- Email: [email protected]
PTH(1-34) suppresses appositional bone formation by cultured rat cranial osteoblasts
Bone Vol. 23, No. 5 November 1998:453– 457
PTH(1-34) Suppresses Appositional Bone Formation by Cultured Rat Cranial Osteoblasts C. GRAY and S. J. JONES Department of Anatomy and Developmental Biology, University College London, London, UK
blasts, and the topography of the machined dentine would be likely to encourage differentiation within the grooves.2,9 We have now studied the effect of short-term exposure of rat calvarial osteoblasts to rat PTH(1-34) (10212, 1029, and 1027 mol/L) using our appositional bone formation assay3 to determine whether intermittent PTH would promote osteogenesis. Unexpectedly, whether the addition was for 24 h only on days 3 or 12, or 24 h on both days 3 and 12, or a pulse for 6 h every 48 h, we obtained suppression of bone formation even at the lowest concentration of added PTH(1-34).
Parathyroid hormone (PTH) provokes cell division of osteoblasts in vitro and is anabolic in vivo when administered intermittently. We studied the effect on bone formation in vitro by rat primary calvarial osteoblasts of the short-term addition of rat PTH(1-34) (10212, 1029, and 1027 mol/L) to the medium. Our aim was to determine whether intermittent PTH(1-34) would promote osteogenesis in an experimental system designed to promote appositional bone formation. Unexpectedly, whether the exposure to PTH(1-34) was for 24 h only on days 3 or 12, or 24 h on both days 3 and 12, or a pulse for 6 h every 48 h, we observed suppression of bone formation even at the lowest concentration of added PTH(134). Our finding that cells which are of the osteoblastic phenotype can be entirely prevented from making appositional bone by high concentrations of PTH(1-34) for 1 day in a week, and have their osteogenic capacity suppressed by lower concentrations even when the PTH(1-34) is administered in brief pulses every other day, suggests that pulses of PTH administered in vivo may not increase bone formation by already differentiated osteoblasts. (Bone 23:453– 457; 1998) © 1998 by Elsevier Science Inc. All rights reserved.
Materials and Methods Calvarial bones were excised from euthanized neonate Sprague Dawley rats. Primary osteoblasts were then released from the bones by sequential enzyme digestion (trypsin/EDTA, followed by 0.2% [w/v] collagenase in Dulbecco’s phosphate-buffered saline [PBS] Sigma), rejecting the first digest, and cultures were established on 7 3 7 mm slices of sperm-whale dentine in which four parallel grooves had been cut on the top surface using a water-cooled diamond saw. The grooves measured approximately 350 mm across and 250 mm deep, and their cross section was close to a hemicircle. Four experiments were conducted. In experiment 1, exposure to added rat PTH(1-34) 1027 mol/L (Sigma) was for 24 h on day 3, day 12, or days 3 and 12. The specimens were fixed at 16 days. Experiment 2 was the same as experiment 1 except that the culture time was 20 days. In experiment 3, rat PTH(1-34) 10212 or 1029 mol/L was added for 6 h every 48 h, and the cultures were fixed at 18 days. Experiment 4 was the same as experiment 3, except that the culture time was 15 days. The PTH(1-34) was dissolved in a carrier solution of tissue-culture-grade water (Sigma) containing 0.1% (w/v) bovine serum albumin (Sigma) and 0.01% (v/v) acetic acid; this carrier solution was added to the control cultures in all cases. The cells were seeded at a density of 2 3 105 cells per 3.5 2 cm culture dish and cultured in MEM with 10% fetal calf serum (FCS) and 2 mmol/L L-glutamine at 37°C in 5% CO2 for up to 4 weeks. L-ascorbic acid 50 mg/mL (Sigma) and b-glycerophosphate 2 mmol/L (Sigma) were added to the PTH(1-34) or carrier from the second day of culture for experiments 1 and 2, and from the first day of culture for experiments 3 and 4. Antibiotic (Gentamicin, Gibco) 75 mg/mL was added during the settling period only (the first 48 h in experiments 1 and 2, and 24 h in experiments 3 and 4). All culture media were replenished every 2–3 days and, when necessary, for the addition or removal of a pulse of PTH(1-34) or carrier solution. Thus, the control specimens in each experiment had the medium changed at the same times as the experimental
Key Words: Parathyroid hormone (1-34); Rat; Osteoblasts; Bone formation; Grooves; In vitro. Introduction We previously found that parathyroid hormone, PTH(1-34) (1028 mol/L and 1027 mol/L), and parathyroid hormone-related protein, PTHrP(1-40) (1029 mol/L and 1028 mol/L), completely abolished bone formation in 4 week cultures of rat calvarial osteoblasts when present throughout culture periods in which medium was renewed three times per week (every 2 or 3 days).10 Pulsed PTH is anabolic in vivo in young and aged animals,12 and recent reports have shown that, whereas continuous exposure of cultured osteoblasts to PTH is catabolic, intermittent pulsing of low concentrations of PTH stimulates the formation of bone nodules.6,15 PTH has different effects on mouse osteoblasts, depending upon their stage of differentiation and the cell density in vitro,7 a finding supported by investigations on bone morphogenetic protein (BMP)-transfected cells.4 This is of particular relevance to the culture protocol we used; that is, we harvested calvarial cells that were possibly already committed preosteoAddress for correspondence and reprints: Professor S. J. Jones, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK. E-mail: [email protected] © 1998 by Elsevier Science Inc. All rights reserved.
453
8756-3282/98/$19.00 PII S8756-3282(98)00129-X
454
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
Bone Vol. 23, No. 5 November 1998:453– 457
Figure 1. Experiment 1. Total length of bone in all grooves per slice at 16 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets. A second control set (white bar on right) experienced medium changes only three times each week (every 2 or 3 days). No bone formed in the specimens pulsed for 24 h on both days 3 and 12.
Figure 2. Experiment 2. Total length of bone in all grooves per slice at 20 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets. No bone formed in the specimens pulsed with PTH for 24 h on day 12, or on both days 3 and 12. (The set pulsed with PTH on both days 3 and 12 had only seven replicates, with one being lost.)
ones. An additional control set in experiment 1 had medium changes three times per week only, at intervals of 2 or 3 days, as was the case for our earlier experiments with continuous exposure to PTH.9 The live cultures were examined daily using an inverted microscope with phase optics. The culture period (from seeding: 15, 16, 18, or 20 days) was terminated 4 days after bone mineralization was first observed in any of the cultures of the experiment. Specimens were fixed at 20°C using 70% ethanol and stored in 70% ethanol at 4°C. The grooved slabs of dentine were stained with alizarin red (CI 58005), as previously described,3 for at least 7 days, and the specimens were then stored in 70% ethanol to elute any excess staining slowly. This ensured that the stain was only present in the newly formed bone, and not masked by the background. We did not differentiate the stain by using acid alcohol as this might cause demineralization and was unnecessary. The specimens were independently randomized and coded so that the measurements could be taken in a blinded manner. The specimens were examined using an Edge R-400 highresolution three-dimensional (3D) microscope (Edge Scientific Instrument) fitted with a Nikon 43 0.13 nA water immersion objective lens and coupled to a color video camera. This was interfaced with a personal computer running an image-analysis package (Fenestra, Kinetic Imaging Ltd.). For each visual field containing one or more whole alizarin red-stained bone loci, a 256-gray-level digital image was stored. A calibration image was also captured and used for scaling the images. Using the calibrated image, a binary image of each bone locus was created, and the program recorded its length (computed maximum chord) and area. The totals of the number, maximum chord lengths, and areas of alizarin redstained bone loci were calculated per specimen. The results were analyzed statistically using MINITAB software by a one-way analysis of variance.
formed per specimen in all the experiments gave similar results and, therefore, only histograms for the lengths are shown. There were eight replicates in each treatment set. No bone formed in any of the cultures to which rat PTH(1-34) (1027 mol/L) was added for 24 h on both day 3 and day 12. The amount of bone formed in the cultures exposed to PTH(1-34) on day 3 was minimal, and five of the eight specimens had no bone. Bone formation was also suppressed when exposure occurred on day 12, although these cultures had already started bone formation before PTH(1-34) or carrier was added. The amount of bone formed in the control cultures that were fed only three times per week did not differ significantly from that formed in the controls, which had the medium changed at the same times as the specimens exposed to added PTH(1-34). This experiment was terminated at 16 days when bone was abundant in the control experiments.
Results Experiment 1: PTH(1-34) pulse for 24 h on day 3, day 12, or days 3 and 12. The results for this experiment are given in Figure 1. The measurements for the areas and lengths of bone
Figure 3. Experiment 3. Total length of bone in all grooves per slice at 18 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets.
Bone Vol. 23, No. 5 November 1998:453– 457
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
455
times. The amount of bone formed at the two concentrations of added PTH(1-34) did not differ significantly. Experiment 4: PTH(1-34) pulse for 6 h every 48 h. The results for experiment 4 are given in Figure 4. There were five replicates in each experimental set. Bone formation had occurred in all specimens by day 15 when the specimens were fixed. There was no significant difference in the amount of bone formed when the cells were exposed to added PTH(1-34) 10212 mol/L, but the addition of PTH(1-34) 1029 mol/L for 6 h every 48 h resulted in a reduction in the amount of bone formed compared with controls, which had medium changes at the same times. The bone formed was morphologically similar in all experimental sets (Figure 5). It was firmly attached to the dentine, and the orientation of the collagen and osteocytes within the bone was parallel to the groove system. The “total length of bone” did not increase as a function of time in the controls of the four experiments because the cells used were primary cells, and each experiment was conducted with a different cell isolation. Figure 4. Experiment 4. Total length of bone in all grooves per slice at 15 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets.
Experiment 2: PTH(1-34) pulse for 24 h on day 3, day 12, or days 3 and 12. The results for this experiment are shown in Figure 2. There were eight replicates in each experimental set except for exposure to added PTH(1-34) on both day 3 and day 12 for which there were seven replicates. No bone formed in any of the specimens exposed on day 12, or on those exposed to PTH(1-34) on both day 3 and day 12. The amount of bone that formed in the specimens exposed on day 3 did not differ statistically from controls, in which two of the eight specimens had no bone mineralization. The experiment was terminated on day 20; bone formation was not as abundant in this experiment as in experiment 1, and did not occur before the addition of PTH(1-34). Experiment 3: PTH(1-34) pulse for 6 h every 48 h. The results for experiment 3 are given in Figure 3. There were eight replicates in each experimental set. Bone formation had occurred in all specimens by day 18 when the specimens were fixed. Exposure to PTH(1-34) 10212 mol/L and 1029 mol/L for 6 h every 48 h resulted in a reduction in the amount of bone formed compared with controls, which had medium changes at the same
Discussion PTH and PTHrP regulate cellular differentiation, proliferation, and development,14 and are now considered to be anabolic skeletal agents when made available periodically rather than continuously in vivo.16 Circulating PTH levels show an endogenous bimodal diurnal pattern with the primary peak at night,1 and small-amplitude pulses of PTH secretion occurring at much shorter intervals.13 As PTH provokes cell division of osteoblasts in vitro,8 the promotion of bone formation in vivo is readily understandable as long as some of the plethora of cells are permitted to differentiate into secretory osteoblasts. We found in previous experiments that PTH, PTH(1-34), and PTHrP(1-40) have similar effects on bone formation in our culture system;10 however, although we renewed the PTH/ PTHrP at longer intervals than what has been proposed for therapeutic administration, we observed complete inhibition rather than an enhancement of bone formation. This we assumed to be due to PTH being present continuously, even though some degradation would have occurred before renewal of the medium.6 Isogai et al.7 found, in addition, that continuous exposure to human PTH(1-34) showed different effects on colony formation by mouse calvarial osteoblasts when added to
Figure 5. Alizarin-stained bone made by rat cranial osteoblasts in grooves, 350 mm wide, cut into dentine (15 day cultures). Left: control culture; right: culture with 1029 mol/L PTH(1-34) added to the medium as a 6 h pulse every 2 days.
456
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
preconfluent and confluent cultures, and surmised that PTH stimulates osteoblast differentiation in immature cells, but inhibits it in more mature cells. Expression of the PTH/PTHrP receptor and cyclic AMP response of the cells to PTH(1-34) occur before mineralization and alkaline phosphatase is detectable, and at a time when bone proteins are barely detectable, in cultures of rat bone marrow cells.11 The cells that we used in our experiments were from the second collagenase digest of the rat calvarium, and likely to be predominately committed osteogenic cells. There is some agreement of the results of recent investigations regarding effects on osteoblasts of intermittent exposure to PTH(1-34) in vitro. Yang et al.14 reported that an exposure time as brief as 15 min every 2–3 days to PTH 10214 to 10210 mol/L was anabolic for fetal rat calvarial cells, whereas higher concentrations or continuous exposure suppressed bone nodule formation. Hryhorenko et al.5 showed that adult rat bone marrow stromal cells subjected to five 15 min pulses of PTH over 10 days reacted anabolically at 10212 and 10210 mol/L. Three 15 min pulses at intervals of 2 days in the middle of the 10 days had a greater effect than doses before day 3 or after day 7. They concluded that pulsatile low doses of PTH were anabolic, whereas higher doses were catabolic. Ishizuya et al.6 found that, for neonate rat calvarial cells, a 1 h pulse of PTH(1-34) 50 ng/mL each 48 h or continuous exposure were catabolic, but a 6 h pulse each 48 h was anabolic, and exposure for 3 h or 24 h had no effect compared with control cultures. Furthermore, only two 48 h cycles were necessary for the effect to be seen in postconfluent cells. They counted the number of cell colonies that stained positive for alkaline phosphatase or appeared brown or black when von Kossa stained. The experiments reported here show that suppression of bone formation can occur when PTH(1-34) 1027 mol/L is added as a single 24 h pulse early in the culture period (experiment 1, day 3), or even if added later (experiment 2, day 12). The concentration we used in these experiments was higher than those considered by Yang et al.15 or Hryhorenko et al.5 to be anabolic in their experiments. Albeit we used only one, or at most two, judicious pulses, our current results are therefore in broad agreement in this aspect, and we have shown that we can prevent osteogenesis over the time-scale of our experiments. The results obtained by Ishizuya et al.6 with intermittent exposures to synthetic PTH(1-34) 50 ng/mL (approximately 1028 mol/L) of different durations are puzzling. Although we employed the same pattern of a 6 h exposure every 48 h, and low concentrations of added PTH(1-34) (either 1029 or 10212 mol/L), we still obtained less bone formation with added hormone than in the controls. The duration of the PTH(1-34) pulse in experiments 3 and 4 (i.e., 6 h) was chosen because it is difficult to adequately control the pH and temperature when medium changes occur in air at very short intervals. Equilibration was, therefore, a small proportion of the pulse duration. It is unlikely that the fetal calf serum we used contained enough PTH to make the overall concentration in the medium significantly higher than the added amount. Ishizuya et al.6 measured the PTH in their medium with fetal calf serum to be about 2 3 10212 mol/L, which is within the normal range for circulating immunoreactive PTH in man. Why concentrations of PTH much lower than those in the physiological range for serum appear to exert an anabolic response in vitro remains unexplained. Daily (5 days in every 7) subcutaneous injections yielding approximately 2 3 1028 mol/L PTH in vivo in aged rats have proved anabolic.12 However, it is not
Bone Vol. 23, No. 5 November 1998:453– 457
possible to determine, from in vivo experiments, which cell population reacts first to the PTH. There may also be other explanations for our results. First, as noted earlier, the primary cell population we harvested probably contained cells already committed to the osteoblastic pathway, and their exposure to PTH(1-34) suppressed further differentiation. Second, the culture system we favor is more representative of postnatal appositional bone formation than early stages of intramembranous ossification, which is simulated in the bone nodule assays, and we measured the amount of mineralized bone. Our finding that cells of the osteoblastic phenotype can be prevented from making appositional bone by high concentrations of PTH(1-34) for 1 day in a week, and have their osteogenic capacity suppressed by lower concentrations even when PTH(1-34) is administered in brief pulses every other day, suggests that pulses of PTH administrated in vivo may not increase bone formation by fully differentiated osteoblasts.
Acknowledgments: The authors thank Maureen Arora for her gracious help. This work was supported by grants from the Wellcome Trust, the Medical Research Council, and the Veterinary Advisory Council of the Horserace Betting Levy Board.
References 1. el-Hajj Fuleihan, G., Klerman, E. B., Brown, E. N., Choe, Y., Brown, E. M. and Czeisler, C. A. The parathyroid hormone is truly endogenous—a general clinical research center study. J Clin Endocrinol Metab 82:281–286, 1997. 2. Gray, C., Boyde, A., and Jones, S. J. Topographically induced bone in vitro: Implications for bone implants and bone grafts. Bone 18:115–123; 1996. 3. Gray, C., Boyde, A., and Jones, S. J. The isolation, culture, and functional assay of osteoclasts and osteoblasts. In: Celis, C. E., Ed. Cell Biology: A Laboratory Handbook, 2nd Ed. (vol. 1). San Diego: Academic; 1998; 142–148. 4. Hollnagel, A., Ahrens, M., and Gross, G. Parathyroid hormone enhances early and suppresses late stages of osteogenic and chondrogenic development in a BMP-dependent mesenchymal differentiation system (C3H10T1⁄2). J Bone Miner Res 12:1993–2004; 1997. 5. Hryhorenko, L., Davies, J., and Sindrey, D. Differentiation between anabolic and catabolic doses of PTH in the mineralization of primary marrow cell cultures on CaP slides. J Bone Miner Res 12(Suppl. 1):S321; 1997. 6. Ishizuya, T., Yokose, S., Hori, M., Noda, T., Suda, T., Yoshiki, S., and Yamaguchi, A. Parathyroid hormone exerts disparate effects on osteoblastic differentiation depending on exposure time in rat osteoblastic cells. J Clin Invest 99:2961–2970; 1997. 7. Isogai, Y., Akatsu, T., Ishizuya, T., Yamaguchi, A., Hori, M., Takahashi N., and Suda, T. Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages. J Bone Miner Res 11:1384 –1393; 1996. 8. Jones, S. J. and Boyde, A. Experimental study of changes in osteoblastic shape induced by calcitonin and parathyroid extract in an organ culture system. Cell Tissue Res 169:449 – 465; 1976. 9. Jones, S. J., Gray, C., and Boyde, A. Simulation of bone resorption-repair coupling in vitro. Anat Embryol 190:339 –349; 1994. 10. Jones, S. J., Gray, C., Boyde, A., and Burnstock, G. Purinergic transmitters inhibit bone formation by cultured osteoblasts. Bone 21:393–399; 1997. 11. Kondo, H., Ohyama, T., Ohya, K., and Kasugai, S. Temporal changes of mRNA expression of matrix proteins and parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor in bone development. J Bone Miner Res 12:2089 –2097; 1997. 12. Qi, H., Li, M., and Wronski, T. J. A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats. J Bone Miner Res 10:948 –955; 1995. 13. Samuels, M. H., Veldhuis, J. D., Kramer, P., Urban, R. J., Bauer, R., and Mundy, G. R. Episodic secretion of parathyroid hormone in postmenopausal women: assessment by deconvolution analysis and approximate entropy. J Bone Miner Res 12:616 – 623; 1997.
Bone Vol. 23, No. 5 November 1998:453– 457 14. Stewart, A. F. PTHrP(1-36) acts as a skeletal anabolic agent for the treatment of osteoporosis. Bone 19:303–306; 1996. 15. Yang, Z.-J., Cheng, V., Barnes, S., Cavalho, L., and Sindrey, D. Pulsatile parathyroid hormone (PTH) treatment increases bone formation in vitro in fetal rat calvarial cell (FRCC) culture system. J Bone Miner Res 12(Suppl 1):S317; 1997. 16. Zhang, L., Takahashi, H. E., Inoue, J., Tanizawa, T., Endo, N., Yamomoto, N., and Hori, M. Effects of intermittent administration of low dose human PTH(1-
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
457
34) on cancellous and cortical bone of lumbar vertebral bodies in adult beagles. Bone 21:501–506; 1997.
Date Received: February 13, 1998 Date Revised: June 25, 1998 Date Accepted: July 9, 1998
PTH(1-34) Suppresses Appositional Bone Formation by Cultured Rat Cranial Osteoblasts C. GRAY and S. J. JONES Department of Anatomy and Developmental Biology, University College London, London, UK
blasts, and the topography of the machined dentine would be likely to encourage differentiation within the grooves.2,9 We have now studied the effect of short-term exposure of rat calvarial osteoblasts to rat PTH(1-34) (10212, 1029, and 1027 mol/L) using our appositional bone formation assay3 to determine whether intermittent PTH would promote osteogenesis. Unexpectedly, whether the addition was for 24 h only on days 3 or 12, or 24 h on both days 3 and 12, or a pulse for 6 h every 48 h, we obtained suppression of bone formation even at the lowest concentration of added PTH(1-34).
Parathyroid hormone (PTH) provokes cell division of osteoblasts in vitro and is anabolic in vivo when administered intermittently. We studied the effect on bone formation in vitro by rat primary calvarial osteoblasts of the short-term addition of rat PTH(1-34) (10212, 1029, and 1027 mol/L) to the medium. Our aim was to determine whether intermittent PTH(1-34) would promote osteogenesis in an experimental system designed to promote appositional bone formation. Unexpectedly, whether the exposure to PTH(1-34) was for 24 h only on days 3 or 12, or 24 h on both days 3 and 12, or a pulse for 6 h every 48 h, we observed suppression of bone formation even at the lowest concentration of added PTH(134). Our finding that cells which are of the osteoblastic phenotype can be entirely prevented from making appositional bone by high concentrations of PTH(1-34) for 1 day in a week, and have their osteogenic capacity suppressed by lower concentrations even when the PTH(1-34) is administered in brief pulses every other day, suggests that pulses of PTH administered in vivo may not increase bone formation by already differentiated osteoblasts. (Bone 23:453– 457; 1998) © 1998 by Elsevier Science Inc. All rights reserved.
Materials and Methods Calvarial bones were excised from euthanized neonate Sprague Dawley rats. Primary osteoblasts were then released from the bones by sequential enzyme digestion (trypsin/EDTA, followed by 0.2% [w/v] collagenase in Dulbecco’s phosphate-buffered saline [PBS] Sigma), rejecting the first digest, and cultures were established on 7 3 7 mm slices of sperm-whale dentine in which four parallel grooves had been cut on the top surface using a water-cooled diamond saw. The grooves measured approximately 350 mm across and 250 mm deep, and their cross section was close to a hemicircle. Four experiments were conducted. In experiment 1, exposure to added rat PTH(1-34) 1027 mol/L (Sigma) was for 24 h on day 3, day 12, or days 3 and 12. The specimens were fixed at 16 days. Experiment 2 was the same as experiment 1 except that the culture time was 20 days. In experiment 3, rat PTH(1-34) 10212 or 1029 mol/L was added for 6 h every 48 h, and the cultures were fixed at 18 days. Experiment 4 was the same as experiment 3, except that the culture time was 15 days. The PTH(1-34) was dissolved in a carrier solution of tissue-culture-grade water (Sigma) containing 0.1% (w/v) bovine serum albumin (Sigma) and 0.01% (v/v) acetic acid; this carrier solution was added to the control cultures in all cases. The cells were seeded at a density of 2 3 105 cells per 3.5 2 cm culture dish and cultured in MEM with 10% fetal calf serum (FCS) and 2 mmol/L L-glutamine at 37°C in 5% CO2 for up to 4 weeks. L-ascorbic acid 50 mg/mL (Sigma) and b-glycerophosphate 2 mmol/L (Sigma) were added to the PTH(1-34) or carrier from the second day of culture for experiments 1 and 2, and from the first day of culture for experiments 3 and 4. Antibiotic (Gentamicin, Gibco) 75 mg/mL was added during the settling period only (the first 48 h in experiments 1 and 2, and 24 h in experiments 3 and 4). All culture media were replenished every 2–3 days and, when necessary, for the addition or removal of a pulse of PTH(1-34) or carrier solution. Thus, the control specimens in each experiment had the medium changed at the same times as the experimental
Key Words: Parathyroid hormone (1-34); Rat; Osteoblasts; Bone formation; Grooves; In vitro. Introduction We previously found that parathyroid hormone, PTH(1-34) (1028 mol/L and 1027 mol/L), and parathyroid hormone-related protein, PTHrP(1-40) (1029 mol/L and 1028 mol/L), completely abolished bone formation in 4 week cultures of rat calvarial osteoblasts when present throughout culture periods in which medium was renewed three times per week (every 2 or 3 days).10 Pulsed PTH is anabolic in vivo in young and aged animals,12 and recent reports have shown that, whereas continuous exposure of cultured osteoblasts to PTH is catabolic, intermittent pulsing of low concentrations of PTH stimulates the formation of bone nodules.6,15 PTH has different effects on mouse osteoblasts, depending upon their stage of differentiation and the cell density in vitro,7 a finding supported by investigations on bone morphogenetic protein (BMP)-transfected cells.4 This is of particular relevance to the culture protocol we used; that is, we harvested calvarial cells that were possibly already committed preosteoAddress for correspondence and reprints: Professor S. J. Jones, Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK. E-mail: [email protected] © 1998 by Elsevier Science Inc. All rights reserved.
453
8756-3282/98/$19.00 PII S8756-3282(98)00129-X
454
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
Bone Vol. 23, No. 5 November 1998:453– 457
Figure 1. Experiment 1. Total length of bone in all grooves per slice at 16 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets. A second control set (white bar on right) experienced medium changes only three times each week (every 2 or 3 days). No bone formed in the specimens pulsed for 24 h on both days 3 and 12.
Figure 2. Experiment 2. Total length of bone in all grooves per slice at 20 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets. No bone formed in the specimens pulsed with PTH for 24 h on day 12, or on both days 3 and 12. (The set pulsed with PTH on both days 3 and 12 had only seven replicates, with one being lost.)
ones. An additional control set in experiment 1 had medium changes three times per week only, at intervals of 2 or 3 days, as was the case for our earlier experiments with continuous exposure to PTH.9 The live cultures were examined daily using an inverted microscope with phase optics. The culture period (from seeding: 15, 16, 18, or 20 days) was terminated 4 days after bone mineralization was first observed in any of the cultures of the experiment. Specimens were fixed at 20°C using 70% ethanol and stored in 70% ethanol at 4°C. The grooved slabs of dentine were stained with alizarin red (CI 58005), as previously described,3 for at least 7 days, and the specimens were then stored in 70% ethanol to elute any excess staining slowly. This ensured that the stain was only present in the newly formed bone, and not masked by the background. We did not differentiate the stain by using acid alcohol as this might cause demineralization and was unnecessary. The specimens were independently randomized and coded so that the measurements could be taken in a blinded manner. The specimens were examined using an Edge R-400 highresolution three-dimensional (3D) microscope (Edge Scientific Instrument) fitted with a Nikon 43 0.13 nA water immersion objective lens and coupled to a color video camera. This was interfaced with a personal computer running an image-analysis package (Fenestra, Kinetic Imaging Ltd.). For each visual field containing one or more whole alizarin red-stained bone loci, a 256-gray-level digital image was stored. A calibration image was also captured and used for scaling the images. Using the calibrated image, a binary image of each bone locus was created, and the program recorded its length (computed maximum chord) and area. The totals of the number, maximum chord lengths, and areas of alizarin redstained bone loci were calculated per specimen. The results were analyzed statistically using MINITAB software by a one-way analysis of variance.
formed per specimen in all the experiments gave similar results and, therefore, only histograms for the lengths are shown. There were eight replicates in each treatment set. No bone formed in any of the cultures to which rat PTH(1-34) (1027 mol/L) was added for 24 h on both day 3 and day 12. The amount of bone formed in the cultures exposed to PTH(1-34) on day 3 was minimal, and five of the eight specimens had no bone. Bone formation was also suppressed when exposure occurred on day 12, although these cultures had already started bone formation before PTH(1-34) or carrier was added. The amount of bone formed in the control cultures that were fed only three times per week did not differ significantly from that formed in the controls, which had the medium changed at the same times as the specimens exposed to added PTH(1-34). This experiment was terminated at 16 days when bone was abundant in the control experiments.
Results Experiment 1: PTH(1-34) pulse for 24 h on day 3, day 12, or days 3 and 12. The results for this experiment are given in Figure 1. The measurements for the areas and lengths of bone
Figure 3. Experiment 3. Total length of bone in all grooves per slice at 18 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets.
Bone Vol. 23, No. 5 November 1998:453– 457
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
455
times. The amount of bone formed at the two concentrations of added PTH(1-34) did not differ significantly. Experiment 4: PTH(1-34) pulse for 6 h every 48 h. The results for experiment 4 are given in Figure 4. There were five replicates in each experimental set. Bone formation had occurred in all specimens by day 15 when the specimens were fixed. There was no significant difference in the amount of bone formed when the cells were exposed to added PTH(1-34) 10212 mol/L, but the addition of PTH(1-34) 1029 mol/L for 6 h every 48 h resulted in a reduction in the amount of bone formed compared with controls, which had medium changes at the same times. The bone formed was morphologically similar in all experimental sets (Figure 5). It was firmly attached to the dentine, and the orientation of the collagen and osteocytes within the bone was parallel to the groove system. The “total length of bone” did not increase as a function of time in the controls of the four experiments because the cells used were primary cells, and each experiment was conducted with a different cell isolation. Figure 4. Experiment 4. Total length of bone in all grooves per slice at 15 days. The control set (white bar on left) had changes of medium that exactly matched the PTH treatment sets.
Experiment 2: PTH(1-34) pulse for 24 h on day 3, day 12, or days 3 and 12. The results for this experiment are shown in Figure 2. There were eight replicates in each experimental set except for exposure to added PTH(1-34) on both day 3 and day 12 for which there were seven replicates. No bone formed in any of the specimens exposed on day 12, or on those exposed to PTH(1-34) on both day 3 and day 12. The amount of bone that formed in the specimens exposed on day 3 did not differ statistically from controls, in which two of the eight specimens had no bone mineralization. The experiment was terminated on day 20; bone formation was not as abundant in this experiment as in experiment 1, and did not occur before the addition of PTH(1-34). Experiment 3: PTH(1-34) pulse for 6 h every 48 h. The results for experiment 3 are given in Figure 3. There were eight replicates in each experimental set. Bone formation had occurred in all specimens by day 18 when the specimens were fixed. Exposure to PTH(1-34) 10212 mol/L and 1029 mol/L for 6 h every 48 h resulted in a reduction in the amount of bone formed compared with controls, which had medium changes at the same
Discussion PTH and PTHrP regulate cellular differentiation, proliferation, and development,14 and are now considered to be anabolic skeletal agents when made available periodically rather than continuously in vivo.16 Circulating PTH levels show an endogenous bimodal diurnal pattern with the primary peak at night,1 and small-amplitude pulses of PTH secretion occurring at much shorter intervals.13 As PTH provokes cell division of osteoblasts in vitro,8 the promotion of bone formation in vivo is readily understandable as long as some of the plethora of cells are permitted to differentiate into secretory osteoblasts. We found in previous experiments that PTH, PTH(1-34), and PTHrP(1-40) have similar effects on bone formation in our culture system;10 however, although we renewed the PTH/ PTHrP at longer intervals than what has been proposed for therapeutic administration, we observed complete inhibition rather than an enhancement of bone formation. This we assumed to be due to PTH being present continuously, even though some degradation would have occurred before renewal of the medium.6 Isogai et al.7 found, in addition, that continuous exposure to human PTH(1-34) showed different effects on colony formation by mouse calvarial osteoblasts when added to
Figure 5. Alizarin-stained bone made by rat cranial osteoblasts in grooves, 350 mm wide, cut into dentine (15 day cultures). Left: control culture; right: culture with 1029 mol/L PTH(1-34) added to the medium as a 6 h pulse every 2 days.
456
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
preconfluent and confluent cultures, and surmised that PTH stimulates osteoblast differentiation in immature cells, but inhibits it in more mature cells. Expression of the PTH/PTHrP receptor and cyclic AMP response of the cells to PTH(1-34) occur before mineralization and alkaline phosphatase is detectable, and at a time when bone proteins are barely detectable, in cultures of rat bone marrow cells.11 The cells that we used in our experiments were from the second collagenase digest of the rat calvarium, and likely to be predominately committed osteogenic cells. There is some agreement of the results of recent investigations regarding effects on osteoblasts of intermittent exposure to PTH(1-34) in vitro. Yang et al.14 reported that an exposure time as brief as 15 min every 2–3 days to PTH 10214 to 10210 mol/L was anabolic for fetal rat calvarial cells, whereas higher concentrations or continuous exposure suppressed bone nodule formation. Hryhorenko et al.5 showed that adult rat bone marrow stromal cells subjected to five 15 min pulses of PTH over 10 days reacted anabolically at 10212 and 10210 mol/L. Three 15 min pulses at intervals of 2 days in the middle of the 10 days had a greater effect than doses before day 3 or after day 7. They concluded that pulsatile low doses of PTH were anabolic, whereas higher doses were catabolic. Ishizuya et al.6 found that, for neonate rat calvarial cells, a 1 h pulse of PTH(1-34) 50 ng/mL each 48 h or continuous exposure were catabolic, but a 6 h pulse each 48 h was anabolic, and exposure for 3 h or 24 h had no effect compared with control cultures. Furthermore, only two 48 h cycles were necessary for the effect to be seen in postconfluent cells. They counted the number of cell colonies that stained positive for alkaline phosphatase or appeared brown or black when von Kossa stained. The experiments reported here show that suppression of bone formation can occur when PTH(1-34) 1027 mol/L is added as a single 24 h pulse early in the culture period (experiment 1, day 3), or even if added later (experiment 2, day 12). The concentration we used in these experiments was higher than those considered by Yang et al.15 or Hryhorenko et al.5 to be anabolic in their experiments. Albeit we used only one, or at most two, judicious pulses, our current results are therefore in broad agreement in this aspect, and we have shown that we can prevent osteogenesis over the time-scale of our experiments. The results obtained by Ishizuya et al.6 with intermittent exposures to synthetic PTH(1-34) 50 ng/mL (approximately 1028 mol/L) of different durations are puzzling. Although we employed the same pattern of a 6 h exposure every 48 h, and low concentrations of added PTH(1-34) (either 1029 or 10212 mol/L), we still obtained less bone formation with added hormone than in the controls. The duration of the PTH(1-34) pulse in experiments 3 and 4 (i.e., 6 h) was chosen because it is difficult to adequately control the pH and temperature when medium changes occur in air at very short intervals. Equilibration was, therefore, a small proportion of the pulse duration. It is unlikely that the fetal calf serum we used contained enough PTH to make the overall concentration in the medium significantly higher than the added amount. Ishizuya et al.6 measured the PTH in their medium with fetal calf serum to be about 2 3 10212 mol/L, which is within the normal range for circulating immunoreactive PTH in man. Why concentrations of PTH much lower than those in the physiological range for serum appear to exert an anabolic response in vitro remains unexplained. Daily (5 days in every 7) subcutaneous injections yielding approximately 2 3 1028 mol/L PTH in vivo in aged rats have proved anabolic.12 However, it is not
Bone Vol. 23, No. 5 November 1998:453– 457
possible to determine, from in vivo experiments, which cell population reacts first to the PTH. There may also be other explanations for our results. First, as noted earlier, the primary cell population we harvested probably contained cells already committed to the osteoblastic pathway, and their exposure to PTH(1-34) suppressed further differentiation. Second, the culture system we favor is more representative of postnatal appositional bone formation than early stages of intramembranous ossification, which is simulated in the bone nodule assays, and we measured the amount of mineralized bone. Our finding that cells of the osteoblastic phenotype can be prevented from making appositional bone by high concentrations of PTH(1-34) for 1 day in a week, and have their osteogenic capacity suppressed by lower concentrations even when PTH(1-34) is administered in brief pulses every other day, suggests that pulses of PTH administrated in vivo may not increase bone formation by fully differentiated osteoblasts.
Acknowledgments: The authors thank Maureen Arora for her gracious help. This work was supported by grants from the Wellcome Trust, the Medical Research Council, and the Veterinary Advisory Council of the Horserace Betting Levy Board.
References 1. el-Hajj Fuleihan, G., Klerman, E. B., Brown, E. N., Choe, Y., Brown, E. M. and Czeisler, C. A. The parathyroid hormone is truly endogenous—a general clinical research center study. J Clin Endocrinol Metab 82:281–286, 1997. 2. Gray, C., Boyde, A., and Jones, S. J. Topographically induced bone in vitro: Implications for bone implants and bone grafts. Bone 18:115–123; 1996. 3. Gray, C., Boyde, A., and Jones, S. J. The isolation, culture, and functional assay of osteoclasts and osteoblasts. In: Celis, C. E., Ed. Cell Biology: A Laboratory Handbook, 2nd Ed. (vol. 1). San Diego: Academic; 1998; 142–148. 4. Hollnagel, A., Ahrens, M., and Gross, G. Parathyroid hormone enhances early and suppresses late stages of osteogenic and chondrogenic development in a BMP-dependent mesenchymal differentiation system (C3H10T1⁄2). J Bone Miner Res 12:1993–2004; 1997. 5. Hryhorenko, L., Davies, J., and Sindrey, D. Differentiation between anabolic and catabolic doses of PTH in the mineralization of primary marrow cell cultures on CaP slides. J Bone Miner Res 12(Suppl. 1):S321; 1997. 6. Ishizuya, T., Yokose, S., Hori, M., Noda, T., Suda, T., Yoshiki, S., and Yamaguchi, A. Parathyroid hormone exerts disparate effects on osteoblastic differentiation depending on exposure time in rat osteoblastic cells. J Clin Invest 99:2961–2970; 1997. 7. Isogai, Y., Akatsu, T., Ishizuya, T., Yamaguchi, A., Hori, M., Takahashi N., and Suda, T. Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages. J Bone Miner Res 11:1384 –1393; 1996. 8. Jones, S. J. and Boyde, A. Experimental study of changes in osteoblastic shape induced by calcitonin and parathyroid extract in an organ culture system. Cell Tissue Res 169:449 – 465; 1976. 9. Jones, S. J., Gray, C., and Boyde, A. Simulation of bone resorption-repair coupling in vitro. Anat Embryol 190:339 –349; 1994. 10. Jones, S. J., Gray, C., Boyde, A., and Burnstock, G. Purinergic transmitters inhibit bone formation by cultured osteoblasts. Bone 21:393–399; 1997. 11. Kondo, H., Ohyama, T., Ohya, K., and Kasugai, S. Temporal changes of mRNA expression of matrix proteins and parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor in bone development. J Bone Miner Res 12:2089 –2097; 1997. 12. Qi, H., Li, M., and Wronski, T. J. A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats. J Bone Miner Res 10:948 –955; 1995. 13. Samuels, M. H., Veldhuis, J. D., Kramer, P., Urban, R. J., Bauer, R., and Mundy, G. R. Episodic secretion of parathyroid hormone in postmenopausal women: assessment by deconvolution analysis and approximate entropy. J Bone Miner Res 12:616 – 623; 1997.
Bone Vol. 23, No. 5 November 1998:453– 457 14. Stewart, A. F. PTHrP(1-36) acts as a skeletal anabolic agent for the treatment of osteoporosis. Bone 19:303–306; 1996. 15. Yang, Z.-J., Cheng, V., Barnes, S., Cavalho, L., and Sindrey, D. Pulsatile parathyroid hormone (PTH) treatment increases bone formation in vitro in fetal rat calvarial cell (FRCC) culture system. J Bone Miner Res 12(Suppl 1):S317; 1997. 16. Zhang, L., Takahashi, H. E., Inoue, J., Tanizawa, T., Endo, N., Yamomoto, N., and Hori, M. Effects of intermittent administration of low dose human PTH(1-
C. Gray and S. J. Jones PTH(1-34) inhibits bone formation in vitro
457
34) on cancellous and cortical bone of lumbar vertebral bodies in adult beagles. Bone 21:501–506; 1997.
Date Received: February 13, 1998 Date Revised: June 25, 1998 Date Accepted: July 9, 1998