Acellular Bone Matrix Research Articles

Treated Pierre Robin Sequence Using Placed Allogenic Acellular Bone Matrix and Mandibular Distraction Osteogenesis in the Neonate

ObjectiveThe aim of the study was to report our experience with placed allogenic acellular bone matrix and mandibular distraction osteogenesis in Pierre Robin sequence (PRS), and explore the role of distraction in the osteogenesis of acellular bone.Materials and MethodsA total of 428 neonates with severe PRS managed with placing allogenic acellular bone and bilateral mandibular distraction osteogenesis were included in the study. The procedure included using oblique-shaped osteotomy, fixing bilateral mandibular distractor, instantly extending a 4–6 mm gap, and placing allogenic acellular bone into the gap. The length of allogenic acellular bone was 4–5 mm. Although the surgical techniques, distraction, and consolidation periods were similar, the allogenic acellular bone matrix we placed was quite different from the traditional distraction. With the technology we used, tracheal intubation could be immediately removed, thus quickly improving breathing conditions compared to traditional methods after the surgery. The jaw extending and oral feeding could begin on the 5th day. The jaw was extended 0.6 mm twice a day until the mandible was overcorrected by 20%.ResultsAll 428 cases included in this study were successfully extubated after the operation, and the difficulty in breathing was instantly relieved. Total mandibular distraction was 15–20 mm. Oral feeding was started at 6 h to 6 days postoperatively, while hospital stay ranged from 18 to 20 days postoperatively. No major complications were reported. Medium to long-term results was good. Mandibular distractors were removed after 3 months.ConclusionsBilateral mandibular distraction osteogenesis combined with placing allogenic acellular bone in the neonate are safe and accurate procedures, which are the primary treatment options for cases of severe PRS. It can be considered that the tension of distraction can promote osteogenesis in acellular bone and thus improve distractive effect of the mandible.

Application of Bovine Acellular Cancellous Bone Matrix in Alveolar Ridge Preservation Following Tooth Extraction

Objective: In this article, we explored the microscopic structure and composition of the decellular-ized cancellous bone matrix of the calf, and established the animal model of Beagle dog extraction. By applying different bone substitute materials in the extraction of teeth, we observed the new collagen-rich in the preservation of the site after tooth extraction. The protein bone matrix maintained the three-dimensional shape of the alveolar ridge compared to other biological materials. Methods: The microstructure of the new collagen-rich bone matrix was observed by scanning electron microscopy. The porous structure, porosity and distribution of collagen fibers were observed. XRD and infrared spectroscopy were used to further detect the inorganic and organic components in the new collagen-rich bone matrix. The premolar extraction and site preservation model of Beagle dogs were constructed. The changes of collagen-rich bone matrix, Bio-oss bone powder, CGF filling and blank control alveolar ridge volume were compared by CBCT. HE staining was used to observe and compare new bone formation, bone remodeling and bone resorption between groups, and to observe the formation of blood vessels, osteogenic mineralization, trabecular bone formation and inflammatory response in different periods. Results: (1) The acellular bone matrix of bovine cancellous bone completely removes the immunogenicity of the cells and has good histocompat-ibility; the pore diameter and porosity closest to the physiological structure, the main component is hydroxyapatite and collagen. (2) Site preservation can reduce the absorption of alveolar ridge following tooth extraction, preserve sufficient bone mass for alveolar fossa, and retain a good width of attachment, which provides further protection for implant surgery. (3) In the post-extraction site preservation, the CGF group has better bone composition than the BABM group and Bio-oss bone powder. Conclusion: Bovine Acellular Cancellous Bone Matrix is a new type of biological bone matrix. The main components are collagen and hydroxyapatite, which can promote bone formation in the extraction socket.

Evaluation of Acellular Bovine Cancellous Bone Matrix Seeded with Foetal Rabbit Osteoblasts for Segmental Bone Defects in Rabbits

The present work was assessed the restoration of segmental bone gap defect in rabbits by application of composite bone grafts. Composite bone grafts were prepared by seeding of rabbit foetal osteoblast on the bovine acellular cancellous bone matrix and evaluated in the segmental gap defect of 20 mm in rabbits. Thirty six adult New Zealand White rabbits of either sex were allotted to three groups of 12 each. Autograft (group A), Acellular cancellous bone matrix (group B) and composite graft seeded with fetal osteoblasts (group C) were implanted in the defects. Radiography, gross observations and histopathology at different intervals were done to evaluate healing. It was concluded that the seeded foetal osteoblasts in the composite grafts augment regeneration of the new bone leading to better integration of graft in host in comparison to bovine acellular cancellous bone matrix graft.

Clinical analysis of acellular dermal matrix and acellular bone matrix in oro-antral fistula repair

This study aimed to investigate the application of acellular dermal matrix and acellular bone matrix in the management of oro-antral fistula. Nine patients with oro-antral fistula (with defect greater than 5 mm×5 mm) after maxillary cyst resection or maxillary molar extraction were selected. The defects were repaired by the simultaneous implantation of acellular dermal matrix and acellular bone matrix. The incisions of nine patients were all primary healing. After 6 months of follow-up, the oro-antral communication healed well, and no symptom such as nasal congestion or runny nose was observed. The clinical and CT examinations confirmed wound healing. The usage of acellular dermal matrix and acellular bone matrix is a reliable repairing method for ora-antral fistula.

Organic composite-mediated surface coating of human acellular bone matrix with strontium

Acellular bone matrix (ACBM) provides an osteoconductive scaffold for bone repair, but its osteoinductivity is poor. Strontium (Sr) improves the osteoinductivity of bone implants. In this study, we developed an organic composite-mediated strontium coating strategy for ACBM scaffolds by using the ion chelating ability of carboxymethyl cellulose (CMC) and the surface adhesion ability of dopamine (DOPA). The organic coating composite, termed the CMC-DOPA-Sr composite, was synthesized under a mild condition, and its chemical structure and strontium ion chelating ability were then determined. After surface decoration, the physicochemical properties of the strontium-coated ACBM (ACBM-Sr) scaffolds were characterized, and their biocompatibility and osteoinductivity were determined in vitro and in vivo. The results showed that the CMC-DOPA-Sr composite facilitated strontium coating on the surface of ACBM scaffolds. The ACBM-Sr scaffolds possessed a sustained strontium ion release profile, exhibited good cytocompatibility, and enhanced the osteogenic differentiation of mesenchymal stem cells in vitro. Furthermore, the ACBM-Sr scaffolds showed good histocompatibility after subcutaneous implantation in nude mice. Taken together, this study provided a simple and mild strategy to realize strontium coating for ACBM scaffolds, which resulted in good biocompatibility and improved osteoinductivity.

Establishment of rabbit radius fracture model and the role of stromal vascular fraction-acellular bone extracellular matrix-chitosan scaffold in promoting the healing of rabbits

Objective To prepare stable structure and good bone induction and biological compatibility of stromal vascular fraction (SVF)-acellular bone extracellular matrix(ABECM)-chitosan composite scaffold study SVF-ABECM-chitosan scaffolds to promote the healing of rabbit radius fracture. Methods Using Triton X-100 and sodium dodecyl sulfate (SDS) acellular method of porcine femur were decellularized, using a solution blending, freeze drying method preparing ABECM-chitosan composite scaffolds. The qualitative analysis of SVF-ABECM-chitosan scaffolds, observe SVF-ABECM-chitosan composite porous scaffold morphology, porosity, mechanical properties and cell scaffold composite culture compatibility.12 adult healthy New Zealand white rabbits were selected, and the fracture model was caused by the fracture gap of about 0.3 cm, which was exposed to the radial shaft of the radial side of the forearm of the rabbit. The SVF-ABECM-chitosan composite scaffold was prepared in the right radius fracture space of each animal, and the left side was the blank control group. The fracture healing was compared between the two sides of the radius fracture model of rabbits by X-ray, biomechanical test and tissue morphology. Results ABECM materials were stained with he and hoechest 33258 fluorescence staining showed no residual cells, immunohistochemical staining of alizarin red staining and type I collagen and bone morphogenetic occurred protein-2 antibody staining positive. Scanning election microscopy (SEM) showed that the scaffolds have a porous structure, the aperture 50-200 μm, porosity of (53.50±4.23)%, dry mechanical test support longitudinal compression modulus is (33.23±14.45) mPa, support wet state of compression modulus of (2.27±1.13) mPa. Cell scaffold composite culture has good compatibility. After 4 weeks and 12 weeks in the experimental group by X-ray films reflect the bone healing was stronger than that of the control group. After 12 weeks, the biomechanical test group in the maximum load, anti bending strength of HA load/displacement parameters and control group comparison differences were statistically significant, and elastic modulus difference were statistically significant (t=7.418, 5.131 and 2.838, P=0.004, 0.009 and 0.027), and elastic modulus difference between the two groups no statistical significance (t=1.455, P=0.064), at 12 weeks after operation, the histomorphometric results showed. In experimental group, the bone and shaping was significantly better than the control group. Conclusion Preparation of ABECM-CS composite porous scaffolds to cells completely, retained the removal of cells from the bone matrix main component, have the appropriate aperture and porosity, cell compatibility is good. SVF-ABECM-chitosan composite scaffold can effectively promote the healing of rabbit radius fracture. Key words: Stromal vascular fraction; Acellular bone matrix; Chitosan; Radius fracture; Rabbit

Induction of neural tissue markers by micronized human spinal cord implants.

The osteoinductive capacity of biological noncellular material has been widely recognized. Studies using bone morphogenetic proteins and acellular bone matrix demonstrate that host mesenchymal cells can be readily transformed into osteoprogenitor cells. The current study sought to determine whether another biological noncellular material, human spinal cord matrix, could induce transformation of host cells into a neural lineage. We demonstrate the formation of neural tissue and the expression of neural-specific lineage markers in host cells colonizing implanted spinal cord fragments and adjacent tissue along with the lack of expression of nonneural lineage markers. These studies demonstrate that the inductive capacity of biological noncellular material is not limited to the osteogenic lineage and suggest that acellular spinal cord matrix could be used to generate host-derived cells for use in neural repair and regeneration.

Injectable thermosensitive PEG–PCL–PEG hydrogel/acellular bone matrix composite for bone regeneration in cranial defects

We have certified that the injectable thermosensitive ABM/PECE composite presented promising potential in bone regeneration benefited from the incorporation of the intrinsic osteoinductive acellular bone matrix (ABM) granules into the poly(ethylene glycol)–poly(ε-capro-lactone)–poly(ethylene glycol) (PEG–PCL–PEG, PECE) hydrogel. In this study, the 12 mm × 8 mm × 2 mm cranial defects of the New Zealand white rabbits were fabricated to evaluate the bone regeneration effect. The ABM/PECE composite was injected into the defect while the pure PECE as control, and the bone regeneration was evaluated at 4, 12 and 20 weeks post-surgery by X-radiological examination, micro-computed tomography examination and histological analysis. In ABM/PECE composite treated group, the new bone formed originally from both the margin and the center of the defect, and the defect region had healed up to 20 weeks. Furthermore, the shadow density of the newly formed bone eventually approximated to host cortical bone. In comparison, the control group was filled with sparing new bone with low-density only from the periphery of the defect. Meanwhile, the quantitative determination of new bone by histomorphometry confirmed the excellent bone regeneration of ABM/PECE composite. All the results suggested that the ABM/PECE composite presented enhanced bone regeneration guidance in rabbit cranial defects.

Nanohydrogels as A Prospective Member of The Nanomedicine Family

modification (to escape from recognition by the reticuloendothelial system) and drug targeting by conjugation of ligand onto the surface of hydrogel nanoparticles to aim the corresponding biomarkers that are overexpressed in the cancer cell membrane. In the past decade, nanohydrogels have been under intensive investigation as a versatile tool for the delivery of advanced biological therapeutics such as proteins, peptides and carbohydrates. Even for oligonucleotides, a few attempts have been made for the delivery of siRNA [3–6]. Physical hydrogels and their biomedical applications have been intensively investigated in recent years. Fang et al. fabricated a honokiol (HK) nanoparticles loaded thermosensitive PEG-poly(e-caprolactone) (PCL)-PEG (PECE) hydrogel (HK-hydrogel), and demonstrated that such a hydrogel composite has outstanding therapeutic effects on malignant pleural effusion. Compared with the use of HK nanoparticles alone, blank hydrogel or normal saline, the HK-hydrogel system has distinct advantages in reducing the number of pleural tumor foci, prolonging survival time and inhibiting angiogenesis of pleural tumors [7]. Yang et al. also found that the thermosensitive PECE hydrogel has a notable efficacy in preventing post-surgical abdominal adhesions [8]. Additionally, Fu et al. tried to enlarge the application of PECE hydrogel in the bone tissue engineering field. Nanohydroxyapatite or acellular bone matrix were incorporated into a PECE hydrogel to form an injectable thermoresponse nano-hydroxyapatite or acellular bone matrix/PECE hydrogel composite [9]. Chemical hydrogels are commonly waterswollen networks of hydrophilic homopolymers or Molecular biomaterials, such as synthetic or natural biodegradable polymers, are the material basis of nanomedicine [1]. Among them, hydrophilic polymers, such as poly(ethylene glycol) (PEG), can be crosslinked to form a hydrogel, which is a 3D network that may absorb water in quantities from a few percent to thousands of times their dry weight. The amount of expansion is determined by the number of crosslinked molecules and the type of composite hydrophilic polymers or copolymers. Hydrogels can be either physical (reversible) gels, when the networks are held together by molecular entanglements and/or secondary forces including ionic or hydrophobic forces, or chemical (permanent) gels, when they are covalently crosslinked networks. Since the 1960s, hydrogels have had wide applications in biomedical engineering, especially in local drug delivery and tissue engineering for tissue repair and regeneration [2]. Recently, nanohydrogels, in other words hydrogel nanoparticles in the nanometer scale from tens to hundreds of nanometers, have become a member of great potential in the nanomedicine family. Among the various nanomedicine formulations, nanohydrogels have shown great advantages for delivery of hydrophilic small-molecule drugs and protein/peptide therapeutics due to their huge loading capacity of water-soluble compounds. Nanohydrogels combine the advantages of hydrogels and nanoparticles for drug formulation and delivery, which include controllable drug release, high stability in physiological media, distinct responsiveness to environmental factors such as pH and temperature, high cellular uptake due to the endocytosis mechanisms, long half-life in circulation by appropriate surface

Interactions of Bone Marrow Stromal Cells with Native and RGD Surface Modified Acellular Bone Matrix: A Biocompatibility Study

Acellular bone matrix (ACM) is a promising biodegradable material scaffold. However, poor cell attachment properties of ACM limit its application. This study investigated the biocompatibility of mesenchymal stem cells (MSCs) and native or surface modified ACM (RGD-ACM) scaffolds in the construction of tissue-engineered bone. Wistar rat bone marrow-derived MSCs were isolated by density gradient centrifugation for in vitro culture. MSCs were seeded on ACM with or without surface modification by RGD peptides to generate tissue-engineered bone for in vitro culture. Significantly higher adhesion of MSCs to RGD-ACM was observed compared with the native material. Detection of active alkaline phosphatase (ALP) indicated intercalation of this protein into the scaffold enhanced cell proliferation. Complete distribution and adhesion of MSCs to the surface of the scaffold was observed by scanning electron microscope. ACM with surface modification by RGD peptides improved the seeding adhesion rates and facilitated the growth efficiency of tissue-engineered bone.

The Poor Osteoinductive Capability of Human Acellular Bone Matrix

Demineralized bone matrix (DBM) has extensive clinical use for bone regeneration because of its osteoinductive and osteoconductive aptitude. It is suggested that the demineralization process in bone matrix preparation is influential in maintaining osteoinductivity; however, relevant investigations, especially into the osteoinductivity of acellular bone matrix, are not often performed. This study addressed the osteoinductive capability of human acellular cancellous bone matrix (ACBM) after subcutaneous implantation in a rat model. The growth and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) seeded in this material were also studied. Without the demineralization process, the ACBM we obtained had an interconnected porous network and the micropores in the surface were clearly exposed. After the ACBM was subcutaneously implanted for 4 months, new osteoid formation was noted but not typical mature bone formation. rBM-MSCs grew well in the ACBM and kept a steady morphology after continuous culture for 28 days. However, no mineralized nodule formation was detected and the expression levels of genes encoding osteogenic markers were significantly decreased. These results demonstrated that human ACBM possess the structural features of native bone and poor osteoinductivity; nonetheless this material helped to preserve the undifferentiated phenotype of rBM-MSCs. Such insights may further broaden our understanding of the application of ACBM for bone regeneration and the creation of stem cell niches.

Biomimetic Construction of Large Engineered Bone Using Hemoperfusion and Cyto-Capture in Traumatic Bone Defect

Due to lack of blood vessel systems, only a few tissues, such as skin, cartilage, and cornea, have been successfully constructed in vivo. Anticoagulative scaffolds have been used in drug-eluting stent systems both in animal studies and clinical therapies, as in the medicinal leech therapy used to salvage venous-congested microvascular free flaps improved perfusion inspired us to tackle this hurdle in bone tissue engineering. We hypothesize that a combination of bone marrow as the blood supply and a heparin/chitosan-coated acellular bone matrix that acts like hirudin, together with a vacuum-assisted closure therapy system, would provide blood perfusion to the scaffold. Using these methods, a biomimetically engineered bone construct would facilitate clinical translation in bone tissue engineering and offer new therapeutic strategies for reconstructing large bone defects if the hypothesis proves to be practical.

Treatment of femoral head osteonecrosis with advanced cell therapy in sheep

The purpose of this study was to evaluate the efficacy of core decompression associated with advanced cell therapy for the treatment of femoral head osteonecrosis in an established sheep model. Early stage osteonecrosis of the right hip was induced cryogenically in 15 mature sheep. At 6 weeks, the sheep were divided into three groups, Group A: core decompression only; Group B: core decompression followed by implantation of an acellular bone matrix scaffold; Group C: core decompression followed by implantation of a cultured BMSC loaded bone matrix scaffold. At 12 weeks, MRI hip studies were performed and then the proximal femur was harvested for histological analysis. In the group of advanced cell therapy, Group C, there was a tendency to higher values of the relative surface of newly formed bone with a mean of 20.3 versus 11.27 % in Group A and 13.04 % in Group B but it was not statistically significant. However, the mean relative volume of immature osteoid was 8.6 % in Group A, 14.97 in Group B, and 53.49 % in Group C (p < 0.05), revealing a greater capacity of osteoid production in the sheep treated with BMSCs. MRI findings were not conclusive due to constant bone edema artifact in all cases. Our findings indicate that a BMCSs loaded bone matrix scaffold is capable of stimulating bone regeneration more effectively than isolated core decompression or in association with an acellular scaffold in a preclinical femoral head osteonecrosis model in sheep.

The poor osteoinductive capability of human acellular bone matrix

Demineralized bone matrix (DBM) has extensive clinical use for bone regeneration because of its osteoinductive and osteoconductive aptitude. It is suggested that the demineralization process in bone matrix preparation is influential in maintaining osteoinductivity; however, relevant investigations, especially into the osteoinductivity of acellular bone matrix, are not often performed. This study addressed the osteoinductive capability of human acellular cancellous bone matrix (ACBM) after subcutaneous implantation in a rat model. The growth and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) seeded in this material were also studied. Without the demineralization process, the ACBM we obtained had an interconnected porous network and the micropores in the surface were clearly exposed. After the ACBM was subcutaneously implanted for 4 months, new osteoid formation was noted but not typical mature bone formation. rBM-MSCs grew well in the ACBM and kept a steady morphology after continuous culture for 28 days. However, no mineralized nodule formation was detected and the expression levels of genes encoding osteogenic markers were significantly decreased. These results demonstrated that human ACBM possess the structural features of native bone and poor osteoinductivity; nonetheless this material helped to preserve the undifferentiated phenotype of rBM-MSCs. Such insights may further broaden our understanding of the application of ACBM for bone regeneration and the creation of stem cell niches.

Synthesis and characterization of injectable, thermosensitive, and biocompatible acellular bone matrix/poly(ethylene glycol)‐poly (ε‐caprolactone)‐poly(ethylene glycol) hydrogel composite

In orthopedic tissue engineering, the extensively applied acellular bone matrix (ABM) can seldom be prefabricated just right to mold the cavity of the diverse defects, might induce severe inflammation on account of the migration of small granules and usually bring the patients great pain in the treatment. In this study, a new injectable thermosensitive ABM/PECE composite with good biocompatibility was designed and prepared by adding the ABM granules into the triblock copolymer poly(ethylene eglycol)-poly(ε-caprolactone)-poly(ethylene eglycol) (PEG-PCL-PEG, PECE). The PECE was synthesized by ring-opening copolymerization and characterized by ¹H NMR. The ABM was prepared by acellular treatment of natural bone and ground to fine granules. The obtained ABM/PECE composite showed the most important absorption bands of ABM and PECE copolymer in FT-IR spectroscopy and underwent sol-gel phage transition from solution to nonflowing hydrogel at 37°C. SEM results indicated that the ABM/PECE composite with different ABM contents all presented similar porous 3D structure. ABM/PECE composite presented mild cytotoxicity to rat MSCs in vitro and good biocompatibility in the BALB/c mice subcutis up to 4 weeks. In conclusion, all the results confirmed that the injectable thermosensitive ABM/PECE composite was a promising candidate for orthopedic tissue engineering in a minimally-invasive way.

Heparin-Chitosan-Coated Acellular Bone Matrix Enhances Perfusion of Blood and Vascularization in Bone Tissue Engineering Scaffolds

Currently, the main hurdle in the tissue engineering field is how to provide sufficient blood supply to grafted tissue substitutes in the early post-transplanted period. For three-dimensional, cell-dense, thick tissues to survive after transplantation, treatments are required for hypoxia, nutrient insufficiency, and the accumulation of waste products. In this study, a biomacromolecular layer-by-layer coating process of chitosan/heparin onto a decellularized extracellular bone matrix was designed to accelerate the blood perfusion and re-endothelialization process. The results of in vitro measurements of the activated partial thromboplastin time supported the theory that the combination of chitosan and heparin could bring both anticoagulation and hemocompatibility to the scaffold. A rabbit bone defect model was established for further evaluation of the application of this kind of surface-modified scaffold in vivo. The final results of computed tomography (CT) perfusion imaging and histological examination proved that this facile coating approach could significantly promote blood perfusion and re-endothelialization in the early post-transplanted period compared with an acellular bone matrix due to its much-improved anticoagulation property.

A novel bilayered scaffold constructed with cartilage extracellular matrix/acellular bone matrix for osteochondral tissue engineering

Objective To fabricate a novel bilayered scaffold constructed with cartilage extracellular matrix (CECM) and acellular bone matrix (ACBM) for osteochondral tissue engineering.Methods The bone layer of the osteochondral scaffold was prepared using canine bone cancellous bone columns, and the cartilage layer was fabricated using CECM.After CECM microfilaments were decellularized, the biphasic scaffolds were fabricated by soaking the ACBM columns into cylindrical silicon moulds with a 30 g/L CECM suspension using simple freeze-drying method.After the scaffolds were cross-linked, the porosity was measureed.MTT test was also done to assess cytotoxicity of the scaffolds.Canine bone marrow-derived mesenchymal stem cells (BMSCs) were induced by chondrogenic medium and seeded into novel scaffold.Cell proliferation and differentiation were analyzed using inverted microscopy, scanning electron microscopy (SEM)and Dead/Live staining method.Results SEM and Micro-CT revealed a 3-D interconnected porous structure, with the CECM pore diameter of 155 ± 34 μm and the porosity of 91.3% ± 2.0%.Cytotoxicity testing with MTT revealed no significant difference in absorbance among different extracts, showing no cytotoxic effect of the scaffold on BMSCs.Inverted microscopy and SEM showed that the novel scaffold could provide a suitable 3-D environment to support the adhesion, proliferation and differentiation of BMSCs to chondroeytes in culture with chondrogenic medium.Confocal microscopy of cell-scaffold constructs revealed cells with green fluorescence.Conclusion Since the novel CECM/ACBM bilayered integrated osteochondral scaffold has good mircostructure, non-toxicity and good biocompatibility, it may be a suitable candidate as an alternative cell-carrier for osteochondral tissue engineering. Key words: Tissue engineering; Extracellular matrix; Bone marrow cells; Scaffold

Adipose-derived stem cells modified genetically in vivo promote reconstruction of bone defects

Bone defects induced by different causes are difficult to replace and repair. We sought to repair bone defects by transplantation of genetically modified adipose-derived stem cells (ADSC) and acellular bone matrix (ACBM). We constructed the biologic material of ACBM and evaluated its mechanical properties, general biocompatibility and biosafety. ADSC isolated from minipigs were cultured in vitro and then transfected by recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) plasmids, respectively. Subsequently, the compounds of ACBM/ADSC/rhBMP-2/rhVEGF were used to repair bone defects of the ulna in minipigs. X-ray examination, radionuclide bone imaging and single photon emission computerized tomography (SPECT) were employed to monitor the therapeutic effects 2, 4, 8 and 12 weeks after operation. Histologic experiments were carried out 12 weeks after operation. ACBM had no or weak antigenicity and the natural mechanical properties of ACBM were preserved. In vitro, ADSC transfected by rhBMP-2 and rhVEGF, respectively, could release rhBMP-2 or rhVEGF for at least 4 weeks. The X-ray, radionuclide bone imaging and SPECT examinations indicated that the compound of ACBM/ADSC/rhBMP-2/rhVEGF had better treatment effects on bone defects compared with the controls. Scaffolds, seed cells and bioactive factors are key points in tissue engineering. This research indicates that ACBM is a good biologic material for tissue repair, and ACBM/ADSC/rhBMP-2/rhVEGF can accelerate bone formation significantly.

Repair of large articular high-load-bearing osteochondral defects with extracellular matrix-derived acellular matrix biphasic scaffold and bone marrow-derived mesenchymal stem cells in a canine model.

Objective To develop an extracellular matrix (ECM)-derived, integrated biphasie scaffold, and to investigate the regeneration potential of the scaffold loaded with chondrogenically induced bonemarrow mesenchymal stem cells (BMSCs) in the repair of large, high-load-bearing osteochoudral defects in a canine model. Methods The biphasie scaffolds were fabricated using cartilage ECM-derived Scaffold (Cartilage layer) and acellular bone matrix (Bone layer) by phase separation technique. Articular high-load-bearing osteochondral defects (4.2 mm diameter×6 mm deep) were created in femoral condyles. The defects were treated with constructs of a biphasic scaffold with chondrogenically induced BMSCs (experimental group) or cell-free scaffolds (control group). The repaired defects were evaluated for gross morphology, histological, and micro-CT analysis at 3 and 6 months post-implantation. Results Statistical analysis demonstrated that the gross and histological grading scores of the experimental group were always higher than those of the control group at both time points and that the scores for the 6-month experimental group was significantly higher than 3--month experimental group, while there was no significantly difference in control group between two time points. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at both 3 and 6 months, with no significant difference between the experimental and control groups. Conclusion The ECM-derived, integrated biphasic scaffold combined with chondrogenically induced BMSCs has high potential in repair of large, high-load-bearing osteochondral defects.

Evaluation of the cytocompatibility of mixed bovine bone

Treatment of bovine bone with peroxides and chaotropic agents aims to obtain an acellular bone matrix that is able to maintain the collagen-apatite complex and a higher mechanical resistance, a mixed biomaterial hereby named mixed bovine bone (MBB). The purpose of this study was to evaluate the cytocompatibility of MBB and cell-MBB interaction. Cell morphology, number of viable cells, ability to reduce methyltetrazolium and to incorporate neutral red upon exposure to different concentrations of the hydrosoluble extract of MBB were assessed in Balb-c 3T3 cells according to ISO 10993-5 standard. The interaction between cells and MBB surface was evaluated by scanning electron microscopy. The water-soluble MBB extracts were cytotoxic and led to cell death possibly due to its effect on mitochondrial function and membrane permeability. Cells plated directly onto the MBB did not survive, although after dialysis and material conditioning in DMEM + 10% FCS, the cells adhered and proliferated onto the material. It may be concluded that, in vitro, water-soluble MBB extracts were cytotoxic. Nevertheless, MBB cytotoxic effect was reverted by dialysis resulting in a material that is suitable for cell based-therapy in the bioengineering field.