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Nanoparticulate Mineral Content In Collagen Scaffolds Increases Osteogenesis Independent Of Exogenous BMP-2 In Rabbit Bone Marrow Stromal Cells
Xiaoyan Ren, MD, PhD1, Justin Maxhimer, MD1, Gina Farias-Eisner, MD1, Daniel W. Weisgerber, BA2, David Bischoff, PhD3, Michael S. Lewis, MD, PhD3, Victor Tu, BA1, Dean T. Yamaguchi, MD, PhD1, Timothy A. Miller, MD1, Brendan A.C. Harley, ScD2, Justine C. Lee, MD, PhD1.
1University of California Los Angeles, Los Angeles, CA, USA, 2University of Illinois Urbana Champaign, Urbana, IL, USA, 3Greater Los Angeles VA Healthcare System, Los Angeles, CA, USA.

PURPOSE:
The success of tissue engineering for bone regeneration depends on the optimal interplay of scaffold technology, growth factors, and cellular material in a deliverable fashion. One barrier to clinical translation is the variable side effects of exogenous growth factors delivered at high concentrations. In this work, we describe a novel mineralized collagen glycosaminoglycan (MC-GAG) scaffold that imparts efficient osteogenesis of rabbit bone marrow stromal cells (rBMSCs) without additional BMP-2 stimulation.
METHODS:
rBMSCs harvested from long bones were cultured in osteogenic media on non-mineralized collagen glycosaminoglycan (Col-GAG) and MC-GAG scaffolds. Gene and protein expression were measured using quantitative real time RT-PCR and western blot analysis. Scaffolds were subjected to histochemical and micro-computed tomographic analyses for mineralization.
RESULTS:
Osteogenic gene expression was found to increase with BMP-2 stimulation at early timepoints. However, as osteogenesis progressed, mineralization of scaffolds via Alizarin red staining and micro-CT scanning demonstrated that MC-GAG was sufficient to result in statistically significant differences in mineralization of rBMSCs in the absence of BMP-2. To investigate the intracellular signaling pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Western blot analysis demonstrated a constitutive Smad1/5 phosphorylation in MC-GAG scaffolds that was slightly increased in response to BMP-2 only at the earliest of timepoints. In contrast, ERK1/2 phosphorylation was decreased in MC-GAG scaffolds. On immunohistochemistry, phosphorylated Smad1/5 demonstrated nuclear translocation.
CONCLUSION:
Mineral content of collagen-GAG scaffolds induces osteogenesis of rabbit BMSCs without exogenous BMP-2 stimulation via a constitutive Smad1/5 activation.


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