Differential response of bone cells isolated by sequential digestion to dichloromethylenebisphonate in culture

Dichloromethylenebisphosphonate (Cl2-MBP), a compound structurally related to inorganic pyrophosphate but resistant to hydrolysis of endogenous phosphatase to yield inorganic phosphate, inhibits bone resorption and soft tissue mineralization in vivo. Previously, we have shown that bone cells isolated from rat calvaria respond profoundly to the exposure of Cl2MBP. To determine whether the cellular effects evoked by Cl2MBP are confined to a particular bone cell type, calvaria from 1 day postnatal rats were subjected to a sequential time-dependent enzyme digestion, yielding five bone cell populations marked by differences in PTH response, alkaline phosphatase activity and collagen, as well as hyaluronic acid synthesis. Culturing these bone cell populations with Cl2MBP revealed that previously observed results found with mixed bone cells (inhibition of cell proliferation, diminution of hyaluronic acid synthesis, and increase in alkaline phosphatase) were limited to cell populations which, according to the isolation scheme, stem from the outer tissue layer(s) of the calvaria. Collagen synthesis, however, was found to be equally increased regardless of cell type. These present results indicate that the action of Cl2MBP on bone may be cell specific.     

Bisphosphonates inhibit 1,25-dihydroxyvitamin D3-induced increase of osteocalcin in plasma of ratsin vivo and in culture medium of rat calvariain vitro

Summary In order to test whether bisphosphonates, which are potent inhibitors of osteoclastic bone resorption, may also act upon osteoblasts, we studied the effect of dichloromethylenebisphosphonate (Cl₂MBP) and 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP) onin vivo levels andin vitro release of osteocalcin, a bone-specific protein produced by osteoblasts. In rats, 161 μmol/kg of Cl₂MBP or 1.61 μmol/kg AHBuBP strongly inhibited the increase of plasma osteocalcin induced by 1,25(OH)₂D₃. The inhibition was measurable within 24 hours after the administration of bisphosphonate and was independent of any change in bone resorption. The effect upon osteocalcin release was also present in calvaria cultures. 250 μM Cl₂MBP strongly inhibited the osteocalcin release induced by 10⁻⁸ M 1,25(OH)₂D₃. In the presence of 1,25(OH)₂D₃, protein synthesis and DNA synthesis were also decreased, whereas in the absence of 1,25(OH)₂D₃, protein synthesis was increased. Thus, bisphosphonates affect the production of a bone-specific protein by osteoblasts in addition to their inhibitory action on osteoclasts.     

Structure-activity relationships of various bisphosphonates

Summary A variety of bisphosphonates with aliphatic side chains of increasing length, as well as 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP, formerly APD), dichloromethylenebisphosphonate (Cl₂MBP, formerly Cl₂MDP), and dibromomethylene bisphosphonate (Br₂MBP, formerly Br₂MDP), were compared in vitro and in vivo to find (a) a possible relationship between structure and activity in order to give some indication about their mechanism(s) of action on bone and (b) the most efficient and safe compound having an effect on bone resorption. Some relationship was found between inhibition of calcium phosphate precipitation in vitro and of mineralization in vivo. No correlation existed, however, between any parameter measured and bone resorption. The number of calvaria cells in culture was decreased by compounds with a chain length greater than 5-C, by AHPrBP, Cl₂MBP, and Br₂MBP. Lactate production by these cells in vitro was increased by the long chain bisphosphonates and AHPrBP, and was decreased by Cl₂MBP. No good correlation existed between the inhibition of bone resorption measured in vitro on calvaria and that seen in vivo on rat tibiae metaphyses. The latter was inhibited the most efficiently by the bisphosphonates longer than 5-C and by AHPrBP; these were 10 times more effective than Cl₂MBP. Taking into account all factors, 1-hydroxypentylidene-1,1-bisphosphonate and AHPrBP seem to be the most active compounds to inhibit bone resorption.     

Treatment of Paget's disease of bone with intravenous 4-amino-1-hydroxybutylidene-1,1-bisphosphonate

Summary 4-amino-1-hydroxybutylidene-1,1-bis-phosphonate (AHButBP) was given intravenously (2.5–25 mg/day for 4 days) to 14 patients with Paget's disease of bone, five of whom had been treated with dichloromethylidene bisphosphonate (Cl₂MBP) 32 months earlier. In the nine patients who had not been treated previously with bisphosphonates, the short course of AHButBP induced a suppression of serum alkaline phosphatase and urinary hydroxyproline values down to 30% of initial values. The biochemical suppression of the disease was sustained for 2–18 months and the time to relapse did correlate to the logarithm of the dose (P<0.001). In the five patients previously treated for Paget's disease, an apparent resistence to treatment with AHButBP was observed. However, in these patients both serum alkaline phosphatase and urinary hydroxyproline fell to or even below the nadir values which had previously been achieved with Cl₂MBP, irrespective of the degree of relapse. Thus the degree of suppression of Paget's disease of bone, achievable after treatment with bisphosphonates, seems to be constant for each patient, such that normal levels of serum alkaline phosphatase and urinary hydroxyproline cannot usually be attained in patients with extremely active disease.     

Effect of interleukin 1 beta on osteoblastic clone MC3T3-E1 cells

Summary The effect of recombinant interleukin 1 Beta (IL-1(β)) was investigated on osteoblastic cell line MC3T3-E1 cloned from mouse calvaria. IL-1(β) stimulated cell proliferation which increased cell number and caused dose-related stimulation of DNA synthesis, with a maximal effect at a concentration of 12.5 U/ml; suppressed alkaline phosphatase activity and collagen synthesis maximally at 0.5 and 62.5 U/ml, respectively; and increased the amount of free [³H] hydroxyproline in the cultures, but the amount was quite low. Prostaglandin E₂ synthesis was also stimulated dose dependently by the presence of IL-1(β), with a maximal increase at 2.5 U/ml, at which concentration the prostaglandin E₂ level in the medium was 1.61±0.10 ng/ml. The increased prostaglandin E₂ synthesis did not affect either the IL-1(β)-mediated change in DNA or collagen synthesis or alkaline phosphatase activity. These results extend the possibility that IL-1(β) is to act as a regulator of bone formation.     

The subcellular distribution of [14C]dichloromethylenebisphosphonate and [14C]1-hydroxyethylidene-1,1-bisphosphonate in cultured calvaria cells

Summary Rat calvaria cells were cultured for 6 days in the presence or absence of [¹⁴C]dichloromethylenebisphosphonate ([¹⁴C]Cl₂MBP) or [¹⁴C]1-hydroxyethylidene-1, 1-bisphosphonate ([¹⁴C]HEBP), after which cell organelles were separated by differential centrifugation. The distribution of protein, glutamate dehydrogenase, acid phosphatase, and 5'-nucleotidase was similar for cells treated or not treated with Cl₂MBP. About 70–80% of the [¹⁴C]Cl₂MBP and [¹⁴C]HEBP was found to be present in the supernatant. This was the only fraction that showed a ratio higher than 1 for the relative specific radioactivity, indicating that the bisphosphonates accumulated mainly in the cytosol. Rapid separation of particulate components and soluble cytoplasm of cells treated with [¹⁴C]Cl₂MBP confirmed this finding, showing that it is unlikely that the result was due to leakage from the organelles. The uptake of [¹⁴C]Cl₂MBP into cells was similar in different cell types. The binding of both bisphosphonates to macromolecules in the medium was 0.1–0.2% and 1–4% in the cells. This binding is not due to metabolic activity of the cells. About 15–20% of [¹⁴C]HEBP and [¹⁴C]Cl₂MBP was modified by the living cells.     

Interaction of 24,25-Dihydroxyvitamin D3 and parathyroid hormone on bone enzymes in vitro

Summary The in vitro effects of vitamin D₃ metabolites, parathyroid extract (PTE), purified parathyroid hormone (bPTH), vitamin A, and heparin on acid and alkaline phosphatases in rat or mouse calvaria in culture were investigated. Results show that: (a) when compared to values found in half calvaria incubated for 24 h in control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with PTE (1 USP/ml), bPTH (4×10⁻⁸M), heparin (5 USP/ml), vitamin A (23 USP/ml), 25-(OH)D₃ (2.5×10⁻¹¹ to 2.5×10⁻⁸M), 24,25-(OH)₂D₃, and 1,25(OH)₂D₃ (2.5×10⁻¹² to 2.5×10⁻⁷M); (b) the presence of 24,25-(OH)₂D₃ at low concentrations in the incubation medium decreases significantly the PTE, bPTH, vitamin A, or heparin induced stimulation of the phosphatase activities. This interaction is also observed when measuringβ glucuronidase and glucose-6-phosphatase activities and⁴⁵Ca release from previously labeled mouse calvaria; (c) a similar activity could not be found with 1,25-(OH)₂D₃ suggesting that 24,25-(OH)₂D₃ may have a specific role in bone metabolism.     

Calcitonin effects on isolated bone cells

Summary Calcitonin decreased calcium uptake in specific rat bone cell populations obtained by sequential collagenase digestions of calvaria. The calcitonin effect on calcium uptake was observed in the same populations that manifested calcitonin-induced increases in cyclic AMP as well as high levels of acid phosphatase and the ability to release⁴⁵Ca from prelabeled devitalized bone. No effect of calcitonin was observed in cell populations that had high levels of alkaline phosphatase and lacked the potential to resorb devitalized bone. These results suggest that changes in cell calcium as well as cyclic AMP may be involved in the mode of action of calcitonin on osteoclast-like cells.     

In vitro effects of aluminum on bone phosphatases: A possible interaction with bPTH and vitamin D3 metabolites

Summary The present study was undertaken to test the in vitro action of aluminum on bone phosphatase activities and the possible interaction of this metal with parathyroid hormone (bPTH) or vitamin D₃ dihydroxymetabolites [1,25- and 24,25(OH)₂D₃). Three-day-old rat calvaria were incubated for 24 h with one of the following: bPTH at 5×10⁻⁸M, 1,25-or 24,25(OH)₂D₃ at 2.5×10⁻⁹M, Al at concentrations ranging from 3×10⁻¹¹M to 6×10⁻⁶M, or their corresponding solvents. Al effects were also investigated when the medium phosphate or calcium concentrations were modified. In some experiments, Al was added simultaneously with bPTH or one of the vitamin D₃ metabolites at the beginning of the 24 h incubation. At the end of all incubations, acid and alkaline phosphatase activities were measured in bone cytoplasmic extract. The results show that: (a) When compared to the value found in half calvaria incubated in a control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with Al (3×10⁻¹¹M to 1.5×10⁻⁶M) as well as with bPTH, 1,25-, or 24,25(OH)₂D₃ and sharply decreased with higher Al concentrations (6×10⁻⁶M). (b) The Al effect on phosphatase activities is modified in a free phosphate or a free calcium medium. (c) The presence of Al at 1.5×10⁻⁶M or 6×10⁻⁶M significantly decreases the bPTH or 1,25(OH)₂D₃-induced stimulation of bone phosphatase activities. (d) A similar interaction could not be found between Al and 24,25-(OH)₂D₃.     

Comparative study of deflazacort,a new synthetic corticosteroid,and dexamethasone on the synthesis of collagen in different rat bone cell populations and rabbit articular chondrocytes

Summary Deflazacort is a new synthetic glucocorticoid which is an oxazoline derivative of prednisolone. In previous studies, it was shown that deflazacort, depending on the test model used, not only showed considerably more antiinflammatory potency than prednisolone in animals but also caused less deleterious effects on bone mineral metabolism than equivalent amounts of other glucocorticoids in man. In this study, we have compared the effects of deflazacort with those of dexamethasone on the synthesis of collagen in various rat bone cell populations and chondrocytes. Three bone cell populations were prepared by sequential time-dependent collagenase treatment of 1-day-old rat calvaria. Each cell population was further purified on a Percoll gradient (10–90%) yielding three populations of which two are different in alkaline and acid phosphatase and response to parathyroid hormone. A 3-day treatment of bone cell populations with deflazacort and dexamethasone (10⁻¹¹-10⁻⁵ M) revealed that both glucocorticoids, although at different concentrations, inhibited collagen synthesis. 21-desacetyl-deflazacort (5β, 11β, 16β)- 11,21-dihydroxy-2′-methyl-5-H-pregna-1-enol[17,16-d]oxazole-3,20-dione), the presumably active form of the steroid, which is formedin vivo after administration, produced nearly identical results as its precursor. Glucocorticoid concentrations at which inhibition was initially observed were 10⁻⁹ M and 10⁻⁷ M for dexamethasone and deflazacort respectively. Inhibition of collagen synthesis was significantly impaired only in cells isolated from bone during early tissue digestion, and not in those obtained during extensive collagenase treatment. Chondrocytes isolated from articular cartilage of 3-month-old rabbits and grown in primary cultures did not respond to either steroid. However, when passed and grown in secondary culture, collagen synthesis was inhibited considerably more by dexamethasone than by deflazacort. It is apparent that both deflazacort and dexamethasone, although at different concentrations, inhibit collagen synthesis in a differential manner. The lesser deterrent effect of deflazacort may be of clinical importance.     

Two biochemical indices of mouse bone formation are increased,in vivo,in response to calcitonin

Summary In a series of four studies, adult female Swiss-Webster mice were used to measure the effects of salmon calcitonin on two biochemical indices of local and systematic bone formation: (1) skeletal alkaline phosphatase activity—in serum and in extracts of calvaria and tibiae, and (2) calvarial collagenase-digestible protein synthesis—measured, acutely, in vitro. Subcutaneous calcitonin doses ranged from 50 to 400 mU/mouse/day (0.95–18.1 U/kg/day), and treatment schedules were continuous (daily) for 2–14 days, acute, or intermittent (2 days/week for 6 weeks). The effects of calcitonin on these bone formation indices (skeletal alkaline phosphatase and collagenase-digestible protein synthesis) were biphasic with respect to dose and treatment time, being increased in response to short-term, low-dose treatment, but not long-term, continuous treatment. The effects of long-term intermittent calcitonin treatment were dose-dependent increases in skeletal alkaline phosphatase in calvaria and serum (r=0.948, P< 0.02, and r=0.960, P< 0.01, respectively).     

Biochemical and histological studies on various bone cell preparations

Summary Four different cell populations—designated PF, OB, OC, and PC—were isolated from calvaria of 18-day-old chick embryos for analysis of the effects of hormones on bone tissue. The cell populations were studied with histological and biochemical methods. Apart from the well-known cell types present in calvaria, a new cell type was found in the noncalcified organic matrix between the osteoblastic layer and the calcified matrix. These cells were provisionally called osteocytic osteoblasts. They represent the “transition state” between osteoblasts and osteocytes. On the basis of histological studies with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM), the PF population was considered to originate primarily from the periosteal fibroblasts, the OB population from the osteoblasts and osteocytic osteoblasts. The population of cells still present in calvaria after removal of periosteal fibroblasts and osteoblasts was called the OC population. This cell population was very much enriched with osteocytes. The fourth isolated population (PC) was a mixed population of fibroblasts, osteoblasts, and preosteoblasts. On exposure to parathyroid hormone (PTH), all four cell populations showed increased lactate production, but only the OB and OC populations displayed increased cAMP production. Prostaglandin E₁ (PGE₁) stimulated cAMP production in both OB and PF cells. From the results of this study it was concluded that PTH receptors are present on all of the cell types studied, but that occupancy of the receptor induces adenylate cyclase stimulation only in osteocytes and fully differentiated osteoblasts.     

Inhibition of bone mineral loss during lactation by Cl2MBP

Summary Pregnant rats were injected subcutaneously with either saline or the bisphosphonate Cl₂MBP (dichloromethylenebisphosphonic acid) at a daily dose of 15 mg P/kg body weight on days 1 through 16 of gestation. Cl₂MBP treatment did not influence maternal body weight nor the number of pups born. When analyzed 1 day after birth, pups from Cl₂MBP-treated rats had a normal body weight but a 10% reduction in carcass calcium (Ca) content. The Cl₂MBP injections were resumed on day 1 postpartum and led to a 10% reduction in pup body weight gain and carcass Ca content at 16 days of age. In saline-injected rats, lactation resulted in slight hypocalcemia, greatly elevated serum levels of 1,25(OH)₂D₃, and loss of bone mineral, as indicated by a reduction in femur ash weight. In nonlactating rats, Cl₂MBP treatment produced slight hypercalcemia but had no effect on serum 1,25(OH)₂D₃ levels or bone mineral content. Compared to lactating rats receiving saline, Cl₂MBP-treated lactating rats were more hypocalcemic and had higher serum 1,25(OH)₂D₃ levels. However, the lactation-induced loss of bone mineral was completely inhibited by Cl₂MBP treatment.     

Calcitonin effects on isolated bone cells

Calcitonin decreased calcium uptake in specific rat bone cell populations obtained by sequential collagenase digestions of calvaria. The calcitonin effect on calcium uptake was observed in the same populations that manifested calcitonin-induced increases in cyclic AMP as well as high levels of acid phosphatase and the ability to release 45Ca from prelabeled devitalized bone. No effect of calcitonin was observed in cell populations that had high levels of alkaline phosphatase and lacked the potential to resorb devitalized bone. These results suggest that changes in cell calcium as well as cyclic AMP may be involved in the mode of action of calcitonin on osteoclast-like cells.     

De novo synthesis of type-I collagen in bone biopsy material

A simple and rapid method was established for the cultivation of bone cell tissue. Human bone tissue derived from orthopaedic surgery was cultivated in the presence of ¹⁴C-proline and β-aminopropionitrile. De novo synthesized collagen was extracted from the tissue and quantified by determination of radioactivity in the purified protein. Measurements of the oxygen consumption of the tissue provided evidence that the physiological conditions for the tissue were optimal. The tissue was vital over a period of as long as 7 days, showing normal respiration and a constant rate of collagen synthesis. The observed levels of alkaline phosphatase and acid phosphatase activity clearly demonstrated that mainly osteoblasts were involved in metabolic activity. The described system is suitable for investigations of bone cell metabolism under quasiphysiological conditions.     

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.     

Mineralized bone nodules formedin vitro from enzymatically released rat calvaria cell populations

Summary Single-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture in the presence of ascorbic acid, Na β-glycerophosphate, and dexamethasone to determine the capacity of these populations to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and β-glycerophosphate or ascorbic acid alone, three-dimensional nodules (∼75 μm thick) covered by polygonal cells resembling osteoblasts could be detected 3 days after confluency. The nodules became macroscopic (up to 3 mm in diameter) after a further 3–4 days. Only in the presence of organic phosphate did they mineralize. Nodules did not develop without ascorbic acid in the medium. Dexamethasone caused a significant increase in the number of nodules. Histologically, nodules resembled woven bone and the cells covering the nodules stained strongly for alkaline phosphatase. Immunolabeling with specific antibodies demonstrated intense staining for type I collagen that was mineral-associated, a weaker staining for type III collagen and osteonectin, and undetectable staining for type II collagen. Nodules did not develop from population I and the number of nodules formed by populations II–V bore a linear relationship to the number of cells plated (r=.99). The results indicated that enzymatically released calvaria cells can form mineralized bone nodulesin vitro in the presence of ascorbic acid and organic phosphate.     

Clonal osteogenic cell lines express myogenic and adipocytic developmental potential

Summary Clonal osteoblastic cell lines were isolated from neonatal rat calvariae and characterized with regard to a number of features associated with authentic osteoblasts. These included elevated alkaline phosphatase activity (relative to fibroblasts), PTH and PGE₂-stimulated increases in cAMP, the predominant synthesis of type 1 collagen, and the production of a mineralized matrixin vitro. By these criteria, five clones with osteoblast-like phenotypes were identified (ROB-C8a, C11, C20, C23, and C26) which varied somewhat in shape, levels of alkaline phosphatase activity, and in responsiveness to PTH and PGE₂, C11, C20, and C23 responded to both effector substances, whereas C8a only responded to PTH and C26 only responded strongly to PGE₂. Upon further examination, two of the clones (C23 and C26) were also found to exhibit significant muscle myotube formation after reaching confluence, and three of the clones (C8a, C11, and C26) showed marked adipocyte differentiation after treatment with dexamethasone. Overall, these data add further supporting documentation to (1) the suspected ontogenetic relationships of osteoblasts to other connective tissue cells, and (2) the concept that osteoblastic cells associated with neonatal rat calvariae are in various stable stages of differentiation and developmental commitment.     

Mineralization and the Expression of Matrix Proteins During In Vivo Bone Development

The in vivo expression of fibronectin, type I collagen, and several non-collagenous proteins was correlated with the development of bone in fetal and early neonatal rat calvariae. Fibronectin was the earliest matrix protein expressed in calvariae, with a peak expression in fetal 16- and 17-day (d) bones. Fibronectin expression coincided with the condensation of preosteoblasts prior to calcification and decreased once bone mineralization commenced. The expression of type I collagen, osteonectin, bone sialoprotein, and alkaline phosphatase mRNAs was found at 17 d. The increase in type I collagen mRNA levels was correlated with a 3.5-fold increase in calcium deposition at 19–20 d. Bone sialoprotein and alkaline phosphatase peaked on fetal 21 d while osteonectin remained at a low level and was localized to the osteoblast layer and the osteocyte lacunae. Osteopontin mRNA levels increased rapidly in neonatal calvariae. After birth, osteonectin and fibronectin were mainly associated with blood vessels. Thus, fibronectin is one of the earliest matrix proteins expressed in calvariae and is rapidly followed by type I collagen, bone sialoprotein, and alkaline phosphatase. Osteocalcin, osteonectin, and osteopontin mRNAs have similar patterns of expression in the developing fetal calvaria, and their synthesis coincided with mineralization. Received: 31 December 1996 / Accepted: 5 June 1997     

Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate

We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.