Mechanical Sensing Element PDLIM5 Promotes Osteogenesis of Human Fibroblasts by Affecting the Activity of Microfilaments.

Xiaolan Huang,Yan Peng,Jingxing Dai,Tingyu Fan,Yuchao Yang,Chujiang Xu,Jun Ouyang,Bing Sun,Shizhen Zhong,Kuanhai Wei,Rongmei Qu,Asmat Ullah Khan,Shutong Wu

doi:10.3390/biom11050759

Abstract

Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.

Highlights

Human skin fibroblasts (HSFs) were obtained from skin dermis, which were purchased from the cell bank of Zhongqiaoxinzhou Company, and human adipose stem cells were extracted from subcutaneous adipose tissue of healthy individuals, using the type I collagenase digestion method as described previously [40]: adipose tissue was digested at 37 ◦ C with 0.1% (w/v) type I collagenase to three times the volume of adipose tissue
To verify the ability of osteogenic differentiation of HSFs, human adipose stem cells (hASCs), which have been proved to be one of the representatives of mesenchymal stem cells with multiple differentiation functions and compared with human mesenchymal stem cells (hMSC), hASCs have a wide range of sources, are easy to obtain, simple separation, and are not restricted by ethics, were induced to indirectly observe the osteogenic ability of fibroblasts. hASCs and HSFs completely adhered to the culture dishes 24 h after inoculation
The osteogenic differentiation ability of HSFs and hASCs was confirmed by ALP staining, which showed that alkaline phosphatase staining increased with the increase of osteogenic induction days, and reached the peak on the 14th day (Figure 1D)

Summary

Introduction

The repair process of bone trauma diseases involves the use of stem cells [3,4,5], and osteoblasts play an important role in the healing process. Among the seed cells from many sources, studies have shown that human fibroblasts exhibit stem cell characteristics and can differentiate into chondrocytes, osteoblasts, and adipocytes [6,7]. Fibroblasts can further differentiate into osteoblasts and maintain a stable phenotype, and play a similar role in osteogenic differentiation as stem cells [8,9], which are widely used as an ideal source of cell seeds in bone tissue engineering [10,11].

Methods

Results

Conclusion