329 CHONDROGENIC POTENTIAL OF PORCINE ADIPOSE-DERIVED STEM CELLS, CHONDROCYTES, PERIOSTEAL CELLS, AND FIBROBLASTS IN A PELLET CULTURE SYSTEM

Abstract

Regenerative medicine has long sought to develop therapies for articular cartilage repair and for enhancing endochondral ossification to address complications of long bone healing. The objective of this study was to determine the chondrogenic potential of porcine primary cell cultures for possible utility in orthopedic tissue engineering applications. Adipose-derived mesenchymal stem cell (ASC), chondrocyte (positive control), periosteal cell, and fibroblast (negative control) primary cell cultures from 8- to 12-month-old Yorkshire pigs were plated at 5000 to 10 000 cells cm^–2 in 75-cm² cell culture flasks using high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with NaHCO₃, 10% fetal bovine serum (FBS), and antimicrobials (penicillin-streptomycin, gentamicin sulfate, and amphotericin B), then incubated at 37°C, 5% CO₂, and 18% O₂. Cells were trypsinized at ~80% confluency and transferred into 15-mL conical tubes at 500 000 cells tube^–1. Suspensions were washed twice by centrifugation in DPBS then condensed via a final centrifugation in 1.0 mL of negative control media (DMEM + 10% FBS) or chondrogenic base media consisting of high-glucose DMEM with 40 µg mL^–1 of proline, 50 µM ascorbic acid-2-phosphate, 100 nM dexamethasone, antimicrobials, and 1× insulin-transferrin-selenium solution added. Chondrogenic additives tested were 2% FBS, Kartogenin (200 nM, 400 nM, or 4 µM), 10 ng mL^–1 of BMP-4, or a combination of 10 ng mL^–1 of BMP-6 + 10 ng mL^–1 of TGFβ-3. Pellets were incubated with media changed every 2 to 3 days for a period of 2 to 4 weeks, fixed with 4% paraformaldehyde in DPBS, and then frozen at –80°C in Neg 50 Frozen Section Medium. Eight-micrometer sections were cut using a cryostat onto charged slides. Histochemical staining was performed using hematoxylin and eosin (H&E) for cell morphology and Safranin O and Alcian Blue for cartilage matrix markers. Immunofluorescent staining was done to detect collagen II and aggrecan with the nuclear marker lamin-C used to assess cell viability. Chondrocyte pellets grown in chondrogenic media, regardless of additive, exhibited cartilage matrix formation with H&E and stained strongly positive with Safranin O, Alcian Blue, and collagen II. The ASC pellets grown in chondrogenic media showed mixed cell morphology and areas of early cartilage matrix formation with H&E and stained faintly positive with Safranin O, Alcian Blue, and collagen II at 3 weeks in culture. Periosteal cell pellets had similar morphology in all conditions and did not stain positive for cartilage matrix markers. Fibroblast pellets did not survive any condition for processing. In conclusion, porcine chondrocytes and ASC were able to form cartilage matrix in a pellet culture system with chondrogenic media regardless of additive, while porcine fibroblasts and periosteal cells showed limited to no chondrogenic potential in the conditions tested.