Changes in the ratio of non-calcified collagen to calcified collagen in human vertebrae with advancing age.

Bone loss associated with aging is associated primarily with a decline in bone formation. To try and further understand the nature of this process we have used a biochemical approach which relies on the fact that osteoid is susceptible to enzymatic degradation whereas calcified collagen is protected by the mineral phase against proteolytic digestion. Our findings show a statistically significant inverse relationship between osteoid and age (r = 0.70 female, r = 0.47 male). A closer relationship was observed when age was related to the ratio of osteoid to bone (r = 0.73 female, r = 0.56 male). In both cases, the observed linear decline begins at an early age and becomes marked with advancing age. Histologic observations illustrate these findings showing decreased osteoid and osteoblasts in the older vertebral specimens compared to the younger ones. Even though the mechanism for osteoid calcification seems to remain unimpaired, the decline of a calcifiable matrix in the presence of normal bone turnover could lead to bone loss.

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.

Acute wound repair in an aged animal: a model for accelerated aging of the microvasculature?

This study of wound repair in the aged rat is based on increased carbohydrate content of various proteins which occurs with aging and is readily seen in the microvasculature (MV). We have used the periodic acid-Schiff (PAS) reaction to identify histochemically the carbohydrate moiety of the glycoproteins found in these blood vessels. In the young rat, as in other young vertebrates, elements of the MV are PAS negative and become increasingly PAS+ beyond the half life span. During acute wound repair in an old animal, the new capillaries and venules are PAS- 2 weeks after injury, moderately PAS+ at 4 weeks, and intensely PAS+ at 8 weeks. Arterioles are present and PAS+ at 6 weeks, and intensively positive at 8 weeks, comparable to vessels remote from the wound site. The MV in wound repair in a young animal remains PAS- throughout healing. Rapid aging of the microvasculature in wound repair in an old animal reproduces histochemically the aging which occurs progressively during the prior 24 months. These histochemical changes may result from successive enzymatic and nonenzymatic glycosylation of the various basement membrane proteins in the microvasculature in both normal aging and wound repair in the aged animal. The latter may serve as a model for study of accelerated aging.

Histochemical characterization of the aging microvasculature in the human and other mammalian and non-mammalian vertebrates by the periodic acid-Schiff reaction.

Prior histochemical studies with the periodic acid-Schiff (PAS) reaction have shown altered biochemical composition in a limited part of the microvasculature (MV) in aging in two species of laboratory animals. We therefore studied, with the PAS reaction, all the components of the MV in multiple tissues from various immature, adult and aged mammals, including human, and immature and aged nonmammalian vertebrates. We now demonstrate that there is an altered biochemical composition of capillaries, arterioles and venules in various tissues with aging. These are first detectable somewhat beyond half the life-span in man (greater than 45 years), marmoset (5 years) and dog (8 years) and seen in old fish, reptiles and birds. The capillary wall is increasingly PAS+; in arterioles there are focal PAS+ areas in the media which increase in size and number with age and become hyalinized masses. The non-muscular venules are increasingly PAS+ apparently due to a polysaccharide staining of connective tissue elements. These histochemical changes in the MV with aging are in the extracellular matrix and appear to be a specific manifestation of aging in vertebrates. The consequences of such changes in MV aging may be important physiologically.

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.

Osteogenesis in bone defects in rats: the effects of hydroxyapatite and demineralized bone matrix.

Hydroxyapatite (HA) and Demineralized Bone Matrix (DBM) are being investigated as potential osteogenic agents with hopes that these substances can be used to induce bone formation in non-union fractures. This study was done to determine the relative effects of HA and DBM implanted as moldable phospholipid composites in bone defects that result in non-unions. We studied 22 ten-month-old Long-Evans male rats with 5.0 mm unilateral radial defects implanted with HA, DBM, and a combination of both substances. Control defects were left unfilled. Eight weeks after implantation, the histological sections demonstrated a decrease in bone formation with HA relative to controls. The HA crystals were encapsulated by connective tissue stroma made up of collagenous elements, fibroblasts, and blood vessels. There were no indications of bone formation within the fibrous stroma. 45Calcium, alkaline phosphatase, and bone gla protein (BGP) assays demonstrated a 16% increase in bone formation in rats implanted with DBM, an 80% decrease in groups implanted with HA (p = 0.01) and an 80% decrease with DBM plus HA (p = 0.01). Radiologic analysis corresponds well with histological and biochemical results. We conclude that osteogenesis in non-union defects is enhanced by the implantation of DBM, while HA interferes with bone formation in the rat model. In the presence of both substances, HA appears to impede new bone growth, negating any positive effects seen with DBM.

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.

Cellular events associated with the induction of bone by demineralized bone.

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.

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.

Ectopic bone formation is enhanced in senescent animals implanted with embryonic cells.

Ectopic bone formation induced by the subcutaneous implantation of demineralized bone matrix (DBM) is very significantly reduced in older Fischer 344 rats. Cells originating from calvaria of 20-day-old embryo donors were introduced into cylinders of DBM sealed at the ends with a Millipore filter or collagen sponges prior to subcutaneous implantation. Cells within the chambers had access to vascular channels that could penetrate through the interstices of the DBM. After four weeks of implantation in 26-month-old rats, the cylinders were full of bone. This bone was assessed by histologic techniques, by calcium and bone gamma-carboxyglutamic acid (gla) protein (BGP) concentrations, and by alkaline phosphatase activity. Cylinders to which no cells were added produced no bone. Bone marrow cells enclosed in similar cylinders or injected weekly at the implantation site also enhanced new bone formation but to a much lesser extent. Embryonic muscle cells formed large amounts of cartilage and less bone. Fibroblasts were inactive in this system. Prior treatment of the DBM with trypsin inhibited the myoblast response but not that of calvaria cells.

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.

Methods in laboratory investigation. Identification and evaluation of histopathology at microelectrode puncture sites.

Tissue puncture techniques using microelectrodes for various measurements have been criticized for producing undetermined degrees of tissue damage. Therefore, a method permitting routine identification of puncture tracks was developed to determine local microelectrode-induced injury. Rabbits were anesthetized and the femoral arteries surgically exposed. A 3-ml mixture of saline-India ink suspension was introduced through an ear vein. Oxygen-sensitive (pO2) microcathodes were advanced into and through the arterial wall at 10- or 20-micron intervals using a stepping microdrive to 150 to 450 micron and then withdrawn. The arteries were fixed in 10% formalin and gelatin embedded, and serial frozen sections (less than or equal to 15 micron) of the microelectrode puncture area were made. We observed within 5 minutes of microcathode withdrawal a dark, punctate, microscopic discoloration within the arterial wall. Histologically, ink distribution within the arterial wall demonstrated an acute permeability change: puncture depths generally less than 300 micron showed ink-lined microelectrode tracks (generally less than 2 micron wide) in the media, and greater puncture depths showed local hemorrhage and focal laminar accumulation of ink which extended from the track. The immediate adjacent area to microelectrode puncture depths less than 300 micron showed an apparent intact internal elastic lamina and media. Therefore, microelectrode damage has been shown to be primarily limited to microelectrode tissue tracks.

Vertebral end-plate changes with aging of human vertebrae.

The present study describes the sequential age changes within the growth and articular layers of the cartilaginous end-plates of vertebrae from humans varying in age from birth to 73 years. There is a gradual reduction in the width of the growth cartilage up to 16-20 years of age. During adulthood and progressing into old age (60-73 years), the end-plates consist of only articular cartilage which undergoes calcification followed by resorption and replacement by bone. Age changes are observed in the arterioles, capillaries, and venules found in the nutrient canals or spaces of the bone adjacent to the cartilage or disc. The calcification of the articular cartilage and vascular changes seen in the older vertebrae would impede the passage of nutrients from the blood to the disc proper. Collagen fibers are observed arising from the older vertebral end-plates to course into the midregion of the disc.

Changes in nerve supply to aging human gingiva.

The innervation of the gingivae of dentulous and edentulous individuals was studied. There was a decrease in the number of nerves in the gingivae of dentulous individuals. The nerves present showed degenerative changes such as fibrosis, fragmentation, and reticulation. In the edentulous gingivae, there was a marked loss of nerves and terminal endings. Nerves observed were restricted to the base of the lamina propria adjacent to the underlying bone.

The maturation and aging of the vertebrae of marmosets.

This study deals with the growth, maturation, and age changes to the cartilaginous end-plates of vertebrae from marmosets (Callithrix jacchus) varying in age from birth to 10 years. The cartilaginous end-plate is divided into an inner growth layer and an outer articular zone that abuts against the intervertebral disc. The growth cartilage gradually narrows and disappears by 1 year of age, when the animal reaches physical maturity. The articular cartilage undergoes changes in collagen and ground substance that leads to its calcification. There was a progressive resorption of the calcified articular cartilage beginning at 3 years of age and continuing throughout the aging period. In the vertebrae of 8- and 10-year-old animals, only a thin layer of calcified cartilage separates the disc from the underlying bone.