The effect of recombinant human osteogenic protein-1 on growth plate repair in a sheep model.

Injuries to the growth plate in children can result in bone bridge formation, which ultimately lead to limb length and angular deformities. The histological and molecular changes associated with growth plate repair following the Langenskiöld procedure, a surgical technique used to remove impeding bone bridges, in conjunction with administration of recombinant human osteogenic protein-1 (rhOP-1) were examined using a sheep model. Following treatment with rhOP-1 there was an increase in the height of the growth plate immediately adjacent to the defect compared to untreated animals. The expression of type I collagen, osteopontin and decorin were observed in the growth plate adjacent to the defect in the untreated animals at day 56, but this response was accelerated in the rhOP-1 treated animals, with these molecules seen as early as day 7. Therefore, treatment with rhOP-1 initiated a complex response that was both chondrogenic and osteogenic in nature.

Factors affecting bovine corneal endothelial cell density in vitro.

AIMS: To examine factors influencing the density and contact inhibition of bovine corneal endothelial cells cultured in vitro. METHODS: Cell counts were performed on bovine corneal endothelial cells cultured for various times in the presence of 10% fetal calf serum, with or without varying concentrations of growth factors, 5% dextran T-500, or 2% chondroitin sulphate, at 32 degrees C or 37 degrees C, and after treatment with beta galactosidase. RESULTS: Both basic fibroblast growth factor (FGFb) and retinal crude extract (RCE), but neither epidermal growth factor (EGF) nor acidic fibroblast growth factor (FGFa), increased endothelial cell density in vitro (p < 0.05). Continuous exposure to RCE resulted in a higher cell density than did a 24 hour pulse (p < 0.01), and higher cell densities were achieved at 37 degrees C than at 32 degrees C (p < 0.0001). In the absence of RCE, dextran T-500 increased cell density modestly (p < 0.05); in the presence of RCE, the addition of dextran T-500 had no effect on final cell density, whereas chondroitin sulphate significantly decreased final cell density (p < 0.01). In the absence of exogenous growth factors, beta galactosidase treatment resulted in a 50% increase in final cell density compared with controls (p < 0.0001). CONCLUSIONS: Bovine corneal endothelial cell growth can be augmented under conditions different from those used in corneal preservation systems. The final cell density in a confluent monolayer can be increased by treatment with beta galactosidase, suggesting that corneal endothelial cells may be contact inhibited through a beta galactosidase sensitive receptor system.

Cryopreservation of rabbit and cat corneas at -18 to -24 degrees C.

A simple method of corneal cryopreservation, in which corneas were frozen at -18 to -24 degrees C, was examined. Rabbit and cat corneas were placed successively in solutions of 50% fetal calf serum in McCarey-Kaufman medium with an increasing glycerol and glucose content. They were then frozen and stored in a -20 degrees C domestic freezer. Rabbit corneas stored in this way were examined in vitro by light and scanning electron microscopy, and both rabbit and cat corneas were also assessed after orthotopic allotransplantation into adult recipient animals. Functional corneal grafts were obtained with rabbit and cat tissue that had been cryopreserved for 3-4 weeks and 1 week, respectively. Endpoint analysis (by light and scanning electron microscopy) of grafts that had survived for 50 days indicated the presence of an intact corneal endothelial monolayer. The corneal endothelium slowly degenerated as the storage time was increased. Importantly, however, the endothelium appeared to withstand the freezing and thawing processes and we conclude that it may be possible to store corneas at temperatures above -196 degrees C, without the need for complex, low-temperature cryopreservation systems.

Metaphyseal factors promote calcium incorporation in physeal chondrocyte cultures.

Our hypothesis is that physiological mineralization within the mammalian growth plate is a consequence of communication between cartilage chondrocytes and cells within metaphyseal bone. To test this hypothesis, chondrocytes were isolated from the proliferative region of the fetal ovine physis and co-cultured with cells or conditioned medium from cells characteristic of those in metaphyseal bone. The mineralization potential of chondrocytes alone and in the presence of other cells or conditioned medium was determined by 45calcium incorporation. Co-culture of chondrocytes with a crude cell isolate from metaphyseal bone resulted in a stimulation of 45calcium incorporation of 93% above that observed in the individual cell populations alone. Conditioned medium from metaphyseal bone cultures also stimulated 45calcium incorporation. This response to conditioned medium was dose-dependent and stable to 90 degrees C. Vascular endothelial cells and conditioned medium from chondrocyte and osteoblast cultures did not stimulate 45calcium incorporation by physeal chondrocytes. Thus, cells found in the metaphyseal bone produce a soluble factor, which promote calcium incorporation by physeal chondrocytes. The source of this factor is not chondrocytic, osteoblastic, or endothelial in origin.

Spatial arrangement of physeal cartilage chondrocytes and the structure of the primary spongiosa.

Using laser confocal microscopy and 5-chloromethyl-fluoresceindiacetate (CMFDA) loading of chondrocytes we have investigated the structure of the ovine physis during late fetal development and its relationship to the structure observed in the primary spongiosa. Chondrocytes within the ovine growth plate form nests that together span the growth plate. We propose that all growth plates may be composed of nests of cells, but that the length of the individual nests changes between growth plates and with gestational age. The continuous column of cells seen within some growth plates is a nest of cells that is in the process of being absorbed by the invading metaphyseal front. Scanning electron microscopy of the mineralized portion of the primary spongiosa revealed structures that were consistent with the hypothesis that the cartilage surrounding the nest structure gives rise to the structure in the primary spongiosa. Although mineralization does not occur between cells within a nest, bands of mineral form between nests in the lower hypertrophic region and around the end of the nest as it reaches the hypertrophic region. This pattern of mineralization around and between nest termini yields the complex three-dimensional network of mineralized trabeculae observed in the primary spongosia.

Donor cornea procurement: six-year review of the role of the eye bank in South Australia.

The Lions Eye Bank of South Australia was established six years ago and has collected corneas from 790 donors. The consent rate is currently 82% of requests made. Two-thirds of donors have been male, with mean donor age/year varying from 54 to 64 years (range two to 93 years). Cardiovascular and respiratory diseases, trauma and haemorrhage account for 80% of all donor deaths. Mean death to enucleation time is five hours. Corneas assessed as being of excellent or very good quality are released preferentially from the bank; those with central endothelial cell counts of less than 1500 cells/mm2 are discarded. Fewer than 1% of donors have returned a positive result for HIV or hepatitis B. Of the 1580 corneas collected by the bank, 863 (55%) have been used for transplantation with a primary non-function rate of 0.46%. The evolving policies, logistics of operation and methodologies employed by the bank are described in detail.