Functional
fibrocartilage regeneration is a bottleneck during bone–tendon
healing, and the currently available tissue-engineering strategies
for fibrocartilage regeneration are insufficient because of a lack
of appropriate scaffold that can load large seeding-cells and induce
chondrogenesis of stem cells. The acellular fibrocartilage scaffold
(AFS) contains active growth factors as well as tissue-specific epitopes
for cell-matrix interactions, which make it a potential scaffold for
tissue-engineered fibrocartilage. A limitation to this scaffold is
that its low porosity inhibits cells loading and infiltration. Here,
inspired by book appearance, we sectioned native fibrocartilage tissue
(NFT) into book-shape to improve cells loading and infiltration, and
then decellularized with four protocols: (1) 2% SDS for 6-h, (2) 2%
SDS for 24-h, (3) 4 SDS for 6-h, (4) 4% SDS for 24-h, followed by
nuclease digestion. The optimal protocol was screened with respect
to microstructures, DNA residence, native ingredients reservation,
and chondrogenic inducibility of the AFS. In vitro studies demonstrated
that this screened scaffold is noncytotoxicity and low-immunogenicity,
allows adipose-derived stromal cells (ASCs) attachment and proliferation,
shows superior chondrogenic inducibility, and stimulates collagen
or glycosaminoglycans secretion. The underlying mechanism for this
chondrogenic inducibility may be related to hedgehog pathway activating.
Additionally, a novel pattern for fabricating tissue-engineered fibrocartilage
was developed to enlarge seeding-cells loading, namely, cell-sheets
sandwiched by book-shaped scaffold. In-vivo studies indicate that
this screened scaffold alone could induce endogenous cells to satisfactorily
regenerate fibrocartilage at 16-week, as characterized by fibrocartilaginous
extracellular matrix (ECM) deposition and good interface integration.
Interleaving this book-shaped AFS with autologous ASCs-sheets significantly
enhanced its ability to regenerate fibrocartilage. Cell tracking demonstrated
that fibrochondrocytes, osteoblasts, and osteocytes in the healing
interface at postoperative 8-week partly originated from the sandwiched
ASCs-sheets. On that basis, we propose the use of this book-shaped
AFS and cell sheet technique for fabricating tissue-engineered fibrocartilage
to improve bone–tendon healing.
The osteogenic differentiation of human bone mesenchymal stromal cells (BMSCs) has been considered as a central issue in fracture healing. Wnt signaling could promote BMSC osteogenic differentiation through inhibiting PPARγ. During atrophic nonunion, Wnt signaling-related factors, WNT5A and FZD3 proteins, were significantly reduced, along with downregulation of Runx2, ALP, and Collagen I and upregulation of PPARγ. Here, we performed a microarray analysis to identify differentially expressed miRNAs in atrophic nonunion tissues that were associated with Wnt signaling through targeting related factors. Of upregulated miRNAs, miR-381 overexpression could significantly inhibit the osteogenic differentiation in primary human BMSCs while increase in PPARγ protein level. Through binding to the 3′UTR of WNT5A and FZD3, miR-381 modulated the osteogenic differentiation via regulating β-catenin nucleus translocation. Moreover, PPARγ, an essential transcription factor inhibiting osteogenic differentiation, could bind to the promoter region of miR-381 to activate its expression. Taken together, PPARγ-induced miR-381 upregulation inhibits the osteogenic differentiation in human BMSCs through miR-381 downstream targets, WNT5A and FZD3, and β-catenin nucleus translocation in Wnt signaling. The in vivo study also proved that inhibition of miR-381 promoted the fracture healing. Our finding may provide a novel direction for atrophic nonunion treatment.
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