Changes in lipids during matrix: induced endochondral bone formation

The changes in lipids occurring during the process of endochondral ossification have been characterized by studying the discrete phases of matrix-induced endochondral bone formation in the rat. Calcium-acidic phospholipid-phosphate complexes were shown to increase in concentration during cartilage calcification (day 9) and to peak in content during early bone formation (days 11-13), the times during which the rate of mineral deposition, as indicated by the change in ash weight was greatest. These data support the hypothesis that the calcium-acidic phospholipid-phosphate complexes play a role in the in vivo initiation of hydroxyapatite deposition. The overall lipid composition of the induced matrix newly formed cartilage (days 7-9) was comparable to that of normal cartilage, with the phospholipid composition matching that of chondrocyte plasma membranes. Times of vascular invasion and formation of marrow cavities were marked by elevated total lipid and triglyceride contents.     

Calcium-acidic phospholipid-phosphate complexes in human atherosclerotic aortas

Summary Since aortic calcification chemically resembles bone mineralization, we tested the hypothesis that a bone mineral nucleator, the Ca-acidic phospholipid-phosphate complexes, is found in atherosclerotic plaques. Calcium-acidic phospholipid-phosphate complexes were isolated from hydroxyapatite-containing calcified plaques and from adjacent nonmineralized areas of adult human aortas. Neonatal aortas, which served as nonmineralized lesion-free controls, contained negligible amounts of the Ca-acidic phospholipid-phosphate complexes. The concentration of complexed acidic phospholipids in the aortic plaques (5 μg/mg demineralized dry wt) was comparable to that found in newly mineralized bone and calcified cartilage. The presence of Ca-acidic phospholipid-phosphate complexes in the nonmineralized regions of the adult aorta, as well as in the mineralized plaques, suggests that this tissue may calcify through mechanisms similar to those involved in bone mineralization.     

Localization of Pigment Epithelium-Derived Factor in Growing Mouse Bone

Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor found in a wide range of fetal and adult tissues, where it is thought to play a role in the regulation of angiogenesis during development. The temporal expression of PEDF during endochondral bone formation has not previously been reported. In this study, we analysed the expression pattern of PEDF in growing mouse hindlimbs from newborn day one through to maturation at week 9, using immunohistochemistry and in situ hybridization. PEDF expression was demonstrated in chondrocytes within the resting, proliferative and upper hypertrophic zones of the epiphyseal growth plate. The pattern of expression was consistent throughout the developmental stages of the mouse. In addition, PEDF was expressed by osteoblasts lining the bone spicules in the ossification zone of metaphyseal bone, as well as by osteoblasts lining cortical periosteum. These novel results demonstrate that PEDF is developmentally expressed in both cartilage and bone cells during endochondral bone formation, and strongly suggest that it may play a regulatory role in the processes of chondrocyte and osteoblast differentiation, endochondral ossification, and bone remodelling during growth and development of long bones.     

Enhancement of Crude Bone Morphogenetic Protein-Induced New Bone Formation and Normalization of Endochondral Ossification by Bisphosphonate Treatment in Osteoprotegerin-Deficient Mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. We investigated both the quantity and quality of heterotopic new bone induced by crude bone morphogenetic protein (BMP) as a means of examining bone metabolism by bisphosphonate administration in OPG^−/− mice. Four weeks after implantation of crude BMP, the volume of heterotopic new bone in OPG^−/− mice without alendronate was significantly less than in wild-type (WT) mice. Alendronate treatment of OPG^−/− mice resulted in enhancement of the volume of heterotopic new bone. Histological findings revealed that WT mice showed normal bone formation with persistent cartilage that was interspersed with islands of bone. In contrast, the cartilage was replaced by trabecular bone and bone marrow adipocytes in OPG^−/− mice without alendronate. However, some cartilage was still present in OPG^−/− mice with alendronate compared to those without alendronate. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG^−/− mice with alendronate than in those without alendronate. These findings suggest that in stimulated osteoclastogenesis without OPG, osteoinductive activity induced by crude BMP is inhibited and endochondral ossification induced by crude BMP is accelerated. On the other hand, alendronate treatment of OPG^−/− mice caused osteoinductive activity induced by crude BMP to increase and endochondral ossification induced by crude BMP to be decelerated. In conclusion, inhibition of stimulated osteoclastogenesis results in the enhancement of new bone formation and normalization of endochondral ossification.     

In vivo analysis of the half-life of the osteoinductive potential of demineralized bone matrix using diffusion chambers

Summary Subcutaneous implantation of demineralized bone matrix (DBM) from rat initiates a sequence of developmental events that results in endochondral bone formation. This investigation examined the modification of the osteoinductive potential of DBM during the intial stages of this developmental cascade. Diffusion chambers (DC), constructed with filters of known pore size, permitting or excluding cells from entering the chambers, and containing DBM were subcutaneously implanted into Long-Evans male rats for specific time periods (1–7 days). DC were recovered and the osteoinductive potential of the matrix from these chambers was then tested by subcutaneous implantation and assaying the resulting day 11 plaque tissue enzymatically for alkaline phosphatase activity, and histologically for evidence of chondrogenesis and osteogenesis. The possible modification of DBM by local systemic factors (enzymatic degra-dation) or contact by polymorphonuclear leukocytes (PMNs) was also investigated. We have concluded from this study that the osteoinductive potential of DBM has a half-life of 5–7 days following implantation and although the enzymes collagenase, elastase, and trypsin abolished this activity, pepsin significantly enhanced it. Culture of PMNs with matrix prior to its implantation appeared to have little effect. Furthermore, during the initial stages of matrix-induced endochondral bone formation, DBM serves as both the instructive inducer and permissive substratum required in this process.     

The role of three genetically distinct collagen types in endochondral ossification and calcification of cartilage.

The role of three genetically distinct collagen types in the formation of endochondral bone and in calcification and resorption of cartilage has been assessed. Using antibodies specific to types I, II and III collagen we have demonstrated in the embryonic chick tibia that endochondral bone formation began with deposition of type III collagen in lacunae of hypertropic chondrocytes by invading bone-marrow-derived cells. This was followed by the deposition of type I collagen, which is the collagenous constituent of endochondral osteoid. At later stages of development endochondral osteoid was found in the epiphysial growth plate in apparently intact lacunae of hypertrophic chondrocytes; this indicated that the latter might contribute to the synthesis of osteoid type I collagen. Immuno-histological staining for collagen types, and von Kossa staining for calcium phosphate on parallel sections, demonstrated that type I and type II collagen matrices were substrates for calcification. Endochondral bone (with type I collagen) was found on scaffolding of both uncalcified and calcified cartilage (with type II collagen), indicating that calcification of endochondral osteoid and of the underlying cartilage occurred independentyl. Spicules of endochondral cancellous bone of a four-week-old chick contained a core of calcified type II collagen.     

In vitro response of neonatal condylar cartilage to simultaneous exposure to the parathyroid hormone fragments 1–34, 28–48, and 53–84 hPTH

Summary Mandibular condylar explants of neonatal ICR mice were maintained as serum-free organ culture systems and were used to study the effects of three synthetic fragments of human parathyroid hormone (hPTH) on the morphology of the organ and its ability to incorporate [³H]thymidine. Forty-eight-hour incubation with hPTH (1–34), at a concentration of 0.5 μg/ml caused an increase of 88% in DNA synthesis and a marked increase in the size of the chondroprogenitor zone. The mitogenic effect of hPTH (1–34) was decreased to 34% over control levels when the fragment hPTH (28–48) was added to the system. However, the addition of the latter fragment brought about a marked enhancement in the mineralization of the cartilaginous extracellular matrix along with the formation of an appreciable amount of new bone. Thede novo osseous tissue was attached to the mineralized cartilage. When the carboxyl-terminal fragment hPTH (53–84) was added together with the other two fragments, the mitogenic effect of hPTH (1–34) was completely abolished and the respective cultures incorporated [³H]thymidine even less than untreated control cultures. Moreover, the addition of hPTH (53–84) to the culture system led to distinct structural features throughout the mineralized hypertrophic cartilage. The latter contained a mixture of cells within an unorganized extracellular matrix. Untreated control cultures lacked such structures, but contained the various cell zones as normally seen in neonatal condylar cartilage. Therefore, it seems reasonable to suggest that each of the three fragments tested induces a biological effect on neonatal cartilage and might be involved in the normal process of endochondral ossification.     

Influence of irradiation on the osteoinductive potential of demineralized bone matrix

Summary Samples of demineralized bone matrix (DBM) were exposed to graduated doses of radiation (1–15 Megarad) (Mrad) utilizing a linear accelerator and then implanted into the thoracic region of Long-Evans rats. Subcutaneous implantation of DBM into allogenic rats induces endochondral bone. In response to matrix implantation, a cascade of events ensues; mesenchymal cell proliferation on day 3 postimplantation, chondrogenesis on day 7, calcification of the cartilagenous matrix and chondrolysis on day 9, and osteogenesis on day 11 resulting in formation of an ossicle containing active hemopoietic tissue. Bone formation was assessed by measuring alkaline phosphatase activity, the rate of mineralization was determined by measuring⁴⁵Ca incorporation to bone mineral, and⁴⁰Ca content measured the extent of mineralization; acid phosphatase activity was used as a parameter for bone resorption. The dose of radiation (2.5 Mrad) currently used by bone banks for sterilization of bone tissue did not destroy the bone induction properties of DBM. Furthermore, radiation of 3–5 Mrad even enhanced bone induction, insofar as it produced more bone at the same interval of time than was obtained from unirradiated control samples. None of the radiation doses used in these experiments abolished bone induction, although the response induced by matrix irradiated with doses higher than 5 Mrad was delayed.