Large Segmental Bone Defects Research Articles

Engraftment of Prevascularized, Tissue Engineered Constructs in a Novel Rabbit Segmental Bone Defect Model.

The gold standard treatment of large segmental bone defects is autologous bone transfer, which suffers from low availability and additional morbidity. Tissue engineered bone able to engraft orthotopically and a suitable animal model for pre-clinical testing are direly needed. This study aimed to evaluate engraftment of tissue-engineered bone with different prevascularization strategies in a novel segmental defect model in the rabbit humerus. Decellularized bone matrix (Tutobone) seeded with bone marrow mesenchymal stromal cells was used directly orthotopically or combined with a vessel and inserted immediately (1-step) or only after six weeks of subcutaneous “incubation” (2-step). After 12 weeks, histological and radiological assessment was performed. Variable callus formation was observed. No bone formation or remodeling of the graft through TRAP positive osteoclasts could be detected. Instead, a variable amount of necrotic tissue formed. Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found. In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed.

Biomechanical stability of novel mechanically adapted open-porous titanium scaffolds in metatarsal bone defects of sheep

Open-porous titanium scaffolds for large segmental bone defects offer advantages like early weight-bearing and limited risk of implant failure. The objective of this experimental study was to determine the biomechanical behavior of novel open-porous titanium scaffolds with mechanical-adapted properties in vivo.Two types of the custom-made, open-porous scaffolds made of Ti6Al4V (Young's modulus: 6–8 GPa and different pore sizes) were implanted into a 20 mm segmental defect in the mid-diaphysis of the metatarsus of sheep, and were stabilized with an osteosynthesis plate. After 12 and 24 weeks postoperatively, torsional testing was performed on the implanted bone and compared to the contralateral non-treated side. Maximum torque, maximum angle, torsional stiffness, fracture energy, shear modulus and shear stress were investigated. Furthermore, bone mineral density (BMD) of the newly formed bone was determined.Mechanical loading capabilities for both scaffolds were similar and about 50% after 12 weeks (e.g., max. torque of approximately 20 Nm). A further increase after 24 weeks was found for most of the investigated parameters. Results for torsional stiffness and shear modulus as well as bone formation depended on the type of scaffold. Increased BMD after 24 weeks was found for one scaffold type but remained constant for the other one.The present data showed the capability of mechanically adapted open-porous titanium scaffolds to function as bone scaffolds for large segmental defects and the influence of the scaffold's stiffness. A further increase in the biomechanical stability can be assumed for longer observation periods of greater than six months.

Repair of large segmental bone defects in rabbits using BMP and FGF composite xenogeneic bone.

The objective of this study was to determine the ability of bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) to repair large segmental radial bone defects in rabbits. We treated calf cancellous bones with 3 mg/L BMP (Group A), 5 μg/L FGF (Group B), or 3 mg/L BMP plus 5 μg/L FGF (Group C). A bone damage model was established using healthy radii from rabbits. The complexes were implanted in the areas of the bone defects in the radii. After successful transplantation, the rabbits underwent radiographic imaging, and bone graft specimens were detected by histopathology methods. Biomechanical indexes were also assessed in order to observe the healing status of the bone defects. Our results indicated that the repair of bone defects was significantly better in Group C compared to the other 2 groups. Therefore, we concluded that combining BMP and FGF significantly promoted bone defect repair and achieved effects that were superior to the use of BMP alone.

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.

Vascularized osseous flaps and assessing their bipartate perfusion pattern via intraoperative fluorescence angiography.

Large segmental bone and composite tissue defects often require vascularized osseous flaps for definitive reconstruction. However, failed osseous flaps due to inadequate perfusion can lead to significant morbidity. Utilization of indocyanine green (ICG) fluorescence angiography has been previously shown to reliably assess soft tissue perfusion. Our group will outline the application of this useful intraoperative tool in evaluating the perfusion of vascularized osseous flaps. A retrospective review was performed to identify those osseous and/or osteocutaneous bone flaps, where ICG angiography was employed. Data analyzed included flap types, success and failure rates, and perfusion-related complications. All osseous flaps were evaluated by ICG angiography to confirm periosteal and endosteal perfusion. Overall 16 osseous free flaps utilizing intraoperative ICG angiography to assess vascularized osseous constructs were performed over a 3-year period. The flaps consisted of the following: nine osteocutaneous fibulas, two osseous-only fibulas, two scapular/parascapular with scapula bone, two quadricep-based muscle flaps, containing a vascularized femoral bone component, and one osteocutaneous fibula revision. All flap reconstructions were successful with the only perfusion-related complication being a case of delayed partial skin flap loss. Intraoperative fluorescence angiography is a useful adjunctive tool that can aid in flap design through angiosome mapping and can also assess flap perfusion, vascular pedicle flow, tissue perfusion before flap harvest, and flap perfusion after flap inset. Our group has successfully extended the application of this intraoperative tool to assess vascularized osseous flaps in an effort to reduce adverse outcomes related to preventable perfusion-related complications.

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair

Objectives The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs. Methods The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining. Key findings An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs. Conclusions According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.

Large defect-tailored composite scaffolds for in vivo bone regeneration.

The discovery of new strategies to repair large segmental bone defects is currently an open challenge for worldwide clinicians. In the treatment of critical-sized bone defects, an alternative strategy to traditional bone grafting is always more frequently the use of tailor-made scaffolds modelled on the final size and shape of the implant site. Here, poly-ε-caprolactone-based composite scaffolds including poly-L-lactic acid continuous fibres and hyaluronan derivates (i.e. HYAFF11®) have been investigated for the peculiar 3D architecture characterized by interconnected macroporous networks and tunable mechanical properties. Composite scaffolds were immersed in simulated body fluid solution in order to support in vivo tissue in-growth. Scaffolds loaded with autologous cells (bone marrow stromal cells) plus platelet-rich plasma and osteoconductive protein such bone morphogenetic protein-7 were also tested to evaluate eventual enhancement in bone regeneration. The morphological and mechanical properties of poly-L-lactic acid-reinforced composite scaffolds have been studied to identify the optimal scaffold design to match the implant-site requirements of sheep metatarsal defects. Dynamic mechanical tests allowed to underline the viscoelastic response of the scaffold - resulting in elastic moduli from 2.5 to 1.3 MPa, suitable to temporarily support the structural function of damaged bone tissue. In vivo preliminary investigations in a sheep model of metatarsus shaft defect also showed the attitude of the scaffold to promote osteogenesis, preferentially in association with bone marrow stromal cell and platelet-rich plasma, even if the highest amount of mature bone was reached in the case of scaffold loaded with human bone morphogenetic protein-7 released via hydrolytic degradation of HYAFF11® phases in the implant site.

Tissue flap transplantation plus bone transportation for repair of massive multi-tissue defects at the leg

Objective To report the clinical effects of treating massive multi-tissue defects at the leg with tissue flap transportation and bone transportation.Methods The clinical data was analyzed retrospectively of 92 patients who had been treated from January 2005 to December 2011 for massive soft tissue and large segmental bone defects at the leg.They were 71 males and 21 females,with a mean age of 38.3 years (range,19 to 56 years).They were treated by herbal dressing on wounds before operation.At the subacute stage,massive skin and large segmental bone defects were treated with skin flap transplantation plus bone transportation and damaged muscular function and large segmental bone defects were managed with musculocutaneous flap transplantation plus bone transportation.After operation,traditional Chinese medication was administered for anticoagulation in all cases.Results All operations were completed successfully without infection.Vascular anastomoses were conducted in free transplantation of 106 pieces of tissue.All transplanted tissues survived except for tip necrosis of one skin flap.On average,each patient received 2.6 operations and had an average duration of 256 days for treatment.The follow-up periods for all the patients ranged from 10 to 48 months (mean,24.6 months).According to Paley criteria,53 cases were rated as excellent,26 as good,9 as fair,and 4 as poor,giving a good to excellent rate of 85.9％.Conclusion As tissue flap transplantation plus bone transportation can restore the function of the leg as much as possible,it is an effective treatment of massive multi-tissue defects at the leg. Key words: Tissue transplantation; Soft tissue defect; Reconstructive surgical procedures; Bone transportation

Citrate-based biphasic scaffolds for the repair of large segmental bone defects.

Attempts to replicate native tissue architecture have led to the design of biomimetic scaffolds focused on improving functionality. In this study, biomimetic citrate-based poly (octanediol citrate)-click-hydroxyapatite (POC-Click-HA) scaffolds were developed to simultaneously replicate the compositional and architectural properties of native bone tissue while providing immediate structural support for large segmental defects following implantation. Biphasic scaffolds were fabricated with 70% internal phase porosity and various external phase porosities (between 5 and 50%) to mimic the bimodal distribution of cancellous and cortical bone, respectively. Biphasic POC-Click-HA scaffolds displayed compressive strengths up to 37.45 ± 3.83 MPa, which could be controlled through the external phase porosity. The biphasic scaffolds were also evaluated in vivo for the repair of 10-mm long segmental radial defects in rabbits and compared to scaffolds of uniform porosity as well as autologous bone grafts after 5, 10, and 15 weeks of implantation. The results showed that all POC-Click-HA scaffolds exhibited good biocompatibility and extensive osteointegration with host bone tissue. Biphasic scaffolds significantly enhanced new bone formation with higher bone densities in the initial stages after implantation. Biomechanical and histomorphometric analysis supported a similar outcome with biphasic scaffolds providing increased compression strength, interfacial bone ingrowth, and periosteal remodeling in early time points, but were comparable to all experimental groups after 15 weeks. These results confirm the ability of biphasic scaffold architectures to restore bone tissue and physiological functions in the early stages of recovery, and the potential of citrate-based biomaterials in orthopedic applications.

Establishment of a bilateral femoral large segmental bone defect mouse model potentially applicable to basic research in bone tissue engineering

BackgroundTo understand the cellular mechanism underlying bone defect healing in the context of tissue engineering, a reliable, reproducible, and standardized load-bearing large segmental bone defect model in small animals is indispensable. The aim of this study was to establish and evaluate a bilateral femoral defect model in mice. Materials and methodsDonor mouse bone marrow mesenchymal stem cells (mBMSCs) were obtained from six mice (FVB/N) and incorporated into partially demineralized bone matrix scaffolds to construct tissue-engineered bones. In total, 36 GFP+ mice were used for modeling. Titanium fixation plates with locking steel wires were attached to the femurs for stabilization, and 2-mm–long segmental bone defects were created in the bilateral femoral midshafts. The defects in the left and right femurs were transplanted with tissue-engineered bones and control scaffolds, respectively. The healing process was monitored by x-ray radiography, microcomputed tomography, and histology. The capacity of the transplanted mBMSCs to recruit host CD31+ cells was investigated by immunofluorescence and real-time polymerase chain reaction. ResultsPostoperatively, no complication was observed, except that two mice died of unknown causes. Stable fixation of femurs and implants with full load bearing was achieved in all animals. The process of bone defect repair was significantly accelerated due to the introduction of mBMSCs. Moreover, the transplanted mBMSCs attracted more host CD31+ endothelial progenitors into the grafts. ConclusionsThe present study established a feasible, reproducible, and clinically relevant bilateral femoral large segmental bone defect mouse model. This model is potentially suitable for basic research in the field of bone tissue engineering.

Numerical optimization of open-porous bone scaffold structures to match the elastic properties of human cortical bone

Treatment of large segmental bone defects, especially in load bearing areas, is a complex procedure in orthopedic surgery. The usage of additive manufacturing processes enables the creation of customized bone implants with arbitrary open-porous structure satisfying both the mechanical and the biological requirements for a sufficient bone ingrowth. Aim of the present numerical study was to optimize the geometrical parameters of open-porous titanium scaffolds to match the elastic properties of human cortical bone with respect to an adequate pore size. Three different scaffold designs (cubic, diagonal and pyramidal) were numerically investigated by using an optimization approach. Beam elements were used to create the lattice structures of the scaffolds. The design parameters strut diameter and pore size ranged from 0.2 to 1.5mm and from 0 to 3.0mm, respectively. In a first optimization step, the geometrical parameters were varied under uniaxial compression to obtain a structural modulus of 15GPa (Young׳s modulus of cortical bone) and a pore size of 800µm was aimed to enable cell ingrowth. Furthermore, the mechanical behavior of the optimized structures under bending and torsion was investigated. Results for bending modulus were between 9.0 and 14.5GPa. In contrast, shear modulus was lowest for cubic and pyramidal design of approximately 1GPa. Here, the diagonal design revealed a modulus of nearly 20GPa. In a second step, large-sized bone scaffolds were created and placed in a biomechanical loading situation within a 30mm segmental femoral defect, stabilized with an osteosynthesis plate and loaded with physiological muscle forces. Strut diameter for the 17 sections of each scaffold was optimized independently in order to match the biomechanical stability of intact bone. For each design, highest strut diameter was found at the dorsal/medial site of the defect and smallest strut diameter in the center. In conclusion, we demonstrated the possibility of providing optimized open-porous scaffolds for bone regeneration by considering both mechanical and biological aspects. Furthermore, the results revealed the need of the investigation and comparison of different load scenarios (compression, bending and torsion) as well as complex biomechanical loading for a profound characterization of different scaffold designs. The usage of a numerical optimization process was proven to be a feasible tool to reduce the amount of the required titanium material without influencing the biomechanical performance of the scaffold negatively. By using fully parameterized models, the optimization approach is adaptable to other scaffold designs and bone defect situations.

Bone Regeneration by the Combined Use of Tetrapod-Shaped Calcium Phosphate Granules with Basic Fibroblast Growth Factor-Binding Ion Complex Gel in Canine Segmental Radial Defects

ABSTRACTThe effect of tetrapod-shaped alpha tricalcium phosphate granules (Tetrabones® [TB]) in combination with basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) on bone defect repair was examined. Bilateral segmental defects 20-mm long were created in the radius of 5 dogs, stabilized with a plate and screws and implanted with 1 of the following: TB (TB group), TB and bFGF solution (TB/f group), and TB and f-IC gel (TB/f-IC group). Dogs were euthanized 4 weeks after surgery. Radiographs showed well-placed TB granules in the defects and equal osseous callus formation in all the groups. Histomorphometry revealed that the number of vessels and volume of new bone in the TB/f-IC group were significantly higher than those in the other groups. However, no significant differences in neovascularization and new bone formation were observed between the TB/f and TB groups. Furthermore, no significant difference in the lamellar bone volume or rate of mineral apposition was observed among groups. These results suggest that increased bone formation might have been because of the promotion of neovascularization by the f-IC gel. Therefore, the combinatorial method may provide a suitable scaffold for bone regeneration in large segmental long bone defects.

The induced membrane technique for treatment of large segmental or cavitary bone defects: How to maximize the success

Large bone defects are a challenging situation for surgeons, either in acute management, either in case of a nonunion or a septic nonunion. Different treatment proposals have been made since a long time using autologous cancellous bone graft, open-air cancellous Papineau grafting, free vascularized bone transfer, bone transport or allograft (1,2,3). Since 2000, the induced membrane technique, so-called the Masquelet technique, has been described for treatment of such very difficult cases that we can express as treatment of critical sized bone defects (4,5). In the following, we’ll try to give as much details as possible for a positive result when using the induced membrane technique. Clinical based algorithm will be emphasized.

Caractéristiques histologiques de la membrane induite selon son site d’implantation : sous-cutané, intra-musculaire ou au niveau de la perte de substance osseuse

Background The induced membrane technique was proposed as a treatment of large segmental bone defects. The influence of the surrounding tissues on its characteristics remains unknown. It is therefore not known which kind of plastic surgery procedure (muscular or facio-cutaneous flap) would optimize bone osteointegration within a bone defect reconstructed using the induced-membrane technique. Hypothesis We hypothesized that membrane characteristics could be influenced by the soft-tissue environment either subcutaneous or muscular. Objective To evaluate the histological characteristics of poly-methylmethacrylate (PMMA)-induced membranes in intramuscular, subcutaneous and bony environment (radius defects) at 2 steps: 1) spacer implantation; 2) secondary bone graft and its subsequent osteintegration after spacer removal. Methods PMMA-induced membranes were obtained in the three sites of 15 rabbits. Subsequent new bone formation was studied in the same environments in 24 other rabbits. Six weeks after the initial implantation, PMMA spacers were replaced with iliac autografs. Animals were euthanized at 2, 4, and 8 weeks postoperatively. Tissue samples were harvested and stained with hematoxylin and eosin. The histological characteristics of the membrane (thickness and microvessel density) and the newly-formed bone (cortical thickness) were quantitatively analyzed. Results The membranes in the subcutaneous sites developed quicker, were thicker and had the lowest microvessel density (P < 0.01). The membranes in the intramuscular sites developed later and were thinner (P < 0.01). The membranes in the osseous defects had the greatest microvessel density (P < 0.01). After bone grafting, induced membranes became thinner and their microvessel density decreased substantially, but maintained better in osseous site. The newly-formed bone that developed in the radius defects, had the thickest cortices (P < 0.01). Conclusions The evolution of membranes induced in the intramuscular and subcutaneous environments was close to that of the bone defect model, although bone formation appeared weaker. Level of evidence Basic science study III.

Histological characteristics of induced membranes in subcutaneous, intramuscular sites and bone defect

The induced membrane technique was proposed as a treatment of large segmental bone defects. The influence of the surrounding tissues on its characteristics remains unknown. It is therefore not known which kind of plastic surgery procedure (muscular or facio-cutaneous flap) would optimize bone osteointegration within a bone defect reconstructed using the induced-membrane technique. We hypothesized that membrane characteristics could be influenced by the soft-tissue environment either subcutaneous or muscular. To evaluate the histological characteristics of poly-methylmethacrylate (PMMA) induced membranes in intramuscular, subcutaneous and bony environment (radius defects) at 2 steps: spacer implantation; secondary bone graft and its subsequent osteintegration after spacer removal. PMMA-induced membranes were obtained in the three sites of 15 rabbits. Subsequent new bone formation was studied in the same environments in 24 other rabbits. Six weeks after the initial implantation, PMMA spacers were replaced with iliac autografts. Animals were euthanized at 2, 4, and 8 weeks postoperatively. Tissue samples were harvested and stained with hematoxylin and eosin. The histological characteristics of the membrane (thickness and microvessel density) and the newly-formed bone (cortical thickness) were quantitatively analyzed. The membranes in the subcutaneous sites developed quicker, were thicker and had the lowest microvessel density (P<0.01). The membranes in the intramuscular sites developed later and were thinner (P<0.01). The membranes in the osseous defects had the greatest microvessel density (P<0.01). After bone grafting, induced membranes became thinner and their microvessel density decreased substantially, but maintained better in osseous site. The newly-formed bone that developed in the radius defects, had the thickest cortices (P<0.01). The evolution of membranes induced in the intramuscular and subcutaneous environments was close to that of the bone defect model, although bone formation appeared weaker.

Differentiation of Rabbit Bone Mesenchymal Stem Cells into Endothelial Cells In Vitro and Promotion of Defective Bone Regeneration In Vivo

Tissue engineering strategies often fail to regenerate bones because of inadequate vascularization, especially in the reconstruction of large segmental bone defects. Large volumes of vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesis are difficult to obtain. In this study, we simulated bone healing by co-culturing differentiated ECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolic acid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairing rabbit mandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells. After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs. Immunohistochemical staining analysis with CD34 showed that the induced cells had the characteristics of ECs and MSC. The induced ECs were co-cultured in vitro, and the induction of MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR) staining experiments were performed, and the Coomassie brilliant blue total protein and ALP activity were measured. The MSCs proliferated and differentiated into osteoblast-like cells through direct contact between the derived ECs and MSCs. The co-cultured cells were seeded on PLGA scaffold to repair 1 cm mandible defects in the rabbit. The effectiveness of the repairs was assessed through soft X-ray and histological analyses. The main findings indicated that MSCs survived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. The results demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis and promoted new bone formation. This study may serve as a basis for the use of in vitro co-culturing techniques as an improvisation to bone tissue engineering for the repair of large bone defects.

Repair of large segmental bone defects: BMP-2 gene activated muscle grafts vs. autologous bone grafting

BackgroundCommon cell based strategies for the treatment of osseous defects require the isolation and expansion of autologous cells. Since this makes such approaches time-consuming and expensive, we developed a novel expedited technology creating gene activated muscle grafts. We have previously shown that large segmental bone defects in rats can be regenerated by implantation of muscle tissue fragments activated by BMP-2 gene transfer.ResultsIn the present study, we compared the bone healing capacities of such gene activated muscle grafts with bone isografts, mimicking autologous bone grafting, the clinical gold standard for treatment of bone defects in patients. Two of 14 male, syngeneic Fischer 344 rats used for this experiment served as donors for muscle and bone. Muscle tissue was harvested from both hind limbs and incubated with an adenoviral vector carrying the cDNA encoding BMP-2. Bone was harvested from the iliac crest and long bone epiphyses. Bone defects (5 mm) were created in the right femora of 12 rats and were filled with either BMP-2 activated muscle tissue or bone grafts. After eight weeks, femora were evaluated by radiographs, micro-computed tomography (μCT), and biomechanical testing. In the group receiving BMP-2 activated muscle grafts as well as in the bone-grafting group, 100% of the bone defects were healed, as documented by radiographs and μCT-imaging. Bone volume was similar in both groups and biomechanical stability of the two groups was statistically indistinguishable.ConclusionsThis study demonstrates that treatment of large bone defects by implantation of BMP-2 gene activated muscle tissue leads to similar bone volume and stability as bone isografts, mimicking autologous bone grafting.

Poor osteoinductive potential of subcutaneous bone cement-induced membranes for tissue engineered bone

Background: Large segmental bone defects remain a challenge for reconstructive surgeons. A two-stage repair strategy may offer a potential solution. Here, we sought to evaluate the osteoinductive potential of bone cement-induced membranes in an ectopic site. Methods: First, bone cements were inserted into the subcutaneous tissues of 16 rabbits to induce membrane formation. After 2, 4, 6 and 8 weeks, the induced membranes were harvested to assess their vascularization and osteoinductive potential. Next, bone cements were subcutaneously inserted into 12 rabbits for 4 weeks. These bone cements were then harvested from the newly formed membranes and replaced with granular porous β-TCP, with or without bone mesenchymal stem cells. New bone formation was then evaluated after 3, 6 and 9 weeks. Results: The highest level of blood vessel formation and bone morphogenetic protein-2 expression in the membranes were found at 4 weeks (p < 0.05). In addition, vascular endothelial growth factor concentration was highest after 2 weeks (p < 0.001), persisting until 8 weeks. However, the results showed little ectopic bone formation at these time points. Conclusion: While bone cement-induced membranes appear to provide a suitable environment for bone formation, they fail to drive osteoinduction in non-osseous sites for the purposes of bone tissue engineering.