Radial Defect Model Research Articles

Synergistic large segmental bone repair by 3D printed bionic scaffolds and engineered ADSC nanovesicles: Towards an optimized regenerative microenvironment

Achieving sufficient bone regeneration in large segmental defects is challenging, with the structure of bone repair scaffolds and their loaded bioactive substances crucial for modulating the local osteogenic microenvironment. This study utilized digital laser processing (DLP)-based 3D printing technology to successfully fabricate high-precision methacryloylated polycaprolactone (PCLMA) bionic bone scaffold structures. Adipose-derived stem cell-engineered nanovesicles (ADSC-ENs) were uniformly and stably modified onto the bionic scaffold surface using a perfusion device, constructing a conducive microenvironment for tissue regeneration and long bone defect repair through the scaffold's structural design and the vesicles' biological functions. Scanning electron microscopy (SEM) examination of the scaffold surface confirmed the efficient loading of ADSC-ENs. The material group loaded with vesicles (PCLMA-BAS-ENs) demonstrated good cell compatibility and osteogenic potential when analyzed for the adhesion and osteogenesis of primary rabbit bone marrow mesenchymal stem cells (BMSCs) on the material surface. Tested in a 15 mm critical rabbit radial defect model, the PCLMA-BAS-ENs scaffold facilitated near-complete bone defect repair after 12 weeks. Immunofluorescence and proteomic results indicated that the PCLMA-BAS-ENs scaffold significantly improved the osteogenic microenvironment at the defect site in vivo, promoted angiogenesis, and enhanced the polarization of macrophages towards M2 phenotype, and facilitated the recruitment of BMSCs. Thus, the PCLMA-BAS-ENs scaffold was proven to significantly promote the repair of large segmental bone defects. Overall, this strategy of combining engineered vesicles with highly biomimetic scaffolds to promote large-segment bone tissue regeneration holds great potential in orthopedic and other regenerative medicine applications.

Polydopamine-coated biomimetic bone scaffolds loaded with exosomes promote osteogenic differentiation of BMSC and bone regeneration.

The repair of bone defects is ideally accomplished with bone tissue engineering. Recent studies have explored the possibility of functional modification of scaffolds in bone tissue engineering. We prepared an SF-CS-nHA (SCN) biomimetic bone scaffold and functionally modified the scaffold material by adding a polydopamine (PDA) coating loaded with exosomes (Exos) of marrow mesenchymal stem cells (BMSCs). The effects of the functional composite scaffold (SCN/PDA-Exo) on BMSC proliferation and osteogenic differentiation were investigated. Furthermore, the SCN/PDA-Exo scaffolds were implanted into animals to evaluate their effect on bone regeneration. SCN biomimetic scaffolds were prepared by a vacuum freeze-drying/chemical crosslinking method. A PDA-functionalized coating loaded with BMSC-Exos was added by the surface coating method. The physical and chemical properties of the functional composite scaffolds were detected by scanning electron microscopy (SEM), energy spectrum analysis and contact angle tests. Invitro, BMSCs were inoculated on different scaffolds, and the Exo internalization by BMSCs was detected by confocal microscopy. The BMSC proliferation activity and cell morphology were detected by SEM, CCK-8 assays and phalloidin staining. BMSC osteogenic differentiation was detected by immunofluorescence, alizarin red staining and qRT‒PCR. Invivo, the functional composite scaffold was implanted into a rabbit critical radial defect model. Bone repair was detected by 3D-CT scanning. HE staining, Masson staining, and immunohistochemistry were used to evaluate bone regeneration. Compared with the SCN scaffold, the SCN/PDA-Exo-functionalized composite scaffold had a larger average surface roughness and stronger hydrophilicity. Invitro, the Exos immobilized on the SCN/PDA-Exo scaffolds were internalized by BMSCs. The BMSC morphology, proliferation ability and osteogenic differentiation effect in the SCN/PDA-Exo group were significantly better than those in the other control groups (p<0.05). The effects of the SCN/PDA-Exo functional composite scaffold on bone defect repair and new bone formation were significantly better than those of the other control groups (p<0.05). In this study, we found that the SCN/PDA-Exo-functionalized composite scaffold promoted BMSC proliferation and osteogenic differentiation invitro and improved bone regeneration efficiency invivo. Therefore, combining Exos with biomimetic bone scaffolds by functional PDA coatings may be an effective strategy for functionally modifying biological scaffolds.

Hollow Hydroxyapatite Microspheres Loaded with rhCXCL13 to Recruit BMSC for Osteogenesis and Synergetic Angiogenesis to Promote Bone Regeneration in Bone Defects.

Bone tissue engineering is a promising method to treat bone defects. However, the current methods of preparing composite materials that mimic the complex structure and biological activity of natural bone are challenging for recruitment of bone marrow mesenchymal stem cells (BMSCs), which affects the application of these materials in situ bone regeneration. Hollow hydroxyapatite microspheres (HHMs) possess a natural porous bone structure, good adsorption, and slow release of chemokines, but have low ability to recruit BMSCs and induce osteogenesis. In this study, The HHM/chitosan (CS) and recombinant human C-X-C motif chemokine ligand 13 (rhCXCL13)-HHM/CS biomimetic scaffolds that optimize bone regeneration and investigated their mechanism of BMSC recruitment and osteogenesis through cell and animal experiments and transcriptomic sequencing. Evaluate the physical characteristics of the HHM/CS and rhCXCL13-HHM/CS biomimetic scaffolds through Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), and the cumulative release curve of rhCXCL13. Transwell migration experiments and co-culture with BMSCs were conducted to study the recruitment ability and osteogenic differentiation of the scaffolds. Transcriptomic sequencing was performed to analyze the osteogenic differentiation mechanism. The osteogenesis and bone healing performance were evaluated using a rabbit radial defect model. SEM demonstrated that the rhCXCL13-HHM/CS scaffold comprised hydroxyapatite microspheres in a porous three-dimensional network. The rhCXCL13 showed excellent sustained release capability. The rhCXCL13-HHM/CS scaffold could recruit BMSCs and induce bone regeneration. Transcriptome sequencing and experimental results showed that the osteogenesis mechanism of rhCXCL13-HHM/CS was through the PI3K-AKT pathway. In vivo, the rhCXCL13-HHM/CS scaffold significantly promoted osteogenesis and angiogenesis at 12 weeks after surgery. The rhCXCL13-HHM/CS scaffold demonstrates excellent potential for BMSC recruitment, osteogenesis, vascularized tissue-engineered bone reconstruction, and drug delivery, providing a theoretical basis for material osteogenesis mechanism study and promising clinical applications for treating large bone defects.

Regenerative engineering of long bones using the small molecule forskolin

Bone grafting procedures have become increasingly common in the United States, with approximately 500,000 cases occurring each year at a societal cost exceeding $2.4 billion. Recombinant human bone morphogenetic proteins (rhBMPs) are therapeutic agents that have been widely used by orthopedic surgeons to stimulate bone tissue formation alone and when paired with biomaterials. However, significant limitations such as immunogenicity, high production cost, and ectopic bone growth from these therapies remain. Therefore, efforts have been made to discover and repurpose osteoinductive small-molecule therapeutics to promote bone regeneration. Previously, we have demonstrated that a single-dose treatment with the small-molecule forskolin for just 24 h induces osteogenic differentiation of rabbit bone marrow-derived stem cells invitro, while mitigating adverse side effects attributed with prolonged small-molecule treatment schemes. In this study, we engineered a composite fibrin-PLGA [poly(lactide-co-glycolide)]-sintered microsphere scaffold for the localized, short-term delivery of the osteoinductive small molecule, forskolin. Invitro characterization studies showed that forskolin released out of the fibrin gel within the first 24 h and retained its bioactivity toward osteogenic differentiation of bone marrow-derived stem cells. The forskolin-loaded fibrin-PLGA scaffold was also able to guide bone formation in a 3-mo rabbit radial critical-sized defect model comparable to recombinant human bone morphogenetic protein-2 (rhBMP-2) treatment, as demonstrated through histological and mechanical evaluation, with minimal systemic off-target side effects. Together, these results demonstrate the successful application of an innovative small-molecule treatment approach within long bone critical-sized defects.

Host type 2 immune response to xenogeneic serum components impairs biomaterial-directed osteo-regenerative therapies

The transformative potential of cells as therapeutic agents is being realized in a wide range of applications, from regenerative medicine to cancer therapy to autoimmune disorders. The majority of these therapies require ex vivo expansion of the cellular product, often utilizing fetal bovine serum (FBS) in the culture media. However, the impact of residual FBS on immune responses to cell therapies and the resulting cell therapy outcomes remains unclear. Here, we show that hydrogel-delivered FBS elicits a robust type 2 immune response characterized by infiltration of eosinophils and CD4+ T cells. Host secretion of cytokines associated with type 2 immunity, including IL-4, IL-5, and IL-13, is also increased in FBS-containing hydrogels. We demonstrate that the immune response to xenogeneic serum components dominates the local environment and masks the immunomodulatory effects of biomaterial-delivered mesenchymal stromal/stem cells. Importantly, delivery of relatively small amounts of FBS (3.2% by volume) within BMP-2-containing biomaterial constructs dramatically reduces the ability of these constructs to promote de novo bone formation in a radial defect model in immunocompetent mice. These results urge caution when interpreting the immunological and tissue repair outcomes in immunocompetent pre-clinical models from cells and biomaterial constructs that have come in contact with xenogeneic serum components.

Mussel-Inspired Gold Nanoparticle and PLGA/L-Lysine-g-Graphene Oxide Composite Scaffolds for Bone Defect Repair.

PurposeInsufficient biological activity heavily restricts the application and development of biodegradable bone implants. Functional modification of bone implants is critical to improve osseointegration and bone regeneration.MethodsIn this study, L-lysine functionalized graphene oxide (Lys-g-GO) nanoparticles and polydopamine-assisted gold nanoparticle (AuNPs-PDA) coatings were applied to improve the biological function of PLGA scaffold materials. The effects of Lys-g-GO nanoparticles and AuNPs-PDA functionalized coatings on the physicochemical properties of PLGA scaffolds were detected with scanning electron microscopy (SEM), contact angle measurement, and mechanical testing instruments. In vitro, the effects of composite scaffolds on MC3T3-E1 cell proliferation, adhesion, and osteogenic differentiation were studied. Finally, a radial defect model was used to assess the effect of composite scaffolds on bone defect healing.ResultsThe prepared AuNPs-PDA@PLGA/Lys-g-GO composite scaffolds exhibited excellent mechanical strength, hydrophilicity and antibacterial properties. In vitro, this composite scaffold can significantly improve osteoblast adhesion, proliferation, osteogenic differentiation, calcium deposition, and other cell behaviour. In vivo, this composite scaffold can significantly promote the new bone formation and collagen deposition in the radial defect site and presented good biocompatibility.ConclusionThe combination of bioactive nanoparticles and surface coatings shows considerable potential to enhance the osseointegration of bone implants.

Effects of topical mechanical stability on the formation of Masquelet membrane in a rabbit radial defect model

The exact mechanism of Masquelet technique is unknown. This study intends to explore the effects of topical mechanical stability on the formation of Masquelet membrane. Segmental radius shaft defect was created in all rabbits, which were filled with polymethylmethacrylate (PMMA) in Non-fixation group, and with PMMA fixed with plates in Fixation group, and subjected to no disposal in control group. The topical stability of PMMA and plates were monitored via X-ray and mechanical test. And the membranes were excised for further Histological, IHC and Western-Blotting analysis 4 and 6 weeks post-operatively. X-ray revealed no sign of plates loosening, or shift of PMMA. Mechanical tests revealed superior topical stability by plates. Pathological examinations suggested that vascularized and osteogenic membranes were formed around PMMA. IHC and Western-Blotting analysis revealed that both Fixation and Non-fixation group exerted significant effects on the expression of Ki67, COL I, and CD31 positive cells, as well as the protein expression of osteogenic (RUNX2, ALP) and angiogenic (VEGFA, TGF-β1) factors. And compared with membrane in Non-fixation group, Fixing PMMA spacer with plates caused a significant increase in osteogenic and angiogenic expression. This study indicates that rigid fixation provided by plate in Masquelet technique positively alters the quality of membrane formed surrounding PMMA, in terms of significantly osteogenic and angiogenic potential.

Guided Bone Regeneration with Ammoniomethacrylate-Based Barrier Membranes in a Radial Defect Model.

Large bone defects pose an unsolved challenge for orthopedic surgeons. Our group has previously reported the construction of a barrier membrane made of ammoniomethacrylate copolymer USP (AMCA), which supports the adhesion, proliferation, and osteoblastic differentiation of human mesenchymal stem cells (hMSCs). In this study, we report the use of AMCA membranes to seclude critical segmental defect (~1.0 cm) created in the middle third of rabbit radius and test the efficiency of bone regeneration. Bone regeneration was assessed by radiography, biweekly for 8 weeks. The results were verified by histology and micro-CT at the end of the follow-up. The AMCA membranes were found superior to no treatment in terms of new bone formation in the defect, bone volume, callus surface area normalized to total volume, and the number of bone trabeculae, after eight weeks. Additional factors were then assessed, and these included the addition of simvastatin to the membrane, coating the membrane with human MSC, and a combination of those. The addition of simvastatin to the membranes demonstrated a stronger effect at a similar radiological follow-up. We conclude that AMCA barrier membranes per se and simvastatin delivered in a controlled manner improve bone regeneration outcome.

Electroactive composite scaffold with locally expressed osteoinductive factor for synergistic bone repair upon electrical stimulation

Tissue engineering is a promising strategy for the repair of large-scale bone defects, in which scaffolds and growth factors are two critical issues influencing the efficacy of bone regeneration. Unfortunately, the broad application of growth factors is limited by their poor stability in the scaffolds. In the present study, the strictly controlled expression of human bone morphogenetic protein-4 (hBMP-4) in the presence of doxycycline is achieved by adding an hBMP-4 gene fragment into a non-viral artificial restructuring plasmid vector (pSTAR) to form the pSTAR-hBMP-4 plasmid (phBMP-4). Furthermore, the controlled release of phBMP-4 is obtained with an electroactive tissue engineering scaffold, generated by combining a triblock copolymer of poly(l-lactic acid)-block-aniline pentamer-block-poly(l-lactic acid) (PLA-AP) with poly(lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA). This PLGA/HA/PLA-AP/phBMP-4 composite scaffold, with controlled gene release and Dox-regulated gene expression upon electrical stimulation, operating synergistically, exhibits an improved cell proliferation ability, enhanced osteogenesis differentiation in vitro, and effective bone healing in vivo in a rabbit radial defect model. Taking these results together, the proposed smart PLGA/HA/PLA-AP/phBMP-4 scaffold lays a solid theoretical and experimental basis for future applications of such multi-functional materials in bone tissue engineering to help patients in need.

Repair of rabbit segmental radial defect by a compound of adipose-derived stem cells with the ink-jet all-core structure composite scaffold

Objective To repair segmental radial defect by adipose-derived stem cells (ADSCs) combined with a porous wrapped-core structure composite scaffold composed of poly(lactic-co-glycolic acid)/β-tricalciumphosphate (PLGA/β-TCP) skeleton ink-jet wrapped with Type I collagen. Methods 1.5 cm segmental radial defects were created in 36 rabbits, then the combination of ADSCs and ink-jet all-core structure composite scaffold (group A) and the combination of ADSCs and PLGA/β-TCP skeleton (group B) were transplanted into the untreated radial defect as blank control group (group C). 12, 24, 48 weeks after implantation, animals were sacrificed and the constructs were harvested and analyzed. Results Imaging, gross observation and histological examination showed that the bone defect in group A was completely repaired, which was significantly superior to the partial repair in group B and not in group C. In group A, synosteosis was formed between the two ends of the critical-size bone defect 12 weeks postoperation; bone integrity was almost finished and bone marrow started a trend of recanalization 24 weeks postoperation; bone defect repair was completely repaired and bone marrow was recanalized 48 weeks later. As compared with group C at 12, 24, and 48 h [(0.58±1.00), (0.58±1.00) and (0.58±1.00) points], Lane-Sandhu indicated that the new bone forming in group B [(1.22±0.59), (4.89±0.8), and (6.18±0.71) points was significantly increased (t=13.500, P<0.05); As compared with group B, the new bone forming in group A [(5.11±0.70), (8.31±0.48), and (12.98±0.60) points] was significantly increased (t=18.000, P<0.05). Conclusion The combination of ADSCs and ink-jet all-core structure composite scaffold exhibits a favorable osteogenesis, biocompatibility and biodegradability during the repair of autologous segmental radial defect model, which clearly demonstrates promise for the treatment of bone defects through tissue engineering. Key words: Ink-jet all-core structure composite scaffold; Adipose-derived stem cells; Tissue engineering; Bone repair

Abstract 14: Large Gap Peripheral Nerve Repair in a Non-Human Primate Model: Improving Outcomes Utilizing Photochemical Tissue Bonding (PTB) with Acellular Nerve Allograft (ANA)

PURPOSE: Segmental peripheral nerve deficits are challenging injuries associated with poor outcomes. While these injuries afflict civilians (e.g. Boston Marathon Bombing), they have become increasingly prevalent in combat trauma since the advent of improvised explosive devices. Standard of care large-gap (>3cm) reconstruction requires nerve autograft. Alternatives are sought in clinical scenarios where donor limb is not available (e.g. multiple extremity trauma) or donor site morbidity (e.g. painful neuroma formation, loss of function) makes autograft use suboptimal. A variety of alternatives have been proposed including allografts and conduits. Photochemical tissue bonding (PTB) as an alternative to traditional suture neurorrhaphy has been extensively studied in the rodent sciatic nerve model. PTB carries several advantages: reduction of needle trauma, prevention of axonal escape, and a water-tight seal to contain neuroregenerative factors and exclude inflammatory invasion. We report a non-human primate study which recapitulates human anatomy, allows for objective quantitative functional outcomes testing, electrophysiology and histomorphometry. The purpose of this study is to evaluate whether PTB can elevate the performance of acellular nerve allograft (ANA) to that of standard of care autograft/suture. METHODS: Nineteen rhesus macaques underwent 4cm proximal radial nerve defect creation in the right upper extremity, the radial nerve transected proximally at the humeral spiral groove, distally prior to the branch to brachioradialis. The radial nerve was selected as it is responsible for an isolated function with no input from other nerves. Three repair techniques were evaluated: n=6 autograft/suture, n=6 ANA/suture, n=7 ANA photosealed in place with light-activated human amnion wraps (PTB). An objective functional outcome test was conceived using an apparatus that accurately measured the degree of wrist extension as a function of time after surgery. Electromyography (EMG) was performed at 0, 120, 240, and 365 days (euthanasia). Histomorphometry and muscle mass retention were analyzed at euthanasia. RESULTS: Average loss of wrist extension was 88.3 ± 8.2° after radial nerve defect creation. Autograft group animals recovered 82.0° of extension by 7 months. Wrist extension recovery was modestly slower, as expected, in the ANA groups, however, by 8 months the ANA/PTB group recovered 63.0° of extension with no difference in recovery of baseline EMG amplitude as compared to controls (Avance/PTB=75.81% vs. autograft/suture=65.06%). If recovery follows current trajectory, we expect ANA/PTB to demonstrate equivalent outcomes to the autograft/suture group at one year. CONCLUSION: This radial nerve defect model improves upon existing animal models by allowing for large nerve gap testing in a primate model more analogous to the clinical large nerve gap injury in humans. ANA/PTB group functional recovery lagged modestly behind autograft/suture (by as long as 8 weeks) but is approaching equivalence at 8 months. EMG recovery is similar at 8 months. This preliminary data confirms PTB as a promising technique to improve outcomes of large nerve gap reconstruction in combination with autograft (previously demonstrated) and with acellular nerve allograft.

Bone Regeneration Induced by Local Delivery of a Modified PTH-Derived Peptide from Nanohydroxyapatite/Chitosan Coated True Bone Ceramics.

A novel PTH-derived peptide, PTHdP, including a repetitive aspartic acid sequence at the C-terminal and phosphorylated serine at the N-terminal has been previously designed. To evaluate its potential as a bone growth factor for bone tissue engineering, true bone ceramics incorporated with nanohydroxyapatite coating and chitosan (CH/TBC) was developed as a desired three-dimensional porous delivery carrier in this study. In vitro results showed that PTHdP could be loaded with high-efficiency and subsequently released in a controlled and sustained manner from CH/TBC. Bioactivity of released PTHdP was retained and able to exert a significant effect on promoting or inhibiting osteogenesis actions when exposed intermittently or continuously, respectively, for MC3T3-E1 cell culture. As evaluated in a critical-size rabbit radial defect model by radiographic and histological examination, the combination of CH/TBC scaffolds with PTHdP exhibited a remarkably stronger capacity to stimulate new bone formation than control and pure CH/TBC groups. These results indicated the novel PTHdP peptide achieved high affinity to bone mineral without interference in bioactivity, and local delivery of PTHdP from apatite materials could be a promising alternative for future bone tissue engineering.

Osteogenesis effects of strontium-substituted hydroxyapatite coatings on true bone ceramic surfaces in vitro and in vivo

To develop bioactive bone graft materials that can induce rapid bone regeneration, a novel biomaterial was synthesized by coating true bone ceramic (TBC) substrates with strontium-substituted nano-hydroxyapatites (SrHA) (Sr concentrations of 0%, 10%, 40%, 100%) through a sol–gel dip-coating approach. All coated TBC scaffolds retained the inherent natural trabecular structure, porosity, compressive strength and simultaneously possessed a micro/nanotopography SrHA layer on the substrate surface. The dimension of the deposited crystal increased and the density of the deposited apatite particles became sparse with increasing Sr content, but a unique HA crystalline phase was observed under all conditions. The modified TBC scaffolds significantly enhanced the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 osteoblasts in vitro. Particularly, the Sr10-TBC group (10 mol% Sr2+ in apatite coating) revealed the highest osteogenic efficacy over the other groups. Three-dimensional CT imaging and histological evaluations on a bilateral critical-sized rabbit radial defect model for 12 weeks showed significant bone formation in the Sr10-TBC implants. The new bone area ratios of the Sr10-TBC group were significantly higher than that of the TBC group. Additionally, Sr10-TBC implants showed faster degradability compared with raw TBC implants during the 12 weeks of implantation. The results indicate that TBC modification with 10% SrHA coating stimulated osteogenesis and could be a promising biomaterial for future bone defect regeneration.

Construction of Radial Defect Models in Rabbits to Determine the Critical Size Defects.

Many studies aimed at investigating bone repair have been conducted through animal models in recent years. However, limitations do exist in these models due to varying regeneration potential among different animal species. Even using the same animal, big differences exist in the size of critical size defects (CSD) involving the same region. This study aimed to investigate the standardization of radial bone defect models in rabbits and further establish more reliable CSD data. A total of 40 6-month-old New Zealand white rabbits of clean grade totaling 80 radial bones were prepared for bone defect models, according to the principle of randomization. Five different sizes (1.0, 1.2, 1.4, 1.7 and 2.0 cm) of complete periosteal defects were introduced under anesthesia. At 12 weeks postoperatively, with the gradual increase in defect size, the grades of bone growth were significantly decreased in all 5 groups. X-ray, CT scans and H&E staining of the 1.4, 1.7, and 2.0-cm groups showed lower grades of bone growth than that of the 1.0 and 1.2-cm groups respectively (P < 0.05). Using rabbit radial defect model involving 6-month-old healthy New Zealand white rabbits, this study indicates that in order to be critical sized, defects must be greater than 1.4 cm.

A resorbable antibiotic-eluting polymer composite bone void filler for perioperative infection prevention in a rabbit radial defect model.

Nearly 1.3 million total joint replacement procedures are performed in the United States annually, with numbers projected to rise exponentially in the coming decades. Although finite infection rates for these procedures remain consistently low, device-related infections represent a significant cause of implant failure, requiring secondary or revision procedures. Revision procedures manifest several-fold higher infection recurrence rates. Importantly, many revision surgeries, infected or not, require bone void fillers to support the host bone and provide a sufficient tissue bed for new hardware placement. Antibiotic-eluting bone void fillers (ABVF), providing both osteoconductive and antimicrobial properties, represent one approach for reducing rates of orthopedic device-related infections. Using a solvent-free, molten-cast process, a polymer-controlled antibiotic-eluting calcium carbonate hydroxyapatite (HAP) ceramic composite BVF (ABVF) was fabricated, characterized, and evaluated in vivo using a bacterial challenge in a rabbit radial defect window model. ABVF loaded with tobramycin eliminated the infectious burden in rabbits challenged with a clinically relevant strain of Staphylococcus aureus (inoculum as high as 107 CFU). Histological, microbiological, and radiographic methods were used to detail the effects of ABVF on microbial challenge to host bone after 8 weeks in vivo. In contrast to the HAP/BVF controls, which provided no antibiotic protection and required euthanasia 3 weeks post-operatively, tobramycin-releasing ABVF animals showed no signs of infection (clinical, microbiological, or radiographic) when euthanized at the 8-week study endpoint. ABVF sites did exhibit fibrous encapsulation around the implant at 8 weeks. Local antibiotic release from ABVF to orthopedic sites requiring bone void fillers eliminated the periprosthetic bacterial challenge in this 8-week in vivo study, confirming previous in vitro results.

Repairing rabbit radial defects by combining bone marrow stroma stem cells with bone scaffold material comprising a core-cladding structure.

We prepared a bone scaffold material comprising a PLGA/β-TCP core and a Type I collagen cladding, and recombined it with bone marrow stroma stem cells (BMSCs) to evaluate its potential for use in bone tissue engineering by in vivo and in vitro experiments. PLGA/β-TCP without a cladding was used for comparison. The adherence rate of the BMSCs to the scaffold was determined by cell counting. Cell proliferation rate was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The osteogenic capability was evaluated by alkaline phosphatase activity. The scaffold materials were recombined with the BMSCs and implanted into a large segmental rabbit radial defect model to evaluate defect repair. Osteogenesis was assessed in the scaffold materials by histological and double immunofluorescence labeling, etc. The adherence number, proliferation number, and alkaline phosphatase expression of the cells on the bone scaffold material with core-cladding structure were significantly higher than the corresponding values in the PLGA/β-TCP composite scaffold material (P < 0.05). An in vivo test indicated that the bone scaffold material with core-cladding structure completely degraded at the bone defect site and bone formation was completed. The rabbit large sentimental radial defect was successfully repaired. The degradation and osteogenesis rates matched well. The bone scaffold with core-cladding structure exhibited better osteogenic activity and capacity to repair a large segmental bone defect compared to the PLGA/β-TCP composite scaffold. The bone scaffold with core-cladding structure has excellent physical properties and biocompatibility. It is an ideal scaffold material for bone tissue engineering.

HBMP-2 and hTGF-β1 expressed in implanted BMSCs synergistically promote the repairing of segmental bone defects

ObjectiveTo evaluate the effects of co-expressing hBMP-2 and hTGF-β31 in BMSCs (bone marrow-derived mesenchymal stem cells) on the repairing process of radial segmental defects in rats. MethodsBMSCs were infected with a high titer recombinant adenovirus carrying hTGF-βl and/or hBMP-2 genes. Expression of exogenous genes in BMSCs was confirmed by RT-PCR and ELISA assays. In vitro effects of exogenous genes were assessed by MTT and ALP activity tests. A left radial defect model was created using 120 SD rats. Genetically modified or unmodified BMSCs were implanted with collagen sponge scaffolds into the 5-mm radial defect. The bone repair process was systematically monitored and evaluated by X-ray examinations, gross anatomic examinations, histological analyses, and biomechanical tests. ResultsExpression of hBMP-2 and hTGF-β1 showed synergistic effects on promoting BMSC proliferation and enhancing ALP activity in vitro. Bone repair assays showed that hBMP-2 and hTGF-β1 promoted the production of chondrocytes and osteoblasts. Implanted BMSCs transfected with both hBMP-2 and hTGF-β1 led to the best bone repair outcome.

A comparison of osteocyte bioactivity in fine particulate bone powder grafts vs larger bone grafts in a rat bone repair model

The osteogenic potential for bone grafts is based on numbers and activities of cells that survive transplantation. In this study, we compared the bioactivity of osteocytes in 300–500μm fine particulate bone powder grafts to 2mm larger bone grafts in a rat radial defect model. Expression levels of bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-β1), alkaline phosphatase (ALP), and collagen I were semi-quantified by both immunohistochemistry and RT-PCR at days 1 and 4, as well as weeks 1, 2, 4, 6 and 10 post-transplantation. Within two weeks post-transplantation, more cells stained positively for BMP-2, TGF-β1, ALP, and collagen I within the bone grafts and in the surrounding tissues in the group transplanted with the fine particulate bone powder grafts than in those with larger bone grafts (P<0.05). The mRNA levels of all four markers in the group transplanted with fine particulate bone powder graft peaked earlier and were expressed more highly than in the larger bone graft group, suggesting that fine particulate bone powder grafts provide more viable and active osteocytes to accelerate bone defect healing than larger bone grafts.

Repair of rabbit segmental radial defect by using bone mesenchymal stem cells into the wrapped-core structure composite scaffold

Objective To repair segmental radial defect by using bone mesenchymal stem cells (BMSCs) cultured in vitro for 3 weeks under osteogenic medium into a porous wrapped-core structure composite scaffold composed of poly(lactic-co-glycolic acid)/β-tricalciumphosphate (PLGA/β-TCP) skeleton wrapped with type Ⅰ collagen.Methods 1.5 cm segmental radial defects were created bilaterally in 36 rabbits,then BMSCs in combination with wrapped-core structure composite scaffold (group A) and BMSCs in combination with PLGA/β-TCP skeleton (group B) were transplanted into the untreated radial defect as blank control group (group C).12,24 and 48 weeks after implantation,12 animals were sacrificed and the constructs were harvested and analyzed.Results In group A,synosteosis formed between the two ends of the critical-size bone defect 12 weeks postoperation; bone integrity was almost finished and bone marrow started a trend of recanalization 24 weeks postoperation ; bone defect repair was completed and bone marrow was recanalized 48 weeks later.As compared with group B (1,4,6 points),and group C (0,0,1point),Lane-Sandhu indicated that the new bone formation in group A (5,8,and 12 points) was significantly increased (P ＜ 0.05,n =4).Conclusion The combination of BMSCs and core-sheath structure composite scaffold exhibits a favorable osteogenesis,biocompatibility and biodegradability during the repair of autologous segmental radial defect model,which clearly demonstrates promise for the treatment of bone defects through tissue engineering. Key words: Wrapped-core structure composite scaffold; Bone mesenchymal stem cells; Segmental bone defects; Bone tissue engineering

Bone regeneration potential of allogeneic or autogeneic mesenchymal stem cells loaded onto cancellous bone granules in a rabbit radial defect model

For developing a clinically effective bone regeneration strategy, we compare the bone regeneration potential of cultured allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) and of autologous BM-MSCs loaded onto allogeneic cancellous bone granule scaffolds. A critical-sized segmental bone defect was made at the mid-shaft of both radiuses in 19 New Zealand White rabbits (NWRs). In the experimental group, allogeneic BM-MSCs loaded onto small-sized allogeneic cancellous bone granules (300~700 um in diameter) were implanted in one side of a bone defect. In the control group, autologous BM-MSCs loaded onto allogeneic cancellous granules were grafted in the other side. Bone regeneration was assessed by radiographic evaluation at 4, 8, 12 and 16weeks post-implantation and by micro-computed tomography (micro-CT) and histological evaluation at 8 and 16weeks. The experimental groups showed lower bone quantity indices (BQIs) than the control groups at 12 and 16weeks (p < 0.05), although no significant difference was observed at 4 and 8weeks (p > 0.05). Micro-CT analysis revealed that both groups had similar mean total bone volume and other parameters including trabecular thickness, number and separation at either 8 or 16weeks. Only bone surface area revealed less area in the experimental group at 16weeks. Histological evaluation of 8-week and 16-week specimens showed similar biologic processes of new bone formation and maturation. There was no inflammatory reaction indicating an adverse immune response in both allogeneic and autologous MSC groups. In conclusion, allogeneic BM-MSCs loaded onto allogeneic cancellous bone granules had comparable bone regeneration potential to autologous BM-MSCs in a rabbit radial defect model.