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PubMed1995

Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells in vitro.

To characterize the mineralized nodules produced by rat periodontal ligament (PDL) cells in vitro, we have studied the synthesis and distribution of mineralized tissue proteins at various stages of nodule formation. PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of ascorbic acid (50 micrograms/ml), dexamethasone (5 microM), and beta-glycerophosphate (10 mM) for 3 weeks. Four stages showing distinct morphological characteristics during development of mineralized nodules were identified. Protein synthesis and deposition of proteins into the matrix were studied during these stages by metabolic labeling with [35S]methionine for 24 hours. Large quantities of SPARC (secreted protein, acidic and rich in cysteine) were synthesized by confluent cells but decreased during the progress of mineralized nodule formation. Two forms of osteopontin (OPN) (67 kDa and 61 kDa) were synthesized in comparable quantities by confluent cells; OPN and bone sialoprotein (BSP) were induced by dexamethasone and represented the major proteins in the mineralized matrix. The 67 kDa form of OPN was the predominant species in the mineralized matrix. Both OPN and BSP were localized by immunogold electron microscopy on globular as well as fused electron-dense structures at sites of tissue mineralization.

Animals↗
PubMed1992

Mechanism of action of beta-glycerophosphate on bone cell mineralization.

Experiments were performed to determine whether beta-glycerophosphate (beta-GP) promoted mineralization in vitro by modulating bone cell metabolic activity and/or serving as a local source of inorganic phosphate ions (Pi). Using MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells in the presence of beta-GP or Pi, we examined mineral formation, lactate generation, alkaline phosphatase (AP) activity, and protein and phospholipid synthesis. Neither beta-GP nor Pi modulated any of the major biosynthetic activities of the bone cells. Thus, we found no change in the levels of phospholipids, and the total protein concentration remained constant. Measurement of lactate synthesis showed that beta-GP did not effect the rate of anaerobic glycolysis. Evaluation of medium Pi levels clearly indicated that beta-GP was hydrolyzed by bone cells; within 24 hours, almost 80% of 10 mM beta-GP was hydrolyzed. It is likely that this local increase in medium Pi concentration promoted rapid mineral deposition. Chemical, energy dispersive X-ray, and Fourier transform infrared analysis of the mineral formed in the presence of beta-GP showed that it was nonapatitic; moreover, mineral particles were also seen in the culture medium itself. Experiments performed with a cell-free system indicated that mineral particles formed spontaneously in the presence of AP and beta-GP and were deposited into a collagen matrix. We conclude that medium supplementation with beta-GP or Pi should not exceed 2 mM. If this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.

Absorptiometry, Photon↗
PubMed1996

Visualization of crystal-matrix structure. In situ demineralization of mineralized turkey leg tendon and bone.

A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal "ghost" in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.

Aged↗
PubMed1992

Low bone mineral density in patients with inflammatory bowel disease.

To assess the prevalence and risk factors for low bone mineral density in inflammatory bowel disease, we studied 61 consecutive patients, mean age 36 +/- 11 years. Twenty-seven had a Crohn's disease and 34 ulcerative colitis (including 13 with ileoanal anastomosis). Three patients, two women and one man (32, 70, and 45 years old, respectively) had vertebral crush fractures. Bone mineral density measured by dual energy x-ray absorptiometry at spine and femoral level was more than 2 SD below normal values in 23% of the patients, all of them having received steroid therapy. Eighteen patients (29%) had never received steroid therapy; their bone mineral density was not different than those who had. Univariate analysis showed a positive correlation between bone mineral density and body weight or oral calcium intakes, and a negative correlation with steroid daily dose. After ileoanal anastomosis, bone mineral density was not different from other groups and showed a positive correlation with time elapsed since coloproctectomy. We concluded that bone mineral density is low in patients with inflammatory bowel disease and exposes them to the risk of bone fracture. Bone mineral density after ileoanal anastomosis may increase with time after surgery.

Absorptiometry, Photon↗
PubMed1996

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage.

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.

Adolescent↗
PubMed1992

Is pelvic bone mineral content assessed through dual energy X-ray absorptiometry an appropriate anatomical area for bone mass estimation in women?

Bibliographic references seem very controversial regarding the most appropriate anatomical area for bone mass estimation. Since some overlapping in the different bone mass measurements among normal and osteoporotic females has been observed, we have studied the bone mineral content of the pelvic bone through DEXA, and have correlated it with the total body bone mineral content, a highly discriminating measure, in order to observe whether pelvic bone mineral may be a useful measure in bone mass assessment. Pelvic and total body bone mineral values did not decrease until menopause in 104 normal premenopausal females aged 20 to 49 years. On the other hand, these values decreased in normal postmenopausal women (n = 44) aged 50 to 65 years (p < 0.001), with a 16% pelvic bone mineral content and an 11% total body bone mineral content decrease. Osteoporotic females (n = 30), showed lower values for both levels than normal postmenopausal ones (p < 0.001), with a 54% pelvic and a 24% total decrease. A 15% overlap was observed when pelvic values between normal postmenopausal and osteoporotic females were compared. The greater percentage decrease in pelvic BMC compared to total body bone mineral content and the lower overlap observed suggest that the pelvis may be an ideal anatomical area for bone mass evaluations.

Absorptiometry, Photon↗
PubMed1982

Focal mineralization defect during disodium etidronate treatment of calcinosis.

The use of disodium etidronate (EHDP) for the treatment of calcinosis is complicated by the threat of drug-induced inhibition of skeletal mineralization. Adults with Paget's disease of bone treated for 6 months with 10-20 mg/kg/day of EHDP have been reported to show both a marked delay in mineralization and a diffuse excess of unmineralized bone matrix. Drug-induced bone disease is, however, a function of growth as well as of the dose and duration of therapy. Therefore, children treated with EHDP may respond differently to the drug-induced mineralization defect. A 10-year-old girl with dermatomyositis developed incapacitating ectopic calcification. After 9 months of therapy with 12 mg/kg/Day of EHDP, a small decrease in the calcinosis was accompanied by a dramatic increase in joint mobility. Bone mineral content of the radial diaphysis showed a failure to gain mineral density as expected with prepubertal growth (8 cm/year). Bone biopsy revealed a patchy excess of osteoid. Although the percentage of osteoid surface labeled by tetracycline was reduced, normal mineralization was evident in the double-labeled areas. In children, the mineralization defect occurring with EHDP treatment may be focal.

Bone Diseases↗
PubMed1975

Ultrastructural observations of amorphous bone mineral in avian bone.

Mineral from medullary bone of three avian species was examined with the electron microscope in order to clarify the ultrastructure of amorphous bone mineral (ABM) in a mineralized tissue. Powders from freeze-dried bone revealed bone mineral with morphology and behavior identical to synthetic amorphous calcium phosphate (ACP). These powders exhibited spherically shaped particles 180--400 A in diameter with uniform electron density when viewed at low beam intensity. Thin sections of embedded freeze-dried bone also revealed spherically shaped particles 100--350 A in diameter with electron beam sensitivity characteristic of ACP. Regions of bone mineral with irregular outline and morphology were observed which closely resemble the discoidal form of synthetic ACP. More electron-dense spherical particles (150 A in diameter) could be seen budding from these regions. Some of these buds exhibited electronlucent centers characteristic of AMB. The inorganic nature of these features of bone mineral was confirmed using ultramicroincineration. Preliminary exploration of a freeze-substitution technique showed spherical bone mineral particles which were similar in morphology to those observed in freeze-dried samples. A limited degree of preservation of cellular material was observed using this freeze-substitution technique.

Animals↗
PubMed1975

The effects of fluoride on the mineralization of embryonic chick tibiae in organ culture.

The effects of various concentrations of fluoride on the mineralization of embryonic chich tibiae were studied in vitro. After 4 days incubation in medium with a physiological Ca x PO4 product (1.8 mM2), fluoride promoted a highly significant dose-related enhancement of bone mineralization. In experiments where the medium Ca x PO4 product was varied by changing either Ca or PO4 while keeping the other constant at 1.8 mM, fluoried at 7.5 muM or at 100 muM affected bone mineralization differbntly at low and high products. At medium Ca x PO4 products below 3 mM2, fluoride significantly enhanced bone mineral deposition compared to paired control bones, while at products above 5 mM2, fluoride significantly inhibited bone mineralization. This dual effect of fluoride was observed in both live bones and bones metabolically inhibited by 1 mM iodoacetic acid. Comparison of the effects of fluoride on the mineralization of live and dead bones suggested that the effects are dependent on the cellular control of Ca x PO4 product in the bone extracellular fluid. These findings support the hypothesis that fluoride may play an important role in governing the rate of mineralization in growing bones.

Animals↗
PubMed1975

Effect of matrix bound phosphate and fluoride on mineralization of dentin.

Completely demineralized bovine dentin mineralized to a high degree, but only after extraction of about half of its organic phosphate. Initiation of mineral formation depended on the concentrations of Ca2 plus, Pi, HCO3 minus and H plus ions in the incubation solution. After nucleation, mineral accretion by the dentin continued in solutions that did not, themselves, initiate mineralization. The nucleation phase, but not the subsequent accretion of mineral, was sensitive to small changes in temperature. Inclusion of 0.05 mM NaF in the solution markedly reduced the amount of mineral deposited during the nucleation phase but apparently enhanced subsequent mineralization.

Acetates↗
PubMed1975

Mineralization of invertebrate cartilage.

Although invertebrate cartilage tissues do not mineralize in nature, it is now reported for the first time that when excised gill cartilage tissue from Limulus (horse shoe crab) is placed in an appropriate incubation medium metastable to hydroxyapatite, mineralization will occur. The mineralization is temperature dependent, and takes place at 37 degrees but not at 20 degrees. Incubations in media metastable to calcite have not produced mineralization. Histologic examination of mineralized tissues showed mineral deposits predominantly within cells, and to a lesser extent in the matrix. X-ray diffraction of the deposited mineral revealed a typical biological hydroxyapatite pattern.

Animals↗
PubMed1995

Transforming growth factor-beta inhibition of mineralization by neonatal rat osteoblasts in monolayer and collagen gel culture.

The latent form of transforming growth factor-beta (TGF-beta) is a component of the extracellular matrix of bone. The active form, when locally injected in vivo, stimulates both inflammation and ectopic bone formation. The present study was undertaken to determine if TGF-beta also stimulated mineralization by isolated rat calvarial osteoblasts cultured in collagen gels. Gels were used because they should mimic in vivo conditions better than classical monolayer culture. Compared to cells in monolayers, osteoblasts cultured in collagen gels exhibited slower growth, but higher alkaline phosphatase activity and mineral deposition. Cultured cells also synthesized the osteoblast-specific marker, osteocalcin. The increase in osteocalcin in cell layers was parallel to the increase in mineral deposition. In the presence of TGF-beta, neither cell growth nor alkaline phosphatase activity increased. Instead, a small decrease occurred in both parameters when compared to untreated cultures. Accumulation of collagen, the major component of the extracellular matrix where mineralization occurs, was similar in untreated and TGF-beta 1-treated cultures. However, 8 pM TGF-beta 1 dramatically suppressed mineral deposition in both types of cultures. Despite TGF-beta 1 stimulating a fourfold increase in lactic acid, the consequent increase in culture medium acidity did not account for the inhibitory effects of TGF-beta 1 on mineralization. These results demonstrate that collagen gel culture is an improved technique over conventional monolayer culture for demonstrating differentiated osteoblast function and sensitivity to TGF-beta 1. TGF-beta 1, at a concentration that has little effect on cell growth, alkaline phosphatase activity, or collagen accumulation, is a potent inhibitor of mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1996

Decreased bone mineralization in children with phenylketonuria under treatment.

Children with phenylketonuria (PKU) obtain a great deal of their protein and mineral intakes from synthetic elemental formulae devoid of phenylalanine. To assess the effect of such diets and/or the disease on bone mineralization, children with PKU were compared to normal children for many parameters of mineral homeostasis and bone mineralization. A total of 11 children with PKU of mean age 10.9 +/- 4.2 years were compared to a large group of normal control children mean age 11.4 +/- 4.2, and an age and sex matched subset (n = 11). Children with PKU had lower serum calcium (9.1 +/- 0.9 vs 10.4 +/- 1.9 mg/dl P < 0.01) amd magnesium (1.67 +/- 1.4 vs 2.07 +/- 0.16 mg/ dl, P < 0.001) but normal values for phosphorus, zinc, and copper. The percentage tubular reabsorption of phosphorus was increased in PKU (93 +/- 3% vs 88 +/- 6%, P < 0.05) suggesting a lower phosphorus intake and/or absorption. Serum 25-hydroxyvitamin D, parathyroid hormone and 1,25 dihydroxyvitamin D were similar in PKU and control children. Serum albumin and lean body mass by dual energy X-ray absorption were not different suggesting that protein intake was adequate. In the 11 pairs, a decreased bone mineral density was seen for the lumbar spine (0.61 +/- 0.15 vs 0.72 +/- 0.24 P < 0.05), and lower extremities (1.56 +/- 0.30 vs 1.87 +/- 0.56 P < 0.05) by paired t-test. Compared to the total controls and the paired controls, decreases were seen in markers of bone formation; bone alkaline phosphatase, (72 +/- 30 vs 126 +/- 43 P < 0.001), osteocalcin (10.7 +/- 3.4 vs 13.1 +/- 2.0 P < 0.05) and procollagen type I carboxyterminal propeptide. No differences were seen in the bone resorption markers tartrate resistant acid phosphatase and urine Ca/Cr. The changes noted could not be related after age correction to serum phenylalanine levels, protein intake, or mineral intakes. It is unclear whether deficits in bone mineralization relate to the disease process itself or its treatment.

Adolescent↗
PubMed2001

Decreased bone mineral density in neurofibromatosis-1 patients with spinal deformities.

A cross-sectional study was carried out to obtain data on the bone mineral density status of a group of neurofibromatosis-1 patients with spinal deformities, and to search for possible accompanying changes in the bone mineral turnover. Neurofibromatosis-1 is a heredofamiliar disorder that is associated with a variety of skeletal anomalies (mostly spinal deformities) in 10-50% of patients. Intraoperatively, a poor vertebral bone quality has been observed. Efforts have been made to identify factors preventing curve progression, to optimize operational planning and to explain the pathomechanism. As part of the preoperative evaluation, dual-energy X-ray absorptiometry was used to assess the bone mineral density of the lumbar spine in 12 patients with neurofibromatosis-1, supplemented by laboratory blood/urine investigations. A significant decrease in bone mineral density of the lumbar spine was measured. An inverse relation was suggested between the severity of scoliosis and the lumbar spine Z-scores. No pivotal alterations were identified in the laboratory measurements. The bony tissue abnormality observed intraoperatively in neurofibromatosis-1 patients may be described as a diminution of the axial bone mineral density. The biochemical parameters do not support the presence of hyperparathyroidism, renal disorders or other associated diseases influencing the bone mineral turnover. The evaluation of bone mineral density in the course of the preoperative planning is proposed in neurofibromatosis-1; the exact background and the role of a possible osteoporosis in the prognosis remain to be elucidated.

Absorptiometry, Photon↗
PubMed2003

Phosphate ions in bone: identification of a calcium-organic phosphate complex by 31P solid-state NMR spectroscopy at early stages of mineralization.

Previous 31P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO(-3)(4) phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO(-2)(4) component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state 31P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state 31P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.

Animals↗
PubMed2003

Targeted overexpression of vitamin D receptor in osteoblasts increases calcium concentration without affecting structural properties of bone mineral crystals.

Increased cross-sectional area and strength of long bones has been observed in transgenic mice with 2-fold (OSV9) and 3-fold (OSV3) elevation of osteoblast vitamin D receptor (VDR) levels. In the present study, mineralization density distributions, including typical calcium content (Ca(Peak)) and homogeneity of mineralization (Ca(Width)) of femoral bone and growth plate cartilage, were determined by quantitative backscattered electron imaging (qBEI). Fourier-transform infrared (FTIR) microspectroscopy was used to examine mineral content, collagen and crystal maturation, and scanning small angle X-ray scattering (scanning-SAXS) for studying mineral particle thickness and alignment. In addition, X-ray diffraction (XRD) of distal tibiae revealed mineral particle c-axis size. In trabecular bone, the increase in Ca(Peak) was significant for both OSV9 (+ 3.14%, P = 0.03) and OSV3 (+ 3.43%, P = 0.02) versus controls with 23.61 +/- 0.45 S.D. wt% Ca baseline values. In cortical bone, Ca(Peak) was enhanced for the OSV3 mice (+ 1.84%, P = 0.02) versus controls with 26.61 +/- 0.28 S.D. wt% Ca, and OSV9 having intermediate values. Additionally, there was significantly increased homogeneity of mineralization as denoted by a reduction of Ca(Width) (-8.4%, P = 0.01) in primary spongiosa. FTIR microspectroscopy, with the exception of an increased collagen maturity in OSV3 trabecular bone (+ 9.9%, P = 0.02), XRD, and scanning-SAXS indicated no alterations in the nanostructure of transgenic bone. These findings indicate that elevation of osteoblastic vitamin D response led to formation of normal bone with higher calcium content. These material properties, together with indications of decreased bone resorption in secondary spongiosa and increased cortical periosteal bone formation, appear to contribute to the improved mechanical properties of their long bones and suggest an important physiological role of the vitamin D-endocrine system in normal bone mineralization.

Animals↗
PubMed2005

Acidosis inhibits bone formation by osteoblasts in vitro by preventing mineralization.

The negative effect of acidosis on the skeleton has been known for almost a century. Bone mineral serves an important pathophysiologic role as a reserve of hydroxyl ions to buffer systemic protons if the kidneys and lungs are unable to maintain acid-base balance within narrow physiologic limits. Extracellular hydrogen ions are now thought to be the primary activation signal for osteoclastic bone resorption, and osteoclasts are very sensitive to small changes in pH within the pathophysiologic range. Herein, we investigated the effects of acidosis on osteoblast function by using mineralized bone nodule-forming primary osteoblast cultures. Osteoblasts harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone. pH was manipulated by addition of 5 to 30 mmol/L HCl and monitored by blood gas analyzer. Abundant, matrix-containing mineralized nodules formed in osteoblast cultures at pH 7.4, but acidification progressively reduced mineralization of bone nodules, with complete abolition at pH 6.9. Osteoblast proliferation and collagen synthesis, assessed by 3H-thymidine and 3H-proline incorporation, respectively, were unaffected by pH in the range 7.4 to 6.9; no effect of acidification on collagen ultrastructure and organization was evident. The apoptosis rate of osteoblasts, assessed by the enrichment of nucleosomes in cell lysates, was also unaffected by pH within this range. However, osteoblast alkaline phosphatase activity, which peaked strongly near pH 7.4, was reduced eight-fold at pH 6.9. Reducing pH to 6.9 also downregulated messenger ribonucleic acid (mRNA) for alkaline phosphatase, but upregulated mRNA for matrix Gla protein, an inhibitor of mineralization. The same pH reduction is associated with two-and four-fold increases in Ca2+ and PO4(3-) solubility for hydroxyapatite, respectively. Our results show that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response. Thus, in uncorrected acidosis, the deposition of alkaline mineral in bone by osteoblasts is reduced, and osteoclast resorptive activity is increased in order to maximize the availability of hydroxyl ions in solution to buffer protons.

Acidosis↗
PubMed2000

Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts.

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (betaGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of betaGP supply were tested. We compared 10 mM betaGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where betaGP was added at day 0. Furthermore, 10 mM betaGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture.

Aged↗