CHAPTER 5 – The Organic Matrix of Bone

Glycoproteins of the connective tissue matrix.

Decellularized adipose tissue microcarriers as a dynamic culture platform for human adipose-derived stem/stromal cell expansion

The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption have been studied in rat tibial diaphyses using electron microscope techniques in an attempt to identify the steps of the resorption process Results suggest that bone resorption occurs in two phases: the first, an extracellular phase, leads to bone matrix fragmentation and partial dissolution, and the second, an intracellular phase, to complete digestion of the breakdown products of the bone matrix The first component of the bone matrix to be attacked by the osteoclast is the ground substance This induces the release of the crystals lying between, and on, the collagen fibrils; any crystals lying within fibrils are released later, when the fibrils break up As this stage proceeds, the collagen fibrils retain their normal intrinsic texture, but gradually loose their lateral aggregation, appearing as individual fibrils (some of them uncovered by crystals), mixed with fragments of fibrils and many free crystals The loosened but otherwise structurally normal collagen fibrils, and their fragments, are strongly argyrophilic Complete dissolution of the disaggregated fibrils occurs outside the cell, both in the resorption zone and in the initial portion of the channels of the ruffled border The free crystals present in the resorption zone and those phagocytosed in cytoplasmic vacuoles are organic-inorganic structures, whose organic component (the crystal ghost) is, at least in part, of proteoglycan nature Dissolution of inorganic material occurs within the cytoplasmic vacuoles of the osteoclasts Results are viewed in relation to the process of bone resorption and, as far as crystal ghosts are concerned, to that of bone calcification A tentative summary of the various steps involved in the mechanism of bone resorption is given

The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption.

Aims Collagen crosslinking treatment of progressive keratoconus using the photosensitiser riboflavin and ultraviolet A light of 370 nm wavelength has been shown to increase significantly the tensile strength of corneal collagen by about 300%. In keratoconus, interlamellar and interfibrillar slippage have been proposed as pathogenetic mechanisms. Therefore, the aim of this study was to assess the impact of collagen crosslinking on the interlamellar cohesive force. Methods 72 post mortem porcine eyes were divided into six different treatment groups: the untreated control group, the standard crosslinking group, the hypo-osmolar crosslinking group, the stromal swelling group, the formaldehyde group and the a-amylase group. An anterior 934 mm strip of 400 mm thickness was prepared using a lamellar rotating microkeratome. For interlamellar cohesive force measurements a splitting plane was created at 50% depth. Forceedistance profiles were recorded using a microcomputer-controlled biomaterial testing machine. Results The mean interlamellar cohesive force was 0.24 N/mm in the untreated control group, 0.26 N/mm in the standard crosslinking group, 0.25 N/mm in the hypoosmolar crosslinking group, 0.23 N/mm in hydrated corneas, 0.27 N/mm in the formaldehyde group without statistically significant difference. Only the values of the a-amylase group were statistically significantly lowered by 31.5% to 0.16 N/mm. Conclusions Surprisingly, corneal crosslinking does not increase the interlamellar cohesive force. In the a-amylase group the cohesive force was mainly decreased because of the digestion of proteoglycans. Crosslinking seems to stabilise only inter- and intrafibrillar, but not interlamellar cohesion.

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Interlamellar cohesion after corneal crosslinking using riboflavin and ultraviolet A light

Different connective tissues were digested with Bacillus subtilis α-amylase in an effort to determine the effects of this enzyme on the glycoprotein-collagen complexes. It was found that in some tissues insoluble collagen could not be attacked by Clostridium histolyticum collagenase unless a glycoprotein fraction, strongly associated with the fibers, was removed. This removal was achieved by α-amylase, thus supporting earlier observations that sugar-containing compounds are strongly bound to collagen. Using alpha amylase an unexpected and interesting result demonstrated that this enzyme(s) releases acid glycosaminoglycans from the various connective tissues. Chemical studies have shown that the α-amylase preparation was not contaminated to any significant extent by hyaluronidase or by a protease.

C. Balduini

Papers

The effects of alpha amylase on collagen-proteoglycans and collagen-glycoprotein complexes in connective tissue matrices.