Bone morphogenetic proteins (BMPs) are currently approved for spinal fusion, tibial fracture repair, and maxillofacial bone regeneration. However, BMP pleiotropism, paradoxical activities on precursor cells, and unexpected side effects at local and ectopic sites may limit their usage. Thus, the need remains for alternative osteoinductive factors that provide more bone-specific activities with fewer adverse effects. Nell-1 [Nel-like molecule-1; Nel (a protein highly expressed in neural tissue encoding epidermal growth factor like domain)] is a novel osteogenic protein believed to specifically target cells committed to the osteogenic lineage. The objective of this project is to incorporate Nell-1 into a moldable putty carrier that can adapt to bony defects and deliver Nell-1 to the local microenvironment. We show here that moldability can be achieved by mixing hyaluronan hydrogel with two types of particles: demineralized bone powder for osteoconductivity, and biomimetic apatite-coated alginate/chitosan microparticles for controlled Nell-1 delivery. Besides enhancing overall osteoconductivity of the carrier, the biomimetic apatite coating also provides a more sustained release (∼ 15% cumulative release over 30 days) and greatly reduces the initial burst release that is observed with non-coated alginate/chitosan microparticles (∼ 40% release after 1 day). The efficacy of Nell-1 delivery from these carriers was evaluated in a rat spinal fusion model against Nell-free carriers as controls. 4 weeks post-implantation, Nell-1 enhanced spinal fusion rates as measured by manual palpation, radiographs, high resolution micro-computerized tomography (μCT), and histology. This moldable putty carrier system appears to be a suitable carrier for promoting osteogenesis, and will be further evaluated in larger animal models over longer periods to follow the remodeling of the regenerated bone.

Bone morphogenetic proteins (BMPs) are widely used as bone graft substitutes in spinal fusion, but are associated with numerous adverse effects. The growth factor Nel-like molecule-1 (Nell-1) is mechanistically distinct from BMPs and can minimize complications associated with BMP therapies. This study evaluates the efficacy of Nell-1 combined with demineralized bone matrix (DBM) as a novel bone graft material for interbody spine fusion using sheep, a phylogenetically advanced animal with biomechanical similarities to human spine. Nell-1 + sheep DBM or Nell-1 + heat-inactivated DBM (inDBM) (to determine the osteogenic effect of residual growth factors in DBM) were implanted in surgical sites as follows: (1) DBM only (control) (n = 8); (2) DBM + 0.3 mg/mL Nell-1 (n = 8); (3) DBM + 0.6 mg/mL Nell-1 (n = 8); (4) inDBM only (control) (n = 4); (5) inDBM + 0.3 mg/mL Nell-1 (n = 4); (6) inDBM + 0.6 mg/mL Nell-1 (n = 4). Fusion was assessed by computed tomography, microcomputed tomography, and histology. One hundred percent fusion was achieved by 3 months in the DBM + 0.6 mg/mL Nell-1 group and by 4 months in the inDBM + 0.6 mg/mL Nell-1 group; bone volume and mineral density were increased by 58% and 47%, respectively. These fusion rates are comparable to published reports on BMP-2 or autograft bone efficacy in sheep. Nell-1 is an independently potent osteogenic molecule that is efficacious and easily applied when combined with DBM.

Nell-1 (Nel-like molecule-1; Nel: protein strongly expressed in neural tissue containing epidermal growth factorlike domain) is a promising osteoblast-specific growth factor for osteoinductive therapies that may circumvent adverse effects, such as nonspecific function and ectopic bone formation, associated with more established osteogenic growth factors such as bone morphogenetic proteins. Beta-tricalcium phosphate (b-TCP), an osteoconductive, biodegradable ceramic biomaterial, has been used successfully to deliver osteoinducers for bone regeneration. The aim of this study was to develop a carrier system for efficiently delivering biologically active Nell-1 protein. After a 40% initial burst release, b-TCP particles retained the majority of adsorbed Nell-1 protein in vitro. To test this system in vivo, L4/L5 spinal fusion was performed in three groups of rats (n ¼ 8 each): (1) 5 mg Nell-1 in b-TCP/demineralized bone matrix putty (DBX); (2) 2.5 mg Nell-1 in b-TCP/DBX; (3) b-TCP/DBX only. Fusion was assessed by radiography, palpation, microcomputed tomography, and histological analysis. After 4 weeks, 75% of Nell-1-treated animals exhibited fusion, with a significant increase in new bone volume, whereas only 25% of Nell-free control animals exhibited fusion. Our findings suggest that b-TCP/DBX can increase both the biochemical stability and biological efficiency of Nell-1 protein.

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