Efficient Decellularization by Application of Moderate High Hydrostatic Pressure with Supercooling Pretreatment

Decellularized tissues are considered superior scaffolds for cell cultures, preserving the microstructure of native tissues and delivering many kinds of cytokines. High hydrostatic pressure (HHP) treatment could remove cells physically from biological tissues rather than chemical methods. However, there are some risks of inducing destruction or denaturation of extracellular matrices (ECMs) at an ultrahigh level of HHP. Therefore, efficient decellularization using moderate HHP is required to remove almost all cells simultaneously to suppress tissue damage. In this study, we proposed a novel decellularization method using a moderate HHP with supercooling pretreatment. To validate the decellularization method, a supercooling device was developed to incubate human dermal fibroblasts or collagen gels in a supercooled state. The cell suspension and collagen gels were subjected to 100, 150, and 200 MPa of HHP after supercooling pretreatment, respectively. After applying HHP, the viability and morphology of the cells and the collagen network structure of the gels were evaluated. The viability of cells decreased dramatically after HHP application with supercooling pretreatment, whereas the microstructures of collagen gels were preserved and cell adhesivity was retained after HHP application. In conclusion, it was revealed that supercooling pretreatment promoted the denaturation of the cell membrane to improve the efficacy of decellularization using static application of moderate HHP. Furthermore, it was demonstrated that the HHP with supercooling pretreatment did not degenerate and damage the microstructure in collagen gels.

Baicalein Alleviates Fibrosis and Inflammation in Scleroderma Through Regulating B Cell Abnormalities

Background Scleroderma (Systemic sclerosis, SSc) is an autoimmune disorder characterized by multisystem extensive fibrosis, vascular changes and immunological dysregulation. B cell abnormalities play an essential role in the fibrotic pathogenesis of scleroderma by promoting autoantibodies and cytokines release as well as modulating the inflammatory processes. Baicalein, a phenolic flavonoid derived from the Chinese herb Scutellaria baicalensis Georgi, has been used in the treatment of the pathological processes of various fibrotic and inflammatory diseases. Here, we aimed to investigate the effect of baicalein on the major pathological characteristics of SSc both in vitro and in vivo, including fibrosis, B cell abnormalities and inflammation. Methods The effect of baicalein on collagen accumulation and expression of fibrogenic markers in human dermal fibroblasts were analyzed. The antifibrotic features of baicalein and its mechanisms were investigated in the bleomycin (BLM)-induced dermal fibrosis mice model by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay and flow cytometry. Results 5-120 µM baicalein significantly abrogated total collagen deposition, decreased soluble collagen secretion, and inhibited the expression of various fibrogenesis molecules, including collagen(COL)1A1, COL1A2, COL3A1, connective tissue growth factor (CTGF), fibronectin, transforming growth factor β1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) in both TGF β1- and PDGF-induced human CCC-ESF-1 dermal fibroblasts. In BLM-induced dermal fibrosis mice model, 25-100 mg/kg baicalein markedly attenuated dermal thickness and collagen accumulation in dose-dependent manners. Baicalein reduced the proportion of B220+ B cells, while increased the percentage of CD27+ memory B cells in the spleen of BLM-induced mice. Consistent with reversing B cell abnormalities, baicalein treatment potently attenuated serum levels of cytokines(IL-1β, IL-2, IL-4, IL6, IL-17A, TNF-α), chemokines (MCP-1, MIP-1β) and autoantibodies (anti-Scl-70, anti-PM-Scl, anti-centromeres and anti-dsDNA). Conclusions Baicalein inhibits TGF-β1 and PDGF- mediated ECM accumulation in human dermal fibroblasts and alleviates the development of experimental dermal fibrosis by reversing B cell abnormalities, reducing autoantibody production and ameliorating inflammation. Baicalein may have the potential to be further developed as a therapeutic candidate against SSc.