Full-length spp24, but not its 18.5-kDa proteolytic fragment, inhibits bone-healing in a rodent model of spine fusion.

BACKGROUND: Growth factors like bone morphogenetic protein (BMP) are used as bone-graft substitutes to enhance bone growth in clinical situations. However, adverse reactions have been associated with BMP use. We developed a synthetic adjuvant therapy based on the sequence of a BMP-binding protein, secreted phosphoprotein-24 (spp24), which enhances the effects of BMPs and ameliorates the adverse reactions. Our hypothesis is that a natural proteolytic fragment of spp24 is identical to an osteogenic protein previously described independently by two investigators. To test this hypothesis, spp24 and a truncated form of spp24 were separately implanted with recombinant human BMP-2 (rhBMP-2) in a rodent model of spine fusion. METHODS: Two isoforms of spp24 were constructed with use of DNA recombinant technology. Spp24 with or without rhBMP-2 were added to collagen sponges and implanted bilaterally between L4 and L5 transverse processes. Radiographs were made biweekly, and spines were explanted after eight weeks. Gross evaluation, microquantitative computed tomography study, and histological analysis were performed to evaluate bone growth. RESULTS: Animals that received full-length spp24 and rhBMP-2 exhibited a complete obliteration of bone growth, while animals with the truncated form in combination with rhBMP-2 exhibited a mild inhibition to bone growth, with bone area measured from radiographs. Manual assessment and gross evaluation of all spines confirmed the results obtained from the bone-area measurements. Microquantitative computed tomography provided three-dimensional visual images of representative specimens, while histological staining of spine tissue displayed cellular evidence of bone formation. CONCLUSIONS: Results from this investigation confirm that the various isoforms of spp24 affect the bone-healing activity of rhBMP-2 in the rat spine fusion model. Thus, proteolytic modification of this protein is a likely mechanism for the regulation of BMP availability in the physiological environment. Future studies will define the roles of these proteins in controlling the activity of BMPs and other members of the transforming growth factor-beta family of cytokines. This information will increase the understanding of normal bone-healing, allowing for the engineering of more effective orthopaedic treatment.

Inflammatory characteristics of rhBMP-2 in vitro and in an in vivo rodent model.

STUDY DESIGN: In vivo and in vitro model. OBJECTIVE: Investigate soft-tissue inflammation caused by rhBMP-2. SUMMARY OF BACKGROUND DATA: Although rhBMP-2 produces excellent rates of fusion in the spine, dysphagia and respiratory compromise have occurred when used in the neck. The mechanism of the swelling and inflammatory response has yet to be fully elucidated. METHODS: ELISA kits (IL-6, IL-10, TNF-α) were used to measure cytokine levels at different concentrations of rhBMP-2. Absorbable collagen sponges were implanted with or without different concentrations of rhBMP-2 into the backs of rats subcutaneously (SC) and intramuscularly (IM). Magnetic resonance imaging was used to measure inflammation at 3 hours and 2, 4, and 7 days. The inflammatory volumes were measured and compared using MIPAV software. Rats were killed after 7 days and studied. RESULTS: IL-6, IL-10, and TNF-α release was dose-dependent. Soft-tissue edema after rhBMP-2 implantation was also dose-dependent, peaking at 3 hours SC, after SC and IM implantations, and on day 2 IM after IM implantation. All formed a granuloma-type mass after SC insertion. The mass was much larger in the 10 and 20 µg/10 µL (high-concentration) groups. The inflammatory response did not diffuse across physiologic barriers (subcutaneous fascia). Both high-dose groups were associated with encapsulated hematomas and a significant increase in the inflammatory zone. CONCLUSION: Swelling and inflammation after rhBMP-2 use are dose-dependent. Swelling may be due to direct contact as well as spread in the plane of access. The causes are a robust inflammatory reaction as well as sterile seroma and encapsulated hematoma formation.

Carboxy terminus of secreted phosphoprotein-24 kDa (spp24) is essential for full inhibition of BMP-2 activity.

Secreted phosphoprotein-24 kDa (spp24) is a bone morphogenetic protein (BMP)-binding protein isolated from bone. It exists in a number of size forms and is hypothesized to function as a BMP latency protein and/or a "slow release" mechanism for BMPs involved in bone turnover and repair. We have examined the hypothesis that proteolytic modification of the C-terminus of spp24 affects its BMP-2-binding properties and bioactivity in the BMP-2-stimulated ectopic bone forming bioassay. Three different size forms of recombinant spp24 that correspond to predicted 18.1 kDa, 16.0 kDa, and 14.5 kDa proteolytic products were compared to full-length (fl) spp24. One of these forms (spp18.1) we hypothesize to be the protein which Urist initially, but apparently inaccurately, called "BMP." Only full-length spp24 completely inhibited BMP-2-induced bone formation. The 18.1 kDa truncated isoform of spp24 which we hypothesize to be Urist's protein did not. The inhibitory capacity of the proteins was correlated with their kinetic constants, assessed by surface plasmon resonance. At the highest, inhibitory, dose of spp24 and its derivatives, k(d) ("stability") best predicted the extent of ectopic bone formation whereas at the lowest dose, which was not inhibitory, k(a) ("recognition") best predicted the extent of ectopic bone formation. We conclude that proteolytic processing of spp24 affects the interaction of this protein with BMP-2 and this affects the function of the protein.

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

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

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

BACKGROUND CONTEXT: Recombinant 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. PURPOSE: The 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 SAMPLE: A total of 80 Lewis rats aged 8-16 weeks were divided into nine groups. STUDY DESIGN/SETTING: Rat spinal fusion model. OUTCOME MEASURES: Radiographs 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. METHODS: Group I animals were implanted with CS alone (negative control); Group II animals with CS containing 10microg rhBMP-2 (positive control); Group III animals with CS containing 3microg rhBMP-2; Group IV animals with CS containing 3microg rhBMP-2 and E-Matrix; Group V animals with CS containing 1microg rhBMP-2; Group VI animals with CS containing 1microg rhBMP-2 and E-Matrix; Group VII animals with CS containing 0.5microg rhBMP-2; Group VIII animals with CS containing 0.5microg rhBMP-2 and E-Matrix; and Group IX animals with CS and E-Matrix without rhBMP-2. RESULTS: Radiographic 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 3microg rhBMP-2 group (V) fused, and one spine in the 0.5microg 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. CONCLUSION: E-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 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.

Comparison of lentiviral and adenoviral gene therapy for spinal fusion in rats.

STUDY DESIGN: Rat spinal fusion model. OBJECTIVE: This study aimed to compare the efficacy of lentiviral gene therapy, and adenoviral gene therapy in inducing spinal fusion in an immune competent rat spinal fusion model. SUMMARY OF BACKGROUND DATA: Recombinant bone morphogenetic proteins (BMPs) have also been used for spinal fusion successfully in clinical trials. However, large doses of BMPs are required to induce adequate bone repair. Hence, regional gene therapy may be a more efficient method to deliver proteins to a specific anatomic site. Recently, lentiviral vectors based on human immunodeficiency virus have been developed for gene therapy. However, lentiviral gene therapy for spinal fusion has not been compared with adenoviral gene therapy. METHODS: Lewis rats were divided into 7 groups. group I, II, III, and IV rats were implanted with a collagen sponge containing rat bone marrow cells (RBMCs) transfected with Lenti-BMP-2, Adeno-BMP-2, Lenti-GFP, Adeno-LacZ, respectively. Group V, VI, and VII rats were implanted with a collagen sponge containing recombinant BMP-2, a collagen sponge containing untransfected RBMCs, and a collagen sponge alone, respectively. The rats were assessed at 4, 6, and 8 weeks after implantation. After sacrificing the rats, their spines were explanted and assessed by manual palpation, high-resolution microcomputed tomography, and histologic analysis. RESULTS: Spinal fusion was observed in all animals in group I, II, and V rats at 8 weeks. None of the rats in groups III, IV, VI, and VII showed spinal fusion. The volumes of the new bone in the area between the L4 and L5 transverse processes were greater in group I rats than in group II, and V rats with a significant difference. CONCLUSION: BMP-2-producing RBMCs developed using lentiviral gene transfer induced more abundant bone within the fusion mass than the RBMCs transduced via adenoviral gene transfer and recombinant protein therapy.

Comparison of human mesenchymal stem cells derived from adipose tissue and bone marrow for ex vivo gene therapy in rat spinal fusion model.

STUDY DESIGN: Rat spinal fusion model. OBJECTIVE: To compare the efficacy of human adipose tissue-derived mesenchymal stem cells (HATDMSCs) and human bone marrow-derived mesenchymal stem cells (HBMDMSCs) transduced with an adenovirus containing the cDNA for bone morphogenetic proteins (BMP)-2 for inducing spinal fusion in an athymic rat model. SUMMARY OF BACKGROUND DATA: Recombinant BMPs have successfully induced spinal fusion in clinical trials. However, large doses are required for adequate bone repair. Regional gene therapy may deliver proteins to specific anatomic sites more efficiently. Gene transfer techniques using HATDMSCs have recently been tested. METHODS: Spinal fusion was performed in rats with different treatments: Group I (n = 10) collagen sponge containing HATDMSCs transfected with adeno-BMP-2, Group II (n = 10) collagen sponge containing HBMDMSCs transfected with adeno-BMP-2, Group III (n = 10) collagen sponge containing recombinant BMP-2 (10 mug), Group IV (n = 6) collagen sponge containing HATDMSCs transfected with adeno-LacZ, Group V (n = 6) collagen sponge containing HBMDMSCs transfected with adeno-LacZ, and Group VI (n = 6) collagen sponge alone. Radiographs were obtained at 4, 6, and 8 weeks. After sacrifice, the rat spines were assessed by manual palpation, microcomputed tomography, and histologic analysis. RESULTS: At 8 weeks, spinal fusion was observed in all Groups I, II, and III rats. 75% (15 of 20) of the gene therapy treatment animals (Groups I and II rats) had spontaneous extension of the fusion to a second level. No Groups IV, V, and VI rats developed fusion. New bone volume was significantly greater in Groups I and II than in Group VI. CONCLUSION: HATDMSCs transfected with adeno-BMP-2 induce abundant bone formation and have a similar posterolateral spinal fusion in rats as similarly genetically modified HBMDMSCs. Both are potential strategies for spinal fusion and may be a more efficient method of obtaining spinal fusion over currently used grafting substances.

Full-length bovine spp24 [spp24 (24-203)] inhibits BMP-2 induced bone formation.

Secreted phosphoprotein 24 kDa (spp24) is a bone matrix protein. It contains a TGF-beta receptor II homology 1 (TRH1) domain. A cyclic, synthetic 19 amino acid peptide (bone morphogenetic protein binding peptide or BBP) based on the sequence of the TRH1 domain enhances BMP-2 induced osteogenesis. Many observations suggest that different size forms of this protein have very different effects (inhibiting or enhancing) on BMP-2 induced osteogenesis. Using the stable recombinant Met(His)(6)-tagged secretory form of full-length (fl) bovine spp24 [Met(His)(6)-spp24 (residues 24-203)] and transgenic (TG) mice expressing fl bovine spp24 (residues 1-203), we have demonstrated that spp24 inhibits BMP-2 induced bone formation. The effects of Met(His)(6)-spp24 (24-203) were determined in the ectopic bone-forming bioassay in male mice. Implantation of 5 microg of BMP-2 stimulated bone formation, assessed densitometrically as bone area and mineral content. When Met(His)(6)-spp24 (24-203) was implanted with BMP-2, it elicited a dose-dependent decrease in BMP-2-medicated ectopic bone formation. When added at a 50-fold excess (w/w), Met(His)(6)-spp24 (24-203) completely ablated the effects of BMP-2, while addition of a 10-fold excess had no effect. Constitutive expression of fl bovine spp24 (1-203) under the control of the osteocalcin promoter in TG female mice reduced femoral and vertebral bone mineral density at 3 months of age and reduced femoral BMD at 8 months of age, but had no effects in male mice, which can exhibit less osteocalcin-promoter driven gene transcription than females. Histomorphometric analysis demonstrated that bone volume and trabecular thickness were lower in TG female mice at 3 months of age than in sex- and age-matched wild type (WT) controls. Thus, fl spp24 and its secretory isoform (Met(His)(6)-spp24 [24-203]), which contain a BMP-binding or TRH1 motif, inhibit ectopic bone formation in male mice and adversely affects BMD and histological parameters related to bone mass and formation in female mice expressing the human transgene. Under these conditions, fl spp24 acts as a BMP antagonist in vivo.