Role of oxygen tension during cartilage formation by periosteum.

Tissue engineering makes regeneration of cartilage possible but requires optimization of culture conditions. The effects of oxygen tension on cartilage metabolism are controversial in the literature, and we could find no information detailing the optimal oxygen concentration for growing new cartilage (neochondrogenesis). Periosteal cells and tissues can be used to grow cartilage in vivo and in vitro. In this study, using a standard periosteal organ culture model, we found that cartilage formation by periosteal explants is affected by the ambient oxygen concentrations. A total of 480 periosteal explants from 30 2-month-old New Zealand White rabbits were cultured in agarose suspension at different oxygen concentrations (1-90%) for 6 weeks. Chondrogenesis, which was analyzed by histomorphometry and quantitative collagen typing, was maximal at 12-15% oxygen. There were no significant differences in chondrogenesis in the range of 12-45%. There was inhibition of cartilage and type-II collagen formation at very high (90%) and very low (1-5%) oxygen concentrations. However, contrary to what some have thought, chondrogenesis is maximal under aerobic conditions. If this is true for systems other than periosteal implants, it would have important implications for growing cartilage in vitro.

Type II collagen quantification in experimental chondrogenesis.

Type II collagen is an excellent indicator of the cartilage phenotype. Accurate quantification with existing methods requires about 100 micrograms of collagen. We have developed a method which allows for accurate quantification of type II collagen in samples as small as 1 microgram (and sample volumes as little as 1 microliters). Types I and II collagen were pepsin purified, cleaved with cyanogen bromide, and dissolved in sample buffer at concentrations of 0.125-200 micrograms/microliters. Volumes of 1 microliter were analyzed by electrophoresis on microgels. The gels were scanned on a laser densitometer and the ratios of the alpha 1 (II)CB10 to the alpha 1(I)CB7,8 plus alpha 1(II)CB11 determined. The cyanogen bromide-derived (CNBr) peptides could be resolved at concentrations as low as 0.25 microgram/microliter. The ideal working concentration for purified collagens was 1-8 micrograms/microliters. Standard mixtures of both purified and non-purified types I and II collagen were analyzed. At a concentration of 1 microgram/microliter the ratio of the bands referred to above was closely related to the relative proportion of type II collagen, in a polynomial fashion. At 8 micrograms/microliters there was an almost perfect linear relationship. The presence of 15-30% type III collagen had < 5% effect on the measurements of type II collagen. The method is simple, reliable, fast and automated. It should have good potential for application in cartilage research as it permits quantitation of type II collagen in extremely small samples of tissue.

Relationship of donor site to chondrogenic potential of periosteum in vitro.

Periosteum has been shown in vitro and in vivo to have a chondrogenic potential that permits it to be used for cartilage regeneration. A useful donor site should have good chondrogenic potential, availability of a large quantity of periosteum, and relative ease of access, and it should be associated with a low rate of morbidity. We hypothesized that the chondrogenic potential of periosteum varies from one bone to another and among different regions of the periosteum from a single bone. A total of 370 periosteal and 37 fascia lata (control) explants were taken from the skull, the ilium, the scapula, the upper, middle, and lower medial proximal tibia, the posterior proximal tibia, and the distal tibia of 2-month-old New Zealand rabbits. The explants were cultured for 6 weeks in agarose/Dulbecco's modified Eagle medium to which 10 ng/ml of transforming growth factor-beta 1 was added during the first 2 weeks. Skeletal muscle and fascia lata were used as controls. In addition, the thickness, cell density, and total cell count of the cambium layer were measured in 24 explants from the donor sites on the ilium and the upper, middle, and lower proximal tibia. At 6 weeks, histomorphometry and quantitative collagen typing were performed. The periosteal donor sites could be grouped into three categories according to chondrogenic potential: ilium (best), scapula and tibia, and skull (no chondrogenesis). The scapular periosteum was slightly better than that from the tibia. Within the tibia, the upper and middle zones of the proximal region were similar and were slightly better than the lower proximal tibia or the distal tibia. The cellularity of the cambium layer correlated positively with the amount of cartilage as a percentage of the total area. The results of this study indicate that iliac periosteum exhibited the best overall chondrogenic potential in vitro but that periosteum from the traditionally used medial proximal tibia also was excellent. Periosteum from the skull was not chondrogenic. The chondrogenic potential of periosteum varies from bone to bone and within the periosteum from one bone. This variation in chondrogenic potential among donor sites may be due to a difference in the total cell count of the cambium layer.

Enhancement of periosteal chondrogenesis in vitro. Dose-response for transforming growth factor-beta 1 (TGF-beta 1).

Transforming growth factor-beta 1 (TGF-beta 1) has been shown to stimulate chondrogenesis in periosteal explants cultured in agarose suspension. In this study, the dose-response curve for such enhancement was measured. Periosteal explants and fascia lata were harvested from two-month-old rabbits, cultured for six weeks with 0, 0.1, 1, 5, 10, 50, or 100 ng/mL TGF-beta 1 in agarose suspension, then analyzed by histomorphometry and quantitative collagen typing. Cartilage was produced by seven of 11 (64%) of the control periosteal explants cultured in agarose suspension without TGF-beta 1. Transforming growth factor-beta 1 enhanced chondrogenesis in a dose-dependent manner in the range 0.1-100 ng/mL. It was most effective at 50 ng/mL. At very high doses (50 and 100 ng/mL) of TGF-beta 1, even fascia lata control explants exhibited chondrogenesis. These data indicate that TGF-beta 1 can induce differentiation toward cartilage production as well as enhance it once it has been initiated.