Chondrogenic differentiation of adipose-derived mesenchymal stem cells induced by L-ascorbic acid and platelet rich plasma on silk fibroin scaffold

Abstract

Articular cartilage is an avascular tissue with limited regenerative property. Therefore, a defect or trauma in articular cartilage due to disease or accident can lead to progressive tissue deterioration. Cartilage tissue engineering, by replacing defective cartilage tissue, is a method for repairing such a problem. In this research, three main aspects—cell, biomaterial scaffold, and bioactive factors—that support tissue engineering study were optimized. Adipose-derived mesenchymal stem cells (ADSC) that become cartilage were grown in an optimized growth medium supplemented with either platelet rich plasma (PRP) or L-ascorbic acid (LAA). As the characterization result, the ADSC used in this experiment could be classified as Mesenchymal Stem Cell (MSC) based on multipotency analysis and cell surface marker analysis. The biomaterial scaffold was fabricated from the Bombyx morii cocoon using silk fibroin by salt leaching method and was engineered to form different sizes of pores to provide optimized support for cell adhesion and growth. Biocompatibility and cytotoxicity evaluation was done using MTT assay to optimize silk fibroin concentration and pore size. Characterized ADSC were grown on the optimized scaffold. LAA and PRP were chosen as bioactive factors to induce ADSC differentiation to become chondrocytes. The concentration optimization of LAA and PRP was analyzed by cell proliferation using MTT assay and chondrogenic differentiation by measuring glycosaminoglycan (GAG) using Alcian Blue at 605 nm wavelength. The optimum silk fibroin concentration, pore size, LAA concentration, and PRP concentration were used to grow and differentiate characterized ADSC for 7, 14, and 21 days. The cell morphology on the scaffold was analyzed using a scanning electron microscope (SEM). The result showed that the ADSC could adhere on plastic, express specific cell surface markers (CD73, CD90, and CD105), and could be differentiated into three types of mature cells. The silk fibroin scaffold made from 12% w/v concentration formed a 500 µm pore diameter (SEM analysis), and was shown by MTT assay to be biocompatible and to facilitate cell growth. The optimum concentrations of the bioactive factors LAA and PRP were 50 µg/mL and 10%, respectively. GAG analysis with Alcian Blue staining suggested that PRP induction medium and LAA induction medium on 12% w/v scaffold could effectively promote not only cell adhesion and cell proliferation but also chondrogenic differentiation of ADSC within 21 days of culture. Therefore, this study provides a new approach to articular tissue engineering with a combination of ADSC as cell source, LAA and PRP as bioactive factors, and silk fibroin as a biocompatible and biodegradable scaffold.

Chondrogenic differentiation of Wharton’s Jelly mesenchymal stem cells on silk spidroin-fibroin mix scaffold supplemented with L-ascorbic acid and platelet rich plasma

“…Overall, cell viability was slightly decreased from day 1 to day 5 and then increased steadily from day 7 until day 14. Initial decrease of cell viability of cells grown on silk scaffold was also observed on Antheraea mylitta silk fibroin scaffold 14 and B.mori silk fibroin scaffold 16,36 . This result was likely caused by cell adaptation and migration on the porous structure of the scaffold 37 .…”

“…The overall pore size formed on scaffold was between the range of NaCl particles used (400-570 μm). Several studies have reported that scaffold with larger pore size; 370-400 μm 27 , 400-500 μm 16,28 were able to support MSCs chondrogenesis better than smaller pore size, mainly due to higher permeability of gases and nutrition from culture medium 29 .…”

“…Addition of LAA at 250 mM (64,1 μg/ml) has been reported to increase proliferation of umbilical cord blood mesenchymal stem cells (UCB-MSC), while high concentration of LAA decreases cell proliferation 43 . Previous studies have also reported www.nature.com/scientificreports/ that supplementation of 10% PRP 16,44,45 or 50 μg/ml LAA 11,34 in the medium were the optimal concentration to support proliferation of MSC. Cells with higher proliferation rate before directed differentiation is known to have correlation with superior chondrogenic differentiation capacity 46 .…”

Mesenchymal stem cells for cartilage regeneration

“…Moreover, proteoglycan production also increased. 49 Parathyroid hormone-related protein (PTHrP) has been reported to perform fundamental roles in cartilage homeostasis, maintaining the chondrocyte phenotype. The effect of PTHrP on MSCs mainly depends on the pattern of application.…”

A novel crosslinker‐free technique toward the fabrication of collagen microspheres

“…In future work, we propose the use of CMs as possible bioactive agent carriers and cell transporters. With extensive work performed looking into adipose-derived mesenchymal stem cells (ADSC) on chondrocyte cells and on the differentiation into chondrocyte cells we feel that CMs are uniquely suited as an injectable transporter for ADSCs into osteoarthritc tissues due to their injectable size and their affinity core cellular adherence(Barlian, Judawisastra, Alfarafisa, Wibowo, & Rosadi, 2018;Manferdini et al, 2013;Zhou et al, 2019). We would like to look at the integration of CMs and ADSCs in a traditional impeller bioreactor and look into the effect of CM-ADSCs on osteoarthritic chondrocyte cells or CM-differentiated ADSCs into chondrocyte cells on osteoarthric articular cartilage.…”