193 HYALURONIC ACID-GLYCIDYL METHACRYLATE HYDROGELS SUPPORT IN VITRO CHONDROGENIC DIFFERENTIATION OF PORCINE ADIPOSE-DERIVED STEM CELLS

Abstract

There is a great need for bioengineered cartilage because of the lack of medical or surgical therapies to improve articular cartilage healing. We hypothesised that porcine adipose-derived stem cells (pASC) can be induced to undergo chondrogenic differentiation within hyaluronic acid (HA) hydrogels. The objective of this study was to develop UV-curable pASC-laden HA hydrogels aimed at application in cartilage tissue engineering. HA was treated with glycidyl methacrylate (GM) to allow chemical gelation of the polymer upon exposure to UV light. 2% HAGM hydrogel was obtained by mixing HAGM with chondrogenic medium consisting of TGFβ, ascorbic acid, ITS+ premix (insulin, transferrin, selenous acid; Cat. No. 354352, BD Biosciences, Franklin Lakes, NJ), sodium pyruvate, and dexamethasone. Passage three-pASC were resuspended in 2% HAGM hydrogel with 2 × 10⁷ cells mL^–1. Twelve-and-one-half (12.5)-μL droplets (micromasses) of this suspension containing 250 000 pASC were placed in 24-well culture plates and incubated for 2 h at 37°C and 5% CO₂ to allow for cell attachment. Subsequently, the cell-laden hydrogels were cured with ~10 mW cm^–2 365-nm UV light for 10 min, covered with 500 μL of chondrogenic medium, and cultured for up to 11 days at 37°C and 5% CO₂. Additionally, pASC micromasses were cultured in chondrogenic medium without loading on 2% HAGM hydrogels as positive controls, and in non-chondrogenic DMEM as negative controls. Samples were collected at 4, 7, and 11 days in to culture for cryopreservation (for immunohistochemistry; IHC) and dimethylmethylene blue (DMMB) assay. IHC on day 11 of culture demonstrated the expression of cartilage specific proteins type-II collagen and aggrecan. On the basis of data from the DMMB assay, chondrogenic differentiation of pASC-laden micromasses in positive controls and 2% HAGM treatments were not different (P > 0.05). This indicates that ASC can produce cartilage equally well under both conditions, supporting the idea that HAGM may be used as a matrix for cartilage formation in vitro and possibly in vivo. In conclusion, using a micromass cell culture system, we demonstrated that 2% HAGM hydrogels support proliferation and chondrogenic differentiation of pASC. Further experiments testing different concentrations of HAGM and UV exposure levels, and larger sample numbers are warranted to further improve this procedure.