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.

Keywords: Bioactive factors; Biomaterial scaffold; Chondrogenesis; Osteoarthritis; Stem cells.

Figure 1. Flow cytometry result of specific ADSC cell surface markers.

Cell expressed positive markers (A) CD90, (B) CD73, (C) CD 105, and (D) negative marker CD45, CD34, CD11b, CD19, HLA-DR.

Figure 2. Multipotency evaluation of ADSC.

(A) Alcian Blue staining, (B) Alizarin Red and (C) Oil Red O in ADSC culture which was induced with differentiation induction medium, and (D) non-staining group. Black arrow showed positive result from each staining group.

Figure 3. Growth curve of ADSC on scaffold in different silk fibroin concentration.

Figure 4. Growth curve of ADSC on scaffolds that have different pore size.

Figure 5. SEM image represented morphology of scaffold.

Scaffold was made from 12% w/v silk fibroin and had 500 µm pore size (Judawisastra & Wibowo, 2017). (l—l) represents pore size.

Figure 6. Morphology of ADSC on scaffold made from 12% w/v silk fibroin and had 500 µm pore size.

Single cell was on (A) day 1 and (B) day 21. Cell population was on (C) day 1 and (D) day 21. Red arrow shows cytoplasm extension, called filopodia. White arrow and the area marked with yellow stripe line show the area covered by cells.

Figure 7. Growth curve of ADSC on scaffold made from 12% w/v Silk Fibroin and had 500 µm pore size.

Figure 8. ADSC population observed on day 21.

There are two types of cell population in (A) monolayer formation and (B) aggregation.

Figure 9. Growth curve of ADSC in L-Ascorbic Acid (LAA) supplemented medium in various concentrations.

Figure 10. Graph of blue colour intensity xomparison from Alcian Blue staining in various LAA concentrations.

Figure 11. Growth curve of ADSC in various Platelet Rich Plasma (PRP) concentration of medium.

Figure 12. Graph of blue colour intensity comparison from Alcian Blue staining in various PRP concentrations.

Figure 13. Graph of glycosaminoglycan (GAG) content in ADSC cultured on scaffold in 50 µg/mL LAA and 10% PRP supplemented medium.