Calcification of implanted vascular tissues associated with elastin in an experimental animal model.

We have previously studied the process of calcification in bioprosthetic porcine heart valves crosslinked with glutaraldehyde. Observations using light microscopy had indicated that calcification of elastic fibers occurs in implanted heart valves, in addition to calcification associated with collagen fibers. To determine the contribution of elastin to the process of calcification, small pieces of rabbit aorta were cross-linked with 0.2% glutaraldehyde, rinsed in buffer, and implanted subcutaneously in young adult male rats. Cross-linked jugular vein implants were included as controls. After an implantation period of 1 month or longer, we observed many areas of calcification in the aortic media associated with elastin and fewer such areas associated with collagen. The elastin-rich aortic tissues accumulated more calcium than venous tissues. Calcium deposits appeared similar in both allogenic and xenogeneic implants. Calcified areas viewed under the electron microscope included intercellular nonfibrous material. Calcified areas involved predominantly the outer layers of elastic fibers. Calcific deposits included needle-like crystals of hydroxyapatite but often consisted of an amorphous flocculant material surrounded by crystals. The close spatial relationship of hydroxyapatite crystals and elastic membranes seen in this study may be relevant to the initiation of dystrophic calcification in glutaraldehyde cross-linked aortic grafts.

Age changes to the anulus fibrosus in human intervertebral discs.

This study deals with age changes in the anulus fibrosus of the lumbar intervertebral discs of human individuals 21-83 years of age. The anular laminas from individuals less than 40 years of age consisted of obliquely orientated collagen fibers exhibiting a pennate arrangement. These fibers were intensely argyrophilic after silver nitrate impregnation. The fibers and surrounding substance appeared light pink after exposure to the periodic-acid-Schiff (PAS) reaction and blue with alcian blue complex. Beginning during middle age and continuing into the eighth decade, there was a progressive degeneration of the laminas. The breakdown of the intact laminas was characterized by the fraying, splitting, and loss of collagen fibers. The newly formed spaces became filled with intense PAS-positive material. In addition, there was a continual deposition of chondroid substance in the anuluses of the aging discs. This phenomenon was not seen in the young disc. These age related changes lead to a loss of integrity to the disc, which may be a factor in disc pathology.

Postmortem degradation of demineralized bone matrix osteoinductive potential. Effect of time and storage temperature.

The osteoinductive growth factors present in demineralized bone are degraded by tissue enzymes. Storage of rat limbs at low temperature (4 degrees C) before harvesting of bones preserves the osteoinductive potential of such factors. Storage at room temperature for more than 24 hours causes the recovered bone matrix to be biologically inactive, presumably as the result of biodegradation.

Morphological changes in the submandibular gland of aging rats.

Age-related histological changes in submandibular glands of 5-, and 24-month-old Sprague-Dawley male rats were compared qualitatively using semi-thin sections of epoxy-embedded glands. Atrophy of acini and granular ducts with a concomitant hyperplasia of intercalated ducts were the dominant features seen in the 15- and 24-month-old glands compared to 5 months. In both aged groups (15 and 24 months) there appeared structures similar to terminal tubules normally seen during development. At 15 months irregular ducts consisting of a mixture of agranular cells and granular cells typical of granular ducts were found in continuity with the hyperplastic intercalated ducts. The significance of these age-related morphological changes remains speculative.

Dystrophic calcification and mineralization during bone induction: biochemical differences.

The calcification of implants of glutaraldehyde-cross linked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity and BGP was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer 344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20 day old embryo donors were introduced into the chambers prior to subcutaneousmplantation. After 4 weeks of implantation in 26 month old rats, the cylinders containing embryonic calvaria or muscle cells were found to be full of bone and/or cartilage.

Biochemical differences between dystrophic calcification of cross-linked collagen implants and mineralization during bone induction.

Ectopic calcification of diseased tissues or around prosthetic implants can lead to serious disability. Therefore, calcification of implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen was compared with mineralization induced by demineralized bone matrix (DBM). Whereas implants of DBM accumulated large amounts of calcium and a bone-specific gamma-carboxyglutamic acid protein (BGP or osteocalcin) following implantation in both young and older rats, implants of cross-linked pericardium calcified with only traces of BGP. Glutaraldehyde-cross-linked DBM failed to calcify after implantation in 8-month-old rats for 2-16 weeks. Implants of cross-linked type I collagen exhibited small calcific deposits 2 weeks postimplantation but calcium content eventually dropped to levels equal to those of soft tissues as the implants were resorbed. The calcium content of DBM implanted in 1- and 8-month-old rats reached comparable levels after 4 weeks, but the BGP content was approximately twice as high in the younger animals than in the older ones. Glutaraldehyde-cross-linked implants of DBM, tendon, and cartilage calcified significantly in young but not in old animals. This form of dystrophic calcification was associated with only trace amounts of BGP. Alkaline phosphatase activity was high in implants of DBM and undetectable in implants of cross-linked collagenous tissues. These results show that implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen calcify to different extents depending upon their origin and the age of the host, and that the mechanism of dystrophic calcification differs significantly from the process of mineralization associated with bone induction as reflected by alkaline phosphatase activity and BGP accumulation.

Light- and electron-microscopic characteristics of artrial smooth muscle cell cultures subjected to hypoxia or carbon monoxide.

Smooth muscle cells from the tunica media of piglet aortae grown under hypoxic conditions undergo the following changes: First, they become modified by partial loss of myofilaments and proliferation of organelles, which are characteristics of young primitive cells. Second, an increase in number of pinocytotic vesicles at and below the cell surface, indicating increased extracellular uptake of material, can be detected. This is followed by accumulation of Oil Red O positive intracytoplasmic granules and vacuoles as well as the subsequent formation of mount-like protrusions. The latter consist of a core of extracellular material and necrotic debris covered with a cap of viable cells. A third feature of the cells subjected to hypoxia is a conspicuous rise in the number of lysosomes. This is considered to be a manifestation of a defense mechanism of the cells to remove undesirable material from cytoplasm. Cells exposed to an atmosphere rich in carbon monoxide exhibit basically the same alterations as those grown under hypoxic conditions; however, formation of mound-like aggregates is less prominent, while the rise in the number of lysosomes is more evident than in the hypoxic cells. The above alterations are similar to changes observed in smooth muscle cells of rabbit with experimental atherosclerosis. It is suggested that whereever the arterial smooth muscle cell is subjected to adverse conditions basically the same mechanism, consisting of dedifferentiation, increased permeability and lysosomal defense reaction, takes place.

The induction of atherosclerotic plaque-like mounds in cultures of aortic smooth muscle cells.

Smooth muscle cells harvested from the tunica media of piglet aortae were maintained in continous culture for 10 months. When grown in the presence of 95% air and 5% CO2 they maintained a mature morphology as evaluated ultrastructurally. As these populations became confluent, the cells became oriented parallel to each other. When grown in the presence of 4% O2, 91% N2, and 5% CO2, this polarized pattern was disrupted. Focal areas of lipid accumulation were observed, succeeded by mound formation at these sites. The mound stained positive with PAS, aldehyde fuchsin, and oil red O. They were surrounded by 2-4 layers of intact cells. The centers of the mound were composed of extracellular material and cell debris.