Healing Of Critical-size Bone Defects Research Articles

Retraction: Bone healing in critical-size defects treated with new bioactive glass/calcium sulfate: A histologic and histometric study in rat calvaria.

Retraction: Nagata, M.J.H., Furlaneto, F.A.C., Moretti, A.J., Bouquot, J.E., Ahn, C.W., Messora, M.R., Fucini, S.E., Garcia, V.G. and Bosco, A.F. (2010), "Bone healing in critical-size defects treated with new bioactive glass/calcium sulfate: A histologic and histometric study in rat calvaria." J. Biomed. Mater. Res., 95B: 269-275. https://doi.org/10.1002/jbm.b.31710. The above article, published on October 8, 2010 in Wiley Online Library (www.wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Jeremy Gilbert, and Wiley Periodicals. The retraction has been agreed due to significant overlap with a previously published article from a similar group of authors (1), as indicated by a third party. An investigation by the publisher and the Editor-in-Chief confirmed the finding of significant overlap, which included the editor's observation that the cranium featured in Figure 1 of the article is identical to Figure 1 in (1), despite a difference in markings. It was agreed by the publisher and editor that the similarities between the two articles are too significant and that the article must be retracted. Maria J.H. Nagata, on behalf of all the co-authors, disagrees with the retraction. (1) Furlaneto, F.A.C., Nagata, M.J.H., Fucini, S.E., Deliberador, T.M., Okamoto, T. and Messora, M.R. (2007), "Bone healing in critical-size defects treated with bioactive glass/calcium sulfate: a histologic and histometric study in rat calvaria." Clinical Oral Implants Research, 18: 311-318. https://doi.org/10.1111/j.1600-0501.2006.01331.x.

Enhanced osteogenic and ROS-scavenging MXene nanosheets incorporated gelatin-based nanocomposite hydrogels for critical-sized calvarial defect repair

The healing of critical-sized bone defects is a major challenge in the field of bone tissue engineering. Gelatin-related hydrogels have emerged as a potential solution due to their desirable properties. However, their limited osteogenic, mechanical, and reactive oxygen species (ROS)-scavenging capabilities have hindered their clinical application. To overcome this issue, we developed a biofunctional gelatin-Mxene nanocomposite hydrogel. Firstly, we prepared two-dimensional (2D) Ti3C2 MXene nanosheets using a layer delamination method. Secondly, these nanosheets were incorporated into a transglutaminase (TG) enzyme-containing gallic acid-imbedded gelatin (GGA) pre-gel solution to create an injectable GGA-MXene (GM) nanocomposite hydrogel. The GM hydrogels exhibited superior compressive strength (44–75.6 kPa) and modulus (24–44.5 kPa) compared to the GGA hydrogels. Additionally, the GM hydrogel demonstrated the ability to scavenge reactive oxygen species (OH- and DPPH radicals), protecting MC3T3-E1 cells from oxidative stress. GM hydrogels were non-toxic to MC3T3-E1 cells, increased alkaline phosphatase secretion, calcium nodule formation, and upregulated osteogenic gene expressions (ALP, OCN, and RUNX2). The GM400 hydrogel was implanted in critical-sized calvarial defects in rats. Remarkably, it exhibited significant potential for promoting new bone formation. These findings indicated that GM hydrogel could be a viable candidate for future clinical applications in the treatment of critical-sized bone defects.

A facile and smart strategy to enhance bone regeneration with efficient vitamin D3 delivery through sterosome technology

The spontaneous healing of critical-sized bone defects is often limited, posing an increased risk of complications and suboptimal outcomes. Osteogenesis, a complex process central to bone formation, relies significantly on the pivotal role of osteoblasts. Despite the well-established osteogenic properties of vitamin D3 (VD3), its lipophilic nature confines administration to oral or muscle injection routes. Therefore, a strategic therapeutic approach involves designing a multifunctional carrier to enhance efficacy, potentially incorporating it into the delivery system. Here, we introduce an innovative sterosome-based delivery system, utilizing palmitic acid (PA) and VD3, aimed at promoting osteogenic differentiation and facilitating post-defect bone regeneration. The delivery system exhibited robust physical characteristics, including excellent stability, loading efficiency, sustained drug release and high cellular uptake efficiency. Furthermore, comprehensive investigations demonstrated outstanding biocompatibility and osteogenic potential in both 2D and 3D in vitro settings. A critical-sized calvarial defect model in mice recapitulated the notable osteogenic effects of the sterosomes in vivo. Collectively, our research proposes a clinically applicable strategy for bone healing, leveraging PA/VD3 sterosomes as an efficient carrier to deliver VD3 and enhance bone regenerative effects.

Evaluation of Two Anorganic Bovine Xenogenous Grafts in Bone Healing of Critical Defect in Rats Calvaria.

The purpose of this study was to provide an evaluation of two different xenogeneic bone substitutes in bone healing of critical-sized bone defects (Ø =5mm) created in rats calvaria. Thirty animals were randomized into 3 groups with one of the following treatments. In the control group (n=10), the defects were filled with blood clots; BO group (n=10), the defects were filled with bovine medullary bone substitute (Bio-Oss®); BF group (n=10), the defects were filled with bovine cortical bone substitute (Bonefill®). All defects were covered with an absorbable membrane. Five animals from each group were euthanized at 30 and 45 days, subsequently histomorphometrical and immunohistochemical analyses were performed. The histomorphometry was used to measure the percentage of new bone formation in the total area of the defect while the immunohistochemistry evaluated the expression of bone immunomarkers for bone morphogenetic protein 2/4 (BMP2/4), osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP). Data was statistically analyzed with a 5% significance level. The results demonstrated that the BO group showed greater bone formation compared to the BF group at 30 days (P<0.05). However, there was no statistically significant difference between the control and BO groups at 30 days (P>0.05). The expression of BMP2/4 and OCN were higher in the BO group at 45 days compared to the BF at 30 and 45 days respectively (P<0.05). In conclusion, even with the higher expression of proteins related to bone formation, there was no difference in new bone formation at 45 days when both anorganic bovine xenogenous grafts were evaluated.

Development of miR-26a-activated scaffold to promote healing of critical-sized bone defects through angiogenic and osteogenic mechanisms

Very large bone defects significantly diminish the vascular, blood, and nutrient supply to the injured site, reducing the bone's ability to self-regenerate and complicating treatment. Delivering nanomedicines from biomaterial scaffolds that induce host cells to produce bone-healing proteins is emerging as an appealing solution for treating these challenging defects. In this context, microRNA-26a mimics (miR-26a) are particularly interesting as they target the two most relevant processes in bone regeneration-angiogenesis and osteogenesis. However, the main limitation of microRNAs is their poor stability and issues with cytosolic delivery. Thus, utilising a collagen-nanohydroxyapatite (coll-nHA) scaffold in combination with cell-penetrating peptide (RALA) nanoparticles, we aimed to develop an effective system to deliver miR-26a nanoparticles to regenerate bone defects in vivo. The microRNA-26a complexed RALA nanoparticles, which showed the highest transfection efficiency, were incorporated into collagen-nanohydroxyapatite scaffolds and in vitro assessment demonstrated the miR-26a-activated scaffolds effectively transfected human mesenchymal stem cells (hMSCs) resulting in enhanced production of vascular endothelial growth factor, increased alkaline phosphatase activity, and greater mineralisation. After implantation in critical-sized rat calvarial defects, micro CT and histomorphological analysis revealed that the miR-26a-activated scaffolds improved bone repair in vivo, producing new bone of superior quality, which was highly mineralised and vascularised compared to a miR-free scaffold. This innovative combination of osteogenic collagen-nanohydroxyapatite scaffolds with multifunctional microRNA-26a complexed nanoparticles provides an effective carrier delivering nanoparticles locally with high efficacy and minimal off-target effects and demonstrates the potential of targeting osteogenic-angiogenic coupling using scaffold-based nanomedicine delivery as a new "off-the-shelf" product capable of healing complex bone injuries.

Evaluation of Vitamin D-enriched Bone Graft in Surgically-induced Critical-sized Bone Defects: An Experimental Study.

Restoration of bone defects in the craniac vault may require the use of autografts, allografts, xenografts, or synthetic grafts. There are promising data that vitamin D may play a positive role in graft incorporation. The purpose of the present study is the evaluation of the impact of vitamin D addition to human-derived bone grafts in the healing of critical-sized bone defects in porcine skulls. Four identical critical-sized defects were created in the calvaria of 8 adult Landrace Large White pigs. The first defect was left blank as control, the second defect was filled with human-derived bone graft, the third defect was filled with human-derived bone graft enriched with a low concentration of vitamin D (2mg/mL), and the fourth defect was filled with human-derived bone graft enriched with a high concentration of vitamin D (10mg/mL). The animals were sacrificed after 12 weeks. Harvested tissue specimens were qualitatively evaluated by histology. New bone formation (bone volume/tissue volume) was quantitatively measured by histomorphometry. Signs of bone formation were evident in all bone sockets. Mean values of the bone volume/tissue volume of the 4 defects were 10.91%, 11.05%, 10.40% and 10.87% respectively, at 12 weeks. In 5 animals, high concentration of vitamin D caused a significant improvement in bone formation in relation to controls. In 3 animals, a high concentration of vitamin D was associated with decreased bone formation compared with controls. No statistical difference was observed in the graft healing among the 4 graft sites ( P > 0.05). The results of this study have shown that the addition of vitamin D to human-derived bone grafts does not have a significant effect on bone formation and graft incorporation in critical-sized bone defects of the porcine calvaria. Further high-quality studies are needed to fully elucidate the role of vitamin D in bone formation and bone graft union.

Effectiveness of Apigenin, Resveratrol, and Curcumin as Adjuvant Nutraceuticals for Calvarial Bone Defect Healing: An In Vitro and Histological Study on Rats

Bone healing is a major clinical issue, especially in bone defects of critical dimensions. Some studies have reported in vivo positive effects on bone healing by some bioactive compounds, such as the phenolic derivatives found in vegetables and plants, such as resveratrol, curcumin, and apigenin. The aim of this work was (1) to analyze in vitro in human dental pulp stem cells the effects of these three natural compounds on the gene expression of related genes downstream to RUNX2 and SMAD5, key factor transcriptions associated with osteoblast differentiation, in order to better understand the positive effects that can occur in vivo in bone healing, and (2) to evaluate in vivo the effects on bone healing of critical-size defects in the calvaria in rats of these three nutraceuticals tested in parallel and for the first time administered by the gastric route. Upregulation of the RUNX2, SMAD5, COLL1, COLL4, and COLL5 genes in the presence of apigenin, curcumin, and resveratrol was detected. In vivo, apigenin induced more consistent significant bone healing in critical-size defects in rat calvaria compared to the other study groups. The study findings encourage a possible therapeutic supplementation with nutraceuticals during the bone regeneration process.

Enhancement of critical-sized bone defect regeneration by magnesium oxide-reinforced 3D scaffold with improved osteogenic and angiogenic properties

• 3D bio-nanocomposite scaffold which have an ideal magnesium ionic microenvironment was developed for critical-sized bony tissue repairing. • Significantly enhanced osteogenic effects were observed over the bio-nanocomposite as compare to the polycaprolactone control. • The rodent femoral and critical-sized cranial defect models were applied to characterize the osteogenesis in vivo . • Magnesium enriched scaffold exhibited remarkably increased bone volume, enhanced angiogenesis, and almost recovered critical-sized defect after an 8-week implantation. The healing of critical-sized bone defects (CSD) remains a challenge in orthopaedic medicine. In recent years, scaffolds with sophisticated microstructures fabricated by the emerging three-dimensional (3D) printing technology have lighted up the treatment of the CSD due to the elaborate microenvironments and support they may build. Here, we established a magnesium oxide-reinforced 3D-printed biocomposite scaffold to investigate the effect of magnesium-enriched 3D microenvironment on CSD repairing. The composite was prepared using a biodegradable polymer matrix, polycaprolactone (PCL), and the dispersion phase, magnesium oxide (MgO). With the appropriate surface treatment by saline coupling agent, the MgO dispersed homogeneously in the polymer matrix, leading to enhanced mechanical performance and steady release of magnesium ion (Mg 2+ ) for superior cytocompatibility, higher cell viability, advanced osteogenic differentiation, and cell mineralization capabilities in comparison with the pure PCL. The in-vivo femoral implantation and critical-sized cranial bone defect studies demonstrated the importance of the 3D magnesium microenvironment, as a scaffold that released appropriate Mg 2+ exhibited remarkably increased bone volume, enhanced angiogenesis, and almost recovered CSD after 8-week implantation. Overall, this study suggests that the magnesium-enriched 3D scaffold is a potential candidate for the treatment of CSD in a cell-free therapeutic approach.

Novel Techniques and Future Perspective for Investigating Critical-Size Bone Defects.

A critical-size bone defect is a challenging clinical problem in which a gap between bone ends will not heal and will become a nonunion. The current treatment is to harvest and transplant an autologous bone graft to facilitate bone bridging. To develop less invasive but equally effective treatment options, one needs to first have a comprehensive understanding of the bone healing process. Therefore, it is imperative to leverage the most advanced technologies to elucidate the fundamental concepts of the bone healing process and develop innovative therapeutic strategies to bridge the nonunion gap. In this review, we first discuss the current animal models to study critical-size bone defects. Then, we focus on four novel analytic techniques and discuss their strengths and limitations. These four technologies are mass cytometry (CyTOF) for enhanced cellular analysis, imaging mass cytometry (IMC) for enhanced tissue special imaging, single-cell RNA sequencing (scRNA-seq) for detailed transcriptome analysis, and Luminex assays for comprehensive protein secretome analysis. With this new understanding of the healing of critical-size bone defects, novel methods of diagnosis and treatment will emerge.

Effect of a Decellularized Omentum Scaffold with Combination of Mesenchymal Stem Cells and Platelet-Rich Plasma on Healing of Critical-Sized Bone Defect: A Rat Model

The high costs and extensive time needed for the treatment of critical-sized bone defects are still major clinical concerns in orthopedic surgery; therefore, researchers continue to look for more cost and time-effective methods. This study aims to investigate the effects of a decellularized omentum scaffold with a combination of platelet-rich plasma (PRP) and mesenchymal stem cells on the healing of critical-sized bone defects. Wistar albino rats (n = 30) were investigated in five groups. Critical-sized bone defects were formed on bilateral radius shafts. No scaffold, decellularized omentum, omentum with PRP and omentum + mesenchymal stem cells was used in group 1 (control group), 2, 3 and 4, respectively. In addition, omentum with a combination of mesenchymal stem cells +PRP was used in group 5. After 6 weeks, both radiological and histological healing were evaluated comparatively among the groups. After the use of a decellularized omentum scaffold, vitality of new cells was maintained, and new bone formation occurred. When compared to the control group, radiological healing was significantly better (p = 0.047) in the omentum and omentum + PRP-treated groups. Furthermore, histological healing was better in the omentum and omentum + PRP-treated groups than the control group (p = 0.001). The use of a decellularized omentum scaffold is suitable in the healing of critical bone defects.

Enhanced Osteogenic Differentiation of Pluripotent Stem Cells via γ-Secretase Inhibition.

Bone healing is a complex, well-organized process. Multiple factors regulate this process, including growth factors, hormones, cytokines, mechanical stimulation, and aging. One of the most important signaling pathways that affect bone healing is the Notch signaling pathway. It has a significant role in controlling the differentiation of bone mesenchymal stem cells and forming new bone. Interventions to enhance the healing of critical-sized bone defects are of great importance, and stem cell transplantations are eminent candidates for treating such defects. Understanding how Notch signaling impacts pluripotent stem cell differentiation can significantly enhance osteogenesis and improve the overall healing process upon transplantation. In Rancourt’s lab, mouse embryonic stem cells (ESC) have been successfully differentiated to the osteogenic cell lineage. This study investigates the role of Notch signaling inhibition in the osteogenic differentiation of mouse embryonic and induced pluripotent stem cells (iPS). Our data showed that Notch inhibition greatly enhanced the differentiation of both mouse embryonic and induced pluripotent stem cells.

In Vitro and In Vivo Evaluation of Nanostructured Biphasic Calcium Phosphate in Granules and Putty Configurations.

Synthetic biphasic calcium phosphate (BCP) granules and powder are biocompatible biomaterials with a well-known capacity for osteoconduction, presenting very satisfactory clinical and histological results. It remains unanswered if the putty configuration impacts the biological response to the material. In this study, we aimed to compare the cytocompatibility and biocompatibility of nanostructured BCP in the putty configuration (moldable nanostructured calcium phosphate, MnCaP) on the healing of critical-sized bone defects (8 mm) in rat calvaria. Cytocompatibility was determined through the viability of fibroblast cells (V-79) to the extracts of different concentrations of MnCaP. Forty-five Wistar rats were randomly divided into three groups (n = 15)—clot, MnCaP, and commercial biphasic calcium phosphate in granules configurations (Nanosynt®)—and subdivided into three experimental periods (1, 3, and 6 months). Histological, histomorphometric, and microtomographic analyses allowed the evaluation of newly formed bone, residual biomaterial, and connective tissue. The in vitro evaluation showed that MnCaP was cytocompatible. The histomorphometric results showed that the Nanosynt® group granted the highest new-formed bone values at six months (p < 0.05), although the biomaterial volume did not differ between groups. The putty configuration was easier to handle, and both configurations were biocompatible and osteoconductive, presented similar biosorption rates, and preserved the calvaria architecture.

The effect of polyethylenglycol gel on the delivery and osteogenic differentiation of homologous tooth germ-derived stem cells in a porcine model.

The aim of this study was to investigate if bone regeneration can be promoted by homologous transplantation of STRO-1 sorted (STRO-1+) porcine tooth germ mesenchymal stem cells (TGSCs) with the combination of polyethylenglycol (PEG)-based hydrogel and biphasic calcium phosphate (BCP) scaffolds. TGSCs were isolated from impacted third molars of domestic pigs. Nine critical-sized defects were created as (1) untreated defect; filled with (2) autogenous bone; (3) BCP + PEG; (4) BCP + PEG + unsorted TGSCs; (5) BCP + unsorted TGSCs; (6) BCP + PEG + STRO-1-sorted TGSCs; (7) BCP + STRO-1-sorted TGSCs; (8) BCP + PEG + osteogenic induced unsorted TGSCs; and (9) BCP + PEG + osteogenic induced STRO-1-sorted TGSCs in 20 domestic pigs. CM-DiI labelling was used to track cells in vivo. Histomorphometric assessment of new bone formation was achieved by toluidine blue O staining and microradiography after 1, 2, 4 and 12 weeks posttransplantation. Complete healing was achieved in all defects although defects with PEG hydrogel presented better bone formation while STRO-1+ and unsorted TGSCs showed similar ability to form new bone after 12 weeks. Transplanted cells were seen in defects where PEG hydrogel was used as carriers in contrast to defects treated with cells and only bone grafts. PEG hydrogel is an efficient carrier for homologous stem cell transplantation. TGSCs are capable of promoting bone healing in critical-sized defects in combination with bone graft and PEG hydrogel. This study provides information about the importance of the delivery vehicle for future translational stem cell delivery approaches.

Use of strontium doping glass-ceramic material for bone regeneration in critical defect: In vitro and in vivo analyses

The in vitro bioactivity and in vivo bone neoformation in critical-size bone defects of a glass-ceramic material containing strontium ions (Sr2+) were evaluated in the present study as well as the antimicrobial effect against oral pathogens. A glass-ceramic bioactive material in powder (CP), based on the composition of S53P4 bioactive glass, was produced by partially replacing calcium with SrO using the sol-gel route. The amount of SrO added was 2.2 wt% (CPSr12.5) and 5.0 wt% (CPSr25). The amount of Sr2+ ions released by the CP increase with time achieving 18 mg/L at 6 h and the release rate decreased at longer times. pH values higher than 10 were obtained in the first 6 h, in agreement with an inhibitory microbial effect. These materials showed in vitro bioactivity, with total surface coated by hydroxyapatite (HA) after 7 days of immersion in simulated body fluid (SBF). The pH of SBF increased rapidly after immersion of CP, reaching a maximum value of 8.72 after 168 h. HA formation was observed in vitro for all samples. On microtomography and histomorphometric analysis, CPSr25 showed higher values than CP without Sr2+ (CPSr0) for bone volume (p = 0.016), density (p = 0.016) and neoformation area (p = 0.025) at 28 days. Histological analysis revealed higher degree of vascularization after 28 days for CPSr25 when compared to CPSr0 (p = 0.003). In addition, CP showed an inhibitory effect on oral pathogens. Substitution of CaO by SrO (CPSr25) presented the best results on the healing of critical-size bone defects, as evidenced by microtomographic and histological analyses. These data confirmed that higher concentrations of Sr2+doped CP materials are potential alternatives to improve bone healing and regeneration in critical-size bone defects.

Autologous Adipose-Derived Stem Cells, Platelet-Rich Plasma, and Fibrin Enhance Healing of Mandibular Bone Defects in Swine

The primary aim of this study was to assess the therapeutic effects of autologous platelet-rich plasma and fibrin scaffolds combined with autologous adipose stem cells (ASCs) for critical-size defects in the pig mandible. Fibrin scaffolds supplemented with calcium hydrogen phosphate and platelet-derived growth factors were hypothesized to accelerate healing in porcine mandible bone compared to ASC-only injections and untreated controls. Three treatments included the use of autologous ASCs from liposuction with the addition of platelet-rich plasma, fibrin scaffolds, or as cell-only controls. All three treatments using ASCs were determined to increase bone mineral density and bone volume fraction compared to untreated controls. In general, the addition of both platelet-rich plasma and fibrin scaffolds to autologous ASCs from liposuction improved bone healing of critical-size defects.

Biodegradable Zwitterion/PLGA Scaffold Enables Robust Healing of Rat Calvarial Defects with Ultralow Dose of rhBMP-2.

Designing smart scaffolds to reduce administration dosage under the premise of functional healing of bone defects to avoid the severe side effects associated with BMP-2 treatments is one of the essential goals in bone tissue engineering. Here, we report a novel biodegradable PLGA/PSBMA composite as the scaffold for bone tissue engineering. The introduction of zwitterionic PSBMA components can alter the intrinsic burst degradation behavior of PLGA and enable a sustained degradation of the scaffold over the time. The PLGA/PSBMA scaffold can sequester rhBMP-2 and enable a sustained release of the sequestered rhBMP-2 with preserved bioactivity. Furthermore, PLGA/PSBMA scaffolds were able to guide robust healing of critical-sized nonunion calvarial defects (5 mm) at an ultralow dose of 400 ng/scaffold, at which level successful healing of critical-sized bone defects has never been reported. These findings indicate the PLGA/PSBMA scaffolds as novel high-efficiency rhBMP-2 delivery vehicles for bone tissue engineering, and the concept of utilizing the material, which is capable of maintaining the bioactivity of the proteins in the preparation of scaffolds, may open a new avenue for the design of smart scaffolds/vehicles for high-efficiency protein/bioactive drug therapies.

Osteogenic Potential of 3-D printed Multilayered Poly L-lactic Acid (PLLA) Scaffold on the Healing of Critical-sized Bone Defect in Dogs

Critical sized bone defect is a major challenge for orthopedic surgeons. These defects result following any pathologic condition leading to massive bone loss. Synthetic and biological based tissue engineered biomaterials and their combinations provide a promising substitute to fill the defect site. The aim of the present study was to evaluate the osteogenic potential of 3-D printed multilayered medical grade PLLA scaffold with collagen and nano hydroxyapatite for the healing of induced critical-sized bone defect in dogs. An experimental study was conducted on 12 skeletally mature male dogs. Critical defect (25mm) was induced into the right femur of all dogs. Dogs were randomly allocated into one of the following groups (4 dogs/group). PLLA scaffold seeded with nano hydroxyapatite and collagen molded on the defect (PLLA/Collagen/nHA group), the second group; the defect seeded with collagen and nano hydroxyapatite (Collagen/nHA group), the third one was left without scaffold or additives (Sham operated group), and the operated animals were left for 12 weeks. Animals were evaluated clinically, radiographically and histopathologicaly. In (PLLA/Collagen/nHA group), all dogs showed an improvement in lameness degree from sever to apparently free from lameness. Radiography showed newly formed bone filling the defect with no inter zone. Histopathology showed more maturation of the newly formed bone in the defect site had occurred as well as defined bone trabeculae in comparing to other groups. In conclusion, 3D printed multilayered medical grade PLLA with collagen and nano hydroxyapatite provide biodegradable osteoconductive scaffold for enhancing the healing of critical sized bone defect.

Burst, Short, and Sustained Vitamin D3 Applications Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells.

Incorporation of 1,25(OH)2 vitamin D3 (vitD3) into tissue-engineered scaffolds could aid the healing of critical-sized bone defects. We hypothesize that shorter applications of vitD3 lead to more osteogenic differentiation of mesenchymal stem cells (MSCs) than a sustained application. To test this, release from a scaffold was mimicked by exposing MSCs to exactly controlled vitD3 regimens. Human adipose stem cells (hASCs) were seeded onto calcium phosphate particles, cultured for 20 days, and treated with 124 ng vitD3, either provided during 30 min before seeding ([200 nM]), during the first two days ([100 nM]), or during 20 days ([10 nM]). Alternatively, hASCs were treated for two days with 6.2 ng vitD3 ([10 nM]). hASCs attached to the calcium phosphate particles and were viable (~75%). Cell number was not affected by the various vitD3 applications. VitD3 (124 ng) applied over 20 days increased cellular alkaline phosphatase activity at Days 7 and 20, reduced expression of the early osteogenic marker RUNX2 at Day 20, and strongly upregulated expression of the vitD3 inactivating enzyme CYP24. VitD3 (124 ng) also reduced RUNX2 and increased CYP24 applied at [100 nM] for two days, but not at [200 nM] for 30 min. These results show that 20-day application of vitD3 has more effect on hASCs than the same total amount applied in a shorter time span.

Repair of segmental rabbit radial defects with Cu/Zn co-doped calcium phosphate scaffolds incorporating GDF-5 carrier.

Repair of segmental bone defects is a challenge in orthopaedics. A bone substitute is a potential solution for this challenge, and angiogenesis and osteogenesis are critical to the performance of scaffold materials. For enhancing angiogenesis and osteogenesis activities of implanted scaffolds, Cu/Zn co-doped calcium phosphate scaffolds carrying GDF-5-release microspheres were prepared and implanted into surgically created critical-sized rabbit radial defects. Radiological examination, histological analysis and biomechanical tests were used to evaluate the bone healing-union. Results showed that, with increasing Cu/Zn concentrations, new bone area, new blood vessel density, and bending failure load all increased significantly. Furthermore, Cu/Zn co-doped scaffolds incorporating GDF-5-release microspheres exhibited further increased angiogenesis and osteogenesis (vs. Cu/Zn co-doped alone), as well as a superior bending failure load. These show that, simultaneous incorporation of trace essential ions and GDF-5 combines pro-angiogenic and pro-osteogenic actions of these bioactive substances, potentially offering an effective approach to assist the healing of critical-sized bone defects.

Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.