Adipose tissue has attracted great interest as an alternative stem cell source with the same multipotent properties as mesenchymal stem cells (MSCs). However, the stromal vascular fraction (SVF) of adipose tissue contains a heterogeneous population of cells, which hamper its regenerative ability. We previously purified human adipose-derived Perivascular Stem Cells (PSCs) from SVF and showed that PSCs were capable of making significantly more bone compared to the SVF when implanted in the mouse muscle. Objectives: To evaluate the effectiveness of PSCs in the healing of mouse critical-size calvarial defects. Methods: Critical size (3mm) defects were created in the parietal bone of 50 adult SCID mice. Defects were either treated with an apatite coated PLGA scaffold alone, scaffold with human SVF or scaffold with human PSCs. Healing was monitored by live MicroCT scans at 0,2,4 and 6 weeks post injury. At 8 weeks postoperative, subjects were sacrificed and high-resolution microCT scanning was performed to observe total bone formation followed by histological analysis. Immunohistochemistry was performed to study expression of key osteogenic markers such as Osteopontin (OPN), Osteocalcin (OCN), Bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF). Results: PSCs resulted in a significantly more healing of the defect sites (69%) as opposed to 25% healing in the SVF models at 6 weeks. H&E staining revealed that the PSC group showed more and higher quality new bone in comparison with the SVF samples. Immunohistochemical staining of the defect sites showed that PSCs induced significant increases in osteogenic growth factor elaboration (BMP2, VEGF), significant evidence of OPN+, OCN+ osteogenesis, and ultimately significant calvarial defect re-ossification. Conclusion: Our results indicate that adipose-derived PSCs are a new cell source for future efforts in skeletal regenerative medicine. Further research to investigate PSCs induced healing in larger animals is needed to further unfold the haling potential of PSCs and understand its mechanism of action.

Previous studies demonstrated that NELL-1 promoted osteogenic differentiation of bone marrowl stromal cells (BMSCs). BMSC differentiation into osteoblasts and the signaling pathways involved are poorly understood. We hypothesized that NELL-1 binds to Integrin and promotes osteogenic differentiation through FAK signaling pathway.

To test this hypothesis, we examined the physical interaction of NELL-1 with Integrin beta-1 and inhibition of NELL-1's cell binding and adhesion using knockdown of Integrin beta-1 siRNA. We also assayed for phosphorylation of FAK as well as expression of osteogenic differentiation markers (OCN, OPN, ALP staining) and adiopogenic differentiation markers (ORO staining) in BMSC.

We found that NELL-1 physically binds to Integrin beta-1 and Integrin beta-1 siRNA knockdown reduced NELL-1 cell binding significantly. NELL-1 stimulated expression of Integrin beta-1 and activated FAK phosphorylation. Finally, NELL-1 enhanced expression of osteogenic genes, whereas it inhibited expression of adiopogenic genes. Taken together, our results suggest that NELL-1 may initiates transduction signals through Integrin, and FAK pathway may play an important role in regulating NELL-1 induced osteogenic and adipogenic differentiation of BMSC.

Bone health can be jeopardized by diseases such as osteoporosis, or by special environmental conditions like microgravity. Both conditions result in reduced bone mineral density (BMD) and an increased risk of bone fractures. NELL-1, a protein that binds integrin β1 and consequently induces Wnt/β-catenin, exhibits both bone-forming and anti-osteoclastic effects. Systemic delivery of NELL-1 via intravenous (IV) injections increases not only murine BMD and percent bone volume, but also new femoral bone formation. However, systemic delivery of NELL-1 under microgravity conditions in future spaceflight experimental studies would be more practical if it were accomplished through the intraperitoneal (IP) route. It is less technique sensitive, safer, and more patient friendly for eventual use by humans. The aim of this study was to experiment the intraperitoneal delivery of NELL-1 to reverse osteoporotic bone loss in mice under normal gravity conditions. First, a post-menopausal osteoporotic model (OVX) was successfully reproduced in BALB/c mice and a significant reduction in BMD was evident by DXA and microCT analyses. Second, two IP dosages of NELL-1 were injected into the OVX mice, resulting in reversed bone loss and positive gains in BMD and BV/TV. In conclusion, NELL-1 can be effectively delivered by systemic intraperitoneal injections to reverse osteoporotic bone loss and regulate bone homeostasis.

Vertebral Implantation of NELL-1 Enhances Bone Formation In Osteoporotic Sheep

By

Michael Ming-Ying Chiang

Master of Science in Oral Biology

University of California, Los Angeles, 2013

Professor Shen Hu, Co-Chair

Professor Xinli Zhang, Co-Chair

Background: Vertebral compression fractures related to osteoporosis greatly afflict the aging population. The most commonly used therapy today is balloon kyphoplasty, a procedure that involves the inflation of a balloon and injection of cement such as polymethyl methacrylate. However, this treatment is far from ideal as it is associated with significant side effects, including local toxicity and rare systemic complications. NELL-1, a secreted osteoinductive factor that possesses both pro-osteogenic and anti-resorptive properties, is a promising candidate for an alternative to current treatment modalities. The use of NELL-1 in balloon kyphoplasty might improve post surgical outcome for over 10 million osteoporotic American who are susceptible to vertebral compression fractures. The present study utilizes the pro-osteogenic properties of recombinant human NELL-1 (rhNELL-1) in lumbar spine balloon kyphoplasty using an osteoporotic sheep model.

Methods: Osteoporosis in N=8 sheep was induced through ovariectomy, dietary depletion of calcium and vitamin D, and steroid administration. After osteoporotic induction was confirmed by dual-energy X-ray absorptiometry (DXA), balloon kyphoplasty was performed in N=6 sheep with significant osteopenia. Sheep were randomly implanted with the control vehicle, comprised of hyaluronic acid (HA), hydroxyapatite, and β-tricalcium phosphate (β-TCP), or the treatment material of rhNELL-1 protein lyophilized onto β-TCP mixed with HA. Analysis of lumbar spine healing was performed by radiographic, histologic, and biomechanical testing.

Results: rhNELL-1 treatment significantly increased lumbar spine bone formation, as determined by bone mineral density, fractional bone volume, and mean cortical width by micro-computed tomography. Histological analysis revealed a significant increase in bone area as well as osteoblast number and decrease in osteoclast number around the implant site. Biomechanical properties of trabecular bone were analyzed using Finite Element Analysis (FEA), demonstrating that rhNELL-1-treatment resulted in a significantly more stress-resistant composition.

Conclusion: Our findings suggest rhNELL-1-based balloon kyphoplasty successfully improved cortical and cancellous bone regeneration in the lumbar spine of osteoporotic sheep. Thus, rhNELL-1 based balloon kyphoplasty represents a potential new anabolic, anti-resorptive local therapy for treating osteoporotic bone loss and/or fractures.

Background: Fibromodulin (FMOD) is a small leucine-rich proteoglycan required for scarless fetal cutaneous wound repair. Interestingly, increased FMOD levels have been correlated with decreased transforming growth factor (TGF)-β1 expression in multiple fetal and adult rodent models. Our previous studies demonstrated that FMOD deficiency in adult animals results in delayed wound closure and increased scar size accompanied by loose package collagen fiber networks with increased fibril diameter. In addition, we found that FMOD modulates in vitro expression and activities of TGF-β ligands in an isoform-specific manner. In this study, we investigated the temporospatial distribution of TGF-β ligands and receptors in both adult FMOD-null and wild-type (WT) mouse wounds to further elucidate how FMOD coordinates TGF-β bioactivity to promote proper cutaneous wound healing.

Methods: Expression profiles of TGF-β ligands and receptors in FMOD-null and WT mice were compared by immunohistochemical staining and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using a full-thickness, primary intention wound closure model.

Results: During the inflammatory stage, elevated inflammatory infiltration accompanied by increased type I TGF-β receptor levels in individual inflammatory cells was observed in FMOD-null wounds. This increased inflammation was correlated with accelerated epithelial migration during the proliferative stage. On the other hand, significantly more robust expression of TGF-β3 and TGF-β receptors in FMOD-null wounds during the proliferative stage was associated with delayed dermal cell migration and proliferation, which led to postponed granulation tissue formation and wound closure and increased scar size. Compared with WT controls, expression of TGF-β ligands and receptors by FMOD-null dermal cells was markedly reduced during the remodeling stage, which may have contributed to the declined collagen synthesis capability and unordinary collagen architecture.

Conclusion: This study demonstrates that a single missing gene, FMOD, leads to conspicuous alternations in TGF-β ligand and receptor expression at all stages of wound repair in various cell types. Therefore, FMOD critically coordinates temporospatial distribution of TGF-β ligands and receptors in vivo, suggesting that FMOD modulates TGF-β bioactivity in a complex way beyond simple physical binding to promote proper wound healing.