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Biomedical subjects

D J Baylink

Publications and source records attributed to D J Baylink.

At least 253 records · Page 14Linked to original sources
PubMed1988

Effect of bone matrix-derived growth factors on skull and tibia in the growing rat.

Aberration in cranial skeletal morphology during growth due to metabolic bone diseases associated with hypocalcemia is related to abnormalities in cranial suture growth. It was found in previous experimental studies that a changed cranial growth pattern induced by hypocalcemia and secondary hyperparathyroidism was dependent on a decreased bone apposition in viscerocranial growth sites. Of interest was the observation that obliterative osteogenesis occurred with this disturbance. In vitro studies have shown that when bone resorption is stimulated by parathyroid hormone, mitogenic factors for bone cells are released from the matrix. The aim of the current study was to elucidate a possible local regulatory mechanism for bone growth especially with regard to cranial growth. Two groups of young rats were injected daily with 150 micrograms and 750 micrograms respectively of bone matrix-derived growth factor preparation (BMDGF) for 2 weeks. By using cephalometric analysis, the BMDGF administration was shown to alter skull morphology during growth. Since the high doses of BMDGF caused markedly more changes than the low dose, the overall effect on craniofacial growth seemed to be dose dependent. The high dose of BMDGF induced a decrease in cortical bone apposition found in this region, as well as in the diaphysis of the tibia. Also a change in position of the viscerocranium in relation to the cranial base was found in the BMDGF-treated rats: the flexure between the viscerocranium and the cranial base being greater compared with the normal skull. This change in relationship seemed to be related to the enhanced bone apposition found in the viscerocranial sutures of BMDGF-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Demonstration of reduced mitogenic and osteoinductive activities in demineralized allogeneic bone matrix from vitamin D-deficient rats.

Osteoinduction is the formation of ectopic bone that follows implantation of demineralized allogeneic bone matrix (DABM) and is believed to be secondary to the release of associated inductive factors from bone matrix. To clarify the role of vitamin D in osteoinduction, we implanted DABM from vitamin D-deficient rats (-D rats) into normal rats (+D rats). Because mitogens and osteocalcin might be involved in osteoinduction, these were measured. Mitogenic activity in extracts from mineralized allogeneic bone matrix (ABM) and DABM from both +D and -D rats was determined with an assay that utilizes monolayer cultures of embryonic chick calvarial cells. Osteocalcin in serum and DABM was measured by radioimmunoassay. DABM from -D rats did not promote osteoinduction as effectively as DABM from +D rats. Resorption of implant matrix from -D rats was diminished compared with resorption of matrix from +D rats (P less than 0.01), and the decrease was attributed to a corresponding decrease in the number of osteoclasts in the implants (P less than 0.02). Bone formation (P less than 0.01) and total implant mineralization (P less than 0.001) were significantly reduced in implants from -D rats, and the reductions corresponded with a decline in the number of osteoblasts (P less than 0.05). Mitogenic activity in DABM from +D rats was only slightly decreased as compared with activity in ABM, but DABM from -D rats contained significantly less activity (P less than 0.001). No mitogenic activity was identified in implants of DABM from either +D or -D rats 3 wk after implantation. Serum osteocalcin was significantly higher in -D as compared with +D animals. In contrast, the concentrations of osteocalcin in DABM from the two groups of animals were not significantly different from each other. These findings indicate that the diminished osteoinductive activity of DABM from -D rats results from deficiency of one or more mitogenic factors that are essential for inducing the proliferation and differentiation of bone cells at the implant site and that osteocalcin does not play a role in this regard.

Animals↗
PubMed1988

Chick osteoblasts contain fluoride-sensitive acid phosphatase activity.

We used histological and biochemical methods to determine the cellular origin of bone matrix fluoride-sensitive acid phosphatase in chicken bone. Embryonic chicken calvariae were embedded in plastic and sections stained for acid phosphatase at various concentrations of substrate and fluoride. Acid phosphatase activity was observed in osteoblasts and osteoclasts but not in fibroblasts. Striking inhibition of osteoblastic acid phosphatase occurred at 100 microM fluoride, a concentration that had no apparent effect on osteoclastic acid phosphatase. Inhibition of osteoblastic and osteoclastic acid phosphatase by fluoride was also examined using extracts of embryonic chicken calvarial cells, mouse osteoblasts (MC3T3-El cell line), and purified chick osteoclasts, respectively. Fluoride is a partial competitive inhibitor of both chicken and mouse osteoblastic acid phosphatases, with apparent inhibition constants of 10-100 microM. These concentrations of fluoride correspond to those that increase bone formation in vitro and in vivo. In contrast, the apparent inhibition constant for fluoride of osteoclastic acid phosphatase was much higher (i.e., 0.5 mM). In summary, this study demonstrates that chicken osteoblasts contain an acid phosphatase that is sensitive to inhibition by low concentrations (i.e., microM) of fluoride.

Acid Phosphatase↗
PubMed1988

The anti-bone-resorptive agent calcitonin also acts in vitro to directly increase bone formation and bone cell proliferation.

The studies summarized in this report were intended to determine whether salmon calcitonin had direct effects on bone formation indices in vitro. The results of these investigations demonstrate acute effects of calcitonin on skeletal tissues derived from embryonic chickens to increase calvarial cell proliferation ([3H]thymidine incorporation into DNA) and bone matrix synthesis ([3H]proline incorporation into collagen, as [3H]hydroxyproline) in intact calvaria and tibiae. The effects of calcitonin on [3H]thymidine incorporation were significant at 1 mU/ml (0.08 nM; P less than 0.05), additive with respect to the action(s) of F (calcitonin increased the maximum effect of F, and F increased the effect of low dose calcitonin; P less than 0.01 for each), associated with an increase in total cell protein (r = 0.82; P less than 0.02), and inversely dependent on osteoblastic differentiation (r = -0.96; P less than 0.005). The effects of calcitonin to increase bone matrix synthesis ([3H]hydroxyproline incorporation, 139% and 155% of untreated control values for tibiae and calvaria, respectively; P less than 0.005 for each) were maximal at approximately 5 mU/ml (0.4 nM) and associated with a proportional increase in alkaline phosphatase activity in the bones (r = 0.71; P less than 0.05 for tibiae). These effects of calcitonin were not dependent on continuous exposure. [3H]Thymidine incorporation was increased in calvarial cells 16 h after a 4-h limited (inductive) exposure to calcitonin (at 3 mU/ml; P less than 0.01). [3H]Proline incorporation in embryonic chicken calvaria was also increased during 3 days of limited exposure (i.e. 4 h/day) to 10 mU/ml calcitonin (P less than 0.02). The proliferative action(s) of calcitonin was not unique to chicken osteoblastline cells. Salmon calcitonin also increased [3H]thymidine incorporation in the transformed murine calvarial cell lines MMB and MC-3T3-E1 and in primary cultures of cells prepared from newborn mouse calvaria (P less than 0.05 for each). Furthermore, these effects were observed at calcitonin doses (3-30 mU/ml) that also decreased murine bone resorption (i.e. 45Ca release from prelabeled neonatal mouse calvaria; P less than 0.01).

Alkaline Phosphatase↗
PubMed1988

Vanadate stimulates bone cell proliferation and bone collagen synthesis in vitro.

We recently proposed a hypothesis for the molecular mechanism of the osteogenic action of fluoride in which it stimulates osteoblast proliferation via the inhibition of an osteoblastic acid phosphatase-like phosphotyrosyl protein phosphatase activity. To test this hypothesis, we investigated whether orthovanadate, a known phosphotyrosyl protein phosphatase inhibitor, would mimic fluoride in the stimulation of bone cell proliferation and bone collagen synthesis in vitro. Orthovanadate inhibited the osteoblastic acid phosphatase activity and stimulated bone cell proliferation at the same low concentrations (i.e. 5-15 microM). At the mitogenic doses, orthovanadate also showed a dose-dependent increase in alkaline phosphatase (a marker of mature osteoblasts) in cultured calvarial cells and stimulated bone collagen synthesis, as measured by the incorporation of [3H]proline and the conversion into [3H] hydroxyproline in organ calvaria cultures. Therefore, orthovanadate stimulated bone formation by increasing the number of mature osteoblasts mediated via stimulation of cell proliferation and differentiation. Orthovanadate was dependent on the presence of a mitogen in cell medium for its mitogenic action in vitro and synergistically potentiated the mitogenic actions on osteoblasts of those growth factors, i.e. insulin, epidermal growth factor, insulin-like growth factor I, and skeletal growth factor, whose mitogenic action involved tyrosyl protein phosphorylation. However, the interaction between orthovanadate and basic fibroblast growth factor, a growth factor that does not appear to involve tyrosyl protein phosphorylation, on bone cell proliferation was additive. In summary, these data are consistent with the hypothesis that inhibition of the osteoblastic phosphotyrosyl protein phosphatases can prolong and/or potentiate the mitogenic actions of growth factors, and thereby stimulates cell proliferation.

Acid Phosphatase↗
PubMed1988

Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures.

The effects of fluoride (20 mumol/L) and bovine bone extract (17 micrograms/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin fibroblasts. The incorporation of [3H]thymidine into DNA was measured 16 hours after the addition of factors. After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity. Fluoride stimulated [3H]thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells. Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately 20 mumol/L. Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells. The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin fibroblasts. These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the growth factors in bovine bone extract primarily stimulate proliferation of bone cells. Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract.

Acid Phosphatase↗
PubMed1987

Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.

The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).

24,25-Dihydroxyvitamin D 3↗
PubMed1987

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix.

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.

Acid Phosphatase↗
PubMed1987

Measurement of bone mineral content in the term and preterm infant.

We tested the best anatomic site, reliability, and reproducibility of single-photon absorptiometric bone density measurements in premature and term newborns. Humerus and radius measurements were compared using a commercially available densitometer. The humerus was a more reliable site of measurement, particularly in the very-low-birth-weight infant. Normal ranges of humerus bone mineral content (BMC) were defined for infants of 24 to 42 weeks' gestational age at birth. Humerus BMC correlated with gestational age and birth weight of patients. We conclude that bone densitometer measurements can be successfully performed, even in very-low-birth-weight infants, when the humerus is used as the measurements site. We define normal humerus BMC values for use in diagnosis and evaluation of efficacy of treatment in infants who are at higher risk for osteopenia of prematurity.

Age Factors↗
PubMed1987

Incidence of hip fractures in osteoporotic women treated with sodium fluoride.

It has been suggested recently that, although fluoride therapy may decrease the occurrence of vertebral fractures, it could increase the risk of hip fractures. To evaluate this possibility, we combined retrospective data from five medical centers that have had a large experience with this therapeutic regimen. In 416 osteoporotic patients who were followed for more than 1,000 patient-years of fluoride treatment, there were 17 nontraumatic hip fractures. This incidence of 1.6% per year is similar to the incidence, 1.9% per year, for 120 of the patients in this series who had been followed prospectively for 3 years prior to initiation of fluoride therapy. The expected incidence for women of the same age in the general community is 0.5% per year. Thus, untreated osteoporotic women are at increased risk for hip fracture, but treatment with fluoride seems neither to decrease nor to increase the incidence of hip fracture substantially.

Aged↗
PubMed1987

Monofluorophosphate is hydrolyzed by alkaline phosphatase and mimics the actions of NaF on skeletal tissues, in vitro.

These studies were intended to assess the osteogenic activity of monofluorophosphate (MFP) in vitro, and to identify the enzyme(s) responsible for MFP hydrolysis-alkaline phosphatase (ALP) and/or acid phosphatase (AcP). ALP and AcP activities were determined by hydrolysis of p-nitrophenylphosphate (PNPP) at pH greater than 8 and pH 5.5, respectively, and MFP hydrolysis was determined, between pH 5.5 and pH 9.0, from measurements of [fluoride ion], using an ion-specific electrode. We found (1) that MFP was an alternative substrate for purified ALP, but not for AcP; (2) that MFPase activity in the embryonic chick resembled ALP, but not AcP, with respect to pH-dependent hydrolysis, sensitivity to effectors (r = 0.98, P less than .001), and tissue distribution (r = 0.96, P less than .001); and (3) that intestinal MFPase activity in the embryonic chick co-purified with ALP activity (r = 0.93, P less than .01) and resembled ALP, but not AcP, in its distribution along the small intestine, being highest in the duodenum and lowest in the distal ileum (r = 0.96, P less than .001). We also found that in vitro exposure to MFP increased (1) the proliferation rate of embryonic chick calvarial cells in serum-free monolayer cultures (i.e., 3[H]-thymidine incorporation into DNA, P less than .001); (2) ALP activity in calvarial cells (P less than .005) and in intact calvaria (P less than .05); and (3) collagen production by intact calvaria (i.e., 3[H]-proline incorporation as 3[H]-hydroxyproline, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1987

Fluoride therapy for osteoporosis: characterization of the skeletal response by serial measurements of serum alkaline phosphatase activity.

Optimum use of fluoride therapy for osteoporosis requires a sensitive and convenient index of the skeletal response to fluoride. Since previous studies had shown that serum alkaline phosphatase activity (SALP) was increased in response to fluoride therapy, we examined serial measurements of SALP in 53 osteoporotics treated with 66 to 110 mg of sodium fluoride (NaF) for 12 to 91 months. SALP was increased in 87% of the subjects during therapy with fluoride. The increase in SALP was thought to reflect the osteogenic action of fluoride based on the findings that SALP correlated with both trabecular bone area (r = .81, P less than .001) and osteoid length (r = .67, P less than .01) in iliac crest biopsies, predicted increased bone density on spinal radiographs in response to fluoride therapy with an 87% accuracy, and predicted decreased back pain in response to fluoride with a 91% accuracy. In addition, the SALP response to fluoride was seen earlier than other therapeutic responses as indicated by the findings that the tau 1/2 for the SALP response (ie, time for 1/2 of the patients to show a significant response) was significantly less (1.2 +/- 0.3 yr) than that for the pain response (1.6 +/- 0.3 yr, P less than .05) or that for the radiographic response (3.7 +/- 0.5 yr, P less than .001). Although most patients responded to fluoride with an increase in SALP, evaluation of the kinetics of the SALP response to fluoride revealed marked interpatient variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

In vitro evidence that bone formation may be coupled to resorption by release of mitogen(s) from resorbing bone.

Bone-derived proteins have been shown to stimulate the proliferation of bone-forming cells and to increase the rate of embryonic bone formation in vitro. The current studies were intended to determine the tissue distribution of bone cell-active mitogen(s) in the embryonic chick, to determine the cellular origin and the target cell specificity of the bone cell-active mitogen(s) in embryonic chick bone, to determine whether the release of mitogenic activity from embryonic chick tibiae was proportional to bone resorption, and to compare mitogenic activities prepared from different skeletal sources, with respect to Mr, chemical stability, and mitogen activity kinetics. A bone cell-active mitogen(s) was identified in extracts of bone and cartilage but not in extracts of muscle, liver, intestine, or brain. (Mitogenic activity was determined as increased incorporation of 3[H]-thymidine into DNA in serum-free, calvarial cell cultures.) Together, the following three observations indicate an osteoblastic origin for the bone cell-active mitogen(s) in chick bone. First, the mitogen content of embryonic chick tibiae increased 4.5-fold, during eight days of serum-free in vitro growth (P less than .005). Second, conditioned medium (CM) from serum-free monolayer cultures of calvarial cells contained bone cell-active mitogen(s), but CM from parallel cultures of skin, liver, and intestinal cells did not. And, finally, the amount of bone cell-active mitogen(s) in calvarial cell CM was correlated with the amount of alkaline phosphatase (ALP) activity per cell, ie, an index of osteoblastic differentiation (r = .92, P less than .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Characterization and assay of tartrate-resistant acid phosphatase activity in serum: potential use to assess bone resorption.

We improved the spectrophotometric assay of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. During development of the assay we found that human serum contains a dialyzable, mixed-type noncompetitive inhibitor(s) of TrACP activity, the effects of which on the assay were substantially lessened by diluting the serum sample with water before assay and increasing the substrate concentration. Hemolysis releases into serum a significant amount of TrACP activity from erythrocytes, which can be inactivated by incubating the serum at 37 degrees C for 1 h before assay. Our improved assay was reproducible (CV = 5%), and measured within 10% of the amount of added bovine skeletal TrACP activity. Preliminary application of the assay revealed that the amount of serum TrACP activity in patients with skeletal diseases differed from normal values and changed in the same direction as the expected change in bone turnover, suggesting that TrACP activity in serum could be useful clinically as a marker of bone metabolism, possibly of bone resorption.

Acid Phosphatase↗