Ibudilast Mitigates Delayed Bone Healing Caused by Lipopolysaccharide by Altering Osteoblast and Osteoclast Activity.

Bacterial infection in orthopedic surgery is challenging because cell wall components released after bactericidal treatment can alter osteoblast and osteoclast activity and impair fracture stability. However, the precise effects and mechanisms whereby cell wall components impair bone healing are unclear. In this study, we characterized the effects of lipopolysaccharide (LPS) on bone healing and osteoclast and osteoblast activity in vitro and in vivo and evaluated the effects of ibudilast, an antagonist of toll-like receptor 4 (TLR4), on LPS-induced changes. In particular, micro-computed tomography was used to reconstruct femoral morphology and analyze callus bone content in a femoral defect mouse model. In the sham-treated group, significant bone bridge and cancellous bone formation were observed after surgery, however, LPS treatment delayed bone bridge and cancellous bone formation. LPS inhibited osteogenic factor-induced MC3T3-E1 cell differentiation, alkaline phosphatase (ALP) levels, calcium deposition, and osteopontin secretion and increased the activity of osteoclast-associated molecules, including cathepsin K and tartrate-resistant acid phosphatase in vitro. Finally, ibudilast blocked the LPS-induced inhibition of osteoblast activation and activation of osteoclast in vitro and attenuated LPS-induced delayed callus bone formation in vivo. Our results provide a basis for the development of a novel strategy for the treatment of bone infection.

Lipoteichoic Acid Accelerates Bone Healing by Enhancing Osteoblast Differentiation and Inhibiting Osteoclast Activation in a Mouse Model of Femoral Defects.

Lipoteichoic acid (LTA) is a cell wall component of Gram-positive bacteria. Limited data suggest that LTA is beneficial for bone regeneration in vitro. Thus, we used a mouse model of femoral defects to explore the effects of LTA on bone healing in vivo. Micro-computed tomography analysis and double-fluorochrome labeling were utilized to examine whether LTA can accelerate dynamic bone formation in vivo. The effects of LTA on osteoblastogenesis and osteoclastogenesis were also studied in vitro. LTA treatment induced prompt bone bridge formation, rapid endochondral ossification, and accelerated healing of fractures in mice with femoral bone defects. In vitro, LTA directly enhanced indicators of osteogenic factor-induced MC3T3-E1 cell differentiation, including alkaline phosphatase activity, calcium deposition and osteopontin expression. LTA also inhibited osteoclast activation induced by receptor activator of nuclear factor-kappa B ligand. We identified six molecules that may be associated with LTA-accelerated bone healing: monocyte chemoattractant protein 1, chemokine (C-X-C motif) ligand 1, cystatin C, growth/differentiation factor 15, endostatin and neutrophil gelatinase-associated lipocalin. Finally, double-fluorochrome, dynamic-labeling data indicated that LTA significantly enhanced bone-formation rates in vivo. In conclusion, our findings suggest that LTA has promising bone-regeneration properties.

[Allogeneic bone transplantation in hip revision surgery : Indications and potential for reconstruction]. / Allogene Knochentransplantation in der Hüftrevisionsendoprothetik : Indikationen und Rekonstruktionsmöglichkeiten.

The increasing number of people having joint replacements will lead to increasing numbers of revision operations. The transplantation of allogeneic bones might reconstruct bone defects and improve long-term anchorage of the implant. A sufficient primary stability of the implanted construct is necessary to achieve osseous incorporation as well as tight contact between the implanted allogeneic and host bones. Transplantation of bone can contribute to downgrading acetabular defects and so avoid bigger reinforcement implants. An improvement of bone stock due to reconstruction of femoral bony defects might also reduce the size of the stem necessary since the indication might be limited in case of extensive bone defects. According to good longterm results of modular revision stems the Impaction-Bone-Grafting has not yet generally been established.

Bioactive coatings on 3D printed scaffolds for bone regeneration: Use of Laponite® to deliver BMP-2 in an ovine femoral condyle defect model.

Biomaterial-based approaches for bone regeneration seek to explore alternative strategies to repair non-healing fractures and critical-sized bone defects. Fracture non-union occurs due to a number of factors resulting in the formation of bone defects. Rigorous evaluation of the biomaterials in relevant models and assessment of their potential to translate towards clinical use is vital. Large animal experimentation can be used to model fracture non-union while scaling-up materials for clinical use. Growth factors modulate cell phenotype, behaviour and initiate signalling pathways leading to changes in matrix deposition and tissue formation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic growth factor, with a rapid clearance time in vivo necessitating clinical use at a high dose, with potential deleterious side-effects. The current studies have examined the potential for Laponite® nanoclay coated poly(caprolactone) trimethacrylate (PCL-TMA900) scaffolds to bind BMP-2 for enhanced osteoinduction in a large animal critical-sized bone defect. An ovine femoral condyle defect model confirmed PCL-TMA900 scaffolds coated with Laponite®/BMP-2 produced significant bone formation compared to the uncoated PCL-TMA 900 scaffold in vivo, assessed by micro-computed tomography (µCT) and histology. This indicated the ability of Laponite® to deliver the bioactive BMP-2 on the PCL-TMA900 scaffold. Bone formed around the Laponite®/BMP-2 coated PCL-TMA900 scaffold, with no erroneous bone formation observed away from the scaffold material confirming localisation of BMP-2 delivery. The current studies demonstrate the ability of a nanoclay to localise and deliver bioactive BMP-2 within a tailored octet-truss scaffold for efficacious bone defect repair in a large animal model with significant implications for translation to the clinic.

Systematic literature review of in vivo rat femoral defect models using biomaterials to improve the induced membrane technique: a comprehensive analysis.

Purpose: The aim of this study was to conduct a systematic literature review analyzing the results of in vivo rat femoral defect models using biomaterials for improving the induced membrane technique (IMT). Methods: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, the PubMed, Embase, and Web of Science databases were searched. Inclusion criteria were studies reporting results of the IMT in in vivo rat femoral critical-sized defect models using a biomaterial possibly combined with molecules. Methodologic quality was assessed with the Animal Research: Reporting In Vivo Experiments guidelines. Results: Twenty studies met the inclusion criteria. Femoral stabilization with plate and screws was the most frequent. Histologic, biomechanical, and/or radiologic analyses were performed. In two-stage strategies, the PMMA spacer could be associated with bioactive molecules to enhance IM growth factor expression and improve bone formation. Modulating the roughness of spacers could increase IM thickness and accelerate its formation. In one-stage strategies, human tissue-derived membranes combined with bone grafting achieved bone formation comparable to a standard IMT. All calcium phosphate grafts seemed to require a functionalization with growth factors or bone marrow mononuclear cells to improve outcomes compared with non-functionalized grafts. Conclusion: This systematic review described the main parameters of the in vivo rat femoral defect models using biomaterials to improve the induced membrane technique. Although the studies included had several methodological limitations that may limit the scope of these conclusions, one- and two-stage strategies reported promising results with biomaterials to improve the IMT.

ATF2-driven osteogenic activity of enoxaparin sodium-loaded polymethylmethacrylate bone cement in femoral defect regeneration.

BACKGROUND: Polymethylmethacrylate (PMMA) bone cement loaded with enoxaparin sodium (PMMA@ES) has been increasingly highlighted to affect the bone repair of bone defects, but the molecular mechanisms remain unclear. We addressed this issue by identifying possible molecular mechanisms of PMMA@ES involved in femoral defect regeneration based on bioinformatics analysis and network pharmacology analysis. METHODS: The upregulated genes affecting the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were selected through bioinformatics analysis, followed by intersection with the genes of ES-induced differentiation of BMSCs identified by network pharmacology analysis. PMMA@ES was constructed. Rat primary BMSCs were isolated and cultured in vitro in the proliferation medium (PM) and osteogenic medium (OM) to measure alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and the expression of RUNX2 and OCN using gain- or loss-of-function experiments. A rat femoral bone defect model was constructed to detect the new bone formation in rats. RESULTS: ATF2 may be a key gene in differentiating BMSCs into osteoblasts. In vitro cell assays showed that PMMA@ES promoted the osteogenic differentiation of BMSCs by increasing ALP activity, extracellular matrix mineralization, and RUNX2 and OCN expression in PM and OM. In addition, ATF2 activated the transcription of miR-335-5p to target ERK1/2 and downregulate the expression of ERK1/2. PMMA@ES induced femoral defect regeneration and the repair of femoral defects in rats by regulating the ATF2/miR-335-5p/ERK1/2 axis. CONCLUSION: The evidence provided by our study highlighted the ATF2-mediated mechanism of PMMA@ES in the facilitation of the osteogenic differentiation of BMSCs and femoral defect regeneration.

Injectable temperature-sensitive hydrogel system incorporating deferoxamine-loaded microspheres promotes H-type blood vessel-related bone repair of a critical size femoral defect.

Insufficient vascularization is a major challenge in the repair of critical-sized bone defects. Deferoxamine (DFO) has been reported to play a potential role in promoting the formation of H-type blood vessels, a specialized vascular subtype with coupled angiogenesis and osteogenesis. However, whether DFO promotes the expression of H-type vessels in critical femoral defects with complete periosteal damage remains unknown. Moreover, stable drug loading systems need to be designed owing to the short half-life and high-dose toxic effects of DFO. In this study, we developed an injectable DFO-gelatin microspheres (GMs) hydrogel complex as a stable drug loading system for the treatment of critical femoral defects in rats. Our results showed that sustained release of DFO in critical femoral defects stimulated the generation of functional H-type vessels. The DFO-GMs hydrogel complex effectively promoted proliferation, formation, and migration of human umbilical vein endothelial cells in vitro. In vivo, the application of the DFO-GMs hydrogel complex expanded the distribution range and prolonged the expression time of H-type vessels in the defect area and was positively correlated with the number of osterix+ cells and new bone tissue. Topical application of the HIF-1α inhibitor PX-478 partially blocked the stimulation of H-type vessels by DFO, whereas the osteogenic potential of the latter was also weakened. Our results extended the local application of DFO and provided a theoretical basis for targeting H-type vessels to treat large femoral defects. STATEMENT OF SIGNIFICANCE: Abundant functional blood vessels are essential for bone repair. The H-type blood vessel is a functional subtype with angiogenesis and osteogenesis coupling potential. A drug loading system with long-term controlled release was first used to investigate the formation of H-type blood vessels in critical femoral defects and promotion of bone repair. Our results showed that the application of DFO-GMs hydrogel complex expanded the distribution range and expression time of H-type vessels, and was positively correlated with the number of osteoblasts and volume of new bone tissue. These results expanded the local application approach of DFO and provide a theoretical basis for targeting H-type vessels to treat large femoral defects.

LL-37-Coupled Porous Composite Scaffold for the Treatment of Infected Segmental Bone Defect.

Increased multiantibiotic-resistant bacteria means that infected bone defects remain a significant challenge to clinics. Great interest has emerged in the use of non-antibiotic antimicrobials to reduce the rate of multiantibiotic-resistant bacterial infection and facilitate bone regeneration. The cationic antimicrobial peptide LL-37 is the sole human cathelicidin and has shown nonspecific activity against a broad spectrum of microorganisms. In this study, we fabricated the poly(lactic-co-glycolic acid)/ß-calcium phosphate/peptide LL-37 (PLGA/TCP/LL-37, PTL) scaffold with low-temperature 3D-printing technology for the treatment of infected segmental bone defects. The prepared scaffolds were divided into three groups: a high LL-37 concentration group (PTHL), low LL-37 concentration group (PTLL) and blank control group (PT). The cytocompatibility and antimicrobial activity of the engineered scaffolds were tested in vitro, and their osteogenesis properties were assessed in vivo in a rat infected bone defect model. We found the fabricated PTL scaffold had a well-designed porous structure that could support a steady and prolonged LL-37 release. Furthermore, the PTHL group showed strong antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) without any inhibition of the proliferation or alkaline phosphatase activity of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. In addition, the infected femoral defects implanted with PTHL group displayed new bone formation in four weeks without any evidence of residual bacteria, which showed similar antibacterial outcomes to the vancomycin and cancellous bone mixture group. In conclusion, the PTHL composite scaffold is a promising non-antibiotic antimicrobial graft with good biodegradability, biocompatibility, and osteogenic capability for infected bone defects.

3D bioprinting of prevascularised implants for the repair of critically-sized bone defects.

For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Micro-computed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. STATEMENT OF SIGNIFICANCE: This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.

Use of three-dimensional-printed custom-made prosthesis to treat unicondylar femoral defect secondary to pathological fracture caused by giant cell tumor.

OBJECTIVE: To evaluate the short-term effectiveness of using a three-dimensional (3D)-printed custom-made prosthesis to repair unicondylar femoral defects. METHODS: We retrospectively reviewed 26 patients with a primary pathological fracture of the distal femur caused by a giant cell tumor. All patients had unicondylar defects involving the articular surface. Twelve patients were treated with a 3D-printed custom-made prosthesis to repair the unicondylar defect (3D-printed group). The other 14 patients were treated with total knee replacement (TKR group). The operation time, blood loss, Musculoskeletal Tumor Society score, range of motion, local recurrence, and complications were statistically compared. RESULTS: The operation time was significantly shorter and the blood loss was significantly less in the 3D-printed group than in the TKR group. The Musculoskeletal Tumor Society scores were significantly higher in the 3D-printed group than in the TKR group from 3 to 24 months postoperatively. The range of motion was significantly better in the 3D-printed group than in the TKR group at 6 and 9 months postoperatively. CONCLUSIONS: 3D-printed custom-made prostheses provide better short-term functional results than does TKR.

Antimicrobial Bioresorbable Mg-Zn-Ca Alloy for Bone Repair in a Comparison Study with Mg-Zn-Sr Alloy and Pure Mg.

Magnesium-zinc-calcium (Mg-Zn-Ca) alloys have attracted increasing attention for biomedical implant applications, especially for bone repair, because of their biocompatibility, biodegradability, and similar mechanical properties to human bone. The objectives of this study were to characterize Mg-2 wt % Zn-0.5 wt % Ca (named ZC21) alloy pins microstructurally and mechanically, and determine their degradation and interactions with host cells and pathogenic bacteria in vitro and in vivo in comparison with the previously studied Mg-4 wt % Zn-1 wt % strontium (named ZSr41) alloy and Mg control. Specifically, the in vitro degradation and cytocompatibility of ZC21 pins with bone marrow derived mesenchymal stem cells (BMSCs) were investigated using both direct culture and direct exposure culture methods. The adhesion density of BMSCs on ZC21 pins (i.e., direct contact) was significantly higher than on pure Mg pins in both in vitro culture methods; the cell adhesion density around ZC21 pins (i.e., indirect contact) was similar to the cell-only positive control in both in vitro culture methods. Interestingly, ZC21 showed a higher daily degradation rate, crack width and crack area ratio in the direct exposure culture than in the direct culture, suggesting different culture methods did affect its in vitro degradation behaviors. When cultured with Gram-positive bacteria methicillin-resistant Staphylococcus aureus (MRSA), ZC21 reduced bacterial adhesion on the surface more significantly than that of ZSr41 and Mg. The in vivo degradation and biocompatibility of the ZC21 pins for bone regeneration were studied in a mouse femoral defect model. The in vivo degradation rate of ZC21 pins was much slower than that of ZSr41 alloy and Mg control pins. After 12 weeks of implantation in vivo, the ZC21 group showed the shortest gap at the femoral defect, indicating that ZC21 pins promoted osteogenesis and bone healing more than ZSr41 and Mg control pins. Overall, the ZC21 alloy is promising for bone repair, while providing antibacterial activities, and should be further studied toward clinical translation.

Decellularized Adipose Tissue Hydrogel Promotes Bone Regeneration in Critical-Sized Mouse Femoral Defect Model.

Critical-sized bone defects fail to heal and often cause non-union. Standard treatments employ autologous bone grafting, which can cause donor tissue loss/pain. Although several scaffold types can enhance bone regeneration, multiple factors limit their level of success. To address this issue, this study evaluated a novel decellularized human adipose tissue (DAT) hydrogel as an alternative. In this study, DAT hydrogel alone, or in combination with adipose-derived stromal/stem cells (ASC), osteo-induced ASCs (OIASC), and hydroxyapatite were tested for their ability to mediate repair of a critical-sized (3 mm) femoral defect created in C57BL/6 mice. Micro-computed tomography results showed that all DAT hydrogel treated groups significantly enhanced bone regeneration, with OIASC + hydroxyapatite treated group displaying the most robust bone regeneration. Histological analyses revealed that all treatments resulted in significantly higher tissue areas with the relative mineralized tissue area significantly increased at 12 weeks; however, cartilaginous content was lowest among treatment groups with OIASC. Immunohistochemical analyses showed that DAT hydrogel enhanced collagen I and osteopontin expression, while the addition of OIASCs to the hydrogel reduced collagen II levels. Thus, DAT hydrogel promotes bone regeneration in a critical-sized femoral defect model that is further enhanced in the presence of OIASCs and hydroxyapatite.

Low intensity pulsed ultrasound promotes the migration of bone marrow- derived mesenchymal stem cells via activating FAK-ERK1/2 signalling pathway.

To investigate the promoting effects and mechanisms of low intensity pulsed ultrasound (LIPUS) on the migration of bone marrow-derived mesenchymal stem cells (BMSCs). The BMSCs migration was researched from cell and animal experiments. In the cell experiment, the BMSCs was treated using LIPUS (30 mW/cm2, 20 min/day, 2 days), and the wound healing and transwell migration were observed. In the animal experiment, the BMSCs labelled with green fluorescent protein (GFP) were injected into rats with femoral defects via the tail vein (1 × 106/mL). The healing of bone was detected using x-ray and sampled for hematoxylin & eosin (H&E) staining and fluorescence microscopy. About the mechanisms, the cellular F-actin of cytoskeleton was stained with FITC-phalloidin. The changes of BMSCs genes after LIPUS treatment were screened using microarray assay and verified using quantitative real-time polymerase chain reaction (qRT-PCR). The biological processes of those genes were predicted by KEGG analysis. The protein expression levels of FAK, ERK1/2 and myosin II related migration were detected using western blotting. The results showed LIPUS promoted the BMSCs migration (p < .05) without significant temperature changes (p > .05) in vitro and in vivo than control group (p < .05). The cytoskeletal rearrangement was carried out, and the ITGA8 gene related with cell migration was found with high expression after LIPUS treatment (p < .05). FAK inhibitor (PF-573228) and ERK1/2 inhibitor (U0126) were proved, in turn, decreased the BMSCs migration induced using LIPUS (p < .05). LIPUS can promote the BMSCs migration in vitro and in vivo, one mechanism may be related to the activation of FAK-ERK1/2 signalling pathways using LIPUS.

Guided Bone Regeneration of Femoral Segmental Defects using Equine Bone Graft: An In-Vivo Micro-Computed Tomographic Study in Rats.

Background and objectives: Guided bone regeneration (GBR) is commonly used for osseous defect reconstruction. The objective of this study was to evaluate in real-time (in-vivo) the efficacy of equine bone graft for GBR in segmental critical-size defects (CSD) of the femur in a rat model. Materials and methods: Following ethical approval, 30 male Wistar-Albino rats (age 12-14 months/weight 450-500 grams) were included. Under general-anesthesia, a mid-diaphyseal segmental CSD (5 mm) was created in the femur and stabilized using titanium Miniplate(4 holes,1.0 mm thickness). Depending upon material used for GBR, animals were randomly divided into three groups(n = 10/per group). Negative control-Defect covered with resorbable collagen membrane(RCM); Positive control-Defect filled with autologous bone and covered by RCM; Equine bone-Defect filled with equine bone and covered by RCM. Real-time in-vivo Micro-CT was performed at baseline, 2, 4, 6 and 8 weeks to determine volume and mineral density of newly formed bone (NFB) and remaining bone graft particles (BGP). Results: In-vivo micro-CT revealed increase in volume and mineral density of NFB within defects from baseline to 8-weeks in all groups. At 8-weeks NFB-volume in the equine bone group(53.24 ± 13.83 mm3; p < 0.01) was significantly higher than the negative control(5.6 ± 1.06 mm3) and positive control(26.07 ± 5.44 mm3) groups. Similarly, NFB-mineral density in the equine bone group(3.33 ± 0.48 g/mm3; p < 0.01) was higher than the other (negative control-0.27 ± 0.02 g/mm3; positive control-2.55 ± 0.6 g/mm3). A gradual decrease in the BGP-volume and BGP-mineral density was observed. Conclusion: The use of equine bone for GBR in femoral segmental defects in rats, results in predictable new bone formation as early as 2-weeks after bone graft placement.

PLGA-linked alendronate enhances bone repair in diaphysis defect model.

Alendronate (ALN) is known as an anti-resorptive drug for the treatment of osteoporosis. Recently, ALN was found to stimulate osteogenic differentiation in mesenchymal stem cells and enhance new bone formation in calvarial bone defects. Previous in vitro and in vivo studies found that the effective concentration of ALN was approximately 1-10   µm. In the present study, a poly (lactic-co-glycolic acid) (PLGA) cross-linked ALN (PLGA-ALN) with a short-term controlled-release property for local application to enhance bone repair was developed. An in vitro drug-release kinetic test showed that PLGA-ALN microspheres released an effective concentration (50-100 nm) of ALN for 9 days. The effect of PLGA-ALN on bone repair was tested in a rat femoral bone defect model. The biomechanical study results showed that the maximal strength, stiffness and energy absorption were significantly increased in the PLGA-ALN group compared with the PLGA group. The microstructure of the newly formed bone at the defect site was analysed using microcomputed tomography. The PLGA-ALN group significantly improved the trabecular bone volume at the defect site compared with the PLGA group. The fibril collagen and immunolocalized bone morphogenetic protein 2 were evident in the newly formed trabecular bone in the PLGA-ALN group. Local use of newly developed PLGA-ALN-enhanced bone repair was attributable to increasing bone matrix formation, which improved the ultrastructure of the newly formed bone and thus increased the biomechanical properties of the repaired bone. It is suggested that PLGA-ALN may be a potential bone graft substitute to enhance bone repair. Copyright © 2016 John Wiley & Sons, Ltd.

In-situ tissue regeneration through SDF-1α driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect.

In-situ tissue regeneration aims to utilize the body's endogenous healing capacity through the recruitment of host stem or progenitor cells to an injury site. Stromal cell-derived factor-1α (SDF-1α) is widely discussed as a potent chemoattractant. Here we use a cell-free biomaterial-based approach to (i) deliver SDF-1α for the recruitment of endogenous bone marrow-derived stromal cells (BMSC) into a critical-sized segmental femoral defect in rats and to (ii) induce hydrogel stiffness-mediated osteogenic differentiation in-vivo. Ionically crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used. Fast-degrading porogens were incorporated to impart a macroporous architecture that facilitates host cell invasion. Endogenous cell recruitment to the defect site was successfully triggered through the controlled release of SDF-1α. A trend for increased bone volume fraction (BV/TV) and a significantly higher bone mineral density (BMD) were observed for gels loaded with SDF-1α, compared to empty gels at two weeks. A trend was also observed, albeit not statistically significant, towards matrix stiffness influencing BV/TV and BMD at two weeks. However, over a six week time-frame, these effects were insufficient for bone bridging of a segmental femoral defect. While mechanical cues combined with ex-vivo cell encapsulation have been shown to have an effect in the regeneration of less demanding in-vivo models, such as cranial defects of nude rats, they are not sufficient for a SDF-1α mediated in-situ regeneration approach in segmental femoral defects of immunocompetent rats, suggesting that additional osteogenic cues may also be required. STATEMENT OF SIGNIFICANCE: Stromal cell-derived factor-1α (SDF-1α) is a chemoattractant used to recruit host cells for tissue regeneration. The concept that matrix stiffness can direct mesenchymal stromal cell (MSC) differentiation into various lineages was described a decade ago using in-vitro experiments. Recently, alginate hydrogels with an optimized stiffness and ex-vivo encapsulated MSCs were shown to have an effect in the regeneration of skull defects of nude rats. Here, we apply this material system, loaded with SDF-1α and without encapsulated MSCs, to (i) recruit endogenous cells and (ii) induce stiffness-mediated osteogenic differentiation in-vivo, using as model system a load-bearing femoral defect in immunocompetent rats. While a cell-free approach is of great interest from a translational perspective, the current limitations are described.

Combination therapy with PTH and DBM cannot heal a critical sized murine femoral defect.

Orthopaedic surgeons continue to search for cost-effective bone graft substitutes to enhance bone repair. Teriparatide (PTH 1-34) and demineralized bone matrix (DBM) have been used in patients to promote bone healing. We evaluated the efficacy of PTH and DBM in healing a critical sized femoral defect in three lineage-specific transgenic mice expressing Col3.6GFPtopaz (pre-osteoblastic marker), Col2.3GFPemerald (osteoblastic marker) and α-SMA-Cherry (pericyte/myofibroblast marker). Mid-diaphyseal defects measuring 2 mm in length were created in the central 1/3 of mice femora using a circular saw and stabilized with an alveolar distractor device and cerclage wires. Three groups were evaluated: Group I, PTH 30 µg/kg injection daily, Group II, PTH 30 µg/kg injection daily + DBM, and Group III, DBM + 30µL saline injection. PTH was given for 28 days or until the time of sacrifice. Animals were sacrificed at 7, 14, 28, and 56 days. Radiographs at the time of sacrifice were evaluated using a 5-point scaled scoring system. Radiographs showed a lack of healing across all treatment groups at all time points: Group I, 1.57 +/- 0.68; Group II, 3.00 +/- 1.29; and Group III, 2.90 +/- 1.03. Bone formation in the defect as measured by radiographic healing score was significantly better at 56 days in Groups II (p = 0.01) and III (p < 0.01) compared to Group I. Across all treatment groups and time points the defects were largely absent of osteoprogenitor cells based on gross observation of frozen histology and quantitation of cellular based histomorphometric parameters. Quantitation of frozen histologic slides showed a limited osteoprogenitor response to PTH and DBM. Our results suggest that the anabolic agent teriparatide is unable to induce healing in a critical sized mouse femoral defect when given alone or in combination with the DBM preparation we used as a local bone graft substitute.

Synthesis and physicochemical, in vitro and in vivo evaluation of an anisotropic, nanocrystalline hydroxyapatite bisque scaffold with parallel-aligned pores mimicking the microstructure of cortical bone.

Scaffolds for bone regeneration are mostly prepared with an isotropic, sponge-like structure mimicking the architecture of trabecular bone. We have developed an anisotropic bioceramic with parallel aligned pores resembling the honeycomb arrangement of Haversian canals of cortical bone and investigated its potential as a scaffold for tissue engineering. Parallel channel-like pores were generated by ionotropic gelation of an alginate-hydroxyapatite (HA) slurry, followed by ceramic processing. Organic components were thermally removed at 650 °C, whereas the pore system was preserved in the obtained HA bioceramic in the processing stage of a bisque. Even without further sintering at higher temperatures, the anisotropic HA bisque (AHAB) became mechanically stable with a compressive strength (4.3 MPa) comparable to that of native trabecular bone. Owing to the low-temperature treatment, a nanocrystalline microstructure with high porosity (82%) and surface area (24.9 m(2)/g) was achieved that kept the material dissolvable in acidic conditions, similar to osteoclastic degradation of bone. Human mesenchymal stem cells (hMSCs) adhered, proliferated and differentiated into osteoblasts when osteogenically induced, indicating the cytocompatibility of the bisque scaffold. Furthermore, we demonstrated fusion of human monocytes to osteoclast-like cells in vitro on this substrate, similar to the natural pathway. Biocompatibility was demonstrated in vivo by implantation of the bisque ceramic into cortical rabbit femur defects, followed by histological analysis, where new bone formation inside the channel-like pores and generation of an osteon-like tissue morphology was observed.

Effect of a ß-TCP collagen composite bone substitute on healing of drilled bone voids in the distal femoral condyle of rabbits.

In this study, we tested the performance and biocompatibility of a composite of ß-tricalcium phosphate (ß-TCP) to collagen as a bone void filler (Cerasorb(®) Ortho Foam) in a rabbit distal femoral condyle model. ß-TCP is a completely resorbable synthetic calcium phosphate and the addition of a collagen matrix couples the osteoconductive effects of the two components. Furthermore, the malleable properties of the implant material during surgical applications for shape control will be enhanced. A critical size defect of 6 mm in diameter and 10 mm in depth was drilled into each distal femur of the rabbits. One hole was filled with the test substance and the other was left empty for control. After 1, 3, and 6 months the animals were killed and the degree of bone healing analyzed. In total, 18 animals were investigated. When the ß-TCP composite was used, histological, histomorphometric, and biomechanical evaluations revealed significantly better bone healing in terms of quantity and quality of the newly formed bone. Moreover, no signs of inflammation were observed in the animals and no allergic or foreign body reaction was noted. This suggests high biocompatibility and osteoconductivity of the investigated material to a bone void in an immune responsive species.

Inflammatory response and bone healing capacity of two porous calcium phosphate ceramics in critical size cortical bone defects.

In the present study, two open porous calcium phosphate ceramics, ß-tricalcium phosphate (ß-TCP), and hydroxyapatite (HA) were compared in a critical-sized femoral defect in rats. Previous comparisons of these two ceramics showed significantly greater osteoinductive potential of ß-TCP upon intramuscular implantation and a better performance in a spinal fusion model in dogs. Results of the current study also showed significantly more bone formation in defects grafted with ß-TCP compared to HA; however, both the ceramics were not capable of increasing bone formation to such extend that it bridges the defect. Furthermore, a more pronounced degradation of ß-TCP was observed as compared to HA. Progression of inflammation and initiation of new bone formation were assessed for both materials at multiple time points by histological and fluorochrome-based analyses. Until 12 days postimplantation, a strong inflammatory response in absence of new bone formation was observed in both ceramics, without obvious differences between the two materials. Four weeks postimplantation, signs of new bone formation were found in both ß-TCP and HA. At 6 weeks, inflammation had subsided in both ceramics while bone deposition continued. In conclusion, the two ceramics differed in the amount of bone formed after 8 weeks of implantation, whereas no differences were found in the duration of the inflammatory phase after implantation or initiation of new bone formation.