Skeletal growth depends upon enchondral ossification in growth plate cartilage, within which chondrocytes undergo well defined stages of maturation. We infused IGF-I or growth hormone (GH), two key regulators of skeletal growth, into hypophysectomized rats and compared their effects on growth plate chondrocyte differentiation using qualitative and quantitative autoradiography, stereology, and incident light fluorescence microscopy. Stem cell cycle time was shortened from 50 to 15 and 8 d after treatment with IGF-I and GH, respectively. Proliferating cell cycle time decreased from 11 to 4.5 and 3 d, and duration of the hypertrophic phase decreased from 6 to 4 and 2.8 d. Average matrix volume per cell at each differentiation stage was similar for normal, hormone-treated, and untreated hypophysectomized groups. Mean cell volume and cell height were significantly reduced by hypophysectomy at the proliferative and hypertrophic stages, but were restored to physiological values by IGF-I and GH. In contrast, cell productivity, i.e., increases in cell volume, height, and matrix production per unit of time, did not reach normal values with either IGF-I or GH, and this parameter was inversely proportional to cell cycle time or phase duration.IGF-I and GH are thus capable of stimulating growth plate chondrocytes at all stages of differentiation, albeit to variable degrees with respect to individual cell activities. Although it is generally accepted that GH acts at both the stem and proliferating phases of chondrocyte differentiation, our data represent the first evidence in vivo that IGF-I is also capable of stimulating stem cells. (J. Clin. Invest. 1994. 93:1078-1086

Normal tissues such as skin, cartilage, and blood and lymphatic vessels are highly tolerant to MBI after entrance doses up to 400 Gy. The striated muscles appeared to be the most sensitive to MBI. Those findings should be taken into consideration in future micro-beam radiation therapy treatment schedules.

The quality of cryosections prepared from high pressure frozen bovine articular cartilage has been recently evaluated by systematic electron diffraction analysis, and vitrification found to be zone-dependent. The lower radial layer was optimally frozen throughout the entire section thickness (150 microns), whereas in the upper radial, transitional and superficial layers this was achieved down to a depth of only approximately 5-50 microns. These differences were found to correlate proportionally with proteoglycan concentration and inversely with water content. In the current investigation, extracellular matrix ultrastructure was examined in high pressure frozen material (derived from the lower radial zone of young adult bovine articular cartilage), by both cryoelectron microscopy of cryosections and by conventional transmission electron microscopy of freeze-substituted and embedded samples. Several novel features were revealed, in particular, the existence of a fine filamentous network; this consisted of elements 10-15 nm in diameter and with a regular cross-banded structure similar to that characterising collagen fibrils. These filaments were encountered throughout the entire extracellular space, even within the pericellular region, which is generally believed to be free of filamentous or fibrillar components. The proteoglycan-rich interfibrillar/filamentous space manifested a fine granular appearance, there being no evidence of the reticular network previously seen in suboptimally frozen material.

Adult cartilage is a unique tissue in that it is avascular and lacks innervation. The chondrocytes are embedded in an extracellular matrix which in most cases occupies 60 to 90% of the tissue volume. The ultrastructure and composition of rat growth plate cartilage has been investigated (1). It was found that during conventional chemical fixation of me tissue (2), about 60% of the extracellular matrix proteoglycans were lost (3). This artefact can be prevented if proteoglycans are precipitated by cationic dyes (eg. ruthenium hexaamine trichloride) during fixation (4) which forms relatively large precipitates by rendering high resolution imaging impossible. High pressure frozen extracellular matrix revealed a fine meshwork which was attributed to proteoglycan distribution (5). A meshwork due to segregation during freezing was not seen in recent experiments where adaequately frozen cartilage was investigated.200μm thick sections of rat growth plate cartilage were excised in a bath of hexadecene (6). The sections were again processed under hexadecene, firstly punched (diameter 1.7mm) and then transferred to an aluminum sandwhich (corresponding in size to the sample).