Risk of fragility fracture is aggravated during bone regeneration processes in osteoporotic sheep

PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 1 / 24 OPEN ACCESS Citation: Toscano-Angulo JJ, Mora-Macías J, Blázquez-Carmona P, Morgaz J, Navarrete-Calvo R, Domínguez J, et al. (2025) Risk of fragility fracture is aggravated during bone regeneration processes in osteoporotic sheep. PLoS One 20(5): e0319910. https://doi.org/10.1371/ journal.pone.0319910 Editor: Furqan A. Shah, University of Gothenburg: Goteborgs Universitet, SWEDEN Received: October 22, 2024 Accepted: February 10, 2025 Published: May 2, 2025 Copyright: © 2025 Toscano-Angulo et al . This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data availability statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the MICIU (Ministry of Science, Innovation and RESEARCH ARTICLE Risk of fragility fracture is aggravated during bone regeneration processes in osteoporotic sheep Juan J. Toscano-Angulo 1*, Juan Mora-Macías2, Pablo Blázquez-Carmona 3, Juan Morgaz4, Rocío Navarrete-Calvo4, Jaime Domínguez1, Esther Reina-Romo1 1 Department of Mechanical and Manufacturing Engineering, Escuela Técnica Superior de Ingeniería, Universidad de Sevilla, Sevilla, Spain, 2 Department of Mining, Mechanical, Energy and Building Engineering, Escuela Técnica Superior de Ingeniería, Universidad de Huelva, Huelva, Spain, 3 Department of Mechanical Engineering and Industrial Design, Escuela Superior de Ingeniería, Universidad de Cádiz, Puerto Real, Spain, 4 Department of Animal Medicine and Surgery, Facultad de Veterinaria, Universidad de Córdoba, Córdoba, Spain * [email protected] Abstract Introduction Bone regeneration processes are associated with a systemic skeletal change in bone quality, increasing the risk of fragility fractures. This condition may be aggravated in osteoporotic patients due to their limited osteogenic capacity. This work evaluates the impairment of the bone quality in osteoporotic sheep during a bone regeneration process. It provides a deeper understanding about the complex multiscale dynamics of bone mineral density, microstructure and chemical composition across different bone tissues, locations and time points. Materials and Methods Osteoporosis was induced in fifteen Merino sheep. A critical-size defect was then created in the sheep’s right hind metatarsus and subsequently regenerated using distraction osteogenesis. The animals were randomly sacrificed during bone regeneration, either on days 40 or 100 after surgery. Computed tomography, micro-computed tomography and chemical composition analyses were conducted on different bone tissues (cortical, trabecular and woven) at several skeletal locations (the operated metatarsus, the contralateral one and the iliac crest) to assess the individual bone quality changes relative to the non-osteoporotic time point. Results After osteoporosis induction, the trabecular tissue experienced a 6.4% reduction in the bone mineral density, while no significant changes were reported in cortical tissue quality. During bone regeneration, the operated bone increased significantly the PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 2 / 24 woven ossification whilst the cortical mineral density decreased by 18.7%. Simultaneously, an early deterioration in the microstructure and chemical composition of the trabecular bone was observed in the iliac crest, persisting over time in non-operated trabecular regions. Conclusions Osteoporosis causes uneven degradation to trabecular tissue quality across different bone locations. Furthermore, the bone regeneration process via bone transport in osteoporotic subjects leads to a systemic skeletal disorder that further impairs the bone quality, surpassing the damage caused by osteoporosis alone. This impairment appears to be intensified by the pre-existing osteoporotic condition. Introduction Osteoporosis is the most common bone disease, affecting approximately 500 million people worldwide [1]. This systemic skeletal disorder is characterized by microarchitectural deterioration of the trabecular bone tissue, decreased bone mineral density (BMD) and chemical compositional changes, leading to increased skeletal fragility and a higher risk of fractures. The clinical outcome of osteoporosis is often fractures from low-energy trauma, known as fragility fractures, which would not typically occur in healthy bones. Globally, 37 million fragility fractures occur annually, affecting one-third of women and one-fifth of men over the age of fifty [2–4]. These fractures result in significant economic cost and major health consequences for patients such as severe pain, dependency and disability. In this context, it has been shown that the risk of fragility fractures increases 5-fold during the first year following an initial fracture at the same or different skeletal sites [5], then gradually decreases from a factor of 2.7 after one year to 1.4 after ten years [6]. Numerous preclinical and clinical studies indicate that, during the bone regeneration, a significant systemic bone quality loss may occur throughout the skeleton, especially in osteoporotic patients, further aggravating the disease [7–11]. In addition, the bone regeneration process is frequently compromised by the poor osteogenic capability of osteoporosis [7,12], resulting in delayed healing, non-unions, bone deformities, chronic pain and even post-surgical complications such as infections [13–15]. In these challenging bone healing situations, distraction osteogenesis via bone transport is a recognized technique, clinically indicated due to its potential to promote bone healing [16–18]. This gold-standard orthopedic procedure consists of gradually displacing a bone fragment, along an osteotomized gap [16,19]. Despite its medical interest, there is a lack of knowledge about the skeletal bone quality alterations of applying such complex techniques in osteoporotic patients. In clinical studies, dual energy X-ray absorptiometry (DXA) is the gold standard method for assessing osteoporosis and osteopenia due to its speed, clinical accessibility and low-radiation exposure. Nevertheless, this method is limited by its two-dimensional assessment of trabecular macroscale or apparent BMD and tends Universities of the Spanish Government) and ERPF/EU (European Regional Development Fund of the European Union) via Grant PID 2023-148828OB-I00 funded by MICIU/ AEI/10.13039/501100011033 and by the ERDF/ EU, received by Esther Reina-Romo and Juan Mora-Macías. Competing interests: The authors have declared that no competing interests exist. PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 3 / 24 to overestimate BMD due to its poor differentiation between trabecular and cortical bone tissues [20,21]. In this context, there are alternative techniques for assessing bone quality that are less common for clinical applications due to their cost, their high levels of radiation or their invasive nature (e.g., bone biopsy extraction). However, these techniques are a great source of knowledge about bone quality in experimental research. In this regard, computed tomography (CT) and micro-computed tomography (μCT) are two widely used three-dimensional and high-resolution techniques for assessing, respectively, the macroscale BMD [22–25], the microscale BMD, and the microstructure of bone samples [26–28], thus offering a multiscale analysis of the bone mineral tissue. In addition, chemical composition analyses of bone samples can provide insights into the bone volumetric composition and stoichiometry of bone tissue as seen in studies on cortical and woven tissues of healthy subjects [29,30]. These techniques could enhance the understanding of bone regeneration in osteoporotic patients, aiding clinicians to develop novel patient-specific treatments and therapies. This could lead to a more effective medical care and follow-up of the disease, reducing recovery time and improving patients’ quality of life, as well as contributing to the development of preventive strategies focused on reducing the risk of refractures or secondary fractures. Osteoporosis clinical and experimental research primarily focuses on understanding the biochemical and mechano-biological mechanisms underlying the disease, identifying risk factors [31–33], improving early detection techniques [1,34–39], developing better drug treatments or therapies for patients [40–43], or preventing the onset of the disease or fragility fracture through nutrition [44–46] or physical exercise [47,48]. A smaller proportion of this effort is dedicated to enhancing bone healing management. In this context, large animal models faithfully represent human bone conditions due to their size and anatomy, enhancing translational relevance and assessment of treatment efficacy in osteoporosis research. Numerous large animal studies have validated or characterized the effect of various osteoporosis induction protocols on bone quality [49–56], providing the temporal evolution data on BMD or microstructure of cortical or trabecular tissues, commonly by DXA and μCT [57–67]. For example, Zhang et al. [61] studied the influence of ovariectomy in sheep after 12 months from surgery by μCT of the trabecular tissue at the lumbar vertebra, femoral neck, mandibular angle and rib. They reported significant differences in trabecular microstructure degradation depending on the skeletal site location. Similarly, Bisazza et al. [57] compared DXA and CT techniques to assess temporal changes in trabecular and cortical BMD at lumbar vertebrae in the sheep osteoporotic model. They concluded that CT provides greater accuracy in detecting changes in BMD and bone microstructure. Nevertheless, few large animals’ studies have evaluated the skeletal bone quality evolution experienced by individuals adding the complexity of bone regeneration [10,11]. In this challenging context, Lill et al. [11] assessed by CT the macroscale BMD changes in cortical and trabecular tissues of the tibia between healthy and osteoporotic sheep groups after a mid-shaft tibia1 osteotomy. They found a significant apparent BMD downtrend, but without giving information about other bone locations or microstructure and chemical composition changes. In contrast, Bindl et al. [10] studied the influence of osteoporosis and right femoral metaphysis bone gap healing on different skeletal locations, providing insights into the microstructural bone quality loss in tibial cortical and trabecular tissues between healthy and osteoporotic sheep groups. However, there is a lack of knowledge regarding the temporal bone quality loss experienced by the subject-specific not only in terms of microstructure, but also in terms of mineral density and chemical composition, during the onset of osteoporosis and the subsequent bone regeneration process. Furthermore, as these studies present a simple fracture healing model, they do not provide information about how osteoporosis influences on bone regeneration processes of major complexity but frequently used in cases of severe trauma, such as the distraction osteogenesis model. In contrast, there are studies that have assessed different bone regeneration processes in healthy large animals, such as fracture healing [68–72] or distraction osteogenesis [73–77]. The woven bone generated within the callus has been characterized through different approaches like nanoindentation [78,79], biomechanical tests [11,80,81], histology [71,79,81,80], CT [11,73,75], finite element analysis from CT reconstructions [80,82] and gait analysis [74,75] among others. However, these studies lack information on the characterization of this immature tissue in osteoporotic subjects. PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 4 / 24 From all of this, it is essential to deepen understanding the skeletal bone quality changes in osteoporotic patients adding the complexity of bone regeneration. As a novelty, the present work will explore this combined condition through in vivo experiments using a large osteoporotic animal model undergoing a bone regeneration process treated by distraction osteogenesis via bone transport. We hypothesize that the osteoporotic bone regeneration process significantly aggravates the systemic bone quality deterioration beyond the baseline effects of osteoporosis, which could be tested by measuring the mineral density, microstructure and chemical composition. In addition, the bone impairment may be intensified due to the presence of the disease. Thus, the study aims to elucidate and compare the bone quality changes in different bone locations of individuals during osteoporosis and osteoporotic bone regeneration. This will be achieved through multiscale imaging techniques (CT and μCT) and chemical composition analyses to explore temporal evolution of specific subject’s skeletons. In this sense, the macroscale and microscale BMD, microstructure, and chemical composition of different bone tissues (cortical, trabecular and woven bones) across different skeletal sites (the operated bone, its contralateral counterpart and a bone far from the operated bone) will be evaluated temporally and individually. Materials and methods This section is organized as follows: firstly, the osteoporotic animal model and the distraction procedure using bone transport (BT) are described. Next, the study design is outlined to quantify the individual temporal impact of osteoporosis and the regeneration of osteoporotic bone defect across different bone locations and tissues. Finally, the methodology followed in the different analyses is described: the apparent or macroscale BMD (BMDCT) data measured by CT, the microscale BMD (BMDμCT) and microstructure data measured by μCT, and chemical composition analysis (ash fraction, volumetric composition, and elemental mass content). Osteoporotic animal model and distraction procedure Osteoporosis was induced in a total of fifteen female merino sheep (weight 60.2 ± 5.6 kg), 2–4 years old using the protocol provided by Zarrinkalam et al. [54]. The animals were selected from a research farm and marked on the wool to avoid confounders. The randomly selection criteria ensures that the specimens are healthy and have proper vaccination and deworming protocols. The sample size was calculated to reduce the number of animals required to a relevant minimum, obtaining significant differences. For this purpose, the insights reported by Bindl et al. [10] was used as a reference, in which similar parameters were analyzed in the same osteoporotic animal model. The sheep were transported to the Clinical Veterinary Hospital of the University of Cordoba, where all in vivo experiments were conducted. This research center has spacious, fenced and partially roofed outdoor facilities, where the animals were housed and cared for. The induction period began with a bilateral ovariectomy. The ovariectomized sheep received periodic intramuscular injections of glucocorticoid (500 mg Solu-Moderín® + 7.8 ml injectable water) every 3 weeks for 33 weeks. Additionally, they were exclusively fed with a calcium-free diet till sacrifice, comprising 12% crude protein, 9% crude fiber, 6.5% crude ash, 2% crude fat, 0% calcium, 0.1% phosphorus and 0.1% sodium. The BT experiments began with a surgical procedure performed on the right hind metatarsus of the ovariectomized animal at week 33 ± 2.5. The sheep was under general anesthesia and intubated during both surgical procedures (BT surgery and ovariectomy), while the body temperature, blood pressure, oxygen levels, exhaled carbon dioxide levels, and electrocardiograms were continuously monitored. As illustrated in Fig 1, an Ilizarov-type external fixator [73–75,83] was initially implanted using Ø4 mm Schanz screws. Once the metatarsus was stabilized, three cross-sectional osteotomies were made using a guided oscillating saw (1.2 mm thickness). These osteotomies created two diaphyseal bone fragments: a 25 mm proximal transportable bone fragment (previously attached to the distractor through two Ø2.5 mm Steinmann pins) and a distal 15 mm bone segment which was removed to generate a critical-size bone defect (Fig 1). Following the bone surgery, the calcium free diet was continued throughout the experimental period, but steroid injections were ceased to PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 5 / 24 preserve postsurgical animal welfare. After one-week latency period, a 15-days distraction phase was carried out, applying a 1 mm/day distal displacement of the transportable bone fragment per day along the 15 mm bone gap. In this way, the naïve tissue formed within the proximal osteotomy was elongated (distraction callus) while the tissue of the critical-size bone defect was compressed (docking site callus). Finally, the animals were randomly sacrificed by an overdose of sodium pentobarbital IV Euthasol® at two different time points during the osteoporotic bone regeneration, at day 40 (n = 5) or at day 100 (n = 5) after the BT surgery. Animal welfare was guaranteed throughout the induction period, surgical interventions and experimental phases following the ARRIVE guidelines, European (63/2010/EU) and national (RD 53/2013) regulations on animal research. The animal ethics of this study was approved by University of Córdoba (Protocol Number: 2021PI/21). Study design at different bone locations As shown in Fig 2, various bone tissue types from three bone locations of the sheep were analyzed: the right hind metatarsus (MO, cortical and woven tissues) as the operated bone, the distal left hind metatarsus metaphysis (MNO, trabecular tissue) as the contralateral counterpart, and the iliac crest (IC, trabecular tissue) as a bone distant from the operated bone. The bone samples were evaluated at different time points: on the day of ovariectomy as a non-osteoporotic time point (Healthy), on the day of the BT surgery (week 33 after the ovariectomy) representing the osteoporotic time point (OP), and on the day of sacrifice, 40 or 100 days after surgery, representing the osteoporotic bone regeneration time point (OP + R40 or OP + R100, respectively). Table 1 shows the time points at which the samples were measured by the different analyses. The influence of osteoporosis and osteoporotic bone regeneration on the bone type and location was evaluated with BMDCT measurements in woven, cortical and trabecular bones by CT. And the analysis through scales and locations were assessed with BMDμCT, microstructure and chemical composition measurements of trabecular bone (see Table 1). For this purpose, μCT and chemical composition analyses were performed in trabecular biopsies extracted from MNO and IC. MNO biopsies were only extracted after sacrificing (OP + R40 or OP + R100), as performing this procedure in vivo could compromise animal welfare. Meanwhile, the CT scans and the IC biopsy extractions were conducted in vivo for Healthy and OP time points during the surgical procedures (under general anesthesia and intubated), and ex vivo for OP + R40 and OP + R100 time points. Unlike the MNO, the IC as distant bone is suitable for in vivo biopsy extractions as it is easily accessible and without major harvesting risk, with abundant trabecular tissue. The biopsies were extracted using a biopsy punch and were preserved at -80ºC in PBS-soaked gauzes. Fig 1. Bone transport (BT) surgery procedure in the right hind metatarsus of an ovariectomized sheep. Fixator implantation and bone defect performance: (A) proximal cross-sectional osteotomy; (B) 25 mm transport fragment; (C) 15 mm critical-size bone defect; (D) Ø4 mm Schanz-screws; (E) Ø2.5 mm Steinmann pins; (F) external fixator frames; (G) non-instrumented fixed bars. https://doi.org/10.1371/journal.pone.0319910.g001 PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 6 / 24 For the different analysis performed on all bone samples, each sheep data reported at the Healthy time point is used as its individual control data. In this way, each sheep’s data at subsequent time points were also normalized by its respective control data, thus providing an individualized temporal evolution of the animal. Macroscale data: BMDCT using CT As illustrated in Fig 3A, CT scans were performed in vivo on the animal under general anesthesia and intubated at the Clinical Veterinary Hospital of the University of Cordoba. All CT images were acquired using Revolution ACT (General Electric, Pekin, China) CT scanner (XYZ voxel size 460–570 x 460–570 x 625 μm/px). A CT phantom QRM-BDC/6–200® (PTW, Freiburg, Germany) was also included to linearly correlate BMDCT (0–800 mg HA/cm3) with the stack’s Hounsfield Units (Fig 3B). All the bone samples (MO, MNO and IC) and the phantom inserts (6 x Ø = 18 mm, h = 200 mm) were individually segmented using the open-source image processing tool ImageJ-Fiji. From each axial cross-sectional slice, a mean and standard deviation value of the BMDCT was estimated at each time point. Intermediate CT scans were taken at weeks 10 and 20 after ovariectomy (10w and 20w time points, respectively) to assess the temporal evolution of BMDCT in MNO Fig 2. Study design scheme. Ovariectomized sheep analyses of the right hind metatarsus cortical and woven bone tissue (MO), the distal metaphysis trabecular tissue of the left hind metatarsus (MNO), and the iliac crest trabecular tissue (IC). https://doi.org/10.1371/journal.pone.0319910.g002 Table 1. Study design overview. Bone assessment Time points Name Interest Tissue type (a) Healthy (b) OP (c) OP + R40 (d) OP + R100 Right Hind Metatarsus (MO) Operated bone Cortical & Woven CT CT CT CT Left Hind Metatarsus (MNO) Contralateral counterpart Trabecular CT CT CT μCT ChC CT μCT ChC Iliac Crest (IC) Distant bone from the operated one Trabecular μCT ChC μCT ChC CT μCT ChC CT μCT ChC Different bone locations (MO, MNO and IC) and bone tissue types (cortical, trabecular and woven) analyses (CT: computed tomography, μCT: micro-computed tomography and ChC: chemical composition) at each time point: (a) Healthy, on the day of ovariectomy; (b) OP, on the day of the BT surgery, 33 weeks after the ovariectomy; (c) OP + R40 and (d) OP + R100, on day 40 and 100 after the BT surgery, respectively. https://doi.org/10.1371/journal.pone.0319910.t001 PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 7 / 24 during the osteoporosis induction phase. The trabecular tissue was segmented without including the cortical tissue in the MNO (13% of total length of the left hind metatarsus) and IC (complete bone). As shown in Fig 3D, different representative regions within the operated metatarsus (MO) were monitored to quantify the local and temporal evolution of the BMDCT in the cortical and woven bone tissues according to each animal’s final CT scan (OP + R40 or OP + R100). Mean and standard deviation values of BMDCT were calculated from three cortical fragments and two bone calluses. The cortical BMDCT was quantified in the proximal, transport and distal fragments (13%, 9% and 12% of the total length of MO, respectively). The woven bone BMDCT was measured in the distraction and docking site calluses (5%, and 0.5% of the total length of MO, respectively). Microscale data: BMDμCT and microstructure using μCT The μCT measurements were taken with the μCT scanner (XYZ voxel size 22.17–34.61 μm/px) model Y. COUGAR SMT (YXLON, Hamburg, Germany) at the University of Seville Research, Technology and Innovation Center (CITIUS). As shown in Figs 4A and 4B, the μCT measurement includes harvested trabecular biopsies from the iliac crest (IC) or contralateral metatarsus (MNO), along with a cylindrical μCT phantom, both fixed to a 3D printed support and placed within Fig 3. Macroscale BMD characterization of the osteoporotic bone. CT assessment of the BMDCT in cortical (operated metatarsus, MO: proximal, transport and distal fragments), in woven (MO: distraction callus, docking site callus) and in trabecular (non-operated metatarsus, MNO). (A) CT measurement of an ovariectomized sheep. (B) CT phantom included in the CT measurement. C) CT XZ (top) and XY (down) measurement projections. (D) Cross-sectional BMDCT throughout the bone distal percentage of MO length (0% proximal, 100% distal) in an ovariectomized sheep at Healthy, OP and OP + R100 time points. https://doi.org/10.1371/journal.pone.0319910.g003 PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 8 / 24 the μCT scanner. The μCT phantom (Ø = 4.5 mm, h = 5 mm) model MicroCT-HAD4.5® (QRM, Moehrendorf, Germany) was included to establish a linear correlation between the BMDμCT of the phantom inserts (0–1200 mg HA/cm³) and the Hounsfield Units of the μCT stack. The μCT measurements were carried out using an open μCT multifocus tube of 25–160 kV voltage and current intensity of 0.01–1 mA. The complete post-processing methodology for the μCT measurements is illustrated in Fig 4C, according to the guidelines for assessment of bone microstructure provided by Bouxsein et al. [84]. First, the BMDμCT of the phantom inserts (5 x Ø = 0.8 mm, 4.5 mm height) was measured using ImageJ-Fiji® by means of the plugging BoneJ. Using the Amira-Avizo® software, all trabecular samples were segmented with a lower threshold of 400 mg HA/cm3 for normalized segmentation value of Hounsfield Units. The threshold was confirmed by visual inspection after checking its capability to adequately discriminate between trabeculae and other components presented in the different bone samples (pores, blood, air, etc.). From the 3D reconstruction of the trabecular volume, the trabecular microstructural parameters were calculated: the trabecular thickness (Tb.Th), the trabecular separation (Tb.Sp), the bone volume per total volume (BV/TV), the trabecular number (Tb.N), and the structure model index (SMI). Finally, the degree of anisotropy (DA), the connectivity density (Conn.D), the fractal dimension (FD), and the mean cross-sectional microscale BMDμCT value of the sample was measured using ImageJ-Fiji® by means of the plugging BoneJ. Chemical composition analyses Chemical composition analyses were performed on the trabecular biopsies from the iliac crest (IC) and the contralateral metatarsus (MNO) after the μCT measurements at the University of Seville Research, Technology and Innovation Center (CITIUS). Firstly, a manual grinding procedure was carried out using a sterilized pestle and mortar to reduce the samples Fig 4. Microscale BMD and microstructure characterization of the osteoporotic trabecular tissue samples. (A) μCT set up. (B) XY μCT stack: IC biopsy and μCT phantom fixed to a 3D printed support (top). (C) μCT measurement procedure. https://doi.org/10.1371/journal.pone.0319910.g004 PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 9 / 24 to a particle size of 1 mm. Subsequently, the total mass mt of the sample was measured. This mass comprises the mass of water mw, the organic mass or organic phase mo and the mineral mass or mineral phase mm, as defined in Eq. 1. mt=mw+mo+mm (1) Afterwards, a drying procedure was applied to the sample by heating it at 105ºC for 1 hour in a BINDER VD 23 vacuum drying chamber (BINDER GmbH, Tuttlingen, Germany). The samples were weighed and heated again at 105ºC every 15 minutes until a constant dry mass md was achieved. md=mo+mm=mt–mw (2) Then, the samples underwent an ashing process in a Nabertherm Muffle Furnace (Nabertherm GmbH, Lilienthal Germany), following the protocol provided by Martínez-Reina et al. [29]: (A) a lineal increase to 250ºC for 30 min; (B) a constant temperature of 250ºC for 1 hour; (C) a lineal increase from 250ºC to 650ºC for 30 min; (D) a constant temperature of 650ºC for 2 hours; (E) sample weighing and 650ºC constant temperature for 30 min until a constant ash mass ma is achieved. The protocol burns the organic phase of the sample, so ma corresponds with the mineral phase, as shown in Eq. 3. ma=mm=md–mo (3) To assess the mineral content, the ash fraction, α, was calculated using Eq. 4. α = m a md = m m mm+mo (4) The volumetric fractions vx of the mass x is obtained by: ν x= Vx Vw+Vo+Vm = m x ρx mw ρ w +mo ρ o +mm ρ m (5) where the density of the mass is ρw = 1 g/cm3, ρo = 1.43 g/cm3 [85] and ρm = 3.12 g/cm3 [29]. Later, an elemental analysis was carried out using a TruSpec Chns Micro analyzer (LECO Corporation, St Joseph, MI, USA) to determine the mass percentage of carbon. After previously dissolving the samples in hydroalcoholic acid, the mass percentage of calcium, phosphorus, magnesium, potassium, sodium and strontium were finally acquired through inductively coupled plasma atomic emission spectrometer Ultima 2 (HORIBA Jobin Yvon, Edison, NJ, USA). Statistical analyses The statistical analyses were carried out using the software tool MATLAB R2023b® (The MathWorks Inc., Natick, MA, USA). A mean and standard deviation value from the individuals was obtained for all parameters measured at every time group (Healthy, OP, OP + 40 and OP + R100) in each analysis (CT, μCT and chemical composition). Simultaneously, a mean and standard deviation value was calculated from the individual data previously normalized by its respective Healthy time point. The underlying absolute and normalized individual data are compiled in the S1-S4 Tables. The time groups outliers were checked and excluded using a Grubbs’ test [86] when p < 0.05. A Shapiro–Wilk test was performed to verify the normality of time groups. The data were analyzed in search of significant differences among time groups using non-parametric tests due to the non-normal distribution (p < 0.05) of the time groups presented in the different analyses (absolute and normalized data). Kruskal-Wallis test followed by Dunn–Sidak post hoc and correction was performed in PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 16 / 24 sheep tibia fracture healing model, with BMDCT reductions of 1.3% and 2.2% at the proximal and distal bone fragments ends, respectively. The greater reductions in the current study are likely due to the higher size of our defect and the influence of induced osteoporosis. Augat et al. [68] proposed that this phenomenon might result from reduced load-bearing capacity in the operated limb following surgery, leading to bone tissue readaptation and resorption [74,83]. This condition induces a bone tissue readaptation in the intervened limb resulting in bone resorption. Another possible explanation that also could lead to an operated bone decreased osteoblastic activity, is the reduction of vascularization in the distal direction due to the osteotomies and the size of the defect [88]. This hypothesis can be supported by the more pronounced degradation of the cortical tissue in the distal direction of the metatarsus (Fig 5A). Augat et al. [68] also observed BMDCT differences although not significant, probably due to the simplicity of the defect generated (2 mm osteotomy). One last hypothesis could be the need for bone mineral to repair the bone defects, which is in line to the mentioned increased level of ossification reported in both calluses at both bone regeneration, especially early in the docking site callus regeneration, as mechanically characterized by Mora-Macías et al. [78] in BT healthy sheep model. In the present study, the mentioned mineral need becomes even more critical due to the poor osteogenic capability caused by the pathology [7,12]. In this context, bone defect regeneration processes could produce a systemic skeletal disorder, making the areas closest to the defect a potential source of mineral resources to contribute to the bone regeneration [68]. However, the transport fragment does not seem to have a significant mineral contribution during the first 40 days after the bone intervention (OP to OP + R40 in Table 2 and Fig 5A). This outcome is probably due to the vascular disconnection of this transport fragment which prevents its minerals from being used as a resource for both adjacent woven mineralization. In contrast, there was a significant loss of BMDCT at 100 days after surgery (OP to OP + R100), suggesting the need for prior angiogenesis and woven bridging to contribute to both callus osteogenesis. Concluding with the macroscale characterization, in the operated bone, the significant cortical BMDCT loss over time and woven mineralization indicates that the mineral has been mobilized for the bone defects regeneration treated by distraction osteogenesis. This need appears to be even more critical due to osteoporosis. In contrast, the influence of this disease alone in the BMDCT seems to be limited to the trabecular tissue of the contralateral bone (Fig 5C). Regarding the microscale analysis of the trabecular tissue performed in the iliac crest by μCT (Table 4 and Figs 6A and 6B), no significant differences were reported after 33 weeks of osteoporosis induction in any of the computed parameters (Healthy to OP). This finding supports previous studies indicating that osteoporosis does not uniformly affect trabecular tissues at different locations [89–91]. In contrast, the iliac crest trabecular microstructure was shown to remodel during the first 40 days after the BT surgery (OP to OP + R40), with a significant increase in the Tb.Sp (67%), and a decrease in the BV/TV (31%), in the Tb.N (28%), and in the Conn.D (54%) in relation to Healthy value. Consequently, the iliac crest trabeculae after the intervention are notably less dense and interconnected, with fewer trabeculae. As far as the authors are concerned, there are no studies assessing the individual microstructural changes of the trabecular tissue throughout a bone defect regeneration process in an osteoporotic sheep model to compare. However, our findings are in line with Bindl et al. [10], who compared osteoporotic and healthy sheep groups, the osteoporotic right femoral fracture healing also induces a decrease in the BV/TV of 32% and the Tb.N of 31%, and a 57% increase in the Tb.Sp at the right tibial trabecular tissue at 56 days post-fracture. In addition, the μCT analysis conducted by Fischer et al. [4] in osteoporotic mice revealed a significant degradation of trabecular microstructure (25% reduction in BV/TV and Tb.N) in the lumbar vertebrae of the femur fractured group compared to the non-fractured group. Regarding the BMDμCT measured 40 days after BT surgery (OP to OP + R40), no significant differences were reported. Unlike CT, which computes an apparent BMD (BMDCT) with trabeculae and pores, μCT has the resolution to analyze the trabeculae focusing on the mineral phase, which appears to maintain the mean BMDμCT while impairing the trabecular iliac crest microstructure. By 100 days post-surgery, the absence of significant differences in BMDμCT and microstructure parameters of the iliac crest with respect to 40 days after surgery (OP + R40 to OP + R100, 60 days difference) suggests that trabecular tissue far enough from the regeneration focus is not affected by the bone regeneration in an osteoporotic subject (Table 4 and Figs 6A and 6B). This is in line with the multiscale BMD results of both trabecular bones (Figs 7A and 7B) and the trabecular microstructure results PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 17 / 24 of the contralateral metatarsus (Table 4). This microstructure was observed to be weaker (higher Tb.Sp, lower Tb.N and Conn.D) than that reported by Blázquez-Carmona et al. [77] in the operated metatarsus trabecular tissue of healthy sheep undergoing a bone lengthening process using distraction osteogenesis. According to this, the osteoclastic activity seems to increase in the osteoporotic trabecular tissue of far non-fractured bones at the early stage, during the first 40 days after the bone surgical intervention (OP to OP + R40). This fact is probably a systemic response of the osteoporotic skeleton to further contribute to the supply of minerals for the bone repair of the operated metatarsus. In addition, the main microscale changes in the trabecular tissue in the non-fractured bones are due to the combination of osteoporosis with the bone callus regeneration process, rather than solely osteoporosis. In terms of chemical composition analyses of the iliac crest trabecular tissue, the ash and volumetric composition temporal evolution results are illustrated in Table 5 and Fig 8A. As far as the authors know, there are no studies in which the volumetric composition is assessed during osteoporosis and osteoporotic bone regeneration (the same for the ash fraction) at the non-fractured trabecular tissue to compare. Considering the differences, the results could be compared with those of Martínez-Reina et al. [29], who analyzed the volumetric composition and elemental mass content percentage of the lamellar (cortical) and woven tissue from the metatarsus of healthy sheep undergoing a BT regeneration process. According to Table 5 and Fig 8A, the parameters measured did not report significant changes at the end of the osteoporosis induction (Healthy to OP), consistent with Bloebaum et al. [92], who found no differences in the ash fraction between young and osteoporotic women groups at different trabecular sites. During the first 40 days after the bone surgical intervention (OP to OP + R40), a significant loss of the ash fraction took place (31.6% with respect to Healthy value). With respect to the volumetric composition of the iliac crest during the bone regeneration (OP to OP + R40 or OP + R100), the significant opposite trends reported in water volumetric fraction (downtrend) and organic phase volumetric fraction (uptrend) seems to indicate a volumetric change comparable to that reported by Martínez-Reina et al. [29] in healthy woven bone. In this immature bone tissue, a high volumetric fraction of organic phase means that it is undergoing a process of mineral change, either absorption or resorption. In addition, a mineral phase loss of volumetric fraction occurred in the early period of osteoporotic bone regeneration, which appears to recover over time. All this information seems to indicate that the osteoporotic bone regeneration stimulates the production of organic matrix in distant non-fractured bones, which suggest the displacement of blood volume to contribute with the inflammation and the transport of minerals. This early loss of mineral phase mass in relation to the mineral phase plus organic phase mass (ash fraction) and water volumetric fraction reflects, once again, a mineral mass resorption and mobilization from the trabecular tissue of the iliac crest to contribute to the osteoporotic regeneration of the operated metatarsus. This hypothesis is consistent with the trabecular iliac crest microstructure parameters degradation discussed above (Table 4 and Figs 6A and 6B). The percentage of the elemental mass content from the mineral phase of the iliac crest trabecular tissue is shown in Table 5 and Fig 8B. While no direct comparisons are available in the literature, Martínez-Reina et al. [29] measured the mass percentages of C, P, and Ca in non-osteoporotic cortical and woven tissue. The comparisons between this study and the current work are limited due to the osteoporosis influence and the differences between these bone tissues with the trabecular tissue, without also analyzing other elements such as Mg, K, Na and Sr. As shown in Fig 8B, the percentage of elemental mass content of C and Mg experienced an increase during the osteoporosis induction (Healthy to OP). An increase in the mass percentage of C (probably from a degradation of the organic matrix) and Mg, indicates an increase in the carbonate content of the hydroxyapatite, which may make it less stable and more fragile and susceptible to an osteoporotic increased reabsorption. However, the elemental mass percentages of Ca, P, K, Na and Sr remained constant, showing that in this trabecular bone the percentage of hydroxyapatite has not changed significantly due to osteoporosis. The most abundant metallic elements in the mass percentage of hydroxyapatite (Ca and P) appear not to change significantly during the osteoporotic bone regeneration (OP to OP + R40 or OP + R100) of the operated metatarsus. However, it cannot be assumed that the mineral mass is not being lost, since the results are described as mass percentage. The same applies to three of the less abundant elements in the bone mineral, C, Na and Sr. In contrast, the mass PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 18 / 24 percentage of K and Mg reported a significant decreasing and increasing trend, respectively, during the osteoporotic bone regeneration process, not reported during the osteoporotic induction period. This fact suggests a metabolic and homeostatic adaptation through the redistribution of secondary elements. In this regard, the increase in Mg could improve the formation of new bone tissue and cellular activity [93], while the decrease in K could indicate its less direct involvement in mineralization, thus focusing on the needs of osteoporotic bone regeneration of the operated bone. With respect to the comparison of the osteoporotic bone regeneration points for both trabecular tissues of non-fractured bones (iliac crest and contralateral metatarsus) in all the analyses (CT, μCT and chemical composition) performed in this work (Tables 4 and 5), no significant differences were found. This fact may indicate that both trabecular tissues have behaved in a similar way between days 40 and 100 after the bone surgery (OP + R40 to OP + R100), prioritizing the osteogenesis of operated bone woven tissues over the recovery of non-fractured trabecular osteoporotic tissue. As for the limitations of the work, it should be noted that not all the analyses presented have been carried out in all the bone tissues and locations considered throughout all the time points (Healthy, OP, OP + R40 or OP + R100) of measurement of the sheep. In the case of μCT scanning and chemical composition analysis, the individual temporal evolution is only completed at the iliac crest, as any in vivo biopsy extractions at the operated or contralateral metatarsus would have compromised the welfare of the animals. In addition, two sheep suffered health difficulties and had to be slaughtered before the BT surgery. Meanwhile, three animals experienced post BT surgery complications that made it necessary to slaughter them before the bone regeneration time point. Thus, these specimens did not provide the complete individual temporal evolution data. The CT scans were limited to a single time point during bone regeneration due to the presence of metal elements in the implanted external fixator, which required post-sacrifice CT scans after fixator removal. Consequently, the comparison between the OP + R40 and OP + R100 time points refer to animals from different sacrifice groups, therefore it is not possible to talk about an individual temporal evolution when comparing both time points. Finally, the influence of osteoporosis during the bone regeneration process could not be determined explicitly since the study does not consider a healthy control group undergoing the same bone regeneration process. In conclusion, this study provides novel quantitative insights into the alterations in bone quality experienced by individual subjects during the onset of osteoporosis combined with bone regeneration processes treated by distraction osteogenesis. Specifically, multiscale BMD, microstructure and chemical composition were assessed in different bone tissues and locations in osteoporotic large animal subjects undergoing a critical-size bone regeneration process. It is determined firstly that osteoporosis alone only seems to significantly impair the bone quality of trabecular tissue heterogeneously depending on the bone localization. Secondly, the osteoporotic bone regeneration process following bone transport interventions results in a systemic skeletal disorder, which is greater than osteoporosis itself, and appears to be intensified by this disease. At the operated bone, this combined condition caused a significant loss of cortical bone quality that was aggravated over time in the distal direction. In addition, the trabecular tissues of far bones were also especially affected during the early stage of bone regeneration, deteriorating their microstructure and altering their chemical composition. This generalized skeletal bone quality impairment seems to be a quick response of the organism to regulate the blood mobilization of bone mineral sources that can contribute to enhance the woven tissue mineralization of the operated bone. This mineral requirement is even more critical in osteoporotic individuals in whom the osteogenic capacity is severely diminished by estrogen deficiency, accelerating bone resorption and decreasing osteoblastic activity. In this sense, the present work reflects the need to study the bone quality by different measurement approaches (multiscale BMD, microstructure, and chemical composition) at different bone locations to evidence the aggravation of the osteoporotic bone regeneration process. In osteoporotic patients who need distraction osteogenesis procedure, it is important to prioritize the continuous monitoring of the bones statistically more prone to a new fracture, especially the already threated bone under regeneration, whose fixation is at high risk of being mechanically and fatally compromised. Therefore, it is crucial the continuous bone quality care and clinical follow-up, especially during the early stage of bone regeneration, to ensure adequate healing, considering the several risks of refractures or secondary fractures. PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 19 / 24 Supporting information S1 Table. Underlying data points of Table 2. Sheep data points of the macroscale BMD characterization in osteoporotic operated bone. (XLSX) S2 Table. Underlying data points of Table 3. Sheep data points of the macroscale BMD characterization in osteoporotic non-fractured trabecular bones. (XLSX) S3 Table. Underlying data points of Table 4. Sheep data points of the microscale BMD and microstructure characterization in osteoporotic non-fractured trabecular bones. (XLSX) S4 Table. Underlying data points of Table 5. Sheep data points of the chemical composition characterization in osteoporotic non-fractured trabecular bones. (XLSX) S5 Table. Statistical analyses of Table 2. P-values of the macroscale BMD characterization in osteoporotic operated bone. (XLSX) S6 Table. Statistical analyses of Table 3. P-values of the macroscale BMD characterization in osteoporotic non-fractured trabecular bones. (XLSX) S7 Table. Statistical analyses of Table 4. P-values of the microscale BMD and microstructure characterization in osteoporotic non-fractured trabecular bones. (XLSX) S8 Table. Statistical analyses of Table 5. P-values of the chemical composition characterization in osteoporotic non-fractured trabecular bones. (XLSX) S9 File. Full ARRIVE 2.0 Guidelines checklist. (PDF) Acknowledgments Grant PID 2020–113790RB-I00 funded by MICIU/AEI/10.13039/501100011033. The authors would like to thank veterinary technicians Juan Ramón Ríos Pérez, Ana Soto Fernández and Álvaro Rincón Panadero for their commitment, effort and involvement in this work. Author contributions Conceptualization: Juan J. Toscano-Angulo, Juan Mora-Macías, Pablo Blázquez-Carmona, Esther Reina-Romo. Data curation: Juan J. Toscano-Angulo. Formal analysis: Juan J. Toscano-Angulo, Pablo Blázquez-Carmona, Esther Reina-Romo. Funding acquisition: Juan Mora-Macías, Esther Reina-Romo. Investigation: Juan J. Toscano-Angulo, Juan Mora-Macías, Pablo Blázquez-Carmona, Juan Morgaz, Esther Reina-Romo. PLOS One | https://doi.org/10.1371/journal.pone.0319910 May 2, 2025 20 / 24 Methodology: Juan J. Toscano-Angulo, Juan Morgaz, Rocío Navarrete-Calvo. Project administration: Esther Reina-Romo. Resources: Juan J. Toscano-Angulo, Juan Mora-Macías, Esther Reina-Romo. Software: Juan J. Toscano-Angulo. 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