Aerogel-Based Materials in Bone and Cartilage Tissue Engineering-A Review with Future Implications

Citation: Lázár, I.; ˇ Celko, L.; Menelaou, M. Aerogel-Based Materials in Bone and Cartilage Tissue Engineering—A Review with Future Implications. Gels 2023,9, 746. https://doi.org/10.3390/ gels9090746 Academic Editor: Shige Wang Received: 9 August 2023 Revised: 9 September 2023 Accepted: 11 September 2023 Published: 13 September 2023 Copyright: © 2023 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/). gels Review Aerogel-Based Materials in Bone and Cartilage Tissue Engineering—A Review with Future Implications István Lázár1,* , Ladislav ˇ Celko 2and Melita Menelaou 3,* 1Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary 2Central European Institute of Technology, Brno University of Technology, Purkynova 656/123, 612 00 Brno, Czech Republic; ladislav[email protected].cz 3Department of Chemical Engineering, Cyprus University of Technology, 30 Arch. Kyprianos Str., Limassol 3036, Cyprus *Correspondence: [email protected] (I.L.); [email protected] (M.M.) Abstract: Aerogels are fascinating solid materials known for their highly porous nanostructure and exceptional physical, chemical, and mechanical properties. They show great promise in various technological and biomedical applications, including tissue engineering, and bone and cartilage substitution. To evaluate the bioactivity of bone substitutes, researchers typically conduct in vitro tests using simulated body fluids and specific cell lines, while in vivo testing involves the study of materials in different animal species. In this context, our primary focus is to investigate the applications of different types of aerogels, considering their specific materials, microstructure, and porosity in the field of bone and cartilage tissue engineering. From clinically approved materials to experimental aerogels, we present a comprehensive list and summary of various aerogel building blocks and their biological activities. Additionally, we explore how the complexity of aerogel scaffolds influences their in vivo performance, ranging from simple single-component or hybrid aerogels to more intricate and organized structures. We also discuss commonly used formulation and drying methods in aerogel chemistry, including molding, freeze casting, supercritical foaming, freeze drying, subcritical, and supercritical drying techniques. These techniques play a crucial role in shaping aerogels for specific applications. Alongside the progress made, we acknowledge the challenges ahead and assess the near and far future of aerogel-based hard tissue engineering materials, as well as their potential connection with emerging healing techniques. Keywords: aerogel; tissue engineering; artificial bone substitution; in vitro and in vivo bioactivity; biodegradation; cartilage regeneration; scaffold; osteogenesis; simulated body fluids; immortalized cell lines 1. Introduction Bone is a rigid tissue with essential functions in providing structural support, protecting vital organs, and enabling movement [ 1 ]. It consists of an organic matrix (20%), primarily made up of type I collagen, a mineral phase (65%) predominantly composed of hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 , HAp], water (10%), and various bioactive factors and cells, mainly osteoblasts and osteoclasts [ 2 ]. Bone has a natural regenerative process that is regulated by biomechanical, cellular, and molecular factors [ 3 ]. Articular cartilage is a thin layer covering the ends of bones, allowing smooth gliding and facilitating proper joint function. The cartilage tissue is a sturdy, flexible avascular structure composed of collagen, proteoglycan, non-collagenous proteins, and water. A unique feature of cartilage is its close connection with the underlying hard subchondral bone, comprising three distinct components: highly mineralized subchondral, intermediate-mineralized calcified, and non-mineralized tissues, separated by a dense tidemark. Gels 2023,9, 746. https://doi.org/10.3390/gels9090746 https://www.mdpi.com/journal/gels Gels 2023,9, 746 2 of 40 Damage to bones and cartilage often occurs due to disease or traumatic injuries. Incidences of boneand cartilage-related disorders have been on the rise, linked to factors like aging, obesity, cancer, and sports-related injuries. These conditions can significantly impact patients’ quality of life, causing pain, reduced mobility, and loss of independence. To address these challenges, the research community has been focusing on regenerative medicine approaches, including the development of biomaterial-based and tissue-engineering solutions. Restoring bone integrity and structure is essential in cases of fractures, skeletal development, or regular physiological reshaping. It involves facilitating the transport, growth, proliferation, and differentiation of osteoprogenitor cells in the injured or defective area. To achieve successful bone repair, a well-designed system is necessary to support the three primary mechanisms of bone regeneration: (a) rapid revascularization, (b) osteogenesis induction, and (c) osteoinduction, which generate new tissue from osteogenic cells. Additionally, the process should promote osteoconduction and encourage cell growth towards the bone surface [ 4 ]. However, there are instances where bone regeneration or repair exceeds the tissue’s capacity for new bone formation. Such cases may include bone deformations, neoplastic diseases, infections, avascular necrosis, and osteoporosis, among others [3]. Addressing bone defects requires orthopedic reconstruction methodologies that involve bone replacement through the implantation of natural or artificial grafts [ 5 ]. Both types of grafts must meet specific criteria, such as high biocompatibility, osteoconduction, and osseointegration [ 6 ]. Moreover, they should exhibit robust mechanical strength, be harmless to the body, and remain stable in the biological environment. Throughout their presence in the body, the grafts must not demonstrate any toxic effects [5]. Recent advancements in bone tissue engineering are centered around the development of structures that can closely mimic the behavior of natural bone in terms of both structure and performance. This involves creating materials with exceptional mechanical reinforcement and a supporting matrix, all while maintaining biocompatibility [ 7 ]. To promote successful regeneration, these materials need to be highly porous, encouraging vascularization into the damaged area and facilitating the migration of osteogenic cells. Biocompatible three-dimensional scaffolds or hydrogels often possess these desirable characteristics [8]. Scaffolds tailored for bone and cartilage tissue engineering must meet specific biological requirements. They should be biocompatible, non-toxic, and biodegradable, capable of seamlessly integrating and interacting with the surrounding environment. Porosity is a key factor, as it enables cellular infiltration and facilitates the transport of essential gases, nutrients, and regulatory factors, all crucial for cell survival. Finding the right balance is crucial, as excessively large pores reduce the surface area available for cell attachment, while overly small pores hinder cell migration and infiltration, and restrict the diffusion of vital nutrients and waste products. Research conducted by Matsiko et al. suggests that the optimal pore sizes for bone and cartilage tissue-engineering applications typically fall within the range of 100–300 µ m [ 9 ]. Various fabrication methods can be employed to create these scaffolds, such as freeze drying, electrospinning, and 3D printing, as studied by Iglesias-Mejuto, García-González, Włodarczyk-Biegun, and del Campo [ 10 , 11 ]. Detailed insights into these methods will be provided in Section 3. In bone and cartilage repair applications, both synthetic and natural polymers have found utility. Synthetic polymers offer versatility in their physical and chemical properties, allowing precise control over molecular weight, degradation time, and hydrophobicity. Prominent examples of synthetic materials employed in such applications, as reported by Puppi et al., include poly L-lactic acid (PLLA), polycaprolactone (PCL), polyglycolic acid (PGA), and polyethylene glycol (PEG) [ 12 ]. These synthetic materials can be used in various forms to create scaffolds of different shapes and sizes. Conversely, natural polymers boast several advantages over their synthetic counterparts. They demonstrate biocompatibility, biodegradability with non-toxic degradation products, and possess bioactive properties that facilitate enhanced cell interactions. Some of the natural polymers used for bone and cartilage repair applications include collagen, silk, Gels 2023,9, 746 3 of 40 gelatin, fibrinogen, elastin, keratin, actin, and myosin. Several examples of polysaccharidebased aerogels exist, such as the crosslinked cellulose nanocrystal aerogels synthesized by Osorio et al. [ 13 ], an alginate aerogel reported by Wu et al. [ 14 ], and the development of a novel high-methoxyl pectin–xanthan aerogel coating on medical-grade stainless steel reported by Horvat et al. [ 15 ]. They can be classified as “bio-aerogels”, which originated from natural, semi-synthetic, and synthetic sources, with promising biomedical applications. The processing steps of the polysaccharide-based aerogels are similar to those applied for silica and other organic counterparts and, most commonly, start with the preparation of gel from an aqueous solution (often called hydrogel or “aquagel”) and the water in the pores of the aquagel is replaced with an alcohol such as methanol, to prepare an “alcogel” and to make possible the drying by supercritical carbon dioxide (scCO 2 ) [ 16 ]. Alternatively, the solvent exchange step can be circumvented if gelation is directly carried out in alcohol. In addition, composite materials such as PEGDA/CNF aerogel–wet hydrogel scaffold (where PEGDA: polyethylene glycol diacrylate; and CNF: cellulose nanofibril) have been proposed to overcome limitations of the single components in a concise review by Kazimierczak and Przekora [4]. Ceramic materials in the form of calcium phosphate have also been studied and proposed as potential candidates for bone regeneration and/or substitution. Hydroxyapatite (HAp), the main inorganic constituent of human bone, has high biocompatibility and osteo-conductivity, rendering it a material of particular interest for bone regeneration [ 3 ]. HAp biomaterials, however, are characterized by poor cell adhesion and difficult ingrowth, thus limiting their therapeutic effect in clinical applications. Also, it is not easy to prepare single-phase HAp porous scaffolds with both high porosity and excellent mechanical properties to be suitable candidates for bone regeneration. Thus, researchers worldwide are working to improve the properties of such materials. An example includes the work of Duan and coworkers who investigated HAp-based composite porous scaffolds instead of “HAp-only” porous scaffolds for such applications. Their experimental results suggest that they prepared a promising material in the form of HAp nanowire aerogel scaffold [17]. Other promising calcium phosphate-based ceramic materials are the a-tricalcium phosphate ( α -Ca 3 (PO 4 ) 2 , α -TCP) and β -tricalcium phosphate ( β -Ca 3 (PO 4 ) 2 , β -TCP). Combining the excellent biocompatibility of β -TCP and the conductivity of carbon aerogels, Tevlek et al. synthesized a β -TCP carbon–aerogel composite material. The biocompatibility of the composite material was evaluated, and their results suggested that composites may also act as promising targets for such applications [ 18 ]. Also, Lin and coworkers developed a β -TCP bioceramic platform coated with carbon aerogel as a novel approach to conquer osteosarcoma in one step [19]. In 2015, Wan et al. proposed mesoporous TiO 2 nanotube materials as a novel 3D porous network-structured scaffold for potential bone tissue engineering. The TiO 2 nanotubes were synthesized using the template-assisted sol–gel method followed by calcination. The scaffold showed an extremely large surface area of 1629 m 2 g −1 and a diameter of less than 100 nm [20]. Repairing cartilage defects remains a significant challenge in the field. While various clinical treatments for cartilage regeneration, such as microfracture, autologous chondrocyte implantation, Pridie perforations, and transplantation of osteochondral plugs have been developed [ 21 ], their success in fully regenerating functional cartilage tissue has been limited. To address these limitations, alternative approaches have been proposed, including the use of cell-loaded scaffold constructs. For successful cartilage regeneration through tissue engineering, an ideal scaffold must possess certain crucial characteristics, similar to those required for bone regeneration. These characteristics include a biomimetic threedimensional (3D) architecture to facilitate cell adhesion, an appropriate porosity to support cell ingrowth, sufficient mechanical strength to maintain its shape, good biocompatibility, and biodegradability, among others. These essential features are key to developing effective strategies for cartilage repair and regeneration. Gels 2023,9, 746 4 of 40 Electrospinning proves to be a highly effective technique for producing composite fibers with varying diameters and arrangements, closely resembling the morphology of the natural extracellular matrix (ECM) found in cartilage tissue, while also possessing suitable mechanical properties. For instance, Feng et al. explored a novel method involving electrospinning cartilage-derived extracellular matrix and polycaprolactone (PCL) composite nanofibrous membranes [ 22 ]. The traditional electrospinning technique primarily produces two-dimensional (2D) fiber membrane materials with minimal thickness and small pores. However, researchers have pursued the electrospinning of multi-component nanofibers to overcome the limitations associated with individual polymers and to cater to specific requirements. These requirements encompass crucial aspects like mechanical strength, biocompatibility, and degradation rate. By finely adjusting the proportion of each component in the composite fibers, these special requirements can be met, as was reviewed in detail by Chen et al. [23]. Various methods for preparing 3D electrospun nanofibrous scaffolds have been extensively researched and published. Examples include multilayering electrospinning, as reported by Zhang et al. [ 24 ] and Chainani et al. [ 25 ], as well as liquid and template-assisted electrospinning and post-treated electrospinning, as explored by Shim and colleagues [ 26 ], among others. In light of these advancements, Chen et al. [ 23 ] and Li et al. [ 27 ] successfully prepared aerogels composed of electrospun gelatin/polylactide (Gel/PLA) or gelatin/polycaprolactone (Gel/PCL) fibers, offering promising potential for cartilage regeneration. Additionally, Wang et al. developed a 3D fibrous aerogel comprising SiO 2 nanofibers with chitosan serving as bonding sites for bone regeneration [ 28 ]. These reports represent a few examples demonstrating the feasibility and potential of fibrous aerogels in the fields of cartilage and bone tissue engineering. Furthermore, inorganic components like hydroxyapatite (HAp) have been widely incorporated into implants for calcified cartilage and subchondral bone regeneration. Meanwhile, glycosaminoglycans (GAG) such as hyaluronic acid (HA) and chondroitin sulfate (CS) are frequently utilized for cartilage regeneration. These materials play crucial roles in enhancing the performance and functionality of tissue-engineering scaffolds. The quest for robust and long-lasting bone regeneration and cartilage tissue remains an important and challenging topic. In light of this, the present review article offers an overview of (composite) aerogel materials, a remarkable category of nanoporous materials with great potential for bone and cartilage repair applications. In the following sections, detailed information on the physical and chemical properties, and the significant role that such aerogels can play in various bone-related biomedical applications will be explored. 2. Aerogel Microstructure The discovery of aerogels dates back to 1931 when Kistler published the first article on the subject in Nature, titled “Coherent expanded aerogels and jellies” [ 29 ]. Kistler’s groundbreaking work involved the successful synthesis of aerogels from silica, achieved through the condensation of sodium metasilicate. He later expanded his research to include aerogels made from alumina, tungsten, nickel tartrate, cellulose, and gelatin. In his definition, aerogels were described as “gels in which the liquid has been replaced by air, with moderate shrinkage of the solid network.” For over 50 years, aerogels received little attention from the scientific community. However, in the last four decades, the interest in these materials has grown exponentially. This surge in interest can be attributed to the diverse range of applications that aerogels offer in various fields. Notably, aerogels provided solutions in catalysis, aerospace, and construction industries, for example. They have also proven valuable in energy-storage devices, solar-steam generation, and medical applications. Aerogels are remarkable porous ultralight solid materials obtained from gels, wherein the liquid component is replaced by a gas, commonly air. These aerogels exhibit several distinctive characteristics, including (a) high porosity (exceeding 90% of the total volume), Gels 2023,9, 746 5 of 40 (b) very low apparent density, (c) very high specific surface area, and (d) high mechanical strength when compared to the density of the material. The term “aerogel” encompasses a broad description of the structure and does not impose specific restrictions on material compositions or synthetic methodologies. Hüsing and Schubert proposed that “aerogels are materials in which the typical pore structures and networks remain remarkably maintained when the pore liquid of a gel is replaced by air” [ 30 ]. This definition better captures the essential characteristic of aerogels, highlighting their porous and highly structured nature, even after the liquid component has been replaced by air. In this regard, aerogel materials exhibit a wide range of classifications, as discussed by Karamikamkar et al. [ 31 ]. These classifications include their appearance, microstructural characteristics, composition, polarity and surface functionality. The most commonly employed and, perhaps, the simplest method for producing aerogels is the well-established sol–gel approach, followed by the specific drying process, known as supercritical drying at or above the supercritical point [ 30 ]. The sol–gel procedure involves two main stages: the formation of a sol and the subsequent transformation into a gel. The last step involves removing the pore liquid through a specialized drying process, leading to the formation of the aerogel. This drying step plays a critical role in shaping the final physical and chemical profile of the aerogel, allowing for precise control over its characteristics and performance. The gelation of inorganic aerogels primarily relies on hydrolysis and condensation processes, while biopolymer aerogels form through the aggregation process. Subsequent to gel formation, liquid extraction from the gel can be achieved using various techniques, resulting in materials classified as xerogels, cryogels, and aerogels [ 32 ]. The drying methods strongly affect the final properties of these materials, and are discussed in Section 3. (Figure 1) Gels 2023, 9, x FOR PEER REVIEW 6 of 39 demonstrated by Randall and coworkers [36]. Theoretical considerations also suggest that improving elastic recovery in silica aerogels can be achieved by including organic flexible linking groups in the silica backbone or by crosslinking the underlying structural gel with silanol groups through reactions with precursors, monomers, or polymers, as described by Lenentis et al. [37]. These methodologies have successfully enhanced the mechanical properties of aerogels while also improving their transparency. Figure 1. Graphical visualization of the general process of making aerogels in a sol–gel process followed by a specific drying technique to provide aerogel materials for biomedical applications. 3. Formulation and Drying Methods 3.1. Formulation Methods 3.1.1. Casting, Molding Monolithic aerogels are crafted through a straightforward casting or molding technique, which stands as the most extensively employed procedure. The constituents for gel formation are poured in a suitable container, allowing the gelation process to reach completion while facilitating subsequent retrieval of the gelled or solidified material devoid of structural compromise. Following this, the material undergoes a series of solvent exchange steps, wherein the original solvent mixture is replaced with an organic solvent compatible with carbon dioxide, such as acetone, methanol, or ethanol. As an alternative route, water-based gels are subjected to freezing and subsequent freeze drying. A viable realization of the casting, solvent exchange, and supercritical drying sequence is elucidated within the literature [38]. 3.1.2. Freeze Casting The freeze-casting process is also frequently used in fabricating porous materials, including aerogels. The technique is thoroughly described in the literature by Li et al. and García-González et al. [39,40]. The aqueous gel is frozen slowly in a segmented pattern before drying. In the process, ice crystals of different sizes are formed in the segmented temperature zones, leading to a patterned meso/macro porosity of the aerogel (cryogel) monoliths after drying, as presented by Tetik and coworkers [41]. This method was used to prepare silk fibroin–silica aerogels by Maleki and coworkers [42] and crosslinked cellulose [13] aerogels for bone substitution by Osorio et al. Figure 1. Graphical visualization of the general process of making aerogels in a sol–gel process followed by a specific drying technique to provide aerogel materials for biomedical applications. To address drawbacks like mechanical issues and limited specific functionalities, various new synthetic approaches have been employed in the production of aerogels. Techniques such as ambient pressure drying and freeze drying have been utilized to tailor the physical, chemical, and biological properties of aerogels, leading to the design and synthesis of hybrid inorganic or organic–inorganic hybrid aerogels. Aerogels stand apart from conventional foams due to their nanometer-scale pores with intricate interconnectivity, resulting in their superior insulating capabilities, being 2–5 times more effective than foams, with low thermal conductivity (0.005–0.1 W/mK), and an ultra-low dielectric constant Gels 2023,9, 746 6 of 40 ( k = 1.0–2.0 ). Such characteristics make aerogels highly appealing for various applications. However, one of the challenges faced by these materials lies in their mechanical properties, which can be limited. For instance, silica aerogels are known for their fragility, hygroscopic nature, and poor mechanical properties, leading to drawbacks in certain applications [31]. To expand the range of applications while preserving the unique properties of aerogels, mechanical reinforcing strategies have been devised. For silica-based aerogels, which represent the most extensively studied family, several methods have been explored in the literature to improve their mechanical properties. A common technique employed for structural reinforcement is prolonged aging time, as utilized by Hong et al., leading to the development of 3D internetworked GA@PDMS (where GA: graphene aerogel; and PDMS: poly(dimethylsiloxane)) [ 33 ]. Another widely employed approach involves surfacecrosslinking of a silica backbone with a polymer. Boday et al. demonstrated the growth of silica aerogel polymer nanocomposites in the presence of poly(methyl methacrylate) (PMMA), while Leventis reported the development of silica aerogels crosslinked with isocyanate-derived polymers [ 34 , 35 ]. In addition, the incorporation of a secondary phase, such as an organic/inorganic phase, embedded in the structure before (or after) gelation, has proven to be an effective strategy for aerogel structural reinforcement, as demonstrated by Randall and coworkers [ 36 ]. Theoretical considerations also suggest that improving elastic recovery in silica aerogels can be achieved by including organic flexible linking groups in the silica backbone or by crosslinking the underlying structural gel with silanol groups through reactions with precursors, monomers, or polymers, as described by Lenentis et al . [ 37 ]. These methodologies have successfully enhanced the mechanical properties of aerogels while also improving their transparency. 3. Formulation and Drying Methods 3.1. Formulation Methods 3.1.1. Casting, Molding Monolithic aerogels are crafted through a straightforward casting or molding technique, which stands as the most extensively employed procedure. The constituents for gel formation are poured in a suitable container, allowing the gelation process to reach completion while facilitating subsequent retrieval of the gelled or solidified material devoid of structural compromise. Following this, the material undergoes a series of solvent exchange steps, wherein the original solvent mixture is replaced with an organic solvent compatible with carbon dioxide, such as acetone, methanol, or ethanol. As an alternative route, water-based gels are subjected to freezing and subsequent freeze drying. A viable realization of the casting, solvent exchange, and supercritical drying sequence is elucidated within the literature [38]. 3.1.2. Freeze Casting The freeze-casting process is also frequently used in fabricating porous materials, including aerogels. The technique is thoroughly described in the literature by Li et al. and García-González et al. [ 39 , 40 ]. The aqueous gel is frozen slowly in a segmented pattern before drying. In the process, ice crystals of different sizes are formed in the segmented temperature zones, leading to a patterned meso/macro porosity of the aerogel (cryogel) monoliths after drying, as presented by Tetik and coworkers [ 41 ]. This method was used to prepare silk fibroin–silica aerogels by Maleki and coworkers [ 42 ] and crosslinked cellulose [13] aerogels for bone substitution by Osorio et al. 3.1.3. Supercritical Foaming In some instances, supercritical carbon dioxide can also generate gelation and macropore formation. The generally used supercritical foaming technique is reviewed in the literature [ 43 , 44 ] and has been successfully applied for the preparation of aerogel–polymer composite scaffolds made from starch and polycaprolactone by Goimil et al. [ 45 ] or from silk fibroin/polycaprolactone by Goimil and coworkers [46]. Gels 2023,9, 746 7 of 40 3.1.4. Stereolithography, 3D Printing Bio-ink technology and 3D printing stand as firmly established and widely embraced methodologies in the biomedical sphere, particularly in scaffold formulation and the provision of intricate structural arrangements. The outcome of a specific tissue replacement or tissue-mimicking application is contingent upon the materials’ intrinsic nature, the 3D architecture of the scaffold, the involved cell types, and the presence or absence of stimulating factors. Computer-aided design tools rapidly generate the blueprint for a 3D framework, subsequently realized using specialized extrusion or syringe-type printers. These printers can utilize a singular bio-ink component capable of light-induced crosslinking, a pliable yet self-supporting paste that undergoes post-printing crosslinking, or a printer with a two-component coaxial head that triggers chemical reactions upon contact, or even a blend thereof. The fabrication of scaffolds for artificial bone or cartilage substitutes presents challenges due to the intricacies of identifying a suitable 3D-printable material. In biomedical practice, nanofibrous bioactive substances are frequently 3D printed and subsequently subjected to freeze drying, transforming them into aerogels or aerogel-like forms to maintain their structural integrity and functionality. An evaluation of the technique’s merits and limitations has been comprehensively compiled by Badhe and colleagues [47]. Iglesias-Mejuto and García-González prepared an alginate–hydroxyapatite 3D-printed aerogel scaffold [ 10 ] as well as sterile dual crosslinked alginate–hydroxyapatite 3D-printed aerogel scaffolds with carbon dioxide gelling and glutaraldehyde crosslinking technology for bone tissue engineering. The as-prepared scaffolds showed enhanced fibroblast migration and good bioactivity; the latter correlated with the hydroxyapatite content [48]. Ng and coworkers developed a technique in which simultaneous 365 nm photocrosslinking and microextrusion 3D printing of the mixture of methacrylated silk fibroin and methacrylated hollow silica nanoparticles provided a mechanically stable scaffold compared to the simple silk fibroin networks. Unidirectional freeze casting provided even more interconnection of the pores, after which the aerogel was made by freeze drying. The as-prepared material is expected to be osteoconductive and osteoinductive bone substitute material that can be loaded with ciprofloxacin or other drugs to treat bone-related diseases [49]. 3.2. Drying Methods Regardless of the specific synthetic methods employed, wet gels undergo diverse drying techniques to transform into aerogel-based materials. Among these approaches, freeze drying and supercritical carbon dioxide drying emerge as the most prevalent. To a somewhat lesser extent, alternative strategies such as subcritical drying, spring-back drying [ 50 ], and ambient pressure drying [ 51 ] have also been explored and subjected to systematic investigation. 3.2.1. Freeze Drying Aqueous gels have been effectively transformed into aerogels through freeze drying, often referred to as cryogels. This method can be directly applied to aqueous gels, eliminating the need for the solvent exchange steps necessary in supercritical drying. An inherent benefit is that even highly heat-sensitive materials can be dried without undergoing decomposition. Thus, the freeze-drying technique has been harnessed to craft aerogels with successful outcomes. Examples include the development of aerogels from nanocellulose-PEGDA by Tang et al. [ 52 ], from rGO-collagen by Bahrami et al. [ 53 ], from rGO network by Asha et al. [ 54 ], from PEGDA-CNF by Sun et al. [ 55 ], from nanocellulose–bioglass by Ferreira et al. [ 56 ], from CA and PCL nanofiber-reinforced chitosan by Zhang et al. [ 57 ], from crosslinked cellulose by Osorio et al. [ 13 ], and from silk fibroin–cellulose developed by Chen and coworkers [ 58 ]. In a number of cases, freeze drying was combined with a freeze-casting/cryotemplating technique. Gels 2023,9, 746 8 of 40 3.2.2. Subcritical Drying Subcritical drying of solvogels or aquagels is a recognized technique, albeit one employed with varying interpretations. It can be conveniently executed using cost-effective equipment at or near atmospheric pressure, or with pressures and temperatures slightly below the critical point. A shared characteristic across all variations is the wet gels’ aging, followed by a solvent exchange step. The drying process takes several hours to a day or two, making subcritical drying comparable in time requirement to freeze drying. When ambient pressure drying is conducted, the resulting solid material can manifest as either an aerogel or a xerogel, contingent upon the solvent and the gel material’s polarity. As an instance, sol–gel-synthesized silica monoliths with chemically modified hydrophobic surfaces can be subjected to the spring-back effect to yield aerogels [50]. The range of conditions and the quality of the dried material depends on the nature of the solvent that fills the pores, as was studied by Kirkbir and coworkers in making aerogels from atmospheric to supercritical conditions [ 51 ]. Shrinkage can be minimized to a few percentages under high-pressure conditions. Lower pressures result in higher shrinkage, which can be extensive at around the atmospheric pressure, as found by Singh and coworkers in making microsphere-based scaffolds for cartilage tissue regeneration [ 59 ]. In that situation, the dried product can be considered more a xerogel than an aerogel, but fairly frequently, it is also called an aerogel. The porosity of the low-pressure dried materials is well under or near 90%, compared to the 95–99% porosity of the supercritically or higher-pressure subcritically dried materials. The shrinkage itself is not necessarily a disadvantage. In some instances, it is a desirable feature to increase the stiffness. Subcritical CO 2 drying was applied to make polymeric microparticles for cartilage engineering by Bhamidipati and coworkers, for example [60]. Subcritical drying was applied by Vazhayal et al. during the synthesis of hierarchically porous aluminosiloxane particles in a sol–gel emulsion process, which was tested as a drug carrier and as an osteoconductive support matrix material for bone tissue engineering. The particles were dried from isopropanol at 50 ◦C under ambient pressure [61]. 3.2.3. Supercritical Carbon Dioxide Drying Supercritical carbon dioxide drying is one of the most widely used techniques to make aerogel materials. It was used in many cases; thus, only a few examples are listed here. The temperature range is approximately 40 to 80–90 ◦ C, and the pressure range is 75–250 bar. This technique was used for the preparation of a wide range of aerogel materials including chitosan-GPTMS by Reyes-Peces et al. [ 62 ], collagen–alginate by Muñoz-Ruíz et al. [ 63 ], alginate–lignin by Quaraishi et al. [ 64 ], alginate by Martins et al. [ 65 ], starch and polycaprolactone by Goimil et al. [45], and silica-TCP-HAp by Lázár et al. [38]. 3.3. Post-Drying Workup and Shaping After the drying process is finished, aerogel materials frequently require further workup, i.e., cutting, mechanical shaping, or thermal treatment to meet the applicationspecific requirements. The most commonly used techniques are graphically summarized in Figure 2. Due to the sensitivity of the fine aerogel structure to any kind of wetting liquids, solidphase post-drying procedures can be used in most cases. Solution-phase soaking, leaching, wet grinding, and melting techniques cannot be applied when the original structure is to be maintained. Gels 2023,9, 746 9 of 40 Gels 2023, 9, x FOR PEER REVIEW 9 of 39 Figure 2. Major types of post-drying thermal or mechanical treatments of aerogels to generate application-specific properties. (A) Burning-out sacrificial porogen materials to provide macropores, (B) annealing of inorganic aerogels to provide increased mechanical strength, (C) high-temperature inert atmosphere carbonization of organic materials to change surface properties, (D) mechanical shaping (turning, drilling, milling) to make customized scaffolds. Due to the sensitivity of the fine aerogel structure to any kind of wetting liquids, solid-phase post-drying procedures can be used in most cases. Solution-phase soaking, leaching, wet grinding, and melting techniques cannot be applied when the original structure is to be maintained. Heat treatment is a simple and convenient way to change the porosity, mechanical strength, dissolvation, and degradation properties of inorganic aerogel-based materials. The process is viable only for thermally stable materials like silica, alumina, TCP, and HAp. Mesoand macropores are generated randomly or pre-arranged by burning out sacrificial porogen template materials at a temperature of a few hundred degrees Celsius (Figure 2A). A further increase in the temperature results in some degree of shrinking of the materials. That may increase the compressive strength and hardness to a high level, decrease the pore diameters, and reduce specific surface areas. Silica aerogel-based TCP composites containing the sacrificial porogen material microcrystalline cellulose or ashless filter paper or highly purified cotton fabric heated in the range of 500–1000 °C preserved their mesoporosity (average pore size: 26–46 nm) along with a decrease in specific surface area (from 400 to 184 m2/g) and a significant increase in compressive strength (from 0.47 to 16 MPa). Biological activities of the heat-treated materials showed the maximum at 800 °C in cell studies and rat critical size calvaria defect model experiments. The preparation and biological activities of the heat-treated materials were presented by Szabó et al., Hegedűs et al., Kuttor et al., Lázár et al., and Hegedűs and coworkers (Figure 2B) [66–70]. Thermosetting polymer-based aerogel materials (resorcinol–formaldehyde, and polybenzoxazine) alone or in composites with other thermally stable material (i.e., TCP) may undergo an inert atmosphere thermal decomposition and carbonization process at a temperature near 1000 °C. The resulting carbon aerogel materials proved to be biocompatible and supported the growth of human osteoblast cells, as pointed out by Dong et al. and Rubinstein and coworkers [19,71] (Figure 2C). Figure 2. Major types of post-drying thermal or mechanical treatments of aerogels to generate application-specific properties. ( A ) Burning-out sacrificial porogen materials to provide macropores, ( B ) annealing of inorganic aerogels to provide increased mechanical strength, ( C ) high-temperature inert atmosphere carbonization of organic materials to change surface properties, ( D ) mechanical shaping (turning, drilling, milling) to make customized scaffolds. Heat treatment is a simple and convenient way to change the porosity, mechanical strength, dissolvation, and degradation properties of inorganic aerogel-based materials. The process is viable only for thermally stable materials like silica, alumina, TCP, and HAp. Mesoand macropores are generated randomly or pre-arranged by burning out sacrificial porogen template materials at a temperature of a few hundred degrees Celsius (Figure 2A). A further increase in the temperature results in some degree of shrinking of the materials. That may increase the compressive strength and hardness to a high level, decrease the pore diameters, and reduce specific surface areas. Silica aerogel-based TCP composites containing the sacrificial porogen material microcrystalline cellulose or ashless filter paper or highly purified cotton fabric heated in the range of 500–1000 ◦ C preserved their mesoporosity (average pore size: 26–46 nm) along with a decrease in specific surface area (from 400 to 184 m 2 /g) and a significant increase in compressive strength (from 0.47 to 16 MPa). Biological activities of the heat-treated materials showed the maximum at 800 ◦ C in cell studies and rat critical size calvaria defect model experiments. The preparation and biological activities of the heat-treated materials were presented by Szabóet al., Heged˝us et al., Kuttor et al., Lázár et al., and Heged˝us and coworkers (Figure 2B) [66–70]. Thermosetting polymer-based aerogel materials (resorcinol–formaldehyde, and polybenzoxazine) alone or in composites with other thermally stable material (i.e., TCP) may undergo an inert atmosphere thermal decomposition and carbonization process at a temperature near 1000 ◦C. The resulting carbon aerogel materials proved to be biocompatible and supported the growth of human osteoblast cells, as pointed out by Dong et al. and Rubinstein and coworkers [19,71] (Figure 2C). High-mechanical-strength materials like heat-treated silica-TCP aerogel composites, successfully used in artificial bone substitution in vivo in animal models, can be implanted in load-bearing positions. A high number of applications, however, would require customized mechanical shaping of the specimens to fit in the shape of the defect. Machining, milling, and drilling can be performed with mechanically sufficiently strong materials Gels 2023,9, 746 16 of 40 Table 1. Cont. Name Properties References Cellulose acetate (CA) Cellulose acetate is a hydrophilic and thermoplastic biodegradable cellulose derivative. It can be conveniently formulated into sheets, nanofibers, etc. CA scaffolds combined with other bioactive molecules, biopolymers, drugs, etc., support endothelial cell migration and adhesion, and do not promote platelet activation. Chemically modified CA mats bolster osteoconduction and osteoinduction and may help bone regeneration. Laboy-López and Frenández; Shaban et al.; Rubenstein et al. [135–137] Cellulose, bacterial cellulose nanofibrils (CNF) Cellulose nanofibers (from plant or bacterial sources) are nontoxic, biocompatible, and biodegradable materials that can be produced in large quantities at low cost. Pristine and chemically modified or crosslinked CNFs have applications in controlled drug delivery, antibacterial wound dressing, and skin and bone tissue engineering. Pandey; Torres et al.; Helenius et al. [138–140] Chitosan Chitosan is an amino group-containing polysaccharide derived from the natural chitin sources by deacetylation. It contains randomly ordered D-glucosamine and N-acetyl-D-glucosamine units. Chitosan is a highly biocompatible and biodegradable material that can be digested by either lysozyme or chitinase enzymes in the body. It is frequently used for drug delivery, antibacterial wound dressing, tissue engineering, and bone substitution purposes, in combination with other biopolymers like PEGDA, PLA, gelatin, and alginate. The higher degree of deacetylation increases the strength of cell membrane interactions and cellular uptake. Rodrigues et al.; Venkatesan and Kim; Bojar et al. [ 141 – 143 ] Collagen, Type-I and II Collagen is a natural fibrous protein with excellent biocompatibility, biodegradability and bioactivity. Type I collagen is the major component of the extracellular matrix and the bones, while Type II collagen can be found in the cartilage tissues. Due to their excellent cellular interactions, both types were applied in bone scaffolds and cartilage repair preparations. Ferreira et al.; Rezvani Ghomi et al.; Kilmer et al. [2,144,145] Gels 2023,9, 746 17 of 40 Table 1. Cont. Name Properties References Gelatin Gelatin is a partly hydrolyzed form of collagen containing interconnecting protein chains. It is isolated from animal skin, bone, or connecting tissues. The amino acid composition and sequence is changing with the origin of the tissue. Gelatin is mostly used with other bioactive polymers, i.e., alginate, chitosan, PLLA, and PCL. In scaffolds, it improves cell adhesion, proliferation, and infiltration. Su and Wang; Peter et al. [146,147] Glycosaminoglycan (GAG) Glycosaminoglycans are long-chained polysaccharides built from repeating disaccharide units. They are present on cell surfaces and in the extracellular matrix. Due to their role in regulating the growth factor signaling, interaction with cytokines, and cell surface receptors, GAGs affect, for instance, the inflammation and cell growth processes. They are used in hydrogels, antibacterial surface layers, and porous scaffolds in tissue engineering. Köwitsch et al. [148] Graphene Graphene nanosheets are made from graphite and consist of only a single layer of carbon atoms. Graphene is biocompatible, although it is not biodegradable. Graphene promotes stem cell growth and proliferation, as well as osteogenic differentiation. High concentrations of pristine graphene may decrease cell viability, but PEGylation may reduce that effect. Due to its electrical conductance, it might find application in the fourth generation of bioactive materials. Dubey et al. [134] Graphene oxide (GO) GO is prepared from graphite or graphene by strong chemical oxidation. Epoxides, hydroxyl, and carboxylic groups are generated on the surface, providing connecting points to anchorage-dependent cells to adhere, spread and function. Berrio et al.; Dubey et al. [8,134] Graphene oxide, reduced (rGO) rGO is made from GO by thermal decomposition or chemical reduction. Epoxide rings are removed, but carboxylic and phenolic groups remain on the perimeter. When combined with collagen type-I, the material becomes mechanically more robust and activates the differentiation of human osteoblast stem cells. Scaffolds made with them could be used in bone substitution. Bahrami et al.; Norahan et al. [53,149] Gels 2023,9, 746 18 of 40 Table 1. Cont. Name Properties References Pectin, Methoxyl pectin Pectin is a highly hydrophilic, biocompatible, and biodegradable natural polysaccharide rich in carboxylic group-containing galacturonic acid. When more than half of the carboxylate groups are in the methyl ester form, the material is called high methoxyl pectin; otherwise, we talk about low methoxyl pectin. High methoxyl pectin can form hydrogels under mildly acidic conditions. Low methoxyl pectins can be crosslinked with calcium ions to make them less polar drug carriers. Pectins are used alone or in combination with other natural polymers in the 3D printing of scaffolds. Martau et al.; Li et al.; Tortorella et al. [131,150,151] Poly(lactic-coglycolic acid) (PLGA) PLGA is a highly biocompatible and biodegradable material approved by the FDA for drug delivery, gene engineering, and biomedical uses. Pristine polyglycolic acid would hydrolyze readily. Thus, it is blended with PLA or other polymers to improve hydrolytic and degradation properties. PLGA is combined with different bioactive materials (TCP, HA, gelatin, etc.) or bone morphogenetic proteins (BMPs) and is extensively used in artificial bone substitution applications to facilitate cell adhesion and proliferation. PLGA can easily be formulated into various matrices, from solid scaffolds to nanofiber mats. Makadia and Siegel; Zhao et al.; Elmowafy et al.; Gentile et al.; Jin et al. [152–156] Poly(lactic acid and poly(L-lactic acid) (PLA and PLLA) PLA is a highly biocompatible and biodegradable thermoplastic polymeric material approved by the FDA for biomedical, drug delivery, and tissue engineering applications. Due to the less polar nature of PLA, it is frequently used in co-polymers with hydrophilic polyglycolic acid to improve hydrolytic behavior. When pristine PLA is used alone in the body, it often induces foreign body reactions. Electrospun PLA-copolymers and their microspheres and nanoparticles provide bioactive materials for drug delivery, wound healing, or bone substitution. PLA is widely used in 3D printing. In the human body, PLA implants degrade significantly slower than polyglycolic acid. Makadia and Siegel; Zhao et al.; Elmowafy et al.; Gentile et al.; DaSilva et al.; Tyler et al.; Böstman and Pihlajamaki [ 152 – 155 , 157 – 159 ] Gels 2023,9, 746 19 of 40 Table 1. Cont. Name Properties References Poly(methyl methacrylate) (PMMA) PMMA is a bioinert polymeric material, the main component of acrylic bone cement. The mechanical properties can be improved by blending, i.e., with polystyrene. PMMA-based bone cement can be injected into the position and cured at room temperature. It can be mixed with antibiotics. PMMA is not biodegradable; it usually works as a spacer in joining implants. Fixation properties can be improved by chemical modification of the PMMA structure and by loading with TCP or other bioactive and degradable materials. PMMA cements are FDA-approved bone graft materials. Arora; Magnan et al. [ 160 , 161 ] Poly(ε-caprolactone) (PCL) PCL is an FDA-approved biocompatible and biodegradable synthetic material for human drug delivery, suture, and adhesion barrier applications. The biodegradation is the slowest among the ester-type bone substitute materials. Thus, PCL is used in long-term implants. Orthopedics frequently combines it with bioactive components like silk fibroin, bioactive glasses, or TCP to improve cell adhesion. It can be formulated by molding, pressing, 3D printing, solution or melt electrospinning. Janmohammadi and Nourbakhsh; Dwivedi et al. [162,163] Polybenzoxazine (PBO) The name polybenzoxazine covers a wide range of polymers in which the benzoxazine/polybenzoxazine moiety is the standard building block. PBO resins are prepared by thermal or catalytic ring opening and polymerization of substituted benzoxazine structures derived from synthetic or natural precursors, i.e., cellulose or chitosan. In thin films, PBOs show good antibacterial and antifungal activity. Carbonization at high temperatures results in carbon foams that offer good biocompatibility. Ghosh et al.; Periyasamy et al.; Thirukumaran et al.; Lorjai et al. [164–167] Poly(ethylene glycol diacrylate) (PEGDA) Ethylene glycol diacrylate alone or combined with other acrylates can be easily polymerized or photopolymerized to PEGDA and copolymers. Crosslinking may increase the mechanical strength. PEGDA is a hydrophilic and low-immunogenic compound suitable for scaffolds and hydrogels. It is a good drug depot, and the drug release profile can be finely tuned. It can be used in bio-inks for 3D printing to provide biocompatible flow-through devices. It forms hydrogels that are used in cartilage tissue regeneration. Rekowska et al.; Warr et al.; Qin et al.; Musumeci et al. [168–171] Gels 2023,9, 746 20 of 40 Table 1. Cont. Name Properties References Silica Silica is a biocompatible, biodegradable, and osteoconductive material. Silica enhances the osteogenic differentiation of stem cells and bone regeneration by promoting Type I collagen formation, stabilization, and matrix mineralization. Porous silica can be combined with various polymers, biomaterials, proteins, enzymes, drugs, and hormones. The surface can be covalently functionalized with bioactive agents. Higher concentrations of nano-silica particles may lead to bioaccumulation and cellular damage. Zhou et al.; Jurkic et al.; Shadjou et al.; Vareda et al. [172–175] Silk fibroin Silk fibroin is a natural protein produced by insects. It is a lightweight but mechanically strong material and can be found, i.e., in spider webs and prepared from the cocoon of the domestic silkworm. Scaffolds made of it are biodegradable, can be functionalized, and support the attachment and growth of cells. In the form of fibers, nanofibers, mats, films, and porous structures, silk fibroin has many applications in cell cultures, tissue engineering, and cartilage tissue regeneration. Nguyen et al.; Wang et al.; Wang et al.; Farokhi et al. [176–179] Starch Starch is a natural polysaccharide consisting of d-glucose units. It is produced mainly from potatoes, manioc, or seeds like rice, wheat, and corn. Starch is an edible, biocompatible, and readily biodegradable material. It supports cell growth on the surface. It can be formulated in different shapes and porosities with biodegradable polymeric materials. By 3D prototyping, custom-shaped bioactive scaffolds are created. Martins et al.; Salgado et al. [180,181] Strontium ranelate (SR) SR is a medical drug to treat osteoporosis in men and women, regardless of age. It is capable of reducing the risk of fracture. Strontium ranelate promotes the osteoblastic differentiation of stem cells, inhibits osteoclasts, and improves the structure of bones. Pilmane et al.; Kaufman et al.; Cianferotti et al. [182–184] Gels 2023,9, 746 21 of 40 Table 1. Cont. Name Properties References Tricalcium phosphate (βTCP, TCP) Beta tricalcium phosphate is the “gold standard” of bone grafts approved by the FDA. It is osteoinductive, biodegradable, and one of the most extensively used bone substitute materials in clinical practice. The physical appearance of TCP covers a wide range, from low-strength porous bodies to hard grafts. TCP shows no adverse effects and maintains normal calcium and phosphate ions level in the blood. The apparent in vivo behavior is affected to some extent by the purity and the way TCP was produced. TCP is insoluble under physiological conditions at pH 7.4 and is dissolved and resorbed by cell-mediated processes. The resorption time is in the 6–24 month range. Lu et al.; Bohner et al.; Tanaka et al.; Gilmann and Jayasuriya [185–188] Xanthan gum Xanthan gum is a biodegradable branched polysaccharide produced in large quantities by industrial fermentation with the bacteria Xanthomonas campestris. The backbone is cellobiose, and the branches contain D-mannoses and D-glucuronic acid. The structure of the chain in solutions can be tuned from coiled to helical by increasing the temperature and the ionic strength. High-molecular-weight xanthan gums, frequently in combination with other biopolymers, have found application in the biomedical field, from drug delivery to bone substitute scaffolds. Petri [189] 5.2. Aerogel-Based Materials for Bone Substitution Given the wide range and diverse compositions of aerogel-based materials utilized in hard-tissue engineering, a systematic classification based on shared properties becomes necessary. The approach adopted here involves categorizing all aerogel-containing structures, except for single-phase aerogels, as composite materials, characterized by distinct physical phase boundaries. This classification proves especially relevant when natural or synthetic polymeric materials, and complex or layered structures are present. While the chemical composition remains the primary determinant, other factors, such as biocompatibility, bioactivity, cellular responses, and tissue reactions, and other parameters are deterministic and discussed in the previous sections. Pore structures, their multi-dimensional orientation, and the arrangement of different scaffold layers also exert significant influence. Table 1 presents the wide array of chemical components utilized in the field of aerogel-based tissue engineering. Their combination can yield numerous materials, the management of which is not always straightforward. Figure 3provides an illustrative representation of potential classes and their interconnections, delineating increasing complexities. Gels 2023,9, 746 22 of 40 Gels 2023, 9, x FOR PEER REVIEW 20 of 39 the chain in solutions can be tuned from coiled to helical by increasing the temperature and the ionic strength. High-molecular-weight xanthan gums, frequently in combination with other biopolymers, have found application in the biomedical field, from drug delivery to bone substitute scaffolds. 5.2. Aerogel-Based Materials for Bone Substitution Given the wide range and diverse compositions of aerogel-based materials utilized in hard-tissue engineering, a systematic classification based on shared properties becomes necessary. The approach adopted here involves categorizing all aerogel-containing structures, except for single-phase aerogels, as composite materials, characterized by distinct physical phase boundaries. This classification proves especially relevant when natural or synthetic polymeric materials, and complex or layered structures are present. While the chemical composition remains the primary determinant, other factors, such as biocompatibility, bioactivity, cellular responses, and tissue reactions, and other parameters are deterministic and discussed in the previous sections. Pore structures, their multi-dimensional orientation, and the arrangement of different scaffold layers also exert significant influence. Table 1 presents the wide array of chemical components utilized in the field of aerogel-based tissue engineering. Their combination can yield numerous materials, the management of which is not always straightforward. Figure 3 provides an illustrative representation of potential classes and their interconnections, delineating increasing complexities. Figure 3. Schematic representation of the major types of chemical composition and structure of aerogel-based artificial bone substitute materials. (A) Homogeneous aerogels structures made of singleor multi-component material [15,17,42,61,62,64,65,71,77]. (B) Nanofibrous materials dried to an aerogel structure [52,55–57,79,190]. (C) Aerogel matrix material containing guest particles and/or fibers [14,18,19,53,63,66–70,80,191]). (D) Polymeric matrices containing guest aerogel particles [45,46,118,192]. (E) Highly complex structures made of aerogels, particles and nanofibers [13,24,27,41,54]. (The different symbols in the figure represent guest particles without further specification.) A single-phase homogeneous material may be a chemically one-component pristine aerogel or a multi-component hybrid aerogel in which the components are mixed at the molecular level. In that meaning, there is no difference between aerogels of organic or Figure 3. Schematic representation of the major types of chemical composition and structure of aerogel-based artificial bone substitute materials. ( A ) Homogeneous aerogels structures made of singleor multi-component material [ 15 , 17 , 42 , 61 , 62 , 64 , 65 , 71 , 77 ]. ( B ) Nanofibrous materials dried to an aerogel structure [ 52 , 55 – 57 , 79 , 190 ]. ( C ) Aerogel matrix material containing guest particles and/or fibers [ 14 , 18 , 19 , 53 , 63 , 66 – 70 , 80 , 191 ]). ( D ) Polymeric matrices containing guest aerogel particles [45,46,118,192]. ( E ) Highly complex structures made of aerogels, particles and nanofibers [ 13 , 24 , 27 , 41 , 54 ]. (The different symbols in the figure represent guest particles without further specification.) A single-phase homogeneous material may be a chemically one-component pristine aerogel or a multi-component hybrid aerogel in which the components are mixed at the molecular level. In that meaning, there is no difference between aerogels of organic or inorganic origin (Figure 3A). Nanofibrous materials from mostly polymeric materials may also be distributed evenly in space by different treatments, forming a gel from which homogeneous aerogels are made by different drying techniques (Figure 3B). Such a homogeneous aerogel phase may serve as the matrix material in which guest particles are distributed (Figure 3C). Polymeric materials may also be combined or fortified with aerogel particles as guests to improve properties (Figure 3D). And finally, all the structures may be evolved into a very complex unit where the matrix and guest functions are combined, and new properties may appear due to the synergistic interaction of materials in the living environment. The way aerogels are made for hard-tissue-engineering purposes depends on the material and the properties of the aerogel phase, as well as the complexity of the structure. However, independently from the nature of the materials and the final complexity of the structures, the common point is that all “pre-aerogels” go through a wet gel state, from which the final aerogel is prepared by a suitable drying technique. The technical implementation of wet gel-making procedures is summarized and shown in Figure 4. The simplest and most traditional way, as mentioned in Section 2, is the sol–gel technique (Figure 1). Gels 2023,9, 746 23 of 40 Gels 2023, 9, x FOR PEER REVIEW 21 of 39 inorganic origin (Figure 3A). Nanofibrous materials from mostly polymeric materials may also be distributed evenly in space by different treatments, forming a gel from which homogeneous aerogels are made by different drying techniques (Figure 3B). Such a homogeneous aerogel phase may serve as the matrix material in which guest particles are distributed (Figure 3C). Polymeric materials may also be combined or fortified with aerogel particles as guests to improve properties (Figure 3D). And finally, all the structures may be evolved into a very complex unit where the matrix and guest functions are combined, and new properties may appear due to the synergistic interaction of materials in the living environment. The way aerogels are made for hard-tissue-engineering purposes depends on the material and the properties of the aerogel phase, as well as the complexity of the structure. However, independently from the nature of the materials and the final complexity of the structures, the common point is that all “pre-aerogels” go through a wet gel state, from which the final aerogel is prepared by a suitable drying technique. The technical implementation of wet gel-making procedures is summarized and shown in Figure 4. The simplest and most traditional way, as mentioned in Section 2, is the sol–gel technique (Figure 1). In the gel-casting process (Figure 4A), the reaction mixture is poured into a mold and allowed to set there. The casting process may be combined with the addition of guest particles, fibers, or nanofibers, followed by crosslinking chemical reactions (Figure 4B). Freeze casting is the way to make controlled bimodal pore size distribution by programmed zone freezing of the solvent in the gelation phase (Figure 4C). Stereolithography processes use chemical crosslinking or photochemical polymerization in special 3D-printing techniques to provide custom shape and geometry of scaffolds with controlled macroporosity (Figure 4D). Nanofibrous gels are made from natural nanofibers or electrospun mats by ball milling in an adequately selected solvent (Figure 4E). A rarely used technique is the supercritical gelation and foaming initiated by a rapid pressure drop of gas-saturated polymeric materials combined with other gel-making steps (Figure 4F). Figure 4. Major types of gel-making techniques leading to simpleor complex-shaped materials prior to drying. (A) Sol–gel process and gel casting, (B) gelling and crosslinking, (C) cryotemplating and freeze casting, (D) stereolithography and 3D printing, (E) ball milling, (F) supercritical CO2 gelation and foaming. (The gel is made with scCO2 then expanded by a rapid pressure drop). Figure 4. Major types of gel-making techniques leading to simpleor complex-shaped materials prior to drying. ( A ) Sol–gel process and gel casting, ( B ) gelling and crosslinking, ( C ) cryotemplating and freeze casting, ( D ) stereolithography and 3D printing, ( E ) ball milling, ( F ) supercritical CO 2 gelation and foaming. (The gel is made with scCO2then expanded by a rapid pressure drop). In the gel-casting process (Figure 4A), the reaction mixture is poured into a mold and allowed to set there. The casting process may be combined with the addition of guest particles, fibers, or nanofibers, followed by crosslinking chemical reactions (Figure 4B). Freeze casting is the way to make controlled bimodal pore size distribution by programmed zone freezing of the solvent in the gelation phase (Figure 4C). Stereolithography processes use chemical crosslinking or photochemical polymerization in special 3D-printing techniques to provide custom shape and geometry of scaffolds with controlled macroporosity (Figure 4D). Nanofibrous gels are made from natural nanofibers or electrospun mats by ball milling in an adequately selected solvent (Figure 4E). A rarely used technique is the supercritical gelation and foaming initiated by a rapid pressure drop of gas-saturated polymeric materials combined with other gel-making steps (Figure 4F). 5.2.1. Single-Component and Hybrid Aerogels Creating biocompatible aerogels for hard-tissue replacement can be achieved through a straightforward approach. One option involves using a single biocompatible or bioactive component, or alternatively, combining multiple such ingredients to form a hybrid structure without macroscopic or micron-level internal phase boundaries. Subsequently, these gels can be dried to aerogels without encountering any constraints in the drying process. This custom formulation allows for the development of the essential macroporous structure crucial for facilitating optimal bone tissue ingrowth. Silica–chitosan hybrid aerogels were synthesized by Perez-Moreno and coworkers in a sol–gel process from TEOS and chitosan with the help of high-power ultrasound. Chitosan improved the mechanical properties of the gels, which were dried with supercritical CO 2 to monoliths with very high specific surface area (786–1072 m 2 /g), and a 0.13–0.20 g/cm 3 density range. The aerogels were tested in simulated body fluid, and found that the surface silanol groups promoted the nucleation and formation of hydroxyapatite crystals on the surface, which is an indication of bioactivity. Human osteoblast cells were cultured on the aerogel surface and immunolabeled to monitor cytoskeletal changes and focal adhesion. The aerogels proved to be osteoconductive and osteoinductive in the cell studies [77]. Gels 2023,9, 746 24 of 40 Maleki et al. synthesized a silica–silk fibroin hybrid aerogel scaffold with honeycomb micromorphology and multiscale porosity manufactured from TEOS and silk fibroin in the presence of hexadecyltrimethylammonium bromide in a one-pot acetic acid-catalyzed sol– gel reaction and unidirectional freeze casting, which controlled the size of the macropores in the ten-micron range. The reason for the combination of silica and silk fibroin was to increase the pore size regime and the mechanical strength synergistically. Mechanical strength increased to a 4–7 MPa Young’s modulus. The as-prepared aerogel proved to be cytocompatible and nonhemolytic, showed no toxicity, and triggered MG63 osteoblast cell attachment and proliferation in 14 days. Implantation of the material in rat femur bone defects resulted in bone formation in 25 days [42]. Polybenzoxazine (PBO) aerogel and its hybrid with resorcinol–formaldehyde (PBORF) were prepared and then carbonized at high temperature by Rubenstein and coworkers. Human calvarial osteoblasts were used in the biological studies. Results showed that PBO aerogel and its combination with RF and the carbonized aerogels are compatible with the osteoblasts. However, PBO-RF aerogel resulted in a low growth rate of cells. Carbonized PBO aerogel had better mechanical properties and high porosity. It proved to be the most advantageous for osteoblast growing, which makes the material a promising candidate for tissue-engineering applications [71]. Horvat and coworkers prepared a methoxyl pectin–xanthan aerogel layer on the surface of medical grade stainless steel from the aqueous solution of high-methoxyl pectin and xanthan in an optimized 1:1 ratio by an absolute ethanol-induced gelation process, after which the gel layer was dried with a continuous flow of supercritical CO 2 . Non-steroidal anti-inflammatory drugs diclofenac sodium and indomethacin were loaded in the aerogel either from the saccharide solution directly or from an ethanol solution in the soaking phase. After drying, their release profiles were determined. The aerogel layer protected the steel surface from corrosion, and the loaded drugs were released in one day. The biocompatibility of the layer material was tested after dissolving the aerogels in a buffer with a human bone-derived osteoblast hFOB cell line. The results showed higher viability and better proliferation of the cells in the aerogel solutions than in the control samples [ 15 ]. Quraishi and coworkers prepared meso–macro porous alginate–lignin hybrid aerogels from a basic solution of alginate and lignin, containing calcium carbonate particles gelled under a CO 2 atmosphere (45 bar for 24 h), then foamed by a controlled release of pressure. The as-prepared gels were subjected to solvent exchange and then CO 2 supercritical drying. The biocompatibility of the materials was tested using a mouse fibroblast-like cell line L929. The aerogels proved to be non-cytotoxic in cell studies compared to tissue culture polystyrene reference. The cell viability was similar to that of the control, and the materials showed good cell adhesion and indicated no negative effect of the lignin component. The alginate–lignin aerogels are good candidates as scaffold materials for further in vivo tissue-engineering studies [64]. Calcium– alginate also served as one of the major components in new alginate–starch aerogels prepared by Martins et al. The wet gels were made from an aqueous solution of sodium alginate and starch in the presence of calcium carbonate particles. The gelation occurred under the acidification effect of high-pressure carbon dioxide. A rapid release of carbon dioxide produced a foamed material that was then dehydrated with anhydrous ethanol and dried to aerogel with supercritical carbon dioxide. The macropore formation sharply depended on the rate of depressurization. In simulated body fluid, the material developed surface hydroxyapatite crystals indicating bioactivity potential, which was attributed to the presence of calcium ions. Cell studies with fibroblast-like cell line L929 showed no cytotoxic effect, and the cells colonized the surface. Thus, the alginate–starch hybrid material may be applied in biomedical research and bone repair [65]. Vazhayal and coworkers synthesized mesochanneled and tunable bimodal pore size distribution aluminosiloxane microspheres from acidic pre-hydrolyzed aluminum isopropoxide sol stabilized with PVA and aminopropyl trimethoxysilane solution injected in ammoniac paraffin oil that initiated self-assembly and solidified the droplets. After Gels 2023,9, 746 25 of 40 fortification of the structure by soaking in TEOS, the microspheres were washed, solvent exchanged, and dried to aerogel under subcritical conditions at 50 ◦ C and ambient pressure. Finally, the microparticles were calcined at 600 ◦ C to provide a pH-responsive, controlledrelease drug carrier material. NSAIDs were adsorbed in the aerogel from hexane solutions, and the preparations were tested for release in simulated gastric and intestinal fluids. The biocompatibility and cytotoxicity of the aerogels were tested in vitro on normal H9c2 cells, while gastric ulceration was tested in vivo on albino male rats. Although it was not tested directly, the authors envisaged utilizing these aerogel microspheres in potential bone tissue engineering [61]. To defeat the mechanical limitations of the traditional hydroxyapatite scaffolds, a new highly porous and elastic single-phase aerogel material made from hydrothermally synthesized and freeze-dried ultra-long hydroxyapatite nanowires was prepared by Huang and coworkers. The biological activity was tested with rat bone marrow mesenchymal stem cells. The results showed that the material promotes cell adhesion, proliferation, and migration of the cells and elevate the expression of osteogenesisand angiogenesis-related genes. The nanowire aerogel scaffold can promote the ingrowth of the new bone and neovascularization in the bone defect region, thus making this a promising material for bone tissue engineering [17]. Osorio and coworkers made sulfate or phosphate half-ester-functionalized cellulose nanocrystals and crosslinked them through the carboxylate derivative with adipic acid dihydrazide. The as-prepared materials were transformed into cryogel by freezing in molds at − 5 ◦ C, and then ice crystals were removed by soaking in absolute ethanol. Finally, the materials were dried with supercritical CO 2 to aerogels. The bioactivity was tested on SaOS-2 cells, and the materials showed an increase in cell metabolism for seven days. A simulated body fluid test showed hydroxyapatite layer formation after the materials were pre-treated with calcium chloride solution. The sulfated aerogel proved to be more advantageous regarding mechanical strength and stability under an aqueous environment. In vivo implantation in the calvaria of male rats showed a significant increase in bioactivity in 12 weeks, proving that the new and flexible materials can facilitate bone ingrowth [13]. Reyes-Pecesetal. combinedchitosanwithhydrolyzing3-glycidoxypropyl-trimethoxysilane (GPTMS) in an acid-catalyzed sol–gel process at 50 ◦ C followed by supercritical CO 2 drying resulting in a mechanically exceptionally strong aerogel material. The crosslinking with GPTMS connects the amino and hydroxyl groups of the polysaccharide chains into a hybrid interconnected silica plus carbohydrate network. In vitro , biocompatibilities were proved by the hydroxyapatite layer formation in simulated body fluid. The in vivo bioactivities were tested on human osteoblast cells. No cytotoxicity was observed; the material induced cell adhesion and the cells showed cytoskeletal rearrangements and elongation with stress fibers [62]. 5.2.2. Nanofiber Aerogels Electrospun PLGA-collagen-gelatin nanofibers combined with Sr-Cu co-doped bioglass fibers and bone morphogenetic protein 2 (BMP-2) were combined in a 3D hybrid nanofiber aerogel network by Weng et al. The new material was tested for cranial bone healing using the critical-size rat calvaria model. The sustained slow-release of BMP-2 proteins from the degradable aerogel increased the rate of bone healing significantly and improved the vascularization. Histopathology data showed a near-complete degradation of the aerogel material in the regenerated tissue [79]. Xu and coworkers transformed electrospun polycaprolactone nanofibers into soft, elastic, and very porous aerogel scaffolds by freeze grinding the nanofibers and then by thermally inducing the self-agglomeration, and the as-prepared gels were freeze-dried. In vitro studies with mouse bone marrow mesenchymal stem cells showed high cell viability. Depending on their elasticities, the materials favored osteogenic or chondrogenic differentiation of the stem cells. In vivo experiments indicated that the highly porous and elastic scaffold can act as a favorable synthetic extracellular matrix for bone and cartilage regeneration [193]. Gels 2023,9, 746 32 of 40 In the near future, the refinement of scaffold designs tailored to specific application sites, coupled with novel material combinations, is poised to amplify their therapeutic efficacy and biomedical utility. Beyond advancements in chemical composition and structural intricacy, the next developments of aerogel bone substitute materials may involve external interactions after implantation, to both bolster and monitor the healing process. Author Contributions: I.L., conceptualization; I.L. and M.M., writing—original draft preparation; I.L., M.M. and L. ˇ C., writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. 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