer cell culture substrates. Biomaterials 2006,27, 2487–2494. [CrossRef][PubMed] 29. Mitchel, J.A.; Hoffman-Kim, D. Cellular scale anisotropic topography guides Schwann cell motility. PLoS ONE 2011,6,e24316.[CrossRef] [PubMed] 30. Bade, N.D.; Xu, T.; Kamien, R.D.; Assoian RK Stebe, K.J. Gaussian curvature directs stress fiber orientation and cell migration. Biophys. J. 2018,114, 1467–1476. [CrossRef][PubMed] 31. Christopherson, G.T.; Song, H.; Mao, H.-Q. The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 2009,30, 556–564. [CrossRef][PubMed] 32. Petrie, R.J.; Koo, H.; Yamada, K.M. Generation of compartmentalized pressure by a nuclear piston governs cell motility in a 3D matrix. Science 2014,345, 1062–1065. [CrossRef][PubMed] 33. Engler, A.J.; Sen, S.; Sweeney, H.L.; Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 2006,126, 677–689. [CrossRef][PubMed] 34. Kilian, K.A.; Bugarija, B.; Lahn, B.T.; Mrksich, M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc. Natl. Acad. Sci. USA 2010,107, 4872–4877. [CrossRef][PubMed] 35. Buxadera-Palomero, J.; Godoy-Gallardo, M.; Molmeneu, M.; Punset, M.; Gil, F.J. Antibacterial Properties of Triethoxysilylpropyl Succinic Anhydride Silane (TESPSA) on Titanium Dental Implants. Polymers 2020,12, 773. [CrossRef][PubMed] 36. Pegueroles, M.; Tonda-Turo, C.; Planell, J.A.; Gil, F.-J.; Aparicio, C. Adsorption of fibronectin, fibrinogen, and albumin on TiO 2 : Time-Resolved Kinetics, Structural Changes, and Competition Study. Biointerphases 2012,7, 48. [CrossRef][PubMed] 37. Guillem-Marti, J.; Cinca, N.; Punset, M.; Cano, I.G.; Gil, F.J.; Guilemany, J.M.; Dosta, S. Porous titanium-hydroxyapatite composite coating obtained on titanium by cold gas spray with high bond strength for biomedical applications. Colloids Surf. B Biointerfaces 2019,180, 245–253. [CrossRef][PubMed] 38. Gil, F.J.; Espinar, E.; Llamas, J.M.; Sevilla, P. Fatigue life of bioactive titanium dental implants treated by means of Grit Blasting and Thermo-Chemical treatment. Clin. Implant Dent. Relat. Res 2014,16, 273–281. [CrossRef][PubMed] 39. Aragoneses, J.; Valverde, N.L.; Fernandez-Dominguez, M.; Mena-Alvarez, J.; Rodriguez, C.; Gil, J.; Aragoneses, J.M. Relevant Aspects of Titanium and Zirconia Dental Implants for Their Fatigue and Osseointegration Behaviors. Materials 2022,15,4036.[CrossRef] Doctoral Thesis Ana Rita Pereira - 149 - Materials 2024,17, 1626 13 of 13 40. Aparicio, C.; Rodriguez, D.; Gil, F.J. The effect of shot blasting and heat treatment on the fatigue behavior of titanium for dental implant applications. Dent. Mater. 2007,23, 486–491. [CrossRef] 41. Orimo, H. The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J. Nippon Med. Sch. 2010,77,4–12. [CrossRef] 42. Vimalraj, S. Alkaline phosphatase: Structure, expression and its function in bone mineralization. Gene 2020,754, 144855. [CrossRef] 43. Manolagas, S.C. Osteocalcin promotes bone mineralization but is not a hormone. PLoS Genet. 2020,16,e1008714.[CrossRef][PubMed] 44. Manolagas, S.C. Birth and death of bone cells: Basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 2000,21, 115–137. [PubMed] 45. Lee, N.K.; Sowa, H.; Hinoi, E.; Ferron, M.; Ahn, J.D.; Confavreux, C.; Dacquin, R.; Mee, P.J.; McKee, M.D.; Jung, D.Y.; et al. Endocrine regulation of energy metabolism by the skeleton. Cell 2007,130, 456–469. [CrossRef][PubMed] 46. Oury, F.; Sumara, G.; Sumara, O.; Ferron, M.; Chang, H.; Smith, C.E.; Hermo, L.; Suarez, S.; Roth, B.L.; Ducy, P.; et al. Endocrine regulation of male fertility by the skeleton. Cell 2011,144, 796–809. [CrossRef][PubMed] 47. Oury, F.; Khrimian, L.; Denny, C.A.; Gardin, A.; Chamouni, A. Maternal and offspring pools of osteocalcin influence brain development and functions. Cell 2013,26, 228–241. [CrossRef][PubMed] 48. Mera, P.; Laue, K.; Wei, J.; Berger, J.M.; Karsenty, G. Osteocalcin is necessary and sufficient to maintain muscle mass in older mice. Mol. Metab. 2016,16, 1042–1047. [CrossRef] 49. Karsenty, G. Update on the biology of osteocalcin. Endocr. Pract. 2017,23, 1270–1274. [CrossRef][PubMed] 50. Ferron, M.; Karsenty, G. Regulation of energy metabolism by bone-derived hormones. In Principles of Bone Biology; Bilezikian, J.P., Martin, T.J., Clemens, T.L., Rosen, C.J., Eds.; Academic Press: Cambridge, MA, USA, 2020; pp. 303–318. 51. Wellendorph, P.; Johansen, L.D.; A Jensen, A.; Casanova, E.; Gassmann, M.; Deprez, P.; Clément-Lacroix, P.; Bettler, B.; BräunerOsborne, H. No evidence for a bone phenotype in GPRC6A knockout mice under normal physiological conditions. J. Mol. Endocrinol. 2008,42, 215–223. [CrossRef][PubMed] 52. Jørgensen, C.V.; Gasparini, S.J.; Tu, J.; Zhou, H.; Seibel, M.J.; Bräuner-Osborne, H. Metabolic and skeletal homeostasis are maintained in full locus GPRC6A knockout mice. Sci. Rep. 2019,9, 5995. [CrossRef][PubMed] 53. Zoch, M.L.; Clemens, T.L.; Riddle, R.C. New insights into the biology of osteocalcin. Bone 2016,82, 42–49. [CrossRef][PubMed] 54. Clemens, T.L.; Karsenty, G. The osteoblast: An insulin target cell controlling glucose homeostasis. J. Bone Miner. Res. 2010,26,677–680. [CrossRef][PubMed] 55. Karsenty, G.; Ferron, M. The contribution of bone to whole-organism physiology. Nature 2012,481, 314–320. [CrossRef][PubMed] 56. Vallet-Regí, M.; Román, J.; Padilla, S.; Doadrio, J.C.; Gil, F.J. Bioactivity and mechanical properties of SiO 2 –CaO–P 2 O 5 glassceramics. J. Mater. Chem. 2005,15, 1353–1359. [CrossRef] 57. Fulzele, K.; Riddle, R.C.; DiGirolamo, D.J.; Cao, X.; Wan, C.; Chen, D.; Faugere, M.-C.; Aja, S.; Hussain, M.A.; Brüning, J.C.; et al. Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition. Cell 2010,142,309–319.[CrossRef] [PubMed] 58. Chekroun, A.; Pujo-Menjouet, L.; Falcoz, S.; Tsuen, K.; Yang, K.Y.-H.; Berteau, J.-P. Theoretical evidence of osteoblast self-inhibition after activation of the genetic regulatory network controlling mineralization. J. Theor. Biol. 2022,537, 111005. [CrossRef][PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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