Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allogra... more Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. Materials & methods: MSC characterization used functional assays, confocal/ scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. Results: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45-CD271 + CD73 + CD90 + CD105 + MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from one gram of cellular allograft were equivalent to approximately 45 ml of BM aspirate. Conclusion: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.
Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following cu... more Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other interferon signalling pathway members as potential immunomodulatory mediators. The CD317 molecule was detected on the surface of CBF-resident cells confirming the gene expression data. Taken together, these data demonstrate that human clinically used CBFs are inherently immunomodulatory and suggest that these viable allografts may be used to deliver therapeutic immunomodulation for immune-related diseases.
BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a new gene... more BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a new generation of viable cellular bone allografts. Allografts consisting of viable cancellous bone - which retains mesenchymal stem cells (MSCs) and osteoprogenitor cells - and demineralized bone matrix (DBM) possess osteoconductive, osteoinductive and osteogenic properties which mimic the healing mechanisms intrinsic to autologous bone grafts.
PURPOSE: To characterize the osteoinductive properties of the cell and tissue components of allograft cellular bone matrix and, where applicable, benchmark to other relevant cell types or commercially available DBM.
METHODS: Cryopreserved cellular bone matrix (Osteocel Plus, NuVasive) and its DBM component were obtained from the tissue processor. The DBM from cellular bone matrix was assessed relative to a DBM fiber putty (Grafton DBM Putty, Osteotech/Medtronic). Expression of BMP Genes: Cellular bone matrix was thawed according to instructions, rinsed with PBS, and then placed into explant culture for 24 hours. RNA was extracted from the cells via homogenization and purification (RNeasy Mini, Qiagen). Gene expression patterns were compared to those found in RNA from primary human fetal osteoblasts and adult fibroblasts using human osteogenesis qPCR arrays. In Vitro Osteoinductivity Assay: Aliquots of DBM digests were added to subconfluent C2C12 myoblast cell cultures. Following 3 days of incubation, cells were lysed, and alkaline phosphatase activity was quantified and normalized to total protein content (AP U/mg protein). BMP ELISA: Bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7) were quantified via enzyme-linked immunosorbent assays (ELISA) from DBM digests/extracts. Rat Ectopic Implantation: 0.3cc of the DBM component of cellular bone matrix was implanted intramuscularly (1 per animal) in the dorsum of athymic rats (n56). Following 8 weeks, animals were imaged via plain radiographs and microCT. Implants were retrieved and processed for histological assessment.
RESULTS: Expression of BMPs previously described as having high osteoinductive potency was markedly upregulated in cellular bone matrix cultures versus osteoblasts and fibroblasts: BMP-2 was up 102x and 5x; BMP-4 was up 14x and 33x; BMP-6 was up 47x and 6x; and BMP-7 was up 55x and 61x, respectively. The DBM from cellular bone matrix induced 11x higher levels of alkaline phosphatase activity versus DBM fiber putty (2206148 vs 20617 AP U/mg protein, p!0.001). Similarly, BMP-2, -4, and-7 concentrations were higher in the DBM from cellular bone matrix versus DBM fiber putty by 2.3x, 2.1x, and 1.1x, respectively. Furthermore, all ectopic implants of the DBM from cellular bone matrix demonstrated mineralization via microCT and the presence of new bone and marrow via histology.
CONCLUSIONS: Cells within allograft cellular bone matrix expressed high levels of BMPs relative to osteoblasts and fibroblasts, complementing previous evidence of an overall upregulation of osteogenic genes in Osteocel Plus-derived cells relative to human bone marrow-derived MSCs. Moreover, osteoinductive capacity was demonstrated using three commonly used assays: in vitro induction of C2C12 cells, BMP quantification via ELISA, and in vivo ectopic implantation. Higher inductive capacity and BMP concentrations were shown versus DBM fiber putty, which may reflect differences in processing, formulation, or storage conditions. Together, the data indicate that both the cell and DBM components contribute to the osteoinductivity of allograft cellular bone matrix which may drive osteogenesis at a fusion site.
FDA DEVICE/DRUG STATUS: Grafton Putty (Approved for this indication).
Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allogra... more Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. Materials & methods: MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. Results: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45-CD271 + CD73 + CD90 + CD105 + MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. Conclusion: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.
ABSTRACT BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a... more ABSTRACT BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a new generation of viable cellular bone allografts. Allografts consisting of viable cancellous bone which retains mesenchymal stem cells (MSCs) and osteoprogenitor cells and demineralized bone matrix (DBM) possess osteoconductive, osteoinductive and osteogenic properties which mimic the healing mechanisms intrinsic to autologous bone grafts. PURPOSE: To characterize the osteoinductive properties of the cell and tissue components of allograft cellular bone matrix and, where applicable, benchmark to other relevant cell types or commercially available DBM. METHODS: Cryopreserved cellular bone matrix (Osteocel Plus, NuVasive) and its DBM component were obtained from the tissue processor. The DBM from cellular bone matrix was assessed relative to a DBM fiber putty (Grafton DBM Putty, Osteotech/Medtronic). Expression of BMP Genes: Cellular bone matrix was thawed according to instructions, rinsed with PBS, and then placed into explant culture for 24 hours. RNA was extracted from the cells via homogenization and purification (RNeasy Mini, Qiagen). Gene expression patterns were compared to those found in RNA from primary human fetal osteoblasts and adult fibroblasts using human osteogenesis qPCR arrays. In Vitro Osteoinductivity Assay: Aliquots of DBM digests were added to subconfluent C2C12 myoblast cell cultures. Following 3 days of incubation, cells were lysed, and alkaline phosphatase activity was quantified and normalized to total protein content (AP U/mg protein). BMP ELISA: Bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7) were quantified via enzyme-linked immunosorbent assays (ELISA) from DBM digests/extracts. Rat Ectopic Implantation: 0.3cc of the DBM component of cellular bone matrix was implanted intramuscularly (1 per animal) in the dorsum of athymic rats (n56). Following 8 weeks, animals were imaged via plain radiographs and microCT. Implants were retrieved and processed for histological assessment. RESULTS: Expression of BMPs previously described as having high osteoinductive potency was markedly upregulated in cellular bone matrix cultures versus osteoblasts and fibroblasts: BMP-2 was up 102x and 5x; BMP-4 was up 14x and 33x; BMP-6 was up 47x and 6x; and BMP-7 was up 55x and 61x, respectively. The DBM from cellular bone matrix induced 11x higher levels of alkaline phosphatase activity versus DBM fiber putty (2206148 vs 20617 AP U/mg protein, p!0.001). Similarly, BMP-2, -4, and-7 concentrations were higher in the DBM from cellular bone matrix versus DBM fiber putty by 2.3x, 2.1x, and 1.1x, respectively. Furthermore, all ectopic implants of the DBM from cellular bone matrix demonstrated mineralization via microCT and the presence of new bone and marrow via histology. CONCLUSIONS: Cells within allograft cellular bone matrix expressed high levels of BMPs relative to osteoblasts and fibroblasts, complementing previous evidence of an overall upregulation of osteogenic genes in Osteocel Plus-derived cells relative to human bone marrow-derived MSCs. Moreover, osteoinductive capacity was demonstrated using three commonly used assays: in vitro induction of C2C12 cells, BMP quantification via ELISA, and in vivo ectopic implantation. Higher inductive capacity and BMP concentrations were shown versus DBM fiber putty, which may reflect differences in processing, formulation, or storage conditions. Together, the data indicate that both the cell and DBM components contribute to the osteoinductivity of allograft cellular bone matrix which may drive osteogenesis at a fusion site. FDA DEVICE/DRUG STATUS: Grafton Putty (Approved for this indication).
It has been proposed that the osteoblastic nature of prostate cancer skeletal metastases is due i... more It has been proposed that the osteoblastic nature of prostate cancer skeletal metastases is due in part to elevated activity of bone morphogenetic proteins (BMPs). BMPs are osteoinductive morphogens, and elevated expression of BMP-6 correlates with skeletal metastases of prostate cancer. In this study, we investigated the expression levels of BMPs and their modulators in prostate, using microarray analysis of cell cultures and gene expression. Addition of exogenous BMP-6 to DU-145 prostate cancer cell cultures inhibited their growth by up-regulation of several cyclin-dependent kinase inhibitors such as p21/CIP, p18, and p19. Expression of noggin, a BMP antagonist, was significantly up-regulated by BMP-6 by microarray analysis and was confirmed by quantitative reverse transcription-polymerase chain reaction and at the protein level. Noggin protein was present in prostate biopsies and localized to the epithelial components of prostate by immunohistochemistry. Recombinant noggin inhibi...
Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following cu... more Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other...
Journal of Histochemistry & Cytochemistry, 2007
Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflamm... more Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflammation, chemotaxis of progenitor cells, mitosis, differentiation of cartilage, and replacement with bone. This multistep cascade is orchestrated by cytokines and morphogens. Members of the interleukin (IL)-17 family, including IL-17B, have been identified in cartilage, but their expression during fracture healing is unknown. In this study, we determined the immunolocalization of cytokines IL-17A and IL-17B, along with the IL-17 receptor (IL-17R) and IL-17 receptor-like protein (IL-17RL), during the sequence of fracture repair in a standard model. The results were extended to developmental changes in the epiphyseal growth plate of long bones. Members of the IL-17 family were localized in chondrocytes in the fracture callus. Moreover, we found significant parallels to the localization of these cytokines and their receptors in chondrocytes during an endochondral differentiation program in th...
To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft an... more To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45(-)CD271(+)CD73(+)CD90(+)CD105(+) MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft repre...
Four fresh human cadaver spines were analyzed during and after disruptive hyperflexion and hypere... more Four fresh human cadaver spines were analyzed during and after disruptive hyperflexion and hyperextension to characterize the pathoanatomy of three-column cervical dissociation. In both flexion and extension, the posterior longitudinal ligament and facet capsules provided the greatest resistance to disruption. At low loading rates, all structures failed through the soft tissues. Three-column disruption caused by either pure flexion or extension resulted in marked elongation of the neural axis, inconsistent with cord survival. Biomechanical studies were carried out in seven additional fresh frozen human cadaver specimens to determine the most rigid method of internally stabilizing three-column cervical dissociations. Specimens were tested in compressive flexion and distractive extension to evaluate stability of anterior, posterior, and combined fixation constructs. Specimens were tested intact, after partial two-column disruption, and after complete three-column dissociation. Posterior wiring provided significantly better flexural stability in two- rather than three-column disruptions. Posterior wiring reduced posterior displacement in two-column partial disruptions to 25% of control. In three-column dissociations, posterior wiring only reduced posterior displacement to 50% of control. In extension, posterior wiring was ineffective in preventing displacement. Anterior plating, used alone, tolerated only 37% of the maximum flexion moment before early failure. On the other hand, combined anterior plating and posterior Roger's wiring reduced posterior displacement in flexion to 20% of control, while reducing anterior displacement in extension to 50% of control. This improvement over the other constructs was statistically significant. In highly unstable cervical injuries, Morscher anterior cervical plates and modified Roger's posterior wire fixation provide a safe, rigid construct that protects neural function while allowing early and aggressive mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)
Although research has determined pedicle screw pullout strengths for normal and osteoporotic bone... more Although research has determined pedicle screw pullout strengths for normal and osteoporotic bone, this study provides the first biomechanical analysis of pedicle screw salvage. Ten fresh frozen human lumbar spines were separated into individual vertebrae; 6.0 x 40 mm pedicle screws were placed in each pedicle; and an axial pullout test was performed to establish control values. Ultimate load, initial stiffness, work, and displacement data were calculated. Each vertebra was reinstrumented with one 7.0 x 40 mm variable screw placement (VSP) screw side by side with either a 7.0 mm Cotrel Dubousset sacral screw (CD) or a 7.0 mm Compact Cotrel Dubousset pedicle screw (CCD). Pullout tests were repeated and compared to control data for individual screws and for each VSP/CD or VSP/CCD pair. Vertebrae were then reinstrumented with 8.0 mm VSP and CD screws and paired pullouts repeated. Statistical analysis was carried out using a paired T test. Analysis of intravertebral and intergroup variation of controls was carried out using a Paired Two Sample T test. The 7.0 mm CCD screws restored pullout strength to 62% of control pullouts; 7.0 mm CD screws, to 85%; 7.0 mm VSP screws, to 99%; 8.0 mm CD screws, to 109%; and 8.0 mm VSP screws, to 148% of control pullouts. The 7.0 mm VSP salvage screws exceeded CD screws in ultimate load by 22.5% (p < .03) and CCD screws by 33.5% (p < .05). The 8.0 mm salvage screws significantly increased pullout relative to both controls and all 7.0 mm salvage screws, with 8.0 mm VSP exceeding 8.0 mm CD by 34% (p < .03). Initial stiffness was also significantly greater in VSP than CD screws. Although applied in a smaller number of vertebrae, 8.0 mm screws sufficiently outperformed smaller screws to provide statistically significant differences. The 7.0 mm VSP salvage screws restored pullout to control levels, roughly equivalent to outcomes previously obtained with unpressurized polymethylmethacrylate (PMMA).
Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allogra... more Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. Materials & methods: MSC characterization used functional assays, confocal/ scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. Results: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45-CD271 + CD73 + CD90 + CD105 + MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from one gram of cellular allograft were equivalent to approximately 45 ml of BM aspirate. Conclusion: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.
Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following cu... more Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other interferon signalling pathway members as potential immunomodulatory mediators. The CD317 molecule was detected on the surface of CBF-resident cells confirming the gene expression data. Taken together, these data demonstrate that human clinically used CBFs are inherently immunomodulatory and suggest that these viable allografts may be used to deliver therapeutic immunomodulation for immune-related diseases.
BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a new gene... more BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a new generation of viable cellular bone allografts. Allografts consisting of viable cancellous bone - which retains mesenchymal stem cells (MSCs) and osteoprogenitor cells - and demineralized bone matrix (DBM) possess osteoconductive, osteoinductive and osteogenic properties which mimic the healing mechanisms intrinsic to autologous bone grafts.
PURPOSE: To characterize the osteoinductive properties of the cell and tissue components of allograft cellular bone matrix and, where applicable, benchmark to other relevant cell types or commercially available DBM.
METHODS: Cryopreserved cellular bone matrix (Osteocel Plus, NuVasive) and its DBM component were obtained from the tissue processor. The DBM from cellular bone matrix was assessed relative to a DBM fiber putty (Grafton DBM Putty, Osteotech/Medtronic). Expression of BMP Genes: Cellular bone matrix was thawed according to instructions, rinsed with PBS, and then placed into explant culture for 24 hours. RNA was extracted from the cells via homogenization and purification (RNeasy Mini, Qiagen). Gene expression patterns were compared to those found in RNA from primary human fetal osteoblasts and adult fibroblasts using human osteogenesis qPCR arrays. In Vitro Osteoinductivity Assay: Aliquots of DBM digests were added to subconfluent C2C12 myoblast cell cultures. Following 3 days of incubation, cells were lysed, and alkaline phosphatase activity was quantified and normalized to total protein content (AP U/mg protein). BMP ELISA: Bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7) were quantified via enzyme-linked immunosorbent assays (ELISA) from DBM digests/extracts. Rat Ectopic Implantation: 0.3cc of the DBM component of cellular bone matrix was implanted intramuscularly (1 per animal) in the dorsum of athymic rats (n56). Following 8 weeks, animals were imaged via plain radiographs and microCT. Implants were retrieved and processed for histological assessment.
RESULTS: Expression of BMPs previously described as having high osteoinductive potency was markedly upregulated in cellular bone matrix cultures versus osteoblasts and fibroblasts: BMP-2 was up 102x and 5x; BMP-4 was up 14x and 33x; BMP-6 was up 47x and 6x; and BMP-7 was up 55x and 61x, respectively. The DBM from cellular bone matrix induced 11x higher levels of alkaline phosphatase activity versus DBM fiber putty (2206148 vs 20617 AP U/mg protein, p!0.001). Similarly, BMP-2, -4, and-7 concentrations were higher in the DBM from cellular bone matrix versus DBM fiber putty by 2.3x, 2.1x, and 1.1x, respectively. Furthermore, all ectopic implants of the DBM from cellular bone matrix demonstrated mineralization via microCT and the presence of new bone and marrow via histology.
CONCLUSIONS: Cells within allograft cellular bone matrix expressed high levels of BMPs relative to osteoblasts and fibroblasts, complementing previous evidence of an overall upregulation of osteogenic genes in Osteocel Plus-derived cells relative to human bone marrow-derived MSCs. Moreover, osteoinductive capacity was demonstrated using three commonly used assays: in vitro induction of C2C12 cells, BMP quantification via ELISA, and in vivo ectopic implantation. Higher inductive capacity and BMP concentrations were shown versus DBM fiber putty, which may reflect differences in processing, formulation, or storage conditions. Together, the data indicate that both the cell and DBM components contribute to the osteoinductivity of allograft cellular bone matrix which may drive osteogenesis at a fusion site.
FDA DEVICE/DRUG STATUS: Grafton Putty (Approved for this indication).
Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allogra... more Aim: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. Materials & methods: MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. Results: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45-CD271 + CD73 + CD90 + CD105 + MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. Conclusion: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.
ABSTRACT BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a... more ABSTRACT BACKGROUND CONTEXT: The development of novel allograft processing methods has produced a new generation of viable cellular bone allografts. Allografts consisting of viable cancellous bone which retains mesenchymal stem cells (MSCs) and osteoprogenitor cells and demineralized bone matrix (DBM) possess osteoconductive, osteoinductive and osteogenic properties which mimic the healing mechanisms intrinsic to autologous bone grafts. PURPOSE: To characterize the osteoinductive properties of the cell and tissue components of allograft cellular bone matrix and, where applicable, benchmark to other relevant cell types or commercially available DBM. METHODS: Cryopreserved cellular bone matrix (Osteocel Plus, NuVasive) and its DBM component were obtained from the tissue processor. The DBM from cellular bone matrix was assessed relative to a DBM fiber putty (Grafton DBM Putty, Osteotech/Medtronic). Expression of BMP Genes: Cellular bone matrix was thawed according to instructions, rinsed with PBS, and then placed into explant culture for 24 hours. RNA was extracted from the cells via homogenization and purification (RNeasy Mini, Qiagen). Gene expression patterns were compared to those found in RNA from primary human fetal osteoblasts and adult fibroblasts using human osteogenesis qPCR arrays. In Vitro Osteoinductivity Assay: Aliquots of DBM digests were added to subconfluent C2C12 myoblast cell cultures. Following 3 days of incubation, cells were lysed, and alkaline phosphatase activity was quantified and normalized to total protein content (AP U/mg protein). BMP ELISA: Bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7) were quantified via enzyme-linked immunosorbent assays (ELISA) from DBM digests/extracts. Rat Ectopic Implantation: 0.3cc of the DBM component of cellular bone matrix was implanted intramuscularly (1 per animal) in the dorsum of athymic rats (n56). Following 8 weeks, animals were imaged via plain radiographs and microCT. Implants were retrieved and processed for histological assessment. RESULTS: Expression of BMPs previously described as having high osteoinductive potency was markedly upregulated in cellular bone matrix cultures versus osteoblasts and fibroblasts: BMP-2 was up 102x and 5x; BMP-4 was up 14x and 33x; BMP-6 was up 47x and 6x; and BMP-7 was up 55x and 61x, respectively. The DBM from cellular bone matrix induced 11x higher levels of alkaline phosphatase activity versus DBM fiber putty (2206148 vs 20617 AP U/mg protein, p!0.001). Similarly, BMP-2, -4, and-7 concentrations were higher in the DBM from cellular bone matrix versus DBM fiber putty by 2.3x, 2.1x, and 1.1x, respectively. Furthermore, all ectopic implants of the DBM from cellular bone matrix demonstrated mineralization via microCT and the presence of new bone and marrow via histology. CONCLUSIONS: Cells within allograft cellular bone matrix expressed high levels of BMPs relative to osteoblasts and fibroblasts, complementing previous evidence of an overall upregulation of osteogenic genes in Osteocel Plus-derived cells relative to human bone marrow-derived MSCs. Moreover, osteoinductive capacity was demonstrated using three commonly used assays: in vitro induction of C2C12 cells, BMP quantification via ELISA, and in vivo ectopic implantation. Higher inductive capacity and BMP concentrations were shown versus DBM fiber putty, which may reflect differences in processing, formulation, or storage conditions. Together, the data indicate that both the cell and DBM components contribute to the osteoinductivity of allograft cellular bone matrix which may drive osteogenesis at a fusion site. FDA DEVICE/DRUG STATUS: Grafton Putty (Approved for this indication).
It has been proposed that the osteoblastic nature of prostate cancer skeletal metastases is due i... more It has been proposed that the osteoblastic nature of prostate cancer skeletal metastases is due in part to elevated activity of bone morphogenetic proteins (BMPs). BMPs are osteoinductive morphogens, and elevated expression of BMP-6 correlates with skeletal metastases of prostate cancer. In this study, we investigated the expression levels of BMPs and their modulators in prostate, using microarray analysis of cell cultures and gene expression. Addition of exogenous BMP-6 to DU-145 prostate cancer cell cultures inhibited their growth by up-regulation of several cyclin-dependent kinase inhibitors such as p21/CIP, p18, and p19. Expression of noggin, a BMP antagonist, was significantly up-regulated by BMP-6 by microarray analysis and was confirmed by quantitative reverse transcription-polymerase chain reaction and at the protein level. Noggin protein was present in prostate biopsies and localized to the epithelial components of prostate by immunohistochemistry. Recombinant noggin inhibi...
Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following cu... more Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other...
Journal of Histochemistry & Cytochemistry, 2007
Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflamm... more Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflammation, chemotaxis of progenitor cells, mitosis, differentiation of cartilage, and replacement with bone. This multistep cascade is orchestrated by cytokines and morphogens. Members of the interleukin (IL)-17 family, including IL-17B, have been identified in cartilage, but their expression during fracture healing is unknown. In this study, we determined the immunolocalization of cytokines IL-17A and IL-17B, along with the IL-17 receptor (IL-17R) and IL-17 receptor-like protein (IL-17RL), during the sequence of fracture repair in a standard model. The results were extended to developmental changes in the epiphyseal growth plate of long bones. Members of the IL-17 family were localized in chondrocytes in the fracture callus. Moreover, we found significant parallels to the localization of these cytokines and their receptors in chondrocytes during an endochondral differentiation program in th...
To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft an... more To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45(-)CD271(+)CD73(+)CD90(+)CD105(+) MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft repre...
Four fresh human cadaver spines were analyzed during and after disruptive hyperflexion and hypere... more Four fresh human cadaver spines were analyzed during and after disruptive hyperflexion and hyperextension to characterize the pathoanatomy of three-column cervical dissociation. In both flexion and extension, the posterior longitudinal ligament and facet capsules provided the greatest resistance to disruption. At low loading rates, all structures failed through the soft tissues. Three-column disruption caused by either pure flexion or extension resulted in marked elongation of the neural axis, inconsistent with cord survival. Biomechanical studies were carried out in seven additional fresh frozen human cadaver specimens to determine the most rigid method of internally stabilizing three-column cervical dissociations. Specimens were tested in compressive flexion and distractive extension to evaluate stability of anterior, posterior, and combined fixation constructs. Specimens were tested intact, after partial two-column disruption, and after complete three-column dissociation. Posterior wiring provided significantly better flexural stability in two- rather than three-column disruptions. Posterior wiring reduced posterior displacement in two-column partial disruptions to 25% of control. In three-column dissociations, posterior wiring only reduced posterior displacement to 50% of control. In extension, posterior wiring was ineffective in preventing displacement. Anterior plating, used alone, tolerated only 37% of the maximum flexion moment before early failure. On the other hand, combined anterior plating and posterior Roger&#39;s wiring reduced posterior displacement in flexion to 20% of control, while reducing anterior displacement in extension to 50% of control. This improvement over the other constructs was statistically significant. In highly unstable cervical injuries, Morscher anterior cervical plates and modified Roger&#39;s posterior wire fixation provide a safe, rigid construct that protects neural function while allowing early and aggressive mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)
Although research has determined pedicle screw pullout strengths for normal and osteoporotic bone... more Although research has determined pedicle screw pullout strengths for normal and osteoporotic bone, this study provides the first biomechanical analysis of pedicle screw salvage. Ten fresh frozen human lumbar spines were separated into individual vertebrae; 6.0 x 40 mm pedicle screws were placed in each pedicle; and an axial pullout test was performed to establish control values. Ultimate load, initial stiffness, work, and displacement data were calculated. Each vertebra was reinstrumented with one 7.0 x 40 mm variable screw placement (VSP) screw side by side with either a 7.0 mm Cotrel Dubousset sacral screw (CD) or a 7.0 mm Compact Cotrel Dubousset pedicle screw (CCD). Pullout tests were repeated and compared to control data for individual screws and for each VSP/CD or VSP/CCD pair. Vertebrae were then reinstrumented with 8.0 mm VSP and CD screws and paired pullouts repeated. Statistical analysis was carried out using a paired T test. Analysis of intravertebral and intergroup variation of controls was carried out using a Paired Two Sample T test. The 7.0 mm CCD screws restored pullout strength to 62% of control pullouts; 7.0 mm CD screws, to 85%; 7.0 mm VSP screws, to 99%; 8.0 mm CD screws, to 109%; and 8.0 mm VSP screws, to 148% of control pullouts. The 7.0 mm VSP salvage screws exceeded CD screws in ultimate load by 22.5% (p < .03) and CCD screws by 33.5% (p < .05). The 8.0 mm salvage screws significantly increased pullout relative to both controls and all 7.0 mm salvage screws, with 8.0 mm VSP exceeding 8.0 mm CD by 34% (p < .03). Initial stiffness was also significantly greater in VSP than CD screws. Although applied in a smaller number of vertebrae, 8.0 mm screws sufficiently outperformed smaller screws to provide statistically significant differences. The 7.0 mm VSP salvage screws restored pullout to control levels, roughly equivalent to outcomes previously obtained with unpressurized polymethylmethacrylate (PMMA).
Anatomic analysis of L4 vertebral morphometry comparing specimens harvested from humans and five ... more Anatomic analysis of L4 vertebral morphometry comparing specimens harvested from humans and five common large animal species. To compare fundamental structural similarities and differences in the vertebral bodies of commonly used experimental animals relative to human vertebrae. Animal models are commonly used for assessment of spine fusion, instrumentation techniques, and vertebral bone biology. Among the animals used, the lumbar vertebrae exhibit considerable anatomic variability. The goal of this study was to determine which of the animals commonly used for spine research is best suited as an anatomic model for the human lumbar spine. Morphometric features of the L4 vertebrae of five common research animals were compared with those of the human L4 vertebrae. Mature canines, immature pigs, mature micropigs, mature dairy goats, and mature sheep were analyzed. These species were chosen because they are commonly selected research animals, and most research facilities do not need to b...
Although research has determined pedicle screw pullout strengths for normal and osteoporotic bone... more Although research has determined pedicle screw pullout strengths for normal and osteoporotic bone, this study provides the first biomechanical analysis of pedicle screw salvage. Ten fresh frozen human lumbar spines were separated into individual vertebrae; 6.0 x 40 mm pedicle screws were placed in each pedicle; and an axial pullout test was performed to establish control values. Ultimate load, initial stiffness, work, and displacement data were calculated. Each vertebra was reinstrumented with one 7.0 x 40 mm variable screw placement (VSP) screw side by side with either a 7.0 mm Cotrel Dubousset sacral screw (CD) or a 7.0 mm Compact Cotrel Dubousset pedicle screw (CCD). Pullout tests were repeated and compared to control data for individual screws and for each VSP/CD or VSP/CCD pair. Vertebrae were then reinstrumented with 8.0 mm VSP and CD screws and paired pullouts repeated. Statistical analysis was carried out using a paired T test. Analysis of intravertebral and intergroup variation of controls was carried out using a Paired Two Sample T test. The 7.0 mm CCD screws restored pullout strength to 62% of control pullouts; 7.0 mm CD screws, to 85%; 7.0 mm VSP screws, to 99%; 8.0 mm CD screws, to 109%; and 8.0 mm VSP screws, to 148% of control pullouts. The 7.0 mm VSP salvage screws exceeded CD screws in ultimate load by 22.5% (p &lt; .03) and CCD screws by 33.5% (p &lt; .05). The 8.0 mm salvage screws significantly increased pullout relative to both controls and all 7.0 mm salvage screws, with 8.0 mm VSP exceeding 8.0 mm CD by 34% (p &lt; .03). Initial stiffness was also significantly greater in VSP than CD screws. Although applied in a smaller number of vertebrae, 8.0 mm screws sufficiently outperformed smaller screws to provide statistically significant differences. The 7.0 mm VSP salvage screws restored pullout to control levels, roughly equivalent to outcomes previously obtained with unpressurized polymethylmethacrylate (PMMA).
Intravenous infusion of E. coli endotoxin at a rate of 4.16 ng/kg/min over 6 hr (total dose 1.5 m... more Intravenous infusion of E. coli endotoxin at a rate of 4.16 ng/kg/min over 6 hr (total dose 1.5 micrograms/kg) in 5 cows in the first trimester of gestation induced abortion between 60 and 72 hr in three cows. Plasma PGF2 alpha levels in the aborting cows increased significantly to 289% of the zero time control (ZTC) at 1 hr and remained elevated for 9 hr. The PGF2 alpha level remained unaffected in the non-aborting cows except at 2 hr. The plasma TxB2 levels were increased by 6 to 18 fold for 6 hr in both the aborting and non-aborting cows relative to their ZTC controls. The 6-keto-PGF1 alpha levels were significantly increased to 2 to 3 fold only in the aborting cows. Plasma cortisol levels were increased maximally to 1,500% of ZTC at 5 hr in the aborting cows. Thereafter, the levels gradually declined but remained significantly elevated for 24 hr. The increases in the cortisol levels in the non-aborting cows were only 280% of ZTC at 5 hr and returned to ZTC value by 12 hr. Plasma progesterone levels in the aborting cows remained unaffected until 12 hr followed by a progressive decline through 18 hr to extremely low levels at 3, 4, and 5 days. Endotoxin-infusion caused hyperglycemia in both aborting and non-aborting cows and lactic acidemia in the aborting cows. Treatment with two doses of flunixin meglumine (FM, 1.1 mg/kg), an inhibitor of cyclooxygenase, 1 hr prior to endotoxin infusion and then 13 hr later, completely prevented the endotoxin-induced abortion and increases in the plasma PGF2 alpha, TxB2 and 6-keto-PGF1 alpha concentrations. The PGE level remained unaffected. Although FM treatment failed to abolish endotoxin-induced increases in the plasma cortisol and lactic acid levels, it effectively prevented marked decreases in the progesterone and increases in the glucose concentrations. It was concluded that the use of FM offers therapeutic promise in preventing bovine abortion caused by endotoxin resulting from bacterial infection during the 1st trimester of gestation.
Rat posterolateral lumbar fusion (PLF) models have been used to assess the safety and effectivene... more Rat posterolateral lumbar fusion (PLF) models have been used to assess the safety and effectiveness of new bone substitutes and osteoinductive growth factors using palpation, radiography, micro-computed tomography (μCT), and histology as standard methods to evaluate spinal fusion. Despite increased numbers of PLF studies involving alternative bone substitutes and growth factors, the quantitative assessment of treatment efficacy during spinal motion has been limited. The purpose of this study was to evaluate the effect of spinal fusion on lumbar spine segment stability during lateral bending using a μCT-based three-dimensional (3D) kinematic analysis in the rat PLF model. Fourteen athymic male rats underwent PLF surgery at L4/5 and received bone grafts harvested from the ilium and femurs of syngeneic rats (Isograft, n=7) or no graft (Sham, n=7). At 8 weeks after the PLF surgery, spinal fusion was assessed by manual palpation, plain radiography, μCT, and histology. To determine lumbar segmental motions at the operated level during lateral bending, 3D kinematic analysis was performed. The Isograft group, but not the Sham group, showed spinal fusion on manual palpation (6/7), solid fusion mass in radiographs (6/7), as well as bone bridging in μCT and histological images (5/7). Compared to the Sham group, the Isograft group revealed limited 3D lateral bending angular range of motion and lateral translation during lateral bending at the fused segment where disc height narrowing was observed. This μCT-based 3D kinematic analysis can provide a quantitative assessment of spinal fusion in a rat PLF model to complement current gold standard methods used for efficacy assessment of new therapeutic approaches.
Therapeutic treatment of intervertebral disc repair using cells. The goal of the study was to tes... more Therapeutic treatment of intervertebral disc repair using cells. The goal of the study was to test the hypothesis that repair of a damaged disc is possible using autologous adipose tissue derived stem and regenerative cells (ADRCs). Degradation resulting from either acute or chronic repetitive disc injury leads to disc degeneration. However, if a damaged disc could be repaired in the early stages, before the onslaught of degradation, then the disc degeneration process may be slowed down. Twelve dogs underwent a partial nucleotomy at 3 lumbar levels (L3-L4, L4-L5, and L5-L6); adjacent levels served as nonoperated controls. The animals (or discs) were allowed to recover from the surgery for 6 weeks. At that time subcutaneous adipose tissue was harvested and ADRCs were isolated. The 3 experimental discs that had undergone a partial nucleotomy were randomized to receive: (1) ADRCs in hyaluronic acid carrier (Cells/HA); (2) HA only; or (3) No Intervention. Assessments of the 3 experimental discs plus the 2 adjacent untouched discs were made using MRI, radiography, histology, and biochemistry. The animals were killed at 6 months and at 12 months. Repair in this study was specifically demonstrated through histology and biochemical analysis. Disc levels receiving ADRCs more closely resembled the healthy controls as evidenced in matrix translucency, compartmentalization of the anulus, and in cell density within the nucleus pulposus. Matrix analysis for Type-II collagen and aggrecan demonstrated evidence of a statistically better regenerative stimulation to the disc provided by ADRCs when compared to either the HA only or no intervention treatments. Autologous adipose tissue derived stem and regenerative cells, as used in this disc injury model, were effective in promoting disc regeneration, as evidenced by disc matrix production and overall disc morphology.
ABSTRACT This biomechanical study of fractures in cadaver vertebrae used specially designed pedic... more ABSTRACT This biomechanical study of fractures in cadaver vertebrae used specially designed pedicle screws to determine screw strains during loading of two different fixation constructs. The authors determined the relative benefit of adding offset sublaminar hooks to standard pedicle screw constructs to reduce screw bending moments and prevent fixation failure and sagittal collapse. Clinical studies have demonstrated a high incidence of early screw failure in short-segment pedicle instrumentation constructs used to treat unstable burst fractures. Strategies to prevent early construct failure include longer constructs, anterior strut graft reconstruction, and use of offset sublaminar hooks at the ends of standard short-segment pedicle instrumentation constructs. Human cadaver spines with an L1 burst fracture were instrumented with a standard short-segment pedicle instrumentation construct using specially instrumented pedicle screws. Mechanical testing was carried out in flexion, extension, side bending, and torsion, and stiffness and screw bending moments were recorded. Offset hooks were applied initially, then removed and testing repeated. Stiffness data were compared to intact and postfracture results, and between augmented and standard constructs. Addition of offset laminar hooks, supralaminar at T11 and infralaminar at L2, to standard short-segment pedicle instrumentation constructs increased stiffness in flexion by 268%, in extension by 223%, in side bending by 161%, and in torsion by 155% (all were significant except torsion). Sublaminar hooks also reduced pedicle screw bending moments to roughly 50% of standard in both flexion and extension (P &lt; 0.05). Supplemental offset hooks significantly increase construct stiffness without sacrificing principles of short-segment pedicle instrumentation, and absorb some part of the construct strain, thereby reducing pedicle screw bending moments and the likelihood of postyield deformation and clinical failure.
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Publications by Tim Moseley
PURPOSE: To characterize the osteoinductive properties of the cell and
tissue components of allograft cellular bone matrix and, where applicable, benchmark to other relevant cell types or commercially available DBM.
METHODS: Cryopreserved cellular bone matrix (Osteocel Plus, NuVasive) and its DBM component were obtained from the tissue processor. The DBM from cellular bone matrix was assessed relative to a DBM fiber putty (Grafton DBM Putty, Osteotech/Medtronic). Expression of BMP Genes: Cellular bone matrix was thawed according to instructions, rinsed with PBS, and then placed into explant culture for 24 hours. RNA was extracted from the cells via homogenization and purification (RNeasy Mini, Qiagen). Gene expression patterns were compared to those found in RNA from primary human fetal osteoblasts and adult fibroblasts using human osteogenesis qPCR arrays. In Vitro Osteoinductivity Assay: Aliquots of DBM digests were added to subconfluent C2C12 myoblast cell cultures. Following 3 days of incubation, cells were lysed, and alkaline phosphatase activity was quantified and normalized to total protein content (AP U/mg protein). BMP ELISA: Bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7) were quantified via enzyme-linked immunosorbent assays (ELISA)
from DBM digests/extracts. Rat Ectopic Implantation: 0.3cc of the
DBM component of cellular bone matrix was implanted intramuscularly
(1 per animal) in the dorsum of athymic rats (n56). Following 8 weeks,
animals were imaged via plain radiographs and microCT. Implants were
retrieved and processed for histological assessment.
RESULTS: Expression of BMPs previously described as having high osteoinductive potency was markedly upregulated in cellular bone matrix cultures versus osteoblasts and fibroblasts: BMP-2 was up 102x and 5x; BMP-4 was up 14x and 33x; BMP-6 was up 47x and 6x; and BMP-7 was up 55x and 61x, respectively. The DBM from cellular bone matrix induced 11x higher levels of alkaline phosphatase activity versus DBM fiber putty (2206148 vs 20617 AP U/mg protein, p!0.001). Similarly, BMP-2, -4, and-7 concentrations were higher in the DBM from cellular bone matrix versus DBM fiber putty by 2.3x, 2.1x, and 1.1x, respectively. Furthermore, all ectopic implants of the DBM from cellular bone matrix demonstrated mineralization via microCT and the presence of new bone and marrow via histology.
CONCLUSIONS: Cells within allograft cellular bone matrix expressed
high levels of BMPs relative to osteoblasts and fibroblasts, complementing previous evidence of an overall upregulation of osteogenic genes in Osteocel Plus-derived cells relative to human bone marrow-derived MSCs. Moreover, osteoinductive capacity was demonstrated using three commonly used assays: in vitro induction of C2C12 cells, BMP quantification via ELISA, and in vivo ectopic implantation. Higher inductive capacity and BMP concentrations were shown versus DBM fiber putty, which may reflect differences in processing, formulation, or storage conditions. Together, the data indicate that both the cell and DBM components contribute to the osteoinductivity of allograft cellular bone matrix which may drive osteogenesis at a fusion site.
FDA DEVICE/DRUG STATUS: Grafton Putty (Approved for this
indication).
Papers by Tim Moseley
PURPOSE: To characterize the osteoinductive properties of the cell and
tissue components of allograft cellular bone matrix and, where applicable, benchmark to other relevant cell types or commercially available DBM.
METHODS: Cryopreserved cellular bone matrix (Osteocel Plus, NuVasive) and its DBM component were obtained from the tissue processor. The DBM from cellular bone matrix was assessed relative to a DBM fiber putty (Grafton DBM Putty, Osteotech/Medtronic). Expression of BMP Genes: Cellular bone matrix was thawed according to instructions, rinsed with PBS, and then placed into explant culture for 24 hours. RNA was extracted from the cells via homogenization and purification (RNeasy Mini, Qiagen). Gene expression patterns were compared to those found in RNA from primary human fetal osteoblasts and adult fibroblasts using human osteogenesis qPCR arrays. In Vitro Osteoinductivity Assay: Aliquots of DBM digests were added to subconfluent C2C12 myoblast cell cultures. Following 3 days of incubation, cells were lysed, and alkaline phosphatase activity was quantified and normalized to total protein content (AP U/mg protein). BMP ELISA: Bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7) were quantified via enzyme-linked immunosorbent assays (ELISA)
from DBM digests/extracts. Rat Ectopic Implantation: 0.3cc of the
DBM component of cellular bone matrix was implanted intramuscularly
(1 per animal) in the dorsum of athymic rats (n56). Following 8 weeks,
animals were imaged via plain radiographs and microCT. Implants were
retrieved and processed for histological assessment.
RESULTS: Expression of BMPs previously described as having high osteoinductive potency was markedly upregulated in cellular bone matrix cultures versus osteoblasts and fibroblasts: BMP-2 was up 102x and 5x; BMP-4 was up 14x and 33x; BMP-6 was up 47x and 6x; and BMP-7 was up 55x and 61x, respectively. The DBM from cellular bone matrix induced 11x higher levels of alkaline phosphatase activity versus DBM fiber putty (2206148 vs 20617 AP U/mg protein, p!0.001). Similarly, BMP-2, -4, and-7 concentrations were higher in the DBM from cellular bone matrix versus DBM fiber putty by 2.3x, 2.1x, and 1.1x, respectively. Furthermore, all ectopic implants of the DBM from cellular bone matrix demonstrated mineralization via microCT and the presence of new bone and marrow via histology.
CONCLUSIONS: Cells within allograft cellular bone matrix expressed
high levels of BMPs relative to osteoblasts and fibroblasts, complementing previous evidence of an overall upregulation of osteogenic genes in Osteocel Plus-derived cells relative to human bone marrow-derived MSCs. Moreover, osteoinductive capacity was demonstrated using three commonly used assays: in vitro induction of C2C12 cells, BMP quantification via ELISA, and in vivo ectopic implantation. Higher inductive capacity and BMP concentrations were shown versus DBM fiber putty, which may reflect differences in processing, formulation, or storage conditions. Together, the data indicate that both the cell and DBM components contribute to the osteoinductivity of allograft cellular bone matrix which may drive osteogenesis at a fusion site.
FDA DEVICE/DRUG STATUS: Grafton Putty (Approved for this
indication).