Critical-sized Segmental Bone Defects Research Articles

The Treatment Efficacy of Bone Tissue Engineering Strategy for Repairing Segmental Bone Defects Under Osteoporotic Conditions.

The potential of increasing bone mass and preventing fractures in osteoporosis using stem cell therapy is currently an area of intense focus. However, there are very little data available regarding the postfracture bony defect healing efficacy under osteoporotic conditions. This study aims to investigate whether critical-sized segmental bone defects in a rabbit model of osteoporosis could be repaired using an allogenic stem cell-based tissue engineering (TE) approach and to investigate the potential influence of osteoporosis on the treatment efficacy. Rabbit fetal bone marrow mesenchymal stem cells (BMSCs) were harvested and expanded in vitro. Decalcified bone matrix (DBM) scaffolds were then seeded with allogenic fetal BMSCs and cultivated in osteogenic media to engineer BMSC/DBM constructs. Critical-sized radial defects were created in ovariectomized (OVX) rabbits and the defects were repaired either by insertion of BMSC/DBM constructs or by DBM scaffolds alone. Also, nonovariectomized age-matched (non-OVX) rabbits were served as control. At 3 months post-treatment under the osteoporotic condition (OVX rabbits), the BMSC/DBM constructs inserted within the defect generated significantly more bone tissue when compared to the DBM scaffold as demonstrated by the X-ray, microcomputed tomography, and histological analyses. In addition, when compared to a normal nonosteoporotic condition (age-matched non-OVX rabbits), the defect treatment efficacy was adversely affected by the osteoporotic condition with significantly less bone regeneration. This study demonstrated the potential of allogenic fetal BMSC-based TE strategy for repairing bone defects in an osteoporotic condition. However, the treatment efficacy could be considerably compromised in the OVX animals. Therefore, a more sophisticated strategy that addresses the complicated pathogenic conditions associated with osteoporosis is needed.

Structure design and manufacturing of layered bioceramic scaffolds for load-bearing bone reconstruction

Bioceramic scaffolds with desired bone regeneration functions have the potential to become real alternatives to autologous bone grafts for reconstruction of load-bearing and critical-sized segmental bone defects. The aim of this paper was to develop a layered scaffold structure that has the biodegradable function of common monolithic scaffolds and adequate mechanical function for surgical fixing and after surgery support. The exemplary case of this study is assumed to be a large-segment tibia or femur bone repair. The layered scaffold structure consists of a macro porous hydroxyapatite–wollastonite layer and a strong dense zirconia matrix dense layer. The bio-functional scaffold layer with interconnected freeze-dried porous structures shows excellent apatite formation, cell attachment, and cell proliferation capabilities. The mechanical functional layer provides a bending strength matching that of the compact bone.

Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels.

Regeneration of load-bearing segmental bone defects is a major challenge in trauma and orthopaedic surgery. The ideal bone graft substitute is a biomaterial that provides immediate mechanical stability, while stimulating bone regeneration to completely bridge defects over a short period. Therefore, selective laser melted porous titanium, designed and fine-tuned to tolerate full load-bearing, was filled with a physiologically concentrated fibrin gel loaded with bone morphogenetic protein-2 (BMP-2). This biomaterial was used to graft critical-sized segmental femoral bone defects in rats. As a control, porous titanium implants were either left empty or filled with a fibrin gels without BMP-2. We evaluated bone regeneration, bone quality and mechanical strength of grafted femora using in vivo and ex vivo µCT scanning, histology, and torsion testing. This biomaterial completely regenerated and bridged the critical-sized bone defects within eight weeks. After twelve weeks, femora were anatomically re-shaped and revealed open medullary cavities. More importantly, new bone was formed throughout the entire porous titanium implants and grafted femora regained more than their innate mechanical stability: torsional strength exceeded twice their original strength. In conclusion, combining porous titanium implants with a physiologically concentrated fibrin gels loaded with BMP-2 improved bone regeneration in load-bearing segmental defects. This material combination now awaits its evaluation in larger animal models to show its suitability for grafting load-bearing defects in trauma and orthopaedic surgery.

Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors

Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60–70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.

Novel osteoinductive photo-cross-linkable chitosan-lactide-fibrinogen hydrogels enhance bone regeneration in critical size segmental bone defects

The purpose of this study was to develop and characterize a novel photo-cross-linkable chitosan-lactide-fibrinogen (CLF) hydrogel and evaluate the efficacy of bone morphogenetic protein-2 (BMP-2) containing a CLF hydrogel for osteogenesis in vitro and in vivo. We synthesized the CLF hydrogels and characterized their chemical structure, degradation rate, compressive modulus and in vitro BMP-2 release kinetics. We evaluated bioactivities of the BMP-2 containing CLF hydrogels (0, 50, 100 and 500ngml−1) in vitro using W-20-17 preosteoblast mouse bone marrow stromal cells and C2C12 mouse myoblast cells. The effect of BMP-2 containing CLF gels (0, 0.5, 1, 2 and 5μg) on bone formation was evaluated using rat critical size segmental bone defects for 4weeks. Fourier transform infrared spectroscopy spectra and scanning electron microscopy images showed chemical and structural changes by the addition of fibrinogen into the chitosan-lactide copolymer. The incorporation of fibrinogen molecules significantly increased the compressive modulus of the hydrogels. The in vitro BMP-2 release study showed initial burst releases from the CLF hydrogels followed by sustained releases, regardless of the concentration of the BMP-2 over 4weeks. Cells in all groups were viable in the presence of the hydrogels regardless of BMP-2 doses, indicating non-cytotoxicity of hydrogels. Alkaline phosphate activity and mineralization of cells exhibited dose dependence on BMP-2 containing CLF hydrogels. Radiography, microcomputed tomography and histology confirmed that the BMP-2 containing CLF hydrogels prompted neo-osteogenesis and accelerated healing of the defects in a dose-dependent manner. Thus the CLF hydrogel is a promising delivery system of growth factors for bone regeneration.

Enhanced repair of a critical-sized segmental bone defect in rabbit femur by surface microstructured porous titanium

Repair of load-bearing bone defects remains a challenge in the field of orthopaedic surgery. In the current study, a surface microstructured porous titanium (STPT) successively treated with H2O2/TaCl5 solution and simulated body fluid was used to repair the critical-sized segmental bone defects in rabbit femur, and non-treated porous titanium (NTPT) and porous biphasic calcium phosphate ceramics (PBCP) were used as control, respectively. A 15mm long implant was positioned in the femoral defect and stabilized by a plate and screws fixation. After implantation into the body for 1, 3 and 6months, X-ray observation confirmed that porous titanium groups (NTPT and STPT) provided better mechanical support than PBCP group at the early stage. However, there was no obvious difference in the formed bony callus between PBCP and STPT groups in the later stage, and they both showed better shape of bony callus than NTPT group. Micro-CT and histomorphometric analysis for the samples of 6-month implantation demonstrated that more new bone formed in the inner pores of PBCP and STPT groups than that in NTPT group. Moreover, the biomechanical tests revealed that STPT group could bear larger compressive load than NTPT and PBCP groups, almost reaching the level of the normal rabbit femur. STPT exhibited the enhanced repairing effect on the critical-sized segmental bone defect in rabbit femur, meaning that it could be an ideal material for the repair of large bone defect in load-bearing site.

Repair of segmental load-bearing bone defect by autologous mesenchymal stem cells and plasma-derived fibrin impregnated ceramic block results in early recovery of limb function.

Calcium phosphate-based bone substitutes have not been used to repair load-bearing bone defects due to their weak mechanical property. In this study, we reevaluated the functional outcomes of combining ceramic block with osteogenic-induced mesenchymal stem cells and platelet-rich plasma (TEB) to repair critical-sized segmental tibial defect. Comparisons were made with fresh marrow-impregnated ceramic block (MIC) and partially demineralized allogeneic bone block (ALLO). Six New Zealand White female rabbits were used in each study group and three rabbits with no implants were used as negative controls. By Day 90, 4/6 rabbits in TEB group and 2/6 in ALLO and MIC groups resumed normal gait pattern. Union was achieved significantly faster in TEB group with a radiological score of 4.50 ± 0.78 versus ALLO (1.06 ± 0.32), MIC (1.28 ± 0.24), and negative controls (0). Histologically, TEB group scored the highest percentage of new bone (82% ± 5.1%) compared to ALLO (5% ± 2.5%) and MIC (26% ± 5.2%). Biomechanically, TEB-treated tibiae achieved the highest compressive strength (43.50 ± 12.72 MPa) compared to those treated with ALLO (15.15 ± 3.57 MPa) and MIC (23.28 ± 6.14 MPa). In conclusion, TEB can repair critical-sized segmental load-bearing bone defects and restore limb function.

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.

Testosterone Delivered with a Scaffold Is as Effective as Bone Morphologic Protein-2 in Promoting the Repair of Critical-Size Segmental Defect of Femoral Bone in Mice

Loss of large bone segments due to fracture resulting from trauma or tumor removal is a common clinical problem. The goal of this study was to evaluate the use of scaffolds containing testosterone, bone morphogenetic protein-2 (BMP-2), or a combination of both for treatment of critical-size segmental bone defects in mice. A 2.5-mm wide osteotomy was created on the left femur of wildtype and androgen receptor knockout (ARKO) mice. Testosterone, BMP-2, or both were delivered locally using a scaffold that bridged the fracture. Results of X-ray imaging showed that in both wildtype and ARKO mice, BMP-2 treatment induced callus formation within 14 days after initiation of the treatment. Testosterone treatment also induced callus formation within 14 days in wildtype but not in ARKO mice. Micro-computed tomography and histological examinations revealed that testosterone treatment caused similar degrees of callus formation as BMP-2 treatment in wildtype mice, but had no such effect in ARKO mice, suggesting that the androgen receptor is required for testosterone to initiate fracture healing. These results demonstrate that testosterone is as effective as BMP-2 in promoting the healing of critical-size segmental defects and that combination therapy with testosterone and BMP-2 is superior to single therapy. Results of this study may provide a foundation to develop a cost effective and efficient therapeutic modality for treatment of bone fractures with segmental defects.

P035 Adipocyte derived stem cells favour healing in a new minipig model of cutaneous radiation syndrome through reepithelialisation and neoangiogenesis

Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60–70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.

The role of adipose-derived stem cells engineered with the persistently expressing hybrid baculovirus in the healing of massive bone defects

Massive segmental defects arising from trauma or tumor resection remain a challenging clinical problem. To repair large, segmental bone defects using adipose-derived stem cells (ASCs) which alone cannot heal massive defects, we hypothesized that sustained expression of factors promoting osteogenesis (BMP2) and angiogenesis (VEGF) provides continuous stimuli to augment the healing. Baculovirus is a vector for gene delivery into stem cells, but it only mediates transient expression. Therefore we developed a dual system whereby one baculovirus expressed FLP recombinase (BacFLP) while the other hybrid baculovirus harbored an Frt-flanking transgene cassette. Within the ASCs transduced with BacFLP and the hybrid baculovirus, the transduction efficiency reached 98% and the FLP/Frt-mediated recombination efficiency approached 46%, leading to cassette excision off the baculovirus genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days. ASCs engineered by the conventional baculovirus transiently expressing BMP2/VEGF (S group) only healed the critical-size (10mm) segmental femoral bone defects in 40% of New Zealand White rabbits at 12 weeks post-implantation, whereas ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-size defects in 12 out of 12 animals in 8 weeks. Compared with the S group, the L group not only accelerated the healing, but also ameliorated the bone quality (metabolism, volume, density, mechanical properties) and angiogenesis, thereby attesting our hypothesis that persistent BMP2/VEGF expression is essential. Use of ASCs engineered by the hybrid BV vector thus holds promise to treat massive segmental defects necessitating sustained stimuli.

Bone Regeneration with BMP-2 Gene-modified Mesenchymal Stem Cells Seeded on Nano-hydroxyapatite/Collagen/ Poly(L-Lactic Acid) Scaffolds

In this study, the capacity of bone morphogenetic protein 2 (BMP-2) gene-transfected bone marrow-derived mesenchymal stem cells (MSCs) in combination with nano-hydroxyapatite/collagen/poly(L-lactic acid) (nHAC/ PLA) to improve the repair of bone defects in rabbit was explored. MSCs from New Zealand White rabbits were cultured and injected with pIRES2-EGFPhBMP-2 or pIRES2-EGFP by electroporation. After the transfer efficiency was determined through the expression of EGFP, the MSCs were seeded on scaffolds to generate an in vitro 3D cell/scaffold construct. The adhesion and proliferation of the MSCs cultured in the scaffold was assessed by SEM. The cellular constructs obtained were allografted into the 15 mm critical-sized segmental bone defects in the radius of New Zealand White rabbits for 12 weeks. The bone regeneration was assessed by radiographical and histological analyses. In vitro, nHAC/PLA facilitated MSC adhesion and proliferation on the scaffold, and gene transfer efficiency reached a maximum of 35.5 ± 3.8%. In vivo, the implantation of BMP-2 transfected MSCs/nHAC/PLA construct significantly enhanced the formation of new bone in the segmental defect, compared to the control groups. This novel 3D BMP-2 transfected MSCs/nHAC/PLA construct has the potential for bone repair by genetic tissue engineering approach.

583. Baculovirus-Engineered Mesenchymal Stem Cells Heal the Critical-Sized Femoral Segmental Bone Defects in Rabbits

583. Baculovirus-Engineered Mesenchymal Stem Cells Heal the Critical-Sized Femoral Segmental Bone Defects in Rabbits

The association of human mesenchymal stem cells with BMP-7 improves bone regeneration of critical-size segmental bone defects in athymic rats

Critical size segmental bone defects are still a major challenge in reconstructive orthopedic surgery. Transplantation of human mesenchymal stem cells (hMSC) has been proposed as an alternative to autogenous bone graft, as MSC can be expanded in vitro and induced to differentiate into bone-regenerating osteoblats by several bone morphogenetic proteins (BMP). The aim of this study was to investigate whether the association of hMSC and BMP-7, with providing the necessary scaffold to fill the bone loss, improved bone regeneration in a rat model of critical size segmental bone defect, compared to treatment with either hMSC or BMP-7 and the matrix. In addition, we tested whether pre-treatment of hMSC with cyclic ADP-ribose (cADPR), an intracellular Ca 2+ mobilizer previously shown to accelerate the in vitro expansion of hMSC (Scarfì S et al, Stem Cells, 2008), affected the osteoinductive capacity of the cells in vivo. X-ray analysis, performed 2, 10 and 16 weeks after transplantation, revealed a significantly higher score in the rats treated with hMSC and BMP-7 compared to controls, receiving either hMSC or BMP-7. Microtomography and histological analysis, performed 16 weeks after transplantation, confirmed the improved bone regeneration in the animals treated with the association of hMSC and BMP-7 compared to controls. Pre-treatment with cADPR to stimulate hMSC proliferation in vitro did not affect the bone regenerating capacity of the cells in vivo. These results indicate that the association of in vitro expanded hMSC with BMP-7 provide a better osteoinductive graft compared to either hMSC or BMP-7 alone. Moreover, cADPR may be used to stimulate hMSC proliferation in vitro in order to reduce the time required to obtain a transplantable number of cells, with no adverse effect on the bone regenerating capacity of hMSC.

Segmental bone regeneration using an rhBMP-2-loaded gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model

We aimed to develop a hybrid scaffold with a porous structure and similar composition as natural bone for the controlled release of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration. We fabricated a gelatin/nanohydroxypatite (nHAP) scaffold by glutaraldehyde chemical cross-linking a gelatin aqueous solution with nHAP granules at a 5:1 ratio (v/w). Then, fibrin glue (FG) mixed with recombinant human BMP-2 (rhBMP-2) was infused into the gelatin/nHAP scaffold and lyophilized to develop an rhBMP-2-loaded gelatin/nHAP/FG scaffold. On scanning electron microscopy, the composite had a 3-D porous structure. The rhBMP-2 release kinetics from the hybrid scaffold was sustained and slow, and release of rhBMP-2 was complete at 40 days. Immunohistochemistry, azo-coupling and alizarin S-red staining were used to study in vitro differentiation of human bone-marrow mesenchymal cells (hBMSCs). Strong positive staining results confirmed that rhBMP-2 released from the scaffold could improve osteocalcin (OCN) and alkaline phosphatase (ALP) expression and calcium deposition formation. RT-PCR results showed significantly high mRNA expression of ALP and OCN in hBM-MSCs cultured on the gelatin/nHAP/FG scaffold with rhBMP-2. DNA assay demonstrated that the scaffold was noncytotoxic and could promote hBMSC proliferation from the components of the hybrid scaffold, not released rhBMP-2. The hybrid scaffolds were then used to repair critical-size segmental bone defects of rabbit radius. Gross specimen, X-ray, bone histomorphology and bone mineral density assay demonstrated that the rhBMP-2-loaded gelatin/nHAP/FG scaffold had good osteogenic capability and could repair the segmental bone defect completely in 12 weeks.

Enhanced effects of BMP‐binding peptide combined with recombinant human BMP‐2 on the healing of a rodent segmental femoral defect

BMP-binding peptide (BBP) enhances the osteogenic activity of recombinant human bone morphogenetic protein-2 (rhBMP-2), but the mechanism underlying the enhancement remains unclear. We aimed to elucidate the potential enhanced efficacy of BBP using critical-sized segmental femoral bone defects in rats. Seventy defects in seven groups of rats were filled with various amounts (0, 2, 5, and 10 microg) of rhBMP-2 with or without 1000 microg BBP. Radiographs were obtained after 4 and 8 weeks. The animals were euthanized at 8 weeks, and femoral specimens were assessed manually, evaluated for bone volume using microcomputed tomography, and subjected to histological or biomechanical analysis. Although 10 microg rhBMP-2 yielded consistent results in terms of bone healing and quality of bone repair across the segmental defect, lower doses of rhBMP-2 failed to induce satisfactory bone healing. However, the combined administration of lower doses of rhBMP-2 and BBP induced the formation of significantly large amounts of bone. Our results suggest that the combined administration of rhBMP-2 and BBP facilitates bone healing and has potential clinical applications.

Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous β-TCP ceramic scaffolds

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic fibroblast growth factor (bFGF) is one of the most prominent osteogenic growth factors that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of growth factors may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of growth factors locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous β tricalcium phosphate (β-TCP) ceramics and allografted into the 15 mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new bFGF gene enhanced tissue engineering strategy could be of potential benefit to accelerate bone healing, especially in defects caused by atrophic nonunion and avascular necrosis of the femoral head.

Bone Regeneration by Modified Gene-Activated Matrix: Effectiveness in Segmental Tibial Defects in Rats

Gene-activated matrix (GAM) is a matrix, such as collagen-containing plasmid vector, that encodes a protein to stimulate tissue regeneration. In the original GAM system, gene transfer efficiency was extremely low. We have recently reported that modifying GAM with calcium-phosphate precipitates (CaP) enhances the efficiency of gene transfer. The purpose of this study was to evaluate the effects of our modified GAM on tissue regeneration. We prepared critical size segmental bone defects in rat tibiae and transplanted GAM consisting of bovine atelocollagen and expression plasmid vector (bmp2), which encodes human BMP2, with or without CaP. The tibiae were later examined radiographically, histologically, and mechanically. Implantation of bmp2-CaP-collagen at 12 microg bmp2 bridged the bone defect at 4 weeks, and the strength of the bone was comparable to that of an intact tibia at 6 weeks. Implantation of bmp2-collagen at the same dose of bmp2 bridged the defect to a smaller extent. Neither collagen alone nor vacant vector-CaP-collagen bridged the defect. These results indicate that our modified GAM with CaP has the potential to be effective in tissue regeneration at lower plasmid DNA doses than used in previous studies.

Tissue-Engineered Bone Repair of Goat-Femur Defects with Osteogenically Induced Bone Marrow Stromal Cells

Tissue engineering can generate bone tissue and has been shown to provide a better means of repairing weight-bearing bone defect. Previous studies, however, have heretofore been limited to the use of nonosteogenically induced bone marrow stromal cells (BMSCs) or the application of slow-degradation scaffolds. In this study, weight-bearing bone was engineered using osteogenically induced BMSCs. In addition, coral was used as a scaffold material, due to its proper degradation rate for the engineering and repair of a goat femur defect. A 25 mm long defect was created at the middle of the right femur in each of 10 goats. The rates of defect repair were compared in an experimental group of ten goats receiving implants containing osteogenically induced BMSCs and in the control group of goats (n = 10) receiving just coral cylinders. In the experimental group, bony union was observed by radiographic and gross view at 4 months, and engineered bone was further remodeled into newly formed cortexed bone at 8 months. There was increased gray density of radiographic rays in the repaired area, which was significantly different (p < 0.05) from that of the control group. H&E staining demonstrated that trabecular bone was formed at 4 months. Moreover, irregular osteon was observed at 8 months. Most importantly, the tissue-engineered bone segment revealed a similarity to the left-side normal femur in terms of bend load strength and bend rigidity, showing no significant difference (p > 0.05). In contrast, the coral cylinders of the control group showed no bone formation. Furthermore, almost complete resorption of the carrier had occurred, being evident at 2 months in the control group. H&E staining demonstrated that a small amount of residual coral particle was surrounded by fibrous tissue at 4 months whereas the residues disappeared at 8 months. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and coral can ideally heal critical-sized segmental bone defects in the weight-bearing area of goats.

Structural and functional healing of critical-size segmental bone defects by transduced muscle-derived cells expressing BMP4.

Our previous studies have shown that muscle-derived cells, including a population of muscle stem cells, transduced with a retroviral vector expressing bone morphogenetic protein 4 (BMP4) can improve the healing of critical-size calvarial defects. However, we did not evaluate the functionality of the healed bone. The purpose of this study was to determine whether primary muscle-derived cells transduced with retroBMP4 can heal a long bone defect both structurally and functionally. Primary muscle-derived cells were genetically engineered to express BMP4 and were implanted into 7-mm femoral defects created in syngeneic rats. Muscle-derived cells transduced with retroLacZ were used in the control group. Bone healing was monitored by radiography, histology, and biomechanical testing at designated time points. Most of the defects treated with muscle-derived cells expressing BMP4 formed bridging callous by 6 weeks after surgery, and exhibited radiographically evident union at 12 weeks after cell implantation. Histological analysis at 12 weeks revealed that the medullary canal of the femur was restored and the cortex was remodeled between the proximal and distal ends of each BMP4-treated defect. In contrast, the defects treated with muscle-derived cells expressing beta-galactosidase displayed nonunion at all tested time points. An evaluation of the maximum torque-to-failure in the treatment group indicated that the healed bones possessed 77 +/- 28% of the strength of the contralateral intact femora. Torsional stiffness and energy-to-failure were not significantly different between the treated and intact limbs. This study demonstrated that primary muscle-derived cells transduced with retroBMP4 can elicit both structural and functional healing of critical-size segmental long bone defects created in rats.