These studies examined the effects of heat-inactivated horse serum, insulin, triiodothyronine (T3), and thyroxine (T4), individually and in combination, on collagen and proteoglycan synthesis by primary cell cultures of articular chondrocytes from immature male rabbits. Insulin concentrations of 25 to 100 ng/ml (4.4 to 17.4 x 10(-9) M) increasingly stimulated collagen and proteoglycan synthesis in the absence of serum. The effects of 25 ng/ml (4.4 x 10(-9) M) insulin or 15% heat-inactivated horse serum on collagen synthesis were similar. Triiodothyronine (10(-10) to 10(-6) M) and T4 (10(-8) to 10(-4) M) also stimulated collagen synthesis in the absence of serum, with peak effects at 10(-8) and 10(-6) M, respectively. Biphasic stimulation of proteoglycan synthesis was obtained with 10(-11) to 10(-7) MT3 (maximum at 10(-8) M) and 10(-8) to 10(-5) M T4 (maximum at 10(-7) M). In these experiments, triiodothyronine was 10 to 100 times more potent than T4 in stimulating cartilage matrix production. The cells retained their chondrocytic phenotype under hormonal stimulation, secreting almost exclusively Type II collagen and large, chondroitin sulfate-rich proteoglycans. The addition of insulin to maximally-stimulating concentrations of either T3 or T4 in serum-free medium further stimulated matrix synthesis, suggesting that these hormones modulate chondrocyte metabolism via multiple biosynthetic/receptor pathways.

Second passage fibroblast-like cells grown from explants of neonatal rat muscle continue to demonstrate fibroblast-like properties for many days when cultured on plastic surfaces. Such cells can be induced to change to a chondrocyte-like mode of expression by the addition of effector materials prepared from bovine cortical bone decalcified with 0.6 N HCl. Other studies show that similar demineralized bone particles and extracts from them have, in vivo, osteoinductive properties. Optimum conditions for this differentiation in monolayer culture were found in the use of 2% fetal calf serum with Dulbecco's modified Eagles medium. At 10% fetal calf serum the chondrogenic changes could not be detected. Light microscopy showed a sequence of morphological changes, after 36 h in culture, which resembled those seen at the beginning of osteogenesis in vivo. Induced cultures showed abundant extracellular proteoglycan production. Isotope incorporation studies showed stimulation of glycosaminoglycan synthesis in response to effector materials in soluble form. Type II collagen could be detected after three days. Electron microscopic analysis of induced and control cultures showed unequivocal evidence for marked production of an extensive extracellular matrix in the region of effector particles. The cells themselves change shape and develop an abundant system of lysosome-like vesicles and a very active, highly engorged endoplasmic reticulum and Golgi apparatus. After nine days in culture, evidence for the formation of a ruthenium red stained structure on the surface of the cells in contact with inductive particles, was observed. The simple monolayer culture system described provides a direct means by which the presence of active chondrogenic fractions may be assessed, and in which the mechanism of action of the effectors can be studied.

The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37 degrees C, yielding 6.8 x 10(6) viable cells per g tissue digested. The addition of 30 microM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p less than 0.05) and doubled viable cell yields to 13.6 x 10(6) per g tissue digested (p less than 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p less than 0.01) and doubled viable cell yield yet again (to 29.9 x 10(6) viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.