The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.

BACKGROUND: Recombinant human bone morphogenetic proteins (rhBMPs) can induce bone formation, but the inability to identify an ideal delivery system limits their clinical application. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone-marrow cells, which allow delivery of the BMP-2 to a specific anatomical site. The autologous BMP-2-producing bone-marrow cells then were used to heal a critical-sized femoral segmental defect in syngeneic rats. METHODS: Femoral defects in five groups of rats were filled with 5 x 10(6) BMP-2-producing bone-marrow cells, created through adenoviral gene transfer (twenty-four femora, Group I); twenty micrograms of rhBMP-2 (sixteen femora, Group II); 5 x 10(6) beta-galactosidase-producing rat-bone-marrow cells, created through adenoviral gene transfer of the lacZ gene (twelve femora, Group III); 5 x 10(6) uninfected rat-bone-marrow cells (ten femora, Group IV); or guanidine hydrochloride-extracted demineralized bone matrix only (ten femora, Group V). Guanidine hydrochloride-extracted demineralized bone matrix served as a substrate in all experimental groups. Specimens that were removed two months postoperatively underwent histological and histomorphometric analysis as well as biomechanical testing. RESULTS: Twenty-two of the twenty-four defects in Group I (BMP-2-producing bone-marrow cells) and all sixteen defects in Group II (rhBMP-2) had healed radiographically at two months postoperatively compared with only one of the thirty-two defects in the three control groups (beta-galactosidase-producing rat-bone-marrow cells, uninfected rat-bone-marrow cells, and guanidine hydrochloride-extracted demineralized bone matrix alone). Histological analysis of the specimens revealed that defects that had received BMP-2-producing bone-marrow cells (Group I) were filled with coarse trabecular bone at two months postoperatively, whereas in those that had received rhBMP-2 (Group II) the bone was thin and lace-like. Defects that had been treated with bone-marrow cells producing beta-galactosidase (Group III), uninfected bone-marrow cells (Group IV), or guanidine hydrochloride-extracted demineralized bone matrix only (Group V) demonstrated little or no bone formation. Histomorphometric analysis revealed a significantly greater total area of bone formation in the defects treated with the BMP-2-producing bone-marrow cells than in those treated with the rhBMP-2 (p = 0.036). Biomechanical testing demonstrated no significant differences, with the numbers available, between the healed femora that had received BMP-2-producing bone-marrow cells and the untreated (control) femora with respect to ultimate torque to failure or energy to failure. CONCLUSIONS: This study demonstrated that BMP-2-producing bone-marrow cells created by means of adenoviral gene transfer produce sufficient protein to heal a segmental femoral defect. We also established the feasibility of ex vivo gene transfer with the use of biologically acute autologous short-term cultures of bone-marrow cells.     

rhBMP-6 stimulated osteoprogenitor cells enhance posterolateral spinal fusion in the New Zealand white rabbit.

BACKGROUND CONTEXT: The nonunion rate after posterolateral spinal fusion can be as high as 35%. This has stimulated interest in the development of techniques for enhancing new bone formation, including the addition of bioactive peptides or the use of cell-based therapies, including genetically modified cells. In previous studies we have demonstrated that exposing autologous, marrow-derived osteoprogenitor cells to a recombinant human bone morphogenetic protein-6 (rhBMP-6) containing extracellular matrix induces osteoblastic differentiation, and that these cells are capable of increasing new bone formation. Growth of autologous cells on a synthetic rhBMP-6 containing matrix yields a population of stimulated osteoprogenitor cells, without the expense of adding large amounts of rhBMP-6 directly, or the risks inherent in the use of genetically altered cells. PURPOSE: This study was performed to evaluate the potential of rhBMP-6 stimulated osteoprogenitor cells (stOPC) to enhance the rate and strength of posterolateral spinal fusion. STUDY DESIGN: Prospective in vivo animal study OUTCOME MEASURES: Radiographic evidence of spinal fusion, biomechanical testing of explanted spines, histological analysis of new bone formation METHODS: Single-level posterolateral spinal arthrodeses were performed in 69 New Zealand white rabbits. Autologous marrow stem cells were concentrated and then plated on an rhBMP-6-rich extracellular matrix synthesized by genetically engineered mouse C3H10T1/2 cells. Animals in Groups I (n=18) and II (n=18) received autografts of 30M and 60M rhBMP-6 stOPCs in guanidine extracted demineralized bone matrix (gDBM), respectively, whereas those in Group III (n=13) received iliac crest bone graft (ICBG). Those in Group IV (n=10) received gDBM, and those in Group V (n=10) underwent decortication only. Assessment of fusion was made with serial radiographs, manual palpation of the explanted spines, and biomechanical testing. The fusion masses from two animals each in Groups I, II, and IV were evaluated histologically. RESULTS: Fifty-three animals were available for analysis at the conclusion of the study. In these animals, the arthrodesis rate was significantly higher after treatment with rhBMP-6 stOPCs (77% for both Groups I and II by palpation) than ICBG, gDBM, or decortication alone (Group III=55%, IV=20% and V=0%, respectively). Similarly, the peak loads to failure of the fusion masses in Groups I and II (212.5+/-37.8 N and 234.6+/-45.7 N) were significantly greater than the corresponding values in the other groups (Group III=155.9+/-36.4N, Group IV=132.7+/-59.9N, and Group V=92.8+/-18.4N), though when only the fused specimens in Groups I, II, and III were compared, only Group II was significantly different than Group III (234.6+/-45.7N and 155.9+/-36.4N, respectively). The fusion masses in the rhBMP-6 stOPC-treated animals were typified by a thin, fusiform cortical shell, newly formed trabecular bone emanating from the decorticated transverse processes, and residual unremodeled gDBM carrier particles. The fusion masses in the gDBM treated bones were morphologically similar, though they contained less newly formed bone. CONCLUSIONS: The use of rhBMP-6 stOPCs in a carrier of gDBM significantly enhanced the rate and strength of single-level posterolateral spinal arthrodeses in the New Zealand white rabbit, compared with ICBG, gDBM, and decortication alone. Our results confirm that the stimulation of marrow-derived osteoprogenitor cells by growing them on a rhBMP-6 containing extracellular matrix is feasible. Further investigation is warranted to determine the relative contribution of rhBMP-6 stimulation and the number of cells implanted, as well as strategies for optimizing the technique for clinical application.     

A porcine collagen-derived matrix as a carrier for recombinant human bone morphogenetic protein-2 enhances spinal fusion in rats

Background contextRecombinant bone morphogenetic proteins (rhBMPs) have been used successfully in clinical trials. However, large doses of rhBMPs were required to induce adequate bone repair. Collagen sponges (CSs) have failed to allow a more sustained release of rhBMPs. Ongoing research aims to design carriers that allow a more controlled and sustained release of the protein. E-Matrix is a injectable scaffold matrix that may enhance rhBMP activity and stimulate bone regeneration.PurposeThe purpose of this study was to test E-Matrix as a carrier for rhBMPs in a CS and examine its feasibility in clinical applications by using a rat spinal fusion model.Patient sampleA total of 80 Lewis rats aged 8–16 weeks were divided into nine groups.Study design/settingRat spinal fusion model.Outcome measuresRadiographs were obtained at 4, 6, and 8 weeks. The rats were sacrificed and their spines were explanted and assessed by manual palpation, high-resolution microcomputed tomography (micro-CT), and histologic analysis.MethodsGroup I animals were implanted with CS alone (negative control); Group II animals with CS containing 10 μg rhBMP-2 (positive control); Group III animals with CS containing 3 μg rhBMP-2; Group IV animals with CS containing 3 μg rhBMP-2 and E-Matrix; Group V animals with CS containing 1 μg rhBMP-2; Group VI animals with CS containing 1 μg rhBMP-2 and E-Matrix; Group VII animals with CS containing 0.5 μg rhBMP-2; Group VIII animals with CS containing 0.5 μg rhBMP-2 and E-Matrix; and Group IX animals with CS and E-Matrix without rhBMP-2.ResultsRadiographic evaluation, micro-CT, and manual palpation revealed spinal fusion in all rats in the BMP-2 and E-Matrix groups (IV, VI, and VIII) and high-dose BMP-2 groups (II and III). Four spines in the 3 μg rhBMP-2 group (V) fused, and one spine in the 0.5 μg rhBMP-2 group (VII) exhibited fusion. No spines were fused in Groups I (CS alone) and IX (E-Matrix alone). The volume of new bone in the area between the tip of the L4 transverse process and the base of the L5 transverse process in Group IV was equivalent to the volumes observed in Group II.ConclusionE-matrix enhances spinal fusion as a carrier for rhBMP-2 in a rat spinal fusion model. The results of this study suggest that E-Matrix as a growth factor carrier may be applicable to spinal fusion and may improve rhBMP-2's activity at the fusion site.     

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.     

Promoting lumbar spinal fusion by adenovirus-mediated bone morphogenetic protein-4 gene therapy

Objective: To determine whether an adenoviral construct containing bone morphogenetic protein-4(BMP-4) gene can be used for lumbar spinal fusion. Methods: Twelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter andβ-galactosidase gene (Ad-β-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9×108 pfu/ml) was directly implanted on the surface of L5-L6 lamina in the experimental group, while Ad-β-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection. Results: In 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified. Conclusions: In vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.     

Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats

The objective of the present study was to assess the ability of bone marrow cells expressing BMP-2 created via lentiviral gene transfer to heal a critical sized femoral defect in a rat model. Femoral defects in Lewis rats were implanted with 5x10(6) rat bone marrow stromal cells (RBMSC) transduced with a lentiviral vector containing either the BMP-2 gene (Group I), the enhanced green fluorescent protein (LV-GFP) gene (Group IV), or RBMSC alone (Group V). We also included femoral defects that were treated with BMP-2-producing RBMSC transduced with lentivirus, 8 weeks after infection (Group III), and a group with 1x10(6) RBMSC transduced with a lentiviral vector with the BMP-2 gene (Group II). All defects (10/10) treated in Group I healed at 8 weeks compared with none of the femora in the control groups (Groups IV and V). In Group II, only one out of 10 femora healed. In Group III, 5 out of 10 femora healed. Significantly higher amounts of in vitro BMP-2 protein production were detected in Groups I, II, and III when compared to that of the control groups (p<0.05). Histomorphometric analysis revealed significantly greater total bone volume in defects in Group I and III when compared to control specimens (p<0.003). Biomechanical testing revealed no significant differences in the healed defects in Groups I and III when compared to intact, nonoperated femora with respect to peak torque and torque to failure. Our results indicate that BMP-2-producing RBMSC created through lentiviral gene transfer have the capability of inducing long-term protein production in vitro and producing substantial new bone formation in vivo.     

The effects of lentiviral gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats

STUDY DESIGN: Rat spinal fusion model. OBJECTIVE: This study aimed to assess the ability of rat bone marrow cells (RBMCs) transfected with bone morphogenetic protein (BMP)-2-containing lentivirus to induce a posterolateral spinal fusion in a rat model. SUMMARY OF BACKGROUND DATA: Spinal arthrodesis is a commonly performed spinal procedure and autograft remains the standard for achieving spinal fusion. However, its procurement is associated with significant morbidity, and the rate of pseudoarthrosis has been reported to be 5% to 43%. Nonunion frequently leads to an unsatisfactory resolution of clinical symptoms and usually results in high medical costs and morbidity as well as the need for additional surgeries. These problems have led surgeons to search for alternative solutions to stimulate bone formation. Recombinant BMPs have also been used successfully in clinical trials. However, large doses of BMPs were required to induce adequate bone repair. The development of a regional gene therapy may be a more efficient method to deliver proteins to a specific anatomic site. Furthermore, adeno-BMP-2-producing rat bone marrow-derived cells have been used successfully to induce posterior spinal fusion. Recently, lentiviral vectors on the basis of human immunodeficiency virus have been developed for gene therapy. Lentiviruses are capable of insertion into the host genome, ensuring a prolonged gene expression. However, safety issues are a major concern when adopting these vectors for clinical use. METHODS: In vitro study, we used RBMCs transfected with lentivirus vectors encoding BMP-2 (Lenti-BMP-2), RBMCs transfected with lentivirus vectors encoding the green fluorescent protein (GFP) (Lenti-GFP), and untransfected RBMCs; the latter 2 were used as controls. Alkaline phosphatase (ALP) staining and ALP activity were compared between the groups to assess the ability of the Lenti-BMP-2-transfected RBMCs to stimulate osteoblastic differentiation. In the rat posterolateral spine fusion model, the experimental study comprised 4 groups. Group 1 comprised 6 animals that were implanted with a collagen sponge containing 5 million RBMCs transfected with Lenti-BMP-2. Group 2 comprised 3 animals that were implanted with a collagen sponge containing 5 million RBMCs transfected with Lenti-GFP. Group 3 comprised 6 animals that were implanted with a collagen sponge containing 5 million untransfected RBMCs. Group 4 comprised 3 animals that were implanted with a collagen sponge alone. The rats were assessed by radiographs obtained at 4, 6, and 8 weeks. After death, their spines were explanted and assessed by manual palpation, high-resolution microcomputerized tomography, and histologic analysis. RESULTS: The ALP staining was significantly greater in the Lenti-BMP-2-transfected RBMCs than in the untransfected RBMCs and the Lenti-GFP-transfected RBMCs. The ALP activity was 3-fold greater in the Lenti-BMP-2-transfected RBMCs than in the untransfected RBMCs and the Lenti-GFP-transfected RBMCs. In the rat spine fusion model, radiographic evaluation, high-resolution microcomputerized tomography, and manual palpation revealed spinal fusion in all the rats in Group 1 at 8 weeks. Groups 2, 3, and 4 comprised the control group. None of the rats in the control group (0 of 12) developed fusion at L4-L5. CONCLUSIONS: The present study demonstrated that BMP-2-producing RBMCs, created through lentiviral gene transfer, induced sufficient spinal fusion. The use of lentiviral vectors that contain the cDNA for BMP-2 will be a novel and promising approach for a spinal fusion strategy.     

Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model

The objective of this study was to compare the efficacy of adenoviral and lentiviral regional gene therapy in a rat critical sized femoral defect model. The healing rates and quality of bone repair of femoral defects treated with syngeneic rat bone marrow cells (RBMCs) transduced with either lentiviral vector (Group I) or adenoviral vector (Group II) expressing bone morphogenetic protein-2 (BMP-2) gene were assessed. RBMCs transduced with the adenoviral vectors produced more than 3 times greater (p < 0.001) BMP-2 when compared to RBMCs transduced with lentiviral vectors in an in vitro evaluation. Serial bioluminescent imaging demonstrated short duration luciferase expression (less than 3 weeks) in defects treated with RBMCs co-transduced with two adenoviral vectors (Group IV; adenovirus expressing BMP-2 and luciferase [Ad-BMP-2 + Ad-Luc]). In contrast, the luciferase signal was present for 8 weeks in defects treated with RBMCs co-transduced with two lentiviral vectors (Group III; lentivirus expressing BMP-2 and luciferase gene [LV-BMP-2 + LV-Luc]). There were no significant differences with respect to the radiological healing rates (p = 0.12) in defects treated with lentiviral versus adenoviral mediated BMP-2 gene transfer. Biomechanical testing of healed Group I femoral specimens demonstrated significantly higher energy to failure (p < 0.05) when compared to Group II defects. Micro CT analysis revealed higher bone volume/tissue volume fraction (p = 0.04) in Group I defects when compared to Group II defects. In conclusion, prolonged BMP-2 expression associated with lentiviral mediated gene transfer demonstrated a trend towards superior quality of bone repair when compared to adenoviral mediated transfer of BMP-2. These results suggest that the bone repair associated with regional gene therapy is influenced not just by the amount of protein expression but also by duration of protein production. This observation needs validation in a more biologically challenging environment where differences in healing rates and quality of bone repair are more likely to be significantly different.     

Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6.

Bone marrow-derived mesenchymal stem cells (BMDMSC) hold promise for targeted osteogenic differentiation and can be augmented by delivery of genes encoding bone morphogenetic proteins (BMP). The feasibility of promoting osteogenic differentiation of BMDMSC was investigated using two BMP genes in monolayer and three-dimensional alginate culture systems. Cultured BMDMSC were transduced with E1-deleted adenoviral vectors containing either human BMP2 or BMP6 coding sequence under cytomegalovirus (CMV) promoter control [17:1 multiplicities of infection (moi)] and either sustained in monolayer or suspended in 1 mL 1.2% alginate beads for 22 days. Adenovirus (Ad)-BMP-2 and Ad-BMP-6 transduction resulted in abundant BMP-2 and BMP-6 mRNA and protein expression in monolayer culture and BMP-2 protein expression in alginate cultures. Ad-BMP-2 and Ad-BMP-6 transduced BMDMSC in monolayer had earlier and robust alkaline phosphatase-positive staining and mineralization and were sustained for a longer duration with better morphology scores than untransduced or Ad-beta-galactosidase-transduced cells. Ad-BMP-2- and, to a lesser degree, Ad-BMP-6-transduced BMDMSC suspended in alginate demonstrated greater mineralization than untransduced cells. Gene expression studies at day 2 confirmed an inflammatory response to the gene delivery process with upregulation of interleukin 8 and CXCL2. Upregulation of genes consistent with response to BMP exposure and osteogenic differentiation, specifically endochondral ossification and extracellular matrix proteins, occurred in BMP-transduced cells. These data support that transduction of BMDMSC with Ad-BMP-2 or Ad-BMP-6 can accelerate osteogenic differentiation and mineralization of stem cells in culture, including in three-dimensional culture. BMP-2-transduced stem cells suspended in alginate culture may be a practical carrier system to support bone formation in vivo. BMP-6 induced a less robust cellular response than BMP-2, particularly in alginate culture.     

Osteoinductivity of commercially available demineralized bone matrix. Preparations in a spine fusion model

BACKGROUND: Although autogenous bone is the most widely used graft material for spinal fusion, demineralized bone matrix preparations are available as alternatives or supplements to autograft. They are prepared by acid extraction of most of the mineralized component, with retention of the collagen and noncollagenous proteins, including growth factors. Differences in allograft processing methods among suppliers might yield products with different osteoinductive activities. The purpose of this study was to compare the efficacy of three different commercially available demineralized bone matrix products for inducing spinal fusion in an athymic rat model. METHODS: Sixty male athymic rats underwent spinal fusion and were divided into three groups of eighteen animals each. Group I received Grafton Putty; Group II, DBX Putty; and Group III, AlloMatrix Injectable Putty. A control group of six animals (Group IV) underwent decortication alone. Six animals from each of the three experimental groups were killed at each of three intervals (two, four, and eight weeks), and the six animals from the control group were killed at eight weeks. At each of the time-points, radiographic and histologic analysis and manual testing of the explanted spines were performed. RESULTS: The spines in Group I demonstrated higher rates of radiographically evident fusion at eight weeks than did the spines in Group III or Group IV (p < 0.05). Manual testing of the spines at four weeks revealed variable fusion rates (five of six in Group I, two of six in Group II, and none of six in Group III). At eight weeks, all six spines in Group I, three of the six in Group II, and no spine in Group III or IV had fused. Histologic analysis of the spines in Groups I, II, and III demonstrated varying amounts of residual demineralized bone matrix and new bone formation. Group-I spines demonstrated the most new bone formation. CONCLUSIONS: This study demonstrated differences in the osteoinductive potentials of commercially available demineralized bone matrices in this animal model.     

Recombinant human bone morphogenetic protein-2 enhances anterior spinal fusion in a thoracoscopically instrumented animal model

BACKGROUND: Thoracoscopically assisted anterior spinal arthrodesis and instrumentation is being used more widely to treat idiopathic scoliosis. However, harvesting autologous bone increases operative time and morbidity. The purpose of this study was to compare autologous iliac crest and rib graft with recombinant human bone morphogenetic protein-2 (rhBMP-2) in thoracoscopically assisted anterior spinal arthrodesis and instrumentation in an animal model. METHODS: Twenty-two pigs underwent thoracoscopically assisted anterior spinal arthrodesis. Each animal had five contiguous thoracic discectomies followed by anterior instrumentation. The animals were randomly assigned to five treatment groups. Group I consisted of control animals that received no graft material; group II, animals treated with autologous rib graft; group III, animals treated with autologous iliac crest graft; group IV, animals treated with an rhBMP-2-composite sponge (collagen-hydroxyapatite-tricalcium phosphate carrier); and group V, animals treated with a composite sponge carrier alone. The animals were killed four months after the procedure, and the spines were harvested. The fusion mass was assessed with use of axial and sagittal computed tomography scans. The spines were tested biomechanically with incremental loads applied in the frontal and axial planes to achieve bending moments of up to 6.0 N-m. Angular motion at each segment was recorded with use of a three-dimensional motion analysis system. Histomorphometric analysis of each undecalcified disc segment was also performed. RESULTS: The fusion grades, according to computed tomography analysis with use of a 4-point grading system in which scores of 3 and 4 indicated a solid fusion, were 0.6 point for group I, 2.1 points for group II, 2.3 points for group III, 3.8 points for group IV, and 0.4 point for group V. Group IV (the rhBMP-2-treated animals) had a higher grade than all of the other groups. Group II (rib graft) and group III (iliac crest) had similar grades, and both were greater than group I (the untreated controls) and group V (composite sponge alone) (p < 0.05). In axial rotation, lateral bending, and flexion-extension, the spines in group IV were stiffer than those in the four other groups (p < 0.05); the spines in groups II and III were similar, and the spines in both of those groups were stiffer than those in groups I and V (the control groups). Histologic analysis demonstrated that the total new-bone area, expressed as a percentage of the total disc space area, was 23.2% in group I, 37.1% in group II, 37.2% in group III, 48.5% in group IV, and 5.9% in group V. Group IV had significantly greater bone formation than all of the other groups (p < 0.001). The animals treated with rib graft (group II) and iliac crest (group III) had a similar amount of bone formation, and it was greater than that in both control groups (p < 0.001). CONCLUSIONS: The rhBMP-2 significantly increased the prevalence and quality of the spinal fusion after thoracoscopically assisted anterior arthrodesis and instrumentation in an animal model compared with that in the other treatment groups and in the controls.     

BMP-silk composite matrices heal critically sized femoral defects

Clinical drawbacks of bone grafting prompt the search for alternative bone augmentation technologies such as use of growth and differentiation factors, gene therapy, and cell therapy. Osteopromotive matrices are frequently employed for the local delivery and controlled release of these augmentation agents. Some matrices also provide an osteoconductive scaffold to support new bone growth. In this study, silkworm-derived silk fibroin was evaluated as an osteoconductive matrix for healing critical sized mid-femoral segmental defects in nude rats. Four treatment groups were assessed over eight weeks: silk scaffolds (SS) with recombinant human BMP-2 (rhBMP-2) and human mesenchymal stem cells (HMSC) that had been pre-differentiated along an osteoblastic lineage ex vivo (Group I; pdHMSC/rhBMP-2/SS); SS with rhBMP-2 and undifferentiated HMSCs (Group II; udHMSC/rhBMP-2/SS); SS and rhBMP-2 alone (Group III; rhBMP-2/SS); and empty defects (Group IV). Bi-weekly radiographs revealed a progressive and similar increase in Group I-III mean defect mineralization through post-operative week (POW) 8. Radiographs, dual energy x-ray absorptiometry, and micro-computed tomography confirmed that Groups I-III exhibited similar substantial and significantly (p<0.05) greater defect mineralization at POW 8 than the unfilled Group IV defects which remained void of bone. No significant differences in Groups I-III defect healing at POW 8 were apparent using these same assays or mechanical testing. Histology at POW 8 revealed moderately good bridging of the parent diaphyseal cortices with woven and lamellar bone bridging islands of silk matrix in Groups I and III. Group II defects possessed comparatively less new bone which was most abundant adjacent to the parent bone margins. Elsewhere the silk matrix was more often enveloped by poorly differentiated loose fibrous connective tissue. Group IV defects showed minimal new bone formation. None of the treatment groups attained the mean mineralization or the mean biomechanical strength of identical defects implanted with SS and pdHMSCs alone in a previous study. However, addition of rhBMP-2 to SS prompted more bone than was previously generated using udHMSC/SS or SS alone. These data imply the clinical potential of silk scaffolds and rhBMP-2 as composite osteopromotive implants when used alone or with select stem cell populations. Additional studies in larger species are now warranted.     

腺病毒介导人骨形态蛋白-7基因感染大鼠髓腔细胞的实验研究

目的 :观察已构建好的腺病毒介导的骨形态蛋白 7基因感染大鼠髓腔内细胞的情况 ,为腺病毒介导BMP 7的进一步基因治疗提供可靠的实验基础。材料和方法 :从人骨肉瘤细胞株U 2OS中获得hBMP 7的cDNA片段 ,插入穿梭质粒后在大肠杆菌内与腺病毒实现同源重组 ,阳性克隆转染 2 93细胞 ,扩增纯化得到大量病毒后注入大鼠椎体髓腔中 ,用冲洗髓腔的方法和流式细胞仪分析来观察注射椎体和上位椎体髓腔内细胞感染腺病毒BMP 7的情况 ,并观察病毒感染的时限。结果 :经体外检测能高效表达活性BMF 7的构建好的AdBMP 7注入椎体髓腔后 ,髓腔冲洗液的荧光显微镜观察 ,术后 1 2d和 2 4d都有感染了带有荧光蛋白标记的髓腔内细胞 ,而且在注射椎体的上位椎体中也出现了感染病毒的细胞。结论 :腺病毒介导的BMP -7基因能够在体内感染髓腔细胞并分泌活性的BMP -7,可以成为基因治疗的有效手段。     

Manipulation of the anabolic and catabolic responses with BMP-2 and zoledronic acid in a rat femoral fracture model

Bone repair involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the fracture site. It has been reported that the optimal time point for single-dose zoledronic acid (ZA) administration systemically increased the strength of bone morphogenetic protein (BMP)-7-mediated callus. However, its repair mechanism during bone fracture healing remains unknown. We aimed to investigate the synergic effect of recombinant human (rh) BMP-2 and ZA in a rat femoral fracture model. Fifty-eight rats were divided into 4 groups. Group I (n = 14) animals were implanted with a carrier alone. Group II (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2. Group III (n = 14) animals were implanted with a carrier and a subcutaneous systemic ZA injection 2 weeks after surgery. Group IV (n = 15) animals were implanted with a carrier containing 1-μg rhBMP-2 and ZA subcutaneous injection 2 weeks after surgery. The rats were euthanized after 6 weeks and their fractured femurs were explanted and assessed by manual palpation, radiographs, and high-resolution micro-computerized tomography (micro-CT) and were subjected to biomechanical and histological analysis. The fusion rates in Group IV (93.3%) were considerably higher than those in Groups I (28.6%), II (53.3%), and III (57.1%). Additionally, the radiographic scores of Group IV were higher than those in Groups I, II, and III. In micro-CT analysis, the tissue volume (TV) of the callus was higher in Group IV than in Groups I and II (p < 0.05). New bone volume (BV) and trabecular spacing (Tb.Sp) also showed essentially the same trend as that of TV. The ratio of BV to TV (BV/TV), the trabecular number (Tb.N), and the trabecular thickness (Tb.Th) was higher in Groups III and IV than in Groups I and II (p < 0.05). In biomechanical analysis, the ultimate loads at failure and stiffness in Groups III and IV were on average higher than those in Groups I and II (p < 0.05), while the energy absorption of Group IV was higher than those of Groups I and II (p < 0.05). The synergic effect of rhBMP-2 and ZA given systemically as a single dose at the optimal time was efficacious for fracture repair and significantly enhanced bone fusion. Our results suggest that this combination facilitates bone healing and has potential clinical application.     

A light and electron microscopic study of ectopic tendon and ligament formation induced by bone morphogenetic protein-13 adenoviral gene therapy.

OBJECT: Bone morphogenetic proteins (BMPs) are involved in the growth and development of many tissues, but it is their role in skeletal development and their unique ability to induce ectopic and orthotopic osteogenesis that have attracted the greatest interest. Expression of the BMP-13 gene is predominantly localized to hypertrophic chondrocytes in regions of endochondral bone formation during development, as well as in mature articular cartilage in the adult. In addition, the application of BMP-13 on a collagen carrier induces neotendon/neoligament formation when delivered subcutaneously or intramuscularly in rodents. The aim of the present study was to determine the histological and ultrastructural changes that occur after the intramuscular injection of a first-generation BMP-13 adenoviral vector. METHODS: Athymic nude rats were injected with 3.75 x 10(10) plaque-forming units of adenovirus (Ad)-BMP-13 or Ad-beta-galactosidase in the thigh musculature, and the region was examined using light and electron microscopy at various time points between 2 days and 100 days postinjection. As early as 2 days after injection of Ad-BMP-13, progenitor cells were observed infiltrating between the transduced muscle fibers. These cells subsequently proliferated, differentiated, and secreted large amounts of collagenous extracellular matrix. By 100 days postinjection, the treated tissue displayed the histological and ultrastructural appearance of neotendon/neoligament, which was clearly demarcated from the surrounding muscle. Small foci of bone and fibrocartilage were also seen within the treated tissue. A short-term bromodeoxyuridine study also demonstrated rapid mesenchymal cell proliferation at the Ad-BMP-13 injection site as early as 48 hours postinjection. At all time points, the control AD-beta-gal injection sites were found to contain only normal muscle, without evidence of inflammation or mesenchymal cell proliferation. CONCLUSIONS: The results of this study indicate that in the future the use of the BMP-13 gene may have therapeutic utility for the healing of tendon and ligament tears and avulsion injuries.     

陶瓷化骨-水凝胶与骨髓基质细胞自体皮下成骨实验

目的观察重组人骨形成蛋白(recombinant human bone morphogenetic protem 2,rhBMP-2)-转化生长因子β(transforming growth factor β,TGF-β)与陶瓷化骨-水凝胶和骨髓基质细胞复合后,植入自体皮下成骨能力的影响.方法用矿化液诱导第1代成年SD大鼠骨髓基质细胞(marrow stromal cells,MSCs)5 d后,应用改良钙钴法染色、Von Kossa染色、免疫组织化学染色观察诱导后的MSCs向成骨细胞分化情况.将收集的细胞(5×106/ml)分成3组,A组加入rhBMP-2(5μg)-TGF-β(0.05μg),B组加入rhBMP-25μg,C组不加生长因子作为空白对照;然后将细胞接种在陶瓷化骨-水凝胶上,植入自体12只SD大鼠皮下,每组6只,每只大鼠3种材料各1块.术后4、8周取材,行组织学观察骨基质形成情况,图像分析测量单位面积骨形成量,免疫组织化学染色观察Ⅰ型胶原、BMP合成情况.结果 MSCs经过5 d矿化诱导后,大部分细胞变小,胞浆突起变短,呈多边形.改良钙钴法染色见胞浆含有棕黑色颗粒的阳性细胞,Ⅰ型胶原免疫织组化学染色见大部分细胞胞核未着色,胞浆呈棕黄色;Von Kossa染色见20 d已经形成矿化结节.动物体内植入后4周,各组均有形状不规则的条索状骨基质形成,图像分析示A组的骨基质面积明显多于B组(P＜0.01);A、B组Ⅰ型胶原平均灰度值明显低于C组(P＜0.01),各组BMP的表达无明显差异(P＞0.05).8周时各组均有形状不规则的条索状、斑块状砖红色骨基质形成,A、B两组成熟骨面积明显多于C组(P＜0.01);Ⅰ型胶原、BMP均有阳性表达,但各组间平均灰度值无明显差异(P＞0.05).结论陶瓷化骨-水凝胶与MSCs复合物植入自体皮下后能形成骨组织,rhBMP 2-TGF-β复合生长因子较单纯rhBMP-2有更强的促进骨形成作用.     

NELL-1 based demineralized bone graft promotes rat spine fusion as compared to commercially available BMP-2 product

Background

Spinal fusion is among the most commonly performed orthopaedic procedures. Unfortunately, current treatments such as autologous bone grafting or recombinant proteins (BMP-2) have numerous clinical shortcomings. Here, we directly compare the efficacy of NELL-1, a novel osteoinductive growth factor, to two currently available treatments, (1) recombinant BMP-2 and (2) iliac crest bone grafting, in a spinal fusion model.

Methods

Twenty-six skeletally mature athymic rats underwent posterolateral spine fusion of L4/L5 vertebrae. Treatment groups included NELL-1 (10 and 50 μg) in a demineralized bone matrix (DBX), as compared to BMP-2 (90 μg) in an absorbable collagen sponge (ACS) or morselized iliac crest bone. Scaffolds without recombinant protein were used as controls. Animals were sacrificed at 4 weeks post-operative and fusion was assessed by manual palpation, radiography [high-resolution X-ray, micro-computed tomography (microCT)], histology (hematoxylin and eosin, Masson’s trichrome) and immunohistochemistry (osteocalcin).

Results

Results showed 100 % fusion in all NELL-1- and BMP-2-treated samples. In contrast, lower rates of fusion were observed in scaffold-only and bone graft treatment groups. MicroCT scans revealed radiographic evidence of fusion among spines treated with NELL-1. Bone bridging was also observed with BMP-2 treatment, but was accompanied by inner radiolucency, suggesting cyst-like bone formation. Histologically, NELL-1-treated grafts showed increased bone formation, endochondral ossification and vascularization. Although BMP-2 treated grafts exhibited increased bone formation and angiogenesis, numerous adipocytes were also observed.

Conclusion

NELL-1-based bone grafts are comparable to BMP-2 + ACS in spinal fusion efficacy. Histological differences were observed however, including robust endochondral ossification with NELL-1 treatment as compared to lipid-filled bone with BMP-2 treatment. These findings suggest NELL-1 based bone grafts show promise for future efforts in skeletal tissue engineering.     

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (β-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.     

BMP-2基因转染的人骨髓基质干细胞复合PLA/PCL支架体外构建组织工程骨

目的　用人骨形态发生蛋白 2腺病毒表达载体 (Ad -BMP - 2 )转染的人骨髓基质干细胞 (hBMSC) ,复合PLA/PCL(聚乳酸 /聚己内酯 )生物降解支架体外构建组织工程骨。方法　用Ad -BMP - 2转染体外培养的成人BMSC ,免疫组化、原位杂交染色和蛋白印迹方法检测细胞BMP - 2的表达 ,并通过流式细胞仪和ALP活性检测分析其对细胞增殖、分化的影响。然后将转染后细胞接种到PLA/PCL支架上 ,扫描电镜观察细胞贴附、生长状况。结果　转染后 ,hBMP - 2基因在mRNA水平和蛋白水平均有表达 ;S期细胞比例和ALP活性明显增高。扫描电镜见转染细胞分布均匀 ,伸展良好。结论　Ad-BMP - 2可高效转染hBMSC ,且促进细胞增殖及成骨转化。转染后细胞在PLA/PCL上生长良好 ,BMP - 2基因治疗的组织工程骨构建成功