Influence of vitamin A on matrix-induced endochondral bone formation

Summary The influence of retinoic acid on matrix-induced endochondral bone differentiation was determined. Retinoic acid was administered during discrete stages of endochondral bone formation, specifically, mesenchymal cell proliferation, chondrogenesis, bone formation, and mineralization. In retinoic acid-treated rats examined on day 3 following matrix implantation, biochemical markers for mesenchymal cell proliferation were about 50% of the controls. Chondrogenesis on day 7, assessed by³⁵SO₄ incorporation into proteoglycans, was 27% of the control. In addition, dissociative extraction of proteoglycans with 4.0 M guanidine-HCl and chromatography on Sepharose CL-2B revealed the synthesis of a smaller molecular weight proteoglycan when compared to controls which exhibited the cartilage-specific type. Osteogenesis and bone mineralization were monitored by alkaline phosphatase activity and⁴⁵Ca incorporation. On day 11 alkaline phosphatase activity was decreased by 40% and⁴⁵Ca incorporation was 48% of the control. These results revealed the multiple foci of the actions of excess vitamin A.     

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.     

Influence of estrogen and progesterone on matrix-induced endochondral bone formation

Summary The influence of estradiol and progesterone, alone or in combination, on the discrete phases of matrix-induced endochondral bone formation was investigated. Administration of estradiol and progesterone in combination increased mesenchymal cell proliferation, as indicated by [³H] thymidine incorporaton into acid precipitable material. However, ornithine decarboxylase (ODC) activity was significantly suppressed by the combination of estradiol and progesterone. Also, this treatment did not influence the³⁵SO₄ incorporation into proteoglycans on day 7. Mineralization of newly induced bone was quantitated by alkaline phosphatase,⁴⁵Ca incorporation into bone mineral and calcium content, and was found to be significantly increased by progesterone alone and in combination with estradiol in both matrix-induced plaques and tibial metaphysis. These results demonstrated the stimulatory role of progesterone in combination with estradiol in bone formation and mineralization.     

Influence of estrogen and progesterone on matrix-induced endochondral bone formation

The influence of estradiol and progesterone, alone or in combination, on the discrete phases of matrix-induced endochondral bone formation was investigated. Administration of estradiol and progesterone in combination increased mesenchymal cell proliferation, as indicated by [3H] thymidine incorporation into acid precipitable material. However, ornithine decarboxylase (ODC) activity was significantly suppressed by the combination of estradiol and progesterone. Also, this treatment did not influence the 35SO4 incorporation into proteoglycans on day 7. Mineralization of newly induced bone was quantitated by alkaline phosphatase, 45Ca incorporation into bone mineral and calcium content, and was found to be significantly increased by progesterone alone and in combination with estradiol in both matrix-induced plaques and tibial metaphysis. These results demonstrated the stimulatory role of progesterone in combination with estradiol in bone formation and mineralization.     

Influence of whole body irradiation and local shielding on matrix-induced endochondral bone differentiation

Summary Subcutaneous implantation of demineralized bone matrix into allogeneic rats induces endochondral dochondral bone formation. We have investigated the effects of irradiation on the sequelae of the interaction of collagenous matrix and mesenchymal cells and on cartilage and bone differentiation. Rats were irradiated in a vertical direction with a midline dose of 850 rad. Radiation entered the rats ventrally while a small area of the upper thorax was locally shielded. After irradiation, bone matrix was implanted in shielded and nonshielded sites, and the implants were studied at various stages. On day 3, [³H]thymidine incorporation, an index of cell proliferation, was inhibited by 70% in the nonshielded sites compared to nonirradiated control rats. The degree of inhibition (35%) was less pronounced in shielded sites. Furthermore, there was recovery of cell proliferation in the shielded sites as opposed to the nonshielded contralateral site. A similar pattern was observed on day 7 as assessed by³⁵SO₄ incorporation into proteoglycans during chondrogenesis. Bone formation and mineralization were quantified on day 11 by alkaline phosphatase activity and⁴⁵Ca incorporation. In nonshielded sites, there was a 73% inhibition of alkaline phosphatase activity. In conclusion, radiation impaired progenitor cell proliferation which resulted in decreased cartilage and bone differentiation. These findings imply that local mesenchymal cells proliferate and differentiate into bone in response to implanted collagenous matrix.     

Bone matrix-induced local bone induction

The sequential cellular changes in the implants in response to collagenous bone matrix-induced local bone formation include: binding of fibronectin to matrix, chemotaxis and attachment of progenitor cells, proliferation and differentiation of progenitor cells into chondrocytes, and finally osteogenesis and marrow differentiation. The cellular origin of osteogenic proteins is not clear. The present study compares the osteogenic potential of demineralized rat and porcine bone matrix by dissociative extraction and reconstitution. Judging from the Sephacryl S-200 gel filtration profiles of the dissociative extracts of rat and porcine matrix, the latter appears to be smaller. Under identical experimental conditions, the rat chondrosarcoma and osteosarcoma were examined for chondrogenic and osteogenic properties and found to be devoid of inductive potential. It is noteworthy that gel filtration fractions of rat chondrosarcoma on Sepharose CL-6B are inhibitory to bone inductive potential of demineralized rat bone matrix.     

Effect of bone marrow mononuclear phagocytes on the bone matrix-induced bone formation in rats

Experimental ulnar bone defects in rats were grafted with freshly isolated whole bone marrow cells; bone marrow mononuclear phagocytes (macrophages); or both types of marrow cell preparations in combination with demineralized bone matrix gelatin (BMG). In the absence of BMG, the osteogenic performance of the marrow cell preparations was superior to that of the macrophages. In the presence of BMG (composite grafts), their osteogenic potential was nearly identical and significantly improved the level of bone formation stimulated by implants of BMG alone. The results encourage speculation and further research on sequential activities of bone marrow monocyte-macrophage (osteoclast) lineages and marrow stromal (osteoprogenitor) cell in bone morphogenetic protein (BMP)-induced regeneration.     

Changes in proline synthetic and degradative enzymes during matrix-induced cartilage and bone formation

Summary Proline biosynthetic and degradative enzymes are unevenly distributed in differentiated mammalian tissues. Activities of the synthetic enzymes are relatively high in collagenous tissues, whereas activities of the degradative enzymes are high in noncollagenous tissues. In order to further characterize tissue-specific proline biosynthesis and degradation, we have determined proline enzyme activities during cartilage and bone formation induced by demineralized bone matrix. We can thus follow temporal changes in enzyme activity in a single tissue as different cell types develop. Ornithine aminotransferase and pyrroline-5-carboxylate reductase have peaks of activity which correlate with maximal type II collagen synthesis by chondrocytes. Both enzymes also are active during bone formation. In contrast, proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at low levels and do not change as new cell types appear. Arginase activity peaks during the first 3 days and then rapidly decreases by the time cartilage and bone formation begin. These observations further substantiate the importance of proline biosynthesis in collagenous tissues. The close correlation between ornithine aminotransferase activity and type II collagen synthesis suggests that the pathway from ornithine to proline may be especially important during formation of type II collagen.     

Changes in proline synthetic and degradative enzymes during matrix-induced cartilage and bone formation.

Proline biosynthetic and degradative enzymes are unevenly distributed in differentiated mammalian tissues. Activities of the synthetic enzymes are relatively high in collagenous tissues, whereas activities of the degradative enzymes are high in noncollagenous tissues. In order to further characterize tissue-specific proline biosynthesis and degradation, we have determined proline enzyme activities during cartilage and bone formation induced by demineralized bone matrix. We can thus follow temporal changes in enzyme activity in a single tissue as different cell types develop. Ornithine aminotransferase and pyrroline-5-carboxylate reductase have peaks of activity which correlate with maximal type II collagen synthesis by chondrocytes. Both enzymes also are active during bone formation. In contrast, proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at low levels and do not change as new cell types appear. Arginase activity peaks during the first 3 days and then rapidly decreases by the time cartilage and bone formation begin. These observations further substantiate the importance of proline biosynthesis in collagenous tissues. The close correlation between ornithine aminotransferase activity and type II collagen synthesis suggests that the pathway from ornithine to proline may be especially important during formation of type II collagen.     

Loss of bone sialoprotein leads to impaired endochondral bone development and mineralization

Bone sialoprotein (BSP) is an anionic phosphoprotein in the extracellular matrix of mineralized tissues, and a promoter of biomineralization and osteoblast development. Previous studies on the Bsp-deficient mouse (Bsp^−/−) have demonstrated a significant bone and periodontal tissue phenotype in adulthood. However, the role of BSP during early long bone development is not known. To address this, early endochondral ossification in the Bsp^−/− mouse was studied. Embryonic day 15.5 (E15.5) wild-type (WT) tibiae showed early stages of ossification that were absent in Bsp^−/− mice. At E16.5, mineralization had commenced in the Bsp^−/− mice, but staining for mineral was less intense and more dispersed compared with that in WT controls. Tibiae from Bsp^−/− mice also demonstrated decreased mineralization and shortened length at postnatal day 0.5 (P0.5) compared to WT bones. There was no detectable difference in the number of tartrate-resistant acid phosphatase-positive foci at P0.5, although the P0.5 Bsp^−/− tibiae had decreased Vegfα expression compared with WT tissue. Due to the shortened tibiae the growth plates were examined and determined to be of normal overall length. However, the length of the resting zone was increased in P0.5 Bsp^−/− tibiae whereas that of the proliferative zone was decreased, with no change in the hypertrophic zone length of Bsp^−/− mice. A reduction in cells positive for Ki-67, an S-phase cell-cycle marker, was noted in the proliferative zone. Decreased numbers of TUNEL-positive hypertrophic chondrocytes were also apparent in the Bsp^−/− tibial growth plates, suggesting decreased apoptosis. Expression of the osteogenic markers Alp1, Col1a1, Sp7, Runx2, and Bglap was reduced in the endochondral bone of the neonatal Bsp^−/− compared to WT tibiae. These results suggest that BSP is an important and multifaceted protein that regulates both chondrocyte proliferation and apoptosis as well as transition from cartilage to bone during development of endochondral bone.     

Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice

BACKGROUND: It is unclear whether platelet-rich plasma is a clinically effective adjunct to osteoinductive agents such as demineralized bone matrix. It contains platelet-derived growth factor (PDGF), which decreases osteoinduction by human demineralized bone matrix in nude-mouse muscle, suggesting that platelet-rich plasma may also have a negative impact. This study tested the hypothesis that platelet-rich plasma reduces demineralized bone matrix-induced bone formation and that this effect varies with donor-dependent differences in platelet-rich plasma and demineralized bone matrix. METHODS: Human platelet-rich plasma was prepared from blood from six men (average age [and standard error of the mean], 29.2 +/- 2.4 years). Platelet numbers were determined, and growth factors were quantified before and after platelet activation. Human demineralized bone matrix from two donors (demineralized bone matrix-1 and demineralized bone matrix-2) was mixed with activated platelet-rich plasma and was implanted bilaterally in the gastrocnemius muscle in eighty male nude mice (eight implants per variable). Fifty-six days after implantation, the hindlimb calf muscles were harvested for histological analysis. Osteoinduction was evaluated with use of a qualitative score and morphometric measurements of ossicle size, new bone formation, and residual demineralized bone matrix. RESULTS: Compared with platelet-poor plasma, platelet-rich plasma preparations exhibited a fourfold increase in the platelet count, a fifteenfold increase in the amount of transforming growth factor-beta, a sixfold increase in the amount of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold increase in the amount of PDGF-AB. Demineralized bone matrix-1 was more osteoinductive than demineralized bone matrix-2, as determined on the basis of a greater ossicle area. The effect of platelet-rich plasma was either neutral or inhibitory depending on the demineralized bone matrix batch. When used with demineralized bone matrix-1, platelet-rich plasma did not alter the qualitative score or overall ossicle size, but it decreased the new bone area. When used with demineralized bone matrix-2, platelet-rich plasma reduced the qualitative score, ossicle area, and new bone area and increased the amount of residual demineralized bone matrix. The effects on osteoinduction also varied with the donor of the platelet-rich plasma. CONCLUSIONS: Platelet-rich plasma decreased the osteoinductivity of demineralized bone matrix implanted in immunocom-promised mice, and the activities of both demineralized bone matrix and platelet-rich plasma were donor-dependent.     

Effect of aluminum and parathyroid hormone on osteoblasts and bone mineralization in chronic renal failure

Bone aluminum, quantitative bone histology, and plasma parathyroid hormone (PTH) were compared in 29 patients undergoing chronic hemodialysis. Histologic techniques included double tetracycline labeling and histochemical identification of osteoclasts and osteoblasts. Bone aluminum was measured chemically by flameless atomic absorption spectrophotometry, and histochemically. When measured chemically, the bone aluminum was 67 +/- 46 (SD) mg/kg dry weight (normal 2.4 +/- 1.2 mg/kg); histochemically, aluminum was present at 2.9 +/- 4.4% of trabecular surface. The biochemical and histochemical results agreed well (r = 0.80, P less than 0.001). No double tetracycline labels were seen at the mineralization front where aluminum was deposited, indicating cessation of mineralization at these sites. The osteoblast surface correlated positively with plasma PTH (r = 0.67, P less than 0.001) and negatively with bone aluminum level (r = -0.42, P less than 0.05). Multiple linear regression showed a correlation of aluminum with osteoblasts additional to that of PTH, consistent with a direct effect of aluminum in depressing osteoblast numbers. Though a relationship between PTH and chemically determined bone aluminum level could not be demonstrated, there was a negative correlation between osteoclast count and aluminum, and the nine patients with severe hyperparathyroid bone disease had lower chemically determined aluminum levels than the other patients. These results suggest that aluminum (a) directly inhibits mineralization, (b) is associated with decreased PTH activity and hence osteoblast numbers, and (c) directly reduces osteoblast numbers. In addition to inducing severe, resistant osteomalacia, aluminum appears to contribute to the mild osteomalacia commonly seen in renal failure, characterized by extensive thin osteoid and low tetracycline and osteoblast surfaces.     

Effect of aluminum and parathyroid hormone on osteoblasts and bone mineralization in chronic renal failure

Summary Bone aluminum, quantitative bone histology, and plasma parathyroid hormone (PTH) were compared in 29 patients undergoing chronic hemodialysis. Histologic techniques included double tetracycline labeling and histochemical identification of osteoclasts and osteoblasts. Bone aluminum was measured chemically by flameless atomic absorption spectrophotometry, and histochemically. When measured chemically, the bone aluminum was 67±46 (SD) mg/kg dry weight (normal 2.4±1.2 mg/kg); histochemically, aluminum was present at 2.9±4.4% of trabecular surface. The biochemical and histochemical results agreed well (r=0.80,P<0.001). No double tetracycline labels were seen at the mineralization front where aluminum was deposited, indicating cessation of mineralization at these sites. The osteoblast surface correlated positively with plasma PTH (r=0.67,P<0.001) and negatively with bone aluminum level (r=−0.42,P<0.05). Multiple linear regression showed a correlation of aluminum with osteoblasts additional to that of PTH, consistent with a direct effect of aluminum in depressing osteoblast numbers. Though a relationship between PTH and chemically determined bone aluminum level could not be demonstrated, there was a negative correlation between osteoclast count and aluminum, and the nine patients with severe hyperparathyroid bone disease had lower chemically determined aluminum levels than the other patients. These results suggest that aluminum (a) directly inhibits mineralization, (b) is associated with decreased PTH activity and hence osteoblast numbers, and (c) directly reduces osteoblast numbers. In addition to inducing severe, resistant osteomalacia, aluminum appears to contribute to the mild osteomalacia commonly seen in renal failure, characterized by extensive thin osteoid and low tetracycline and osteoblast surfaces.     

Platelet-derived growth factor enhances demineralized bone matrix-induced cartilage and bone formation

Summary Subcutaneous implantation of demineralized bone matrix induces the local formation of cartilage and bone. In this study we have investigated the influence of adding various growth factors to the implant. Cartilage formation was monitored by measuring collagen II mRNA levels, and bone formation in the implant was assessed from alkaline phosphatase activity and calcium content. Supplements of the platelet-derived growth factor to implants in older rats increased and production of mRNA for collagen II, alkaline phosphatase activity, and the calcium content of the implant, whereas the other growth factors tested were without effect. The data suggest that under some conditions bone induction is submaximal and can be increased by local supplement of platelet-derived growth factor (PDGF). The present observations may have important therapeutic implications in the treatment of nonunions of fractures and impaired bone formation in the aged.     

Dystrophic calcification and mineralization during bone induction: biochemical differences

The calcification of implants of glutaraldehyde-crosslinked 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 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 subcutaneous implantation. After 4 weeks of implantation in 26-month-old rats, the cylinders containing embryonic calvaria or muscle calls were found to be full of bone and/or cartilage.     

DSPP effects on in vivo bone mineralization

Dentin sialophosphoprotein has been implicated in the mineralization process based on the defective dentin formation in Dspp null mice (Dspp-/-). Dspp is expressed at low levels in bone and Dspp-/- femurs assessed by quantitative micro-computed tomography (micro-CT) and Fourier transform infrared spectroscopic imaging (FTIRI) exhibit some mineral and matrix property differences from wildtype femurs in both developing and mature mice. Compared to wildtype, Dspp-/- mice initially (5 weeks) and at 7 months had significantly higher trabecular bone volume fractions and lower trabecular separation, while at 9 months, bone volume fraction and trabecular number were lower. Cortical bone mineral density, area, and moments of inertia in Dspp-/- were reduced at 9 months. By FTIRI, Dspp-/- animals initially (5 months) contained more stoichiometric bone apatite with higher crystallinity (crystal size/perfection) and lower carbonate substitution. This difference progressively reversed with age (significantly decreased crystallinity and increased acid phosphate content in Dspp-/- cortical bone by 9 months of age). Mineral density as determined in 3D micro-CT and mineral-to-matrix ratios as determined by 2D FTIRI in individual cortical and trabecular bones were correlated (r(2)=0.6, p<0.04). From the matrix analysis, the collagen maturity of both cortical and trabecular bones was greater in Dspp-/- than controls at 5 weeks; by 9 months this difference in cross-linking pattern did not exist. Variations in mineral and matrix properties observed at different ages are attributable, in part, to the ability of the Dspp gene products to regulate both initial mineralization and remodeling, implying an effect of Dspp on bone turnover.     

Platelet-derived growth factor inhibits demineralized bone matrix-induced intramuscular cartilage and bone formation. A study of immunocompromised mice

BACKGROUND: Platelet-derived growth factor (PDGF) has been proposed as a therapeutic agent to promote bone-healing. The purpose of this study was to examine the effect of PDGF on the ability of human demineralized bone matrix to induce bone formation in a nude-mouse muscle-implantation model. We also examined whether platelet-rich plasma, which contains PDGF, also modulates osteoinduction in this model. METHODS: Human demineralized bone matrix, previously shown to be osteoinductive in the calf muscles of nude mice, was mixed with PDGF-BB (0, 0.1, 1, and 10 microg/10 mg of demineralized bone matrix) and was implanted bilaterally in the calf muscles of immunocompromised (nu/nu) mice (six mice in each group). Heat-inactivated demineralized bone matrix was used as a control. Tissue was harvested at fourteen, twenty-eight, and fifty-six days after implantation. Platelet-rich plasma was prepared from the blood of a healthy donor with use of the Harvest PRP preparation device, activated with thrombin, and mixed with active and inactive demineralized bone matrix. Fifty-six days post-implantation, tissues were harvested. Osteoinduction was assessed with use of a qualitative scoring system and with quantitative histomorphometry. RESULTS: Cartilage was present at fourteen days in all tissues that had received an implant, but the amount decreased as the PDGF concentration increased. PDGF reduced bone formation at twenty-eight days in a dose-dependent manner. This inhibitory effect was resolved by fifty-six days, except in tissues in which demineralized bone matrix and 10 microg of PDGF had been implanted. In sites treated with 10 microg of PDGF, the area of new bone was decreased and the area of bone marrow was reduced at twenty-eight and fifty-six days. PDGF also appeared to retard resorption of demineralized bone matrix in a dose-dependent manner. Platelet-rich plasma reduced osteoinduction by human demineralized bone matrix that had high osteoinductive activity and had no effect on osteoinduction by demineralized bone matrix with low activity. CONCLUSIONS: PDGF inhibits, in a dose-dependent manner, intramuscular osteoinduction and chondrogenesis by demineralized bone matrix in immunocompromised mice. Platelet-rich plasma also reduces the osteoinductivity of active demineralized bone matrix.     

Quantitative analysis on the rate of secondary bone mineralization

The microradiographic-photometric method of studying the X-ray absorption, and the microhardness testing technique were concurrently applied to investigate the rate of secondary mineralization of bone of known age in the osteons of young immature and adult dogs.The results of the two series of measurement show a close agreement. They indicate that the rate of secondary mineralization, (a) slowly and progressively decreases with time in each osteon, (b) undergoes little variations in the various osteons of each subject independently of the reconstruction rate characteristic of each skeletal region, and (c) markedly decreases with the ageing of the animal.This work is dedicated with great respect to Prof. O. M. Olivo in honour of his 75th birthday.