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Dura Mater Cells Stimulate Human Adipose-Derived Stromal Cells to Undergo Osteogenic Differentiation via Paracrine BMP Signaling in Skeletal Repair
Benjamin Levi, MD1, Emily R. Nelson, BS1, Jeong Hyun, MD1, Shuli Li, PhD1, Daniel Montoro, BS1, Min Lee, PhD2, George Commons, MD1, Michael T. Longaker, MD1.
1Stanford University, Stanford, CA, USA, 2UCLA, Los Angeles, CA, USA.
Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair in a calvarial defect are not fully understood. In this study, we examined the potential for dura mater cells to stimulate hASCs to enhance repair of a mouse calvarial defect.
hASCs were engrafted onto an osteoinductive scaffold into 4mm calvarial defects over an intact dura mater, over a surgically disrupted dura mater or over a permeable or semipermeable membrane, functioning to separate ASCs from the dura mater. Methods of analysis included microcomputed tomography, histology, in situ hybridization and qRT-PCR. Next, the in vitro interaction between hASCs and mouse dural cells was assessed by conditioned media and co-culture assays. Medium was supplemented with BMP modifiers, including anti BMP-2,and 4.
Significant calvarial healing was observed when hASC engrafted defects, in comparison to defects where the dura mater was removed or a barrier was placed between the dura and hASCs (*P < 0.05). Mouse dura mater cells enhanced hASC osteogenesis, including: 1) increased expression of bone markers in the middle of the defect away from the wound edge (or putative hASC:mOB junction) by in situ hybridization, as well as, 2) increased host mouse osteogenic gene expression by species-specific qRT-PCR. In vitro, dura mater cells produced BMP by Western blot. Using conditioned media or co-culture assays, dura mater cells stimulated hASC osteogenic differentiation, accompanied by activation of BMP signaling. BMP loaded scaffolds were able to rescue those defects with disrupted dura mater as shown by microCT and histology.
Human ASCs heal critical sized athymic mouse calvarial defects when in contact with the underlying dura mater. This is, at least in part, via stimulation of hASCs by the underlying dura mater cells. Our studies suggest that dura-derived BMP signaling may play a paracrine role in hASC skeletal repair and BMP loaded scaffolds can be used to augment healing in a calvarial defect with injured dura.
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