The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells

Annulus fibrosus （AF） tissue engineering has recently received increasing attention as a treatment for intervertebral disc 0VD） degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibroua polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived-stem/progenitor cells （AFSCs）. Random fibrous scaffolds, also prepared via electrospinningy were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of coUagen-I and aggrecan. The gene expression and protein production of coUagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.

Comparison of phenotype characteristics of rat annulus fibrosus cells cultured on flexible silicone membrane and in plastic plate

Objective：To compare the phenotype characteristics of rat annulus fibrosus （AF） cells cultured on flexible silicone membranes and those in plastic plates. Methods：The morphology of AF cells cultured in different suhstrates was examined. Proteoglycan was stained by toluidine blue. Contents of collagen type Ⅰ, collagen type Ⅰ and aggrecan mRNAs were determined by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of integrin β1 was monitored by flow cytometry. By using propidium iodide （PI）, the cell cycle in AF cells was analyzed. Cell adhesion to silicone membrane was also measured. Results：The AF cells cultured on different suhstrates were morphologically undistinguishable. Toluidine blue staining showed that there was also no difference between AF cells cultured on these 2 substrates. They still had the same expression levels of collagen type Ⅰ , collagen type Ⅰ, aggrecan mRNAs, and integrin β1. No significant difference was observed in the distribution of the cell cycle. AF cells grew well on silicone membrane. Conclusion：AF cells cultured on flexible silicone membrane maintain the stability of phenotype and may he appropriate for further studying the metabolic responses to mechanical stimuli at the cellular level.

Role of calcium signaling in down-regulation of aggrecan induced by cyclic tensile strain in annulus fibrosus cells

Objective:To study the role of intracellular calcium signal pathway in the down-regulation of aggrecan induced by cyclic tensile strain in the annulus fibrosus cells. Methods:The expression of aggrecan mRNA and core protein were respectively detected with RT-PCR and western blot after the channels transmitting calcium ions were blocked with EGTA, gadolinium and verapamil. Results :EGTA. gadolinium and verapamil partially prevented the effects of cyclic tensile strain on the expression of aggrecan in annulus fibrosus cells. Conclusion:The calcium signaling is involved in the down-regulation of proteoglycan resulting from cyclic tensile strain in the annulus fibrosus cells.

Decoding the annulus fibrosus cell atlas by scRNA-seq to develop an inducible composite hydrogel:A novel strategy for disc reconstruction

Low back pain is one of the most serious public health problems worldwide and the major clinical manifestation of intervertebral disc degeneration(IVDD).The key pathological change during IVDD is dysfunction of the annulus fibrosus(AF).However,due to the lack of an in-depth understanding of AF biology,the methods to reconstruct the AF are very limited.In this study,the mice AF cell atlas were decoded by single-cell RNA sequencing to provide a guide for AF reconstruction.The results first identify a new population of AF cells,fibrochondrocyte-like AF cells,which synthesize both collagen Ⅰ and collagen Ⅱ and are potential functional cells for AF reconstruction.According to the dual features of the AF extracellular matrix,a composite hydrogel based on the acylation of methacrylated silk fibroin with methacrylated hyaluronic acid was produced.To obtain the ability to stimulate differentiation,the composite hydrogels were combined with a fibrochondrocyte-inducing supplement.Finally,reconstruction of the AF defects,by the novel AF stem cell-loaded composite hydrogel,could be observed,its amount of chondroid matrices recovered to 31.7% of AF aera which is significantly higher than that in other control groups.In summary,this study decodes the AF cell atlas,based on which a novel strategy for AF reconstruction is proposed.