Parathyroid Hormone Modulates the Response of Osteoblast-Like Cells to Mechanical Stimulation

Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca²⁺]_i. Although PTH did not significantly change [Ca²⁺]_i levels, flow and PTH did produce a significantly greater [Ca²⁺]_i response and increased the number of responding cells than is found in fluid shear alone. The [Ca²⁺]_i response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca²⁺]_i by modulating the mechanosensitive channel. Received: 5 October 1999 / Accepted: 15 February 2000     

Parathyroid Hormone Secretory Response to EDTA-Induced Hypocalcemia in Black and White Premenopausal Women

One consistent racial difference in mineral homeostasis is increased efficiency of renal calcium conservation in blacks which could account, in part, for differences in bone density and fracture risk. Since parathyroid hormone (PTH) is the major regulator of calcium homeostasis, we investigated its secretion in black and white women in response to hypocalcemia. Two hour EDTA infusions (50 mg/kg) were performed in 34 premenopausal women (17 black, 17 white). Blood was sampled at 30-minute intervals during the infusion, at 60-minute intervals for 3 more hours, and at 24 hours. Serum ionized calcium decreased identically in both groups with a nadir at 2 hours and returned to baseline within 24 hours. Serum 1-84 PTH levels rose similarly in both groups with a peak PTH level that was slightly higher in black women, and on average, slightly earlier than that in white women. Serum PTH levels remained elevated in both groups at 24 hours with no overall group differences in PTH response. In black, but not white women, serum 25OHD levels correlated negatively with both basal PTH and peak PTH level, achieved with infusion. Serum 1,25(OH)₂D levels rose and osteocalcin levels decreased, with no group differences. We conclude that overall, premenopausal black women show no clear differences in PTH secretory activity to an EDTA-induced hypocalcemic stimulus. Basal vitamin D status appeared to be a determinant of the degree of the PTH response in black women, with the peak PTH level being inversely correlated with levels of 25OHD. Since we have previously shown that the skeleton contributes less to acute calcium needs in blacks than in whites, the lack of a racial difference in PTH secretory responsivity suggests that calcium homeostasis is more likely maintained in blacks through greater PTH sensitivity at extraskeletal sites, such as the kidney. Received: 31 August 1998 / Accepted: 12 March 1999     

Influence of Antiresorptive Agent OST-766 on Signal Transduction Pathways Involved in Parathyroid Hormone Action

The effects of OST-766, an inhibitor of vacuolar H⁺-ATPase activity, on adenylyl cyclase and phospholipase C activity were explored in the osteoblast cell line ROS 17/2.8. In fresh homogenates of ROS 17/2.8 cells, OST-766 inhibited adenylyl cyclase activity (ACA) in response to guanine nucleotide and forskolin but had no effect on basal ACA. OST-766 enhanced the basal generation of IP2, but not that formed in response to Ca²⁺ or guanine nucleotides. In marked contrast, incubation of intact ROS 17/2.8 cells with OST-766 for at least 48 hours resulted in an increase in basal ACA as well as in response to PTH, guanine nucleotides and forskolin. Under similar conditions, the compound also increased IP1, IP2 and IP3 generation in response to guanine nucleotides and Ca²⁺. Levels of the guanine nucleotide binding proteins Gs and Gi were also increased in OST-766-treated cells. The results suggest that the actions of this H⁺-ATPase inhibitor include effects on osteoblasts through PTH-sensitive signal transduction pathways. Received: 20 August 1997 / Accepted: 7 August 1998     

Parathyroid Hormone (1-34) Receptor-Binding and Second-Messenger Response in Rat Incisor Odontoblasts

Even though indirect evidence indicates that PTH exerts an anabolic effect on dentinogenesis, the existence of PTH receptors and any second-messenger response in odontoblasts have not been demonstrated. The aim of this study was to investigate whether rat incisor odontoblasts express PTH receptors, and to identify which second messenger pathway the hormone may activate. Odontoblasts were dissected from rat incisors. Amino-terminal (1-34) fragment rat PTH [rPTH(1-34)] conjugated to fluorescein isothiocyanate visualized receptor sites on the cell surface. Upon incubation of odontoblasts with rPTH(1-34), cAMP formation was increased. However, no fluctuations in intracellular calcium activity were observed upon rPTH(1-34) stimulation when using Fura-2 as a Ca²⁺ probe. In long-time incubations, stimulation with PTH(1-34) upregulated APase activity. The results demonstrate that rPTH(1-34) evokes an anabolic response in dentinogenically active odontoblasts, and that this may be mediated through the protein kinase A/cAMP pathway, whereas no indications for Ca²⁺ as a second messenger were evident. Received: 12 March 1997 / Accepted: 26 June 1997     

Association Study of Parathyroid Hormone Gene Polymorphism and Bone Mineral Density in Japanese Postmenopausal Women

Association of BST B1 restriction fragment length polymorphism (RFLP) of the parathyroid hormone (PTH) gene with bone mineral density (BMD) was examined in 383 healthy postmenopausal women in Japan who were unrelated. The RFLP was represented as B or b, the capital letter signifying the presence of and the small letter the absence of restriction site for BST B1. The frequency of each genotype—BB, Bb, and bb—was 82.5%, 16.7%, and 0.8%, respectively. When we statistically compared age, years after menopause, body height, and body weight between the BB genotype and the Bb genotype groups, there was no significant difference between the groups. However, the lumbar BMD and the score of BMD adjusted for age and body weight (Z score) were significantly lower in the group of genotype Bb than in the BB: 0.859 ± 0.019 g/cm² versus 0.925 ± 0.011 (mean ± SE, P= 0.01) and −0.412 ± 0.138 versus 0.067 ± 0.082 (mean ± SE, P= 0.01). In addition, the Z score of total body BMD in the Bb genotype group was lower than that in the BB group. Comparison of serum and urinary biochemical bone metabolic markers suggested that the subjects with Bb genotype might be in a relatively higher state of bone turnover than those with BB genotype. These results suggest that the polymorphism in the PTH gene would be a useful genetic marker for lower BMD and the susceptibility for osteoporosis. Received: 19 March 1998 / Accepted: 24 June 1998     

Gastric Fundectomy in the Rat: Effects on Mineral and Bone Metabolism, with Emphasis on the Gastrin–Calcitonin–Parathyroid Hormone–Vitamin D Axis

In humans, gastric surgery results in in osteopenia via mechanisms that are insufficiently understood; surgery-induced changes in the hormonal axes involving the stomach, thyroid, and the parathyroids may play a role. To study this in more detail, we evaluated calcium (Ca), magnesium (Mg), and phosphorus (P) metabolism as well as physical, chemical, and histomorphometric bone parameters in rats rendered hypergastrinemic by fundectomy (FX). In independent experiments, the response to an oral Ca challenge was investigated in intact rats versus FX, and in thyroidectomized versus thyroid-intact FX rats. Sixteen weeks following FX, body weight was approximately 80% that of sham-operated controls. In urine, P excretion was elevated fivefold, the pH was significantly decreased, and cAMP excretion was elevated as compared with controls; serum parathyroid hormone (PTH), calcitonin, 25OHD, Ca, Mg, and P were normal; gastrin and 1,25(OH)₂D were elevated. On the basis of bone ash mineral content, FX rats developed significant osteopenia, and histomorphometry indicated only slightly elevated bone turnover and mineralization. Following oral Ca, thyroid-intact FX rats developed hypercalcemia, serum gastrin decreased, and calcitonin increased significantly; in thyroidectomized FX rats, calcitonin remained at baseline levels although there was a similar degree of hypercalcemia; PTH decreased during the hypercalcemic period in both groups. Serum gastrin did not correlate with calcitonin or PTH, and in multivariate regression analysis the only predictor of serum 1,25(OH)₂D was urinary phosphorus. It was concluded that in the FX rat (1) osteopenia is not caused by intestinal Ca malabsorption, vitamin D, Ca deficiency, or secondary hyperparathyroidism; (2) osteopenia may be related to PTH-independent urinary hyperexcretion of P, followed by a rise of serum 1,25(OH)₂D; (3) the existence of endocrine axes among gastrin, calcitonin, and PTH cannot be substantiated. FX osteopenia appears to be related to gastric acid abolition, and the reactive hypergastrinemia probably stabilizes the mass and turnover of bone. Received: 12 August 1997 / Accepted: 26 January 1998     

Increased Serum 1,25-Dihydroxyvitamin D after Growth Hormone Administration is not Parathyroid Hormone-Mediated

To assess the effects of growth hormone (GH) on serum 1,25-dihydroxyvitamin D [1,25(OH)₂D], we performed the following prospective crossover study in six healthy, young, adult, white men. During each of two admissions for 2? days to a general clinical research center, subjects were placed on a daily dietary calcium intake of 400 mg. Serum calcium, phosphorus, 1,25(OH)₂D, immunoreactive intact parathyroid hormone (PTH), insulin-like growth factor I (IGF-I), IGF binding protein 3 (IGFBP3), tubular reabsorption of phosphate (TRP), and maximum tubular reabsorption of phosphate (TMP/GFR) were measured. Recombinant human GH (rhGH, Humatrope) (25 μg/kg/day subcutaneously for 1 week) was administered prior to and during one of the admissions. Results are expressed as mean ± SEM. Whereas serum 1,25(OH)₂D (58.9 ± 7.7 versus 51.6 ± 7.4 pg/ml, P < 0.01), serum phosphorus (4.5 ± 0.1 versus 3.7 ± 0.1 mg/dl, P < 0.01), TRP (92.0 ± 0.5 versus 87.8 ± 0.7 mg/dl, P < 0.005), TMP/GFR (4.6 ± 0.1 versus 3.5 ± 0.2, P < 0.005), and urinary calcium (602 ± 49 versus 346 ± 25 mg/day, P < 0.001) increased significantly, serum PTH decreased significantly (19.9 ± 1.9 versus 26.8 ± 4.0 pg/ml, P < 0.05) and serum calcium did not change when subjects received rhGH. These findings indicate that in humans, GH affects serum 1,25(OH)₂D independently of circulating PTH and that this effect is mediated by IGF-I. We propose, therefore, that one potential mechanism by which GH stimulates increases in bone mass is via modest increases in serum 1,25(OH)₂D. Received: 2 May 1996 / Accepted: 18 October 1996     

The Hypotensive Actions of Osteogenic and Nonosteogenic Parathyroid Hormone Fragments

Parathyroid hormone (PTH), hPTH-(1-84), and its hPTH-(1-34), hPTH-(1-31)NH₂, and hPTH-(1-30)NH₂ fragments reduced the tail artery pressure in anesthetized female Sprague-Dawley rats by 42.4–67.1% within about 1 minute after injection into a femoral vein, but reduced the pressure by only 8.5–36.2% 2–19 minutes after subcutaneous injection. hPTH-(1-84) and hPTH-(1-34) stimulate both adenylyl cyclase and phospholipase-C in their target cells, but the hypotensive action must have been stimulated specifically by adenylyl cyclase activation, because hPTH-(1-30)NH₂ and hPTH-(1-31)NH₂, which can only stimulate adenylyl cyclase, were potently hypotensive when injected intravenously whereas hPTH-(7-84), which can only stimulate phospholipase-C, was not significantly hypotensive when injected intravenously. Since PTH's osteogenic action is also mediated by adenylyl cyclase stimulation, it was expected that the hypotensive response might be used to screen new PTH constructs for possible osteogenicity. Indeed, the osteogenic activities of subcutaneously injected hPTH-(1-31)NH₂, hPTH-(1-34), and hPTH-(1-84) correlated closely to their hypotensive activities, with hPTH-(1-34) being much more hypotensive and significantly more osteogenic than the other two molecules. hPTH-(1-31)NH₂ and hPTH-(1-84) were equally osteogenic and hypotensive. However, this correlation broke down with hPTH-(1-30)NH₂ which does not stimulate bone formation, but in the present study it stimulated adenylyl cyclase and reduced tail artery pressure almost as much as hPTH-(1-31)NH₂ and hPTH-(1-34). Nevertheless, the ability to significantly reduce arterial pressure is a common property of osteogenic PTH and PTH fragments and is thus a rapidly determinable preliminary indicator of in vivo bioactivity of PTH fragments. Received: 2 May 1996 / Accepted: 9 August 1996     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

Parathyroid Hormone-Related Protein (1–37) Induces cAMP Response in Human Osteoblast-Like Cells

During recent years parathyroid hormone-related protein (PTHrP) research has been focused on the physiological functions of different fragments of the PTHrP molecule. Here we demonstrate that PTHrP (1–37) induced cyclic adenosine monophosphate (cAMP) response in primary human osteoblast-like cells, which were well characterized by the presence of alkaline phosphatase activity and osteocalcin production after stimulation with 1,25-dihydroxyvitamin D₃. However, there was no cAMP response to PTHrP (58–77). Furthermore, the response to PTHrP (1–37) was dose dependent, with a significant increase at 1 nM. The presence of PTHrP (1–37)-induced cAMP response in human osteoblast-like cells implies that aminoterminal PTHrP fragments may exert important functions in the bone. Received: 27 January 1997 / Accepted: 26 June 1997     

Calcium Bioavailability and Parathyroid Hormone Acute Changes after Oral Intake of Dairy and Nondairy Products in Healthy Volunteers

This study was designed to compare calcium bioavailability and serum parathyroid hormone acute changes after oral intake of 500 mg of elemental calcium from liquid milk, yogurt, calcium-citrate-enriched powdered milk or a calcium carbonate pill; or after intake of soybean imitation-milk. After a 12-h fast, blood samples were drawn both at baseline and 1, 2, 3 and 4 h after an oral intake of the above-mentioned products, which were ingested together with a light neutral breakfast. The administration order of the study products was randomly assigned to each of 19 healthy young volunteers (11 females, 8 males). The baseline serum concentrations of ionized calcium, phosphorus and intact parathyroid hormone (iPTH) were normal. Calcium-citrate-enriched powdered milk induced a significant increase in serum ionized calcium (p<0.001) and a significant and continuous decrease in serum iPTH concentration (p<0.001). Yogurt and the calcium carbonate pill induced a similar but less significant effect, increasing serum ionized calcium (p<0.05) and decreasing serum iPTH (p<0.01). Liquid milk only induced a significant change in serum ionized calcium and iPTH concentration during the first 2 h; this effect was lost during the following 2 h. In conclusion, our study suggests the possibility that the addition of calcium citrate to powered milk may improve calcium bioavailability and enhance the inhibitory effect on serum iPTH in the assayed conditions. Received: 15 September 1998 / Accepted: 15 January 1999     

Differences in Bone Turnover and Skeletal Response to Thyroid Hormone Treatment Between Estrogen-Depleted and Repleted Rats

This study was undertaken to compare the effect of supraphysiological doses of thyroxine (T4) on bone metabolism in SHAM and OVX young adult rats. Female Sprague Dawley rats (220 ± 2 g, approx. 5 months of age) were divided into four groups of eight animals each. The animals were intraperitoneally injected 6 days per week with vehicle (Vh): 0.001 N NaOH/0.9% NaCl (SHAM+Vh and OVX+Vh) or 250 μg of thyroxine/kg/day (SHAM+T4 and OVX+T4) during a 5-week period. Serum T4 and osteocalcin (BGP), urinary pyridinolines (Pyr), and creatinine (creat) were determined. At the beginning and at end of the experiment, skeletal bone mineral content (BMC), bone mineral density (BMD), and area (A) of the total skeleton, femur, spine, and whole tibia, as well as proximal, middle, and distal areas of the tibia were assessed by dual X-ray absorptiometry (DXA) in an ultra-high-resolution mode. T4 treatment of the SHAM rats did not induce significant changes in BGP level or Pyr/creat excretion compared with the SHAM+Vh control group. However, these two biochemical bone markers significantly increased due to T4 treatment in OVX rats compared with both OVX+Vh and SHAM+T4 groups (P < 0.05 and P < 0.001, respectively). The OVX+T4 group had a significantly lower ΔBMD than SHAM+T4 rats in all studied regions (P < 0.05) except for the middle tibia region. OVX+T4 groups presented a significantly lower ΔBMC and ΔA compared with SHAM+T4 animals (P < 0.001). OVX+T4 rats significantly impaired the ΔBMD in the femur (P < 0.01), spine (P < 0.05), whole (P < 0.05) and middle (P < 0.05) tibia whereas T4 treatment of SHAM rats only affected, significantly, the whole (P < 0.05) and the proximal tibia region (P < 0.01). T4 treatment affects bone growth in young adult rats. The effect is significantly greater in the estrogen-depleted than in the estrogen-repleted state. The bone site most adversely affected by T4 treatment depends on the estrogen status. The proximal tibia (principally trabecular bone) was the most affected area in estrogen-repleted rats. Conversely, in OVX rats, the middle tibia (principally cortical bone) presented the greatest decrease in bone density. Received: 20 May 1999 / Accepted: 4 February 2000     

Genetic Variation and Covariation of Parathyroid Hormone Levels and Bone Density in the Human Population

The present study was an attempt to evaluate the relative importance of familial/genetic factors in interindividual variation of plasma concentrations of parathyroid hormone (PTH) and bone mineral density (BMD). We also examined to what extent common genetic and environmental factors may be involved in covariation between the hormone concentrations and BMD levels. Ninety-five nuclear pedigrees (consisting of 187 males and 168 females, aged 18–91 and 18–86 years old, respectively), from several small villages in the Chuvasha Autonomy, Russia, were assessed for PTH, sex hormones, and BMD. PTH plasma levels were measured in duplicate by immunoradiometric assay using an N-tact PTH SP kit. Standard roentgenography was done from the second and third phalanges of the middle finger on both hands for assessment of compact and cancellous bone BMD separately. The present study clearly confirmed the results of the previous genetic analyses of BMD which indicated that between 47% and 60% of the total variance of BMD, adjusted for sex and age effects, were attributable to genetic factors. Genetic factors also contributed significantly to interindividual variation of PTH. Constraining these additive genetic effects to zero dramatically increased the likelihood ratio (P < 0.001), indicating that at least 30% of the hormone plasma variation was attributable to genetic sources. The results of bivariate decomposition analysis were not clear cut. Two types of bivariate analyses showed that PTH-BMD genetic correlations according to sex and between the opposite sexes were consistently negative, but only marginally significant. Received: 21 July 1998 / Accepted: 30 September 1999     

Effect of Avicatonin (Chicken Carbocalcitonin) on Galvanic Skin Response: A Randomized, Prospective, Double-Blind, Controlled Study for an Objective Assessment of Pain

In an attempt to objectively evaluate the analgesic effect of avicatonin (chicken carbocalcitonin), galvanic skin response (GSR) was recorded in 18 patients with osteoporosis or osteopenia and backache in a randomized, prospective, double-blind, controlled study. Backache on examination and in daily living was assessed weekly by scores utilizing a questionnaire. After two measurements 1 week apart on induction of backache with a maximum anterior flexion of the back from a supine position, either 20 units of avicatonin or inactive placebo was intramuscularly injected once a week for 4 consecutive weeks. In the avicatonin group but not in the placebo group, the area under the curve (AUC) of GSR tracing was decreased, giving a significant difference between the avicatonin and placebo groups after the second week. The pain score obtained by questionnaire decreased in both groups, suggesting a placebo effect. Galvanic skin response may provide a breakthrough to the objective and reliable evaluation of the biological response to pain which could not be accomplished by questionnaires based on subjective impression markedly influenced by emotional and psychological factors. Received: 3 March 1999 / Accepted: 30 September 1999     

Age-Related Changes in the Expression of Cadherin-11, the Mesenchyme Specific Calcium-Dependent Cell Adhesion Molecule

Cadherin-11 is a calcium-dependent cell adhesion molecule that is expressed in cells of the mesenchymal lineage during embryonic development. In this study we show, for the first time, that cadherin-11 gene is expressed in the bone marrow and bone cells obtained from rabbits of various age groups. Furthermore, a quantitative measurement of gene expression revealed that cadherin-11 was expressed in young rabbits (6 week-old: open epiphysis) at a level of 6.7 × 10⁵± 0.7 × 10⁵ molecules; in mature rabbits (8–10 month-old: closed epiphysis) at 11 × 10⁵± 0.9 × 10⁵ molecules; and in aged rabbits (4–5 year-old) at a level of 1.2 × 10⁵± 0.2 × 10⁵ molecules/μg total RNA. The relative level of cadherin-11 gene expression in mature rabbit marrow was found to be approximately 50% greater than in young rabbits. However, aged animals showed a reduction in cadherin-11 specific gene expression of greater than 900% as compared with mature animals. Age-related changes in bone remodeling/turnover lead to reduced bone density and high fracture risk, and since cadherins play a crucial role in tissue morphogenesis, this marked decrease may represent an index of the aging process in bone. Received: 2 January 1997 / Accepted: 29 September 1997     

Bisphosphonate Maintains Parathyroid Hormone (1-34)-Induced Cortical Bone Mass and Mechanical Strength in Old Rats

This study was designed to determine the fate of new parathyroid hormone (PTH)-induced cortical bone after withdrawal of PTH treatment, and to evaluate whether subsequent treatment with a bisphosphonate would influence this. Six groups of 21-month-old rats were used: a baseline group killed at the beginning of the experiment, three groups injected with human PTH (1-34) (62 μg/kg) daily for 8 weeks (day 1-56), then one group was killed and the other two groups were injected for another 8 weeks (day 57-112) with either saline or bisphosphonate (risedronate 5 μg/kg twice a week). Two control groups were injected with vehicle for the first 8 weeks, then one group was killed and the other group injected with saline the next 8 weeks. All animals were labeled with tetracycline and calcein on day 35 and day 49 of the experiment, respectively. PTH increased periosteal (35%) and in particular endosteal mineralizing surfaces (188%), mineral appositional rates, and bone formation rates at the femur diaphysis, leading to an increase in cortical cross-sectional area of 31%. Withdrawal of PTH induced a fast and pronounced endosteal bone resorption whereas risedronate prevented this resorption. No differences were seen in apparent density of dry defatted bone and ash among the groups. PTH increased the mechanical strength of the femur diaphysis; ultimate load increased by 64% and ultimate stress by 25%. A pronounced decrease in mechanical strength and competence was found after withdrawal of PTH: ultimate load decreased by 31% and ultimate stress by 21%. Risedronate, however, prevented this decrease in mechanical strength and competence in these 2-year-old rats. Received: 13 March 1997 / Accepted: 22 August 1997     

Molecular Characterization of Gene Expression Changes in ROS 17/2.8 Cells Cultured in Diffusion Chambers In Vivo

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3–10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3–28 days and by 56 days was 4–5 fold above control levels. In 3–10 days, c-jun expression increased 1.6–1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10–56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to ≤28% of preimplantation values. In contrast, OPN mRNA levels increased up to 7-fold by day 10 and thereafter, returned to 1.7-fold above control values. COL1A1 mRNA decreased 2-fold at day 3 and increased to 3.5-, 1.6-, and 2.8-fold above control at days 10, 28, and 56, respectively. MMP-9 levels increased 5- to 10-fold by days 3–10, but fell to undetectable levels by 28–56 days. These results indicate that the formation of mineralized matrix (bone nodules) seen in the 56-day DC of ROS 17/2.8 cells was preceded by coordinate temporal expression of IEGs, matrix proteins, and matrix-modifying enzymes. Additionally, these results substantiate that measurement of molecular parameters in tissues formed by cells incubated in DC in vivo may be a useful predictor of the osteogenic process. Received: 6 February 1998 / Accepted: 9 December 1998     

Expression of the parathyroid hormone receptor and correlation with other osteoblastic parameters in fetal rat osteoblasts

Primary fetal rat calvarial cell cultures were examined for the expression of different osteoblastic parameters at the single cell level and in the whole population. The presence of the parathyroid hormone (PTH) receptor was studied by employing receptor autoradiography. After 3 days of culture, 10% of the cells expressed the PTH receptor. Immunolocalization of osteocalcin in 3-day-old cell cultures was found to be strongly correlated with the presence of the PTH receptor. Alkaline phosphatase (APase) localization in 3-day-old cultures correlated with only 69% of the PTH receptor expressing cells. Our results show that in 3-day-old rat calvarial cell cultures, only about 10% of the cells show markers of osteoblastic differentiation. The presence of the PTH receptor is strongly correlated with the presence of osteocalcin, but less with the presence of APase, indicating that it is the mature osteoblast that expresses the PTH receptor. After 7 days of culture, most receptor labeling, APase, and osteocalcin expression was found in multilayered areas of cells (nodules). Received: 28 February 1995 / Accepted: 27 July 1995     

Comparison of In Vitro Response to Growth Hormone by Chondrocytes from Mandibular Condyle Cartilage of Young and Old Mice

In several aspects the features of aging resemble those of the state of growth hormone (GH) deficiency. Alterations of bone density and increased incidence of osteoporosis are some of the characteristics of aging that are similar to the findings in GH-deficient adults. Osteoarthritis (OA), a nonfatal condition predominating in old age, is characterized by the slowly progressive destruction of articular cartilage of joints. OA lesions are observed in the temporomandibular joint of ICR mice aged 7 months and older. The aim of the present study was to compare the response of chondrocytes from mandibular condyle cartilage of young and old mice to GH and to ascertain whether chondrocytes of old animals could still be stimulated to proliferate and to synthesize matrix components under the direct effect of GH in vitro. Mandibular condyles from young (3-month-old) and old (20-month-old) female ICR mice were cultured up to 72 hours in the presence of recombinant rat GH (0.1–100 ng/ml). ³H-Thymidine and ³⁵S-sulfate were introduced during the last 24 hours of culture. Administration of GH appeared to increase only slightly the incorporation of ³H-thymidine in cartilage from young compared with the response in cartilage from old animals which was much more significant at 24 hours and 48 hours in culture. The same pattern was seen for the effect of GH on the incorporation of ³⁵S-sulfate. Cartilage from young animals responded only slightly to GH, whereas a significant response was observed in cartilage from old animals after 24 and 48 hours in vitro. DNA contents were significantly elevated at all time intervals in both old and young animals, yet more significantly in cultures from old animals. In contrast, the activities of both alkaline and acid phosphatases were elevated at all time intervals in cultures from young animals, whereas no induction was observed in cultures from old animals. Tissue sections from cultures of old animals treated with GH (10 ng/ml, 48 hours) revealed atypical hypertrophic cells along the articular surface and also dark staining along the cartilage-bone interface. Exposure to tetracycline (10 mg/ml, 24 hours) resulted in an induced fluorescence, indicating enhanced mineralization in this region. Results of this study indicate that mandibular condyle cartilage from OA old mice responds in vitro to the addition of GH via anabolic activities of cell proliferation, sulfated proteoglycan synthesis, and possibly enhanced mineralization. Received: 29 March 1996 / Accepted: 31 December 1996