Synergistic Effect of NELL-1 and an Ultra-Low Dose of BMP-2 on Spinal Fusion.

Bone morphogenetic protein 2 (BMP-2) is widely used in spinal fusion but it can cause adverse effects such as ectopic bone and adipose tissue in vivo. Neural epidermal growth factor like-like molecule-1 (NELL-1) has been shown to suppress BMP-2-induced adverse effects. However, no optimum carriers that control both NELL-1 and BMP-2 releases to elicit long-term bioactivity have been developed. In this study, we employed polyelectrolyte complex (PEC) as a control release carrier for NELL-1 and BMP-2. An ultra-low dose of BMP-2 synergistically functioned with NELL-1 on bone marrow mesenchymal stem cells osteogenic differentiation with greater mineralization in vitro. The osteoinductive ability of NELL-1 and an ultra-low dose of BMP-2 in PEC was investigated in rat posterolateral spinal fusion. Our results showed increased fusion rate, bone architecture, and improved bone stiffness at 8 weeks after surgery in the combination groups compared with NELL-1 or BMP-2 alone. Moreover, the formation of ectopic bone and adipose tissue was negligible in all the PEC groups. In summary, dual delivery of NELL-1 and an ultra-low dose of BMP-2 in the PEC control release carrier has greater fusion efficiency compared with BMP-2 alone and could potentially be a better alternative to the currently used BMP-2 treatments for spinal fusion. Impact Statement In this study, polyelectrolyte complex was used to absorb neural epidermal growth factor like-like molecule-1 (NELL-1) and bone morphogenetic protein 2 (BMP-2) to achieve controlled dual release. The addition of NELL-1 significantly reduced the effective dose of BMP-2 to 2.5% of its conventional dose in absorbable collagen sponge, to produce solid spinal fusion without significant adverse effects. This study was the first to identify the efficacy of combination NELL-1 and BMP-2 in a control release carrier in spinal fusion, which could be potentially used clinically to increase fusion rate and avoid the adverse effects commonly associated with conventional BMP-2.

Bone Regeneration by Controlled Release of Bone Morphogenetic Protein-2: A Rabbit Spinal Fusion Chamber Molecular Study.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used in spine fusion surgery. However, high doses of rhBMP-2 delivered with absorbable collagen sponge (ACS) have led to inflammation-related adverse conditions. Polyelectrolyte complex (PEC) control release carrier can substantially reduce the rhBMP-2 dose and complication without compromising fusion. The molecular events underlying controlled release and their effects on spinal fusion remain unknown. In this study, a rabbit interbody spinal fusion chamber was designed to provide a controlled environment for profiling molecular events during the fusion process. Study groups included Group 1, PEC with 100 µg rhBMP-2; Group 2, ACS with 100 µg rhBMP-2; Group 3, ACS with 300 µg rhBMP-2; Group 4, autologous bone graft; and Group 5, empty chamber. Manual palpation, microcomputed tomography, and histological analysis showed that Group 1 and 3 achieved bone fusion, while the other groups showed no signs of fusion. Gene expression profiling showed robust induction of osteogenic markers in Groups 1 and 3, with modulated early induction of inflammatory genes in the PEC group. Delivery of 100 µg rhBMP-2 with ACS (Group 2) resulted in less upregulation of osteogenic genes, increased inflammatory genes expression, and upregulation of osteoclastic genes compared to Group 1. These results suggest that the manner of BMP-2 release at the interbody spinal defect site could dictate the balance of in-situ osteogenic and antiosteogenic activities, affecting fusion outcomes. The molecular evidence supports PEC for sustained release of BMP-2 for spinal interbody fusion, and the feasibility of employing this novel interbody spinal fusion chamber for future molecular studies. Impact Statement A radiolucent rabbit interbody spinal fusion chamber was developed to study the molecular events during spinal fusion process. The gene expression profile suggests that control release of bone morphogenetic protein-2 (BMP-2) resulted in lower inflammatory and osteoclastic activities, but elicited higher osteogenic activities, while burst release of BMP-2 resulted in predominantly inflammation and osteoclastogenesis with minimum osteogenic activity. This study provides the molecular evidence that underscores the regeneration outcomes from the two different BMP-2 delivery systems. This spinal fusion chamber could be used for future molecular studies to optimize carrier design for spinal fusion.

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

BACKGROUND CONTEXT: The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. PURPOSE: We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. STUDY DESIGN/SETTING: A prospective study using a rodent model of posterolateral spinal fusion was carried out. PATIENT SAMPLE: Thirty-six syngeneic Fischer rats comprised the patient sample. METHODS: Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. RESULTS: Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and ß-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). CONCLUSION: In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.

Novel Protamine-Based Polyelectrolyte Carrier Enhances Low-Dose rhBMP-2 in Posterolateral Spinal Fusion.

STUDY DESIGN: A rodent posterolateral spinal fusion model. OBJECTIVE: This study evaluated a protamine-based polyelectrolyte complex (PEC) developed to use heparin in enhancing the biological activity of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) in spinal fusion. SUMMARY OF BACKGROUND DATA: rhBMP-2 is commonly regarded as the most potent bone-inducing molecule. However, poor pharmacokinetics and short in vivo half-life means that large amounts of the bioactive growth factor are required for consistent clinical outcomes. This has been associated with a number of adverse tissue reactions including seroma and heterotopic ossification. Glycosaminoglycans including heparin are known to stabilize rhBMP-2 bioactivity. Previous studies with poly-L-lysine (PLL) and heparin-based PEC carriers amplified the therapeutic efficacy of low-dose BMP-2. However, questions remained on the eventual clinical applicability of relatively cytotoxic PLL. In the present study, a protamine-based PEC carrier was designed to further enhance the safety and efficacy of BMP-2 by delivering lower dose within the therapeutic window. METHODS: A polyelectrolyte shell was deposited on the surface of alginate microbead templates using the polycation (protamine)/polyanion (heparin) layer-by-layer polyelectrolyte self-assembly protocol. rhBMP-2 was loaded onto the outermost layer via heparin affinity binding. Loading and release of rhBMP-2 were evaluated in vitro. The bone-inductive ability of 20-fold reduction of rhBMP-2 with the different carrier vehicle was evaluated using a posterolateral spinal fusion model in rats. RESULTS: In vitro uptake and release analysis, protamine-based PEC showed higher uptake and significantly enhanced control release than PLL-based PEC (P < 0.05). In vivo implantation with protamine-based and PLL-based PEC showed better fusion performances than absorbable collagen sponge-delivered same dose of rhBMP-2, and negative control group through manual palpation, micro-computed tomography, and histological analyses. CONCLUSION: Solid posterolateral spinal fusion was achieved with 20-fold reduction of rhBMP-2 when delivered using protamine-based PEC carrier in the rat posterolateral spinal fusion model. LEVEL OF EVIDENCE: N/A.