The matrix of endochondral bone differs from the matrix of intramembranous bone

Summary Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induced cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM.     

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.     

The relationship between metabolic status and endochondral calcification in the process of bone development following decalcified bone matrix implantation

Experimental bone formation induced by decalcified bone matrix is an excellent model in vivo for studying endochondral bone formation. This study investigated the relationship between metabolic status and the onset of initial calcification in a decalcified matrix implanted subcutaneously in rat to characterize changes at discrete phases after implantation. Morphological evaluation and analyses of acid soluble mineral contents revealed that calcification took place on day 12 in regions of hypertrophic cartilage which were newly produced in the bone matrix. Levels of inorganic phosphate ions increased from day 6 and plateaued on day 9. Alkaline phosphatase activities peaked on day 12, and thereafter decreased with the progression of calcification. Energy charge ratio (ECR), which was considered to be an index reflecting the energy status of cells, was found to have shifted to lower values with the onset of calcification. These data support the hypothesis that the metabolic status of cells is closely associated with the beginning of calcification.     

Extracellular matrix alterations during endochondral ossification in humans

Immunohistochemical methods were employed to examine alterations in the cartilage extracellular matrix constituents associated with endochondral ossification in humans. The distributions of chondroitin 4- and 6-sulfate and keratan sulfate proteoglycan (PG) determinants, cartilage PG link protein, collagen types I and II, and fibronectin were determined in iliac crest growth-plate specimens using the avidin-biotin-horseradish peroxidase system. Collagen type II was distributed throughout the growth plate, providing a framework within which chondrocytes divided and formed clusters of differentiating (hypertrophic) cells. The septa between these clusters and their subchondral extensions into underlying bone trabeculae were rich in PG, PG link protein, and collagen type II and resembled the extracellular matrix of reserve cartilage. The territorial matrix associated with the differentiating cells within the clusters contained reduced amounts of collagen type II, PG link protein, and possibly cartilage PG. Collagen type I and fibronectin were detected within the cytoplasm of the maturing and degenerating cells, and fibronectin localized intensely to the pericellular matrix envelopes of these cells. These alterations presumably facilitate the degradation of the matrix associated with the cell clusters by invading vascular tissue, while the septa, which retain the characteristics of more typical cartilage matrix, are not degraded and firmly anchor the cartilage to the subchondral bone.     

Bone lengthening osteogenesis,a combination of intramembranous and endochondral ossification: an experimental study in sheep

We evaluated the morphological features of the newly formed tissue in an experimental model of tibial callotasis lengthening on 24 lambs, aged from 2 to 3 months at the time of operation. A unilateral external fixator prototype Monotube Triax^® (Stryker Howmedica Osteonics, New Jersey) was applied to the left tibia. A percutaneous osteotomy was performed in a minimally traumatic manner using a chisel. Lengthening was started 7 days after surgery and was continued to 30 mm. The 24 animals were randomly divided into three groups of 8 animals each: in Group 1, lengthening took place at a rate of 1 mm/day for 30 days; in Group 2, at a rate of 2 mm/day for 15 days; in Group 3, at a rate of 3 mm/day for 10 days. In each group, 4 animals were killed 2 weeks after end of lengthening, and the other 4 animals at 4 weeks after end of lengthening. To assess bony formation in the distraction area, radiographs were taken every 2 weeks from the day of surgery. To study the process of vascularization, we used Spalteholz’s technique. After killing, the tibia of each animal was harvested, and sections were stained with hematoxylin and eosin, Masson’s trichrome, and Safranin-O. Immunohistochemistry was performed, using specific antibodies to detect collagens I and II, S100 protein, and fibronectin. A combination of intramembranous and endochondral ossification occurred together at the site of distraction. Our study provides a detailed structural characterization of the newly formed tissue in an experimental model of tibial lengthening in sheep and may be useful for further investigations on callotasis.     

Role of proteoglycans in endochondral ossification: Inhibition of calcification

Summary Proteoglycans from bovine nasal septa and from the Swarm rat chondrosarcoma were isolated as aggregates (PGC) and as monomers (PGS). Portions of the PGC preparations were degraded with cathepsin D or chondroitinase AC. Chondroitin sulfates were isolated by differential precipitation from alkaline digests of the PGS from bovine nasal septa. The effects of these preparations at concentrations up to 2 mg/ml on the precipitation of tricalcium phosphatein vitro at pH 7.8 in 16 hours at 25°C were ascertained. To this end, the amounts of calcium and phosphate in the precipitates and in the supernates were determined. The PGC preparations were found to be very effective inhibitors; in the presence of 2 mg/ml, precipitate did not form. The PGS preparations were less effective than the PGC preparations; in the presence of 2 mg/ml, about 20% as much calcium phosphate precipitated as in their absence. The chondroitinase AC-degraded preparations at concentrations equivalent to 2 mg/ml of the PGC preparations were approximately as effective as the PGS preparations. On the other hand, the cathepsin D-degraded PGC preparations and the chondroitin sulfate chains were relatively poor inhibitors. Although the viscosity of the solutions may have influenced the rate at which the precipitates settled to the bottom of the bubes, the amounts of the tricalcium phosphate formed were related to the composition and concentration of the proteoglycan preparations.     

Changes in endochondral bone elongation rates during pregnancy and lactation in rats

Summary The rates of endochondral bone elongation during pregnancy and lactation in rats have been studied. The rate of growth at the distal femoral epiphyseal growth plate was measured using fluorescent bone markers. Endochondral growth rates were substantially increased in pregnant animals when compared with age-matched, nonmated controls. There were also increases in growth plate thickness, hypertrophic cell lacunar height, and the calculated rate of cell production during pregnancy. At parturition, this growth trend was reversed and during lactation there were significant decreases in endochondral growth rates. There were also corresponding decreases in growth plate thickness, hypertrophic cell lacunar height, and the calculated rate of cartilage cell production. These results indicate that significant changes occur in maternal endochondral growth rates during the reproductive cycle in rats.     

Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing.

Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data.

In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group.

In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.     

Immunofluorescent localization of structural collagen types in endochondral fracture repair

A nonimmobilized rat tibial fracture model of endochondral osseous repair was examined for the unique localizations of specific collagen genetic types. At various stages of the healing process, the demineralized callus was reacted with immunofluorescent antibodies directed against the type specific forms of matrix collagen. Type III collagen rapidly appeared (day 8-10) and remained in the primitive mesenchymal callus until remodeled. It was particularly prominent in the highly vasoformative regions and the pericallus encapsulation but not present in preexisting cortical and neoformed lamellar bone. The type II collagen, a marker of cartilage, was uniquely located only in areas of chondroid differentiation and calcification. Type II collagen was absent from all bone and was not identified beneath the repairing intact periosteum. The differentiating chondrocytes synthesized type II collagen on an underlayer of type III collagen already within the mesenchymal matrix. From these studies of genetically unique collagen markers, it appears that only in areas of motion or anoxia does an intermediate of chondroid tissue appear. The utilization of specific type II and type III collagen immunofluorescent antibodies has facilitated the understanding of the fracture repair process and has acted as an indicator for unique matrix components.     

Influence of magnesium depletion on matrix-induced endochondral bone formation

Summary The effect of magnesium deficiency on bone cell differentiation and bone formation was investigated using in vivo matrix-induced endochondral ossification as a test system. Demineralized bone matrix was implanted subcutaneously in young (35-day-old) male Long-Evans rats that had been fed a semisynthetic Mg-deficient diet (50 ppm Mg) for 7 days. Plasma Mg levels were reduced to 25–30% of control values at that time. Control rats were pairfed the same diet, supplemented to contain 1000 ppm Mg. The implants were harvested 7, 9, 11, 15, and 20 days after implantation and analyzed for Mg and Ca content,⁴⁵Ca incorporation, and alkaline phosphatase levels. At each stage, plaques (implants) removed from Mg-deficient rats showed retardation in cartilage and bone differentiation and matrix calcification. Magnesium content was markedly reduced when compared to the control plaques. Histological appearance of the matrix-induced plaques confirmed the retardation in bone development and mineralization suggested by the chemical indicators. Most marked was the virtual absence of bone marrow in 20-day-old plaques in Mg-depleted rats. These data show that bone cell differentiation can occur in a severely Mg-depleted environment, although the onset of mineralization and bone remodeling was delayed and bone marrow differentiation was impaired.     

Induction of endochondral bone by demineralized bone matrix from diabetic rats

Summary In this investigation we examined the osteoinductive potential of demineralized bone matrix derived from chronically diabetic (streptozotocin-induced) rats. Long-Evans rats (28–31 days) were made diabetic with a single injection of streptozotocin (65 mg/kg) and provided food and waterad lib for 2 months. Diaphyseal shafts of femurs and tibias removed from the diabetic rats and their sibling controls were dehydrated, pulverized, sieved to 74–420 μm particles, and demineralized Matrix was then bioassayed for its ability to induce endochondral bone on day 11 following subcutaneous implantation over the thorax of Long-Evans rats. The resulting plaques of tissue were subjected to histological analysis, determination of alkaline phosphatase activity, and calcium content. Bone matrix derived from diabetic animals proved to be a significantly better inducer of endochondral bone than did control matrix.     

Matrix metalloproteinases and bone

Matrix metalloproteinases (MMPs) are members of a family of zinc-dependent proteolytic enzymes. Several of the MMPs are expressed at high levels in bone and cartilage in mammals including humans and mice and are capable of cleaving native, undenatured collagens with long uninterrupted triple helices; these MMPs therefore potentially function as collagenases in vivo. Several MMPs expressed in the skeleton appear to function in endochondral ossification during embryonic development and in modeling and remodeling of bone postnatally and later in life. Different functions of MMPs have been elucidated through observations of spontaneous mutations in MMP genes in humans and of targeted mutations in Mmp genes and collagen (substrate) genes in mice. Potential mechanisms to account for effects of these mutations are considered in this review.     

NDST-1 modulates BMPR and PTHrP signaling during endochondral bone formation in a gene knockout model

GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST-1), a member of the enzyme family catalyzing the first modification step in the biosynthesis of heparan sulfate (HS), was knocked out in mice to investigate its role in embryonic development. NDST-1 null mice exhibited delayed endochondral bone formation including shortened calcified zones in limbs, delayed chondrocyte and osteogenetic differentiation, and increased chondrocyte proliferation. In situ HS binding assay revealed that the binding ability of bone morphogenetic protein (BMP) -2, -4, and -6 to endogenous HS was decreased in mutant phalanges, while that of fibroblast growth factor-1 (FGF-1) was not affected. Up-regulation of BMPR-IA, Phospho-Smad1 (P-Smad1) and parathyroid-hormone related protein (PTHrP), but not the Indian hedgehog, Gli1, Gli3, Patched, and FGFR-3, was observed. Furthermore, block of BMPR signaling with noggin rescued the delayed chondrocyte hypertrophic differentiation in NDST-1 (−/−) mice and recovered the expression of both P-Smad1 and PTHrP proteins. These results suggested that NDST-1-dependent heparan sulfate might negatively modulate BMP and its downstream PTHrP signaling, and thus affect endochondral bone development.     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

Extraction of a calcium-phospholipid-phosphate complex from bone

A calcium-phospholipid-phosphate complex with a constant 1∶1 calcium to total phosphate molar ratio is shown to exist in rabbit and calf bone. This complex, which may be involved in the transport and depositon of bone mineral, appears to constitute a significantly greater proportion of the lipids of younger bone than of more mature bone. The complex was isolated by a modified Folch extraction employing ultrasonic disruption of cellular material. Evidence is presented to show that the complex is a natural constituent of bone rather than one created artifcatually during extraction.     

Changes in proteoglycan aggregates during cartilage mineralization

Summary The dimensions of proteoglycan aggregates, aggregated monomers, and nonaggregated monomers, and the proportion of aggregated monomers found in the different zones of bovine rib growth plate have been defined by the electron microscopic monolayer technique. Growth plates were divided into the following 1 mm thick transverse slices; the hypertrophic zone, the lower proliferative zone, the upper proliferative zone, a transitional zone, and epiphyseal cartilage. Proteoglycans prepared by associative extraction followed by equilibrium density gradient centrifugation under associative conditions were examined by electron microscopy. Proteoglycan aggregate size decreased sharply in the lower proliferative and hypertrophic zones, as indicated by decreases in hyaluronate filament length and in the number of monomers per aggregate. Aggregated proteoglycan monomers did not show evidence of proteolytic degradation. Nonaggregated monomers were shorter than aggregated monomers, but their mean length did not decrease in the lower proliferative and hypertrophic zones. However, the proportion of nonaggregated monomers increased in these zones. Thus, before the cartilage matrix mineralized in the lower proliferative zone and as the cartilage matrix began to mineralize in the hypertrophic zone, proteoglycan aggregate size decreased and the proportion of aggregated monomers decreased. These changes in matrix proteoglycans may be one of the events that allow cartilage mineralization.     

Changes in osteonectin distribution and levels are associated with mineralization of the chicken tibial growth cartilage

Summary Osteonectin is a calcium-binding matrix protein thought to play a role in regulating calcium distribution in a variety of biologic processes. To examine its role in endochondral bone formation, we examined the distribution of the protein during mineralization of the chicken tibial growth cartilage, using immunohistochemistry and immunoelectron microscopy. The synthesis of osteonectin was also determined in chondrocyte populations isolated from premineralizing and mineralizing regions of growth cartilage and assayed in short-term culture. The resuls show that a very low level of osteonectin is detectable in the resting, proliferating, and early hypertrophic zones of growth cartilage; in these zones, osteonectin is largely cell-associated. In contrast, a large amount of osteonectin is present in the mineralizing zone where it is associated with the matrix. Biosynthetic data from short-term culture experiments indicate, however, that osteonectin is synthesized and secreted by chondrocytes from both premineralizing and mineralizing zones. As indicated by immunoprecipitation, Northern hybridization,in vitro translation of hybrid-selected messenger RNA (mRNA), and electrophoretic analysis, osteonectin synthesized by chondrocytes of the premineralizing zones is not obviously different in structure from that synthesized by chondrocytes of the mineralizing zone. We conclude that osteonectin is a product of chondrocytes in each zone of growth cartilage but accumulates only in the mineralizing zone. The high affinity of the protein for calcium could favor its retention in calcifying matrix.