Increased Osteoclast-Like Cells Formation in Long-Term Bone Marrow Cultures from Patients with a Spinal Cord Injury

Patients with a spinal cord section loose a significant amount of bone. After paraplegia, bone loss occurs below the lesional level and is the more dramatic in iliac bones and in the metaphyseal area of long bones. A peak of urinary calcium and hydroxyprolinuria is observed approximately 6 weeks after their lesion. To further understand the mechanisms underlying the bone damage, we used long-term bone marrow cultures to compare osteoclast-like (OCL-like) cell formation above and below the lesional level. Seven paraplegic, one quadriparetic, one quadriplegic patients and five normal subjects were investigated. Six weeks after their spinal cord section, the number of OCL-like cells formed in iliac bone marrow cultures was significantly greater than those formed in sternal bone marrow cultures for all paraplegic patients tested. No significant differences were seen between iliac and sternal bone marrow cultures for the quadriparetic, the quadriplegic patient, or for the five normal subjects. Conditioned media (CM) from iliac marrow of paraplegic patients increased OCL-like cell formation in normal bone marrow cultures. IL-1, TNF-alpha, IL-6, and PGE₂ were measured in the CM after 3 weeks of culture. IL-6 was found to be significantly higher in iliac CM compared with sternal CM in six out of seven paraplegic patients. In two patients, addition of an anti-IL-6 monoclonal antibody to the marrow cultures significantly decreased the number of OCL-like cells formed at 3 weeks. We conclude that paraplegia caused by a cord section locally induces an increase in the capacity of progenitors to form OCL-like cells in long-term bone marrow cultures. A locally increased IL-6 production in the marrow below the lesional level could be partly responsible for this observation. Received: 25 July 1997 / Accepted: 5 March 1998     

Does Colchicine Really Induce Bone Formation in the Rodent Bone Marrow?

Intravenous injection of a single dose of colchicine into inbred strains of BALB/c and CFW/L1 mice and into WAG rats did not effect rapid intramedullar bone formation and resorption, as has been claimed by the research group from Tokyo Medical and Dental University [14–17]. The applied doses of colchicine arrested metaphase during the first 4 hours postadministration and were noxious for hemopoietic tissue (necrosis of bone marrow was evident in 2 and 4 day specimens), but on longitudinal, serial sections of long bones there was no evidence of stimulation of osteogenesis at any point in time (2–26-day specimens). It is postulated that the system of ectopic osteogenesis by colchicine injection is not reproducible in mice and WAG rats, and the apparently osteogenic effect of colchicine, observed by the Ogura group [14–17], was mistakenly described as congenital osteopetrosis. Received: 10 May 1996 / Accepted: 31 December 1996     

Basic Fibroblast Growth Factor in the Presence of Dexamethasone Stimulates Colony Formation, Expansion, and Osteoblastic Differentiation by Rat Bone Marrow Stromal Cells

Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, β-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect. Received: 17 January 1997 / Accepted: 30 July 1998     

Expression of BMP-2 by Rat Bone Marrow Stromal Cells in Culture

To investigate the role of bone morphogenetic protein (BMP-2) in ossifying rat bone marrow stromal cell cultures, we determined the population of fibroblast-like stromal cells that expressed BMP-2 immunocytochemically (anti-rhBMP-2 monoclonal antibody), and compared that to alkaline phosphatase (AP) and collagen synthesis formed in culture over a 4-week period in control and dexamethasone-supplemented mineralizing media. In control media, the percentage of BMP-2-positive stromal cells (BMP-2⁺) increased from 12 to 25% within the first 4 days of culture. In mineralizing media, the level of BMP-2⁺ cells was significantly increased (43–44%). The intensity of immunostaining gradually increased with time. The levels of AP were undetectable at 1 week in both control and mineralizing media, but increased gradually over the next 2 weeks and peaked at 3 weeks. ALP levels were significantly greater in cultures grown in mineralizing medium (P < 0.05 at 3 weeks, P < 0.01 at 4 weeks). Collagen synthesis peaked and was significantly greater at 3 weeks (P < 0.05) in cultures grown in mineralizing medium. The levels of AP and collagen synthesis most closely reflected the changes in the percentage of BMP-2⁺ cells from 7 to 28 days. Though these changes may reflect a primary action of BMP-2 on marrow osteoprogenitor-like stromal cells, they do not exclude a mechanism that involves the induction of other members of the BMP family known to stimulate AP and collagen synthesis. We conclude that BMP-2 expression in cultures of fibroblast-like marrow stromal cells is enhanced when those cells are induced to become osteoblasts by exposure to dexamethasone. Received: 30 October 1997 / Accepted: 24 June 1998     

Different Behavior of Human Osteoblast-Like Cells Isolated from Normal and Heterotopic Bone In Vitro

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM β-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3–6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells. Received: 29 March 1996 / Accepted: 21 May 1997     

Reduction of Bone Mass in Women after Bone Marrow Transplantation

Osteoporosis is a common disease among patients undergoing transplantation. Its prevalence and complications have been well described in solid organ recipients, especially kidney, liver, and heart. However, studies in bone marrow transplantation (BMT) are scarce. Among the mechanisms invoked in the pathogenesis of BMT osteoporosis are the baseline disease, the use of immunosuppressive drugs and, more remarkably, secondary hypogonadism. We present a study of 27 women who underwent BMT, all of them suffering ovarian failure. We studied different biochemical markers of bone formation/resorption and also evaluated the presence of osteopenia/osteoporosis by dual energy X-ray absorptiometry (DXA) of the lumbar spine. Osteopenia was observed in nine patients (33%) and osteoporosis in another five (18%), according to the World Health Organization criteria. We also detected a subgroup showing elevation of several bone turnover biochemical markers, indicating high osseous remodeling. A remarkable increase in urine hydroxyproline/creatinine was detected in 95% of cases, although an explanation is lacking. We outline a reasonable therapeutic approach for osteoporosis in BMT emphasizing the need to monitor these patients after transplantation. Received: 11 January 1996 / Accepted: 25 October 1996     

A Human Homolog of the 150 kD Avian Osteoclast Membrane Antigen Related to Superoxide Dismutase and Essential for Bone Resorption is Induced by Developmental Agents and Opposed by Estrogen in FLG 29.1 Cells

Osteoclast development from hematopoietic bone marrow precursors is associated with the expression of various enzymes, receptors, adhesion molecules, and other specialized components. Among these is a novel 150 kD superoxide dismutase-related membrane glycoprotein, originally identified by its reaction with the anti-osteoclast monoclonal antibody 121F. This antigen is uniquely restricted to osteoclasts in bone, universally present on osteoclasts from multiple species, induced during osteoclast differentiation in vitro and in ovo, and required at high levels for avian osteoclastic bone pit resorption. Expression of a comparable human antigen was investigated using human leukemic FLG 29.1 cells capable of differentiating towards an osteoclast-like phenotype. Phorbol ester, 1,25 (OH)₂ vitamin D₃, and osteoblast-derived soluble factors elicited dose and time-dependent inductions of this antigen as measured by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical staining, coincident with their display of multiple other osteoclastic features. Synergistic interactions of these modulators led to further elevations in the ultimate expression levels of this antigen, although not to the full extent associated with in vivo-formed avian osteoclasts. The potent antiresorptive hormone 17β-estradiol, but not its inactive α isomer, partially suppressed the phorbol ester-induced elevation of the 121F antibody-reactive antigen in FLG 29.1 cells as it does in avian osteoclast-like cells. Characterization of the human antigen isolated from FLG 29.1 cells by 121F immunoaffinity purification demonstrated that this regulated membrane component was synthesized by these human cells, more abundant following their differentiation into osteoclast-like cells, and similar biochemically and immunologically to the 150 kD integral membrane glycoprotein previously described from avian osteoclasts. Therefore, this report is the first documentation that human osteoclast-like FLG 29.1 cells express, in a developmentally regulated fashion, a homolog of the specific 150 kD avian osteoclast surface antigen that is related to superoxide dismutase, a protective free radical scavenging enzyme and is essential for osteoclastic bone resorption. Received: 1 April 1996 / Accepted: 19 July 1996     

Bone Sialoprotein (BSP) is a Crucial Factor for the Expression of Osteoblastic Phenotypes of Bone Marrow Cells Cultured on Type I Collagen Matrix

In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were harvested from rat femur and cultured on type I collagen matrix gels in medium containing 15% fetal calf serum, neither β-glycerophosphate nor glucocorticoid. Cells showed osteoblastic phenotypes (high alkaline phosphatase activity, osteocalcin synthesis, and responsiveness against parathyroid hormone) on collagen matrix gels at week 3 after the inoculation, and simultaneously, BSP was detected in the conditioned medium by Western blotting using an anti-BSP monoclonal antibody. However, cells in the conventional culture dishes did not show osteoblastic phenotypes during the experimental period. To investigate the physiological function of BSP in osteoblastic differentiation, bone marrow cells were cultured on collagen matrix with an anti-BSP monoclonal antibody for 3 weeks. This treatment suppressed the expression of the osteoblastic phenotypes, and the effect of the antibody was abolished by the addition of bovine bone BSP. Furthermore, bovine bone BSP stimulated the expression of osteoblastic phenotypes of bone marrow cells. Our results indicate that BSP plays a crucial role in the expression of osteoblastic phenotypes of bone marrow cells. Received: 17 February 1999 / Accepted: 14 December 1999     

A Novel Human Bone Marrow Stroma-Derived Cell Line TF274 Is Highly Osteogenic In Vitro and In Vivo

A novel, immortalized, human bone marrow stroma-derived cell line TF274 is described which has the ability to form bone both in vitro and in vivo. Under basal conditions these cells expressed alkaline phosphatase (ALP) and type I collagen genes which are characteristic of the osteoblast phenotype. ALP levels were upregulated in the presence of osteotropic agents such as parathyroid hormone (PTH), transforming growth factor beta (TGF-β), and BMP-2. In addition, PTH also increased cAMP levels in these cells. The capacity of these cells to form bone in vitro was evaluated by culturing them in the presence of L-ascorbic acid and β-glycerophosphate. Matrix mineralization in these cultures was assessed by Alizarin Red staining and increased ⁴⁵Ca uptake. Under these conditions mineralized nodule formation was observed in less than 2 weeks. Northern analysis of TF274 cells at various times during the mineralization process indicated a temporal expression of the osteocalcin gene that is typically associated with differentiating osteoblasts. The osteogenic nature of TF274 cells was confirmed in vivo using the severe combined immunodeficient (SCID) mouse model. Antibodies to human leukocyte antigens (HLA), class I antigens, and human OK^a blood group antigen were used to demonstrate that the lesions formed were of human origin. By 21 days, the lesion consisted of a homogeneous focus of ALP-positive cells containing areas of mineralized bone lined with tartarate-resistant acid phosphatase (TRAP) positive osteoclasts. Thus, the TF274 cells exhibit osteogenic potential both in vitro and in vivo. This immortalized cell line represents a consistent source of cells that can be used to study human osteoblast differentiation both in vitro and in vivo. Received: 30 July 1997 / Accepted: 23 January 1998     

The Role of Osteoblast Density and Endogenous Interleukin-6 Production in Osteoclast Formation From the Hemopoietic Stem Cell Line FDCP-MIX C2GM in Coculture With Primary Osteoblasts

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C₂GM was shown to be strongly dependent on osteoblast density. In cocultures of C₂GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 × 10⁴ cells/cm²), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 × 10⁴ cells/cm²). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear. Received: 26 June 1997 / Accepted: 14 November 1997     

Alendronate Reduces Adhesion of Human Osteoclast-like Cells to Bone and Bone Protein-Coated Surfaces

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40–45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as α_Vβ₃ integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration. Received: 8 August 1997 / Accepted: 27 January 1998     

Effects of Human Tumor Cell Lines on Local New Bone Formation In Vivo

Although some tumors cause osteolytic lesions, there are some that stimulate new bone formation. This is an important phenomenon because the responsible mechanisms probably represent an aberration of normal physiological bone formation, and identifying the factors involved in the process may lead to new therapies for various bone diseases. To clarify our understanding of the potential mechanism responsible, we compared and quantitated the extent of new bone formation stimulated by human tumors (HeLa, Hep-2, AV-3, FL, WISH and KB), some of which have osteogenic activity in vivo [2]. Tumor cells were injected over the calvaria of nude mice to examine formation of new bone. The tumor cells produced three histologically distinct patterns of new bone growth: (1) WISH and KB stimulated appositional bone growth adjacent to periosteal bone surfaces; (2) HeLa and Hep2 induced new bone growth over calvarial surface even when distant from the tumor mass; (3) FL stimulated bone formation adjacent to periosteum as well as ectopic bone formation in sites distant from bone. All tumors except AV3 induced mean new bone thickness >100 μm, and Hep-2 cells produced bone 330 μm thick. PCR and Northern blot analysis of mRNA isolated from cultured tumor cells revealed that all cell lines expressed mRNA for TGFβ, (fibroblast growth factor) FGF-1, FGF-2, and IGF-I, and most cell lines produced mRNA for PDGF. Only FL expressed large amounts of mRNA for BMP2. In serum-free conditioned media from Hep2 and HeLa cells purified by heparin affinity chromatography, we have identified FGF-1, FGF-2, and PDGF by immunodetection with specific antibodies. Our results show that new bone growth caused by these tumors is likely due to the production of bone growth factors by the tumor cells, and that the overall effects on bone may be due to several factors working in concert. Received: 15 January 1996 / Accepted: 3 May 1996     

The Resorption of Vital and Devitalized Bone In Vitro: Significance for Bone Grafts

Several studies have suggested that devitalized bone is less satisfactory than live tissue for surgical grafting purposes because an initial resorption step, prior to new formation, is lacking. We have compared the osteoclastic resorption of cultured bone containing living osteocytes with that of similar bone in which the osteocytes were dead. In experiment I, transverse slices cut from freshly harvested adult rabbit femora were either placed in phosphate buffered saline (Set 1) or subjected to freezing and thawing (Set 2). In experiment II, a heated set (Set 3) was prepared in addition. All slices were cultured with osteoclasts for 24 hours, eight slices per set being seeded with bone cells in experiment I and three per set in experiment II. The areas and volumes of resorption pits formed during the culture period were measured using reflection confocal microscopy. In both experiments, the mean values for the areas of the pits were smaller in the bone containing live osteocytes (P < 0.03, Mann Whitney test), and in experiment II the volumes of the pits in Set 1 were smaller than those in Set 3 (P < 0.0001, Mann Whitney test). However, in neither experiment was there a significant difference between the Sets in the volume:area ratios (mean depths) of the pits. The findings show that devitalized bone is resorbed by osteoclasts at least as readily as bone containing vital osteocytes in vitro, and indicate that if grafted devitalized bone resorbs less well in vivo it is not because the bone tissue is intrinsically resistant to osteoclastic resorption. Received: 25 November 1997 / Accepted: 24 June 1998     

Species Differences in Growth Requirements for Bone Marrow Stromal Fibroblast Colony Formation In Vitro

The marrow stromal fibroblast (MSF) population has been shown to include precursor cells for at least five types of connective tissue: bone, cartilage, adipose tissue, fibrous tissue, and hematopoiesis-supporting reticular stroma. In this study, growth requirements for MSF colony formation were studied in vitro. In order to exclude the influence of nonadherent cells, after a period of initial adhesion of bone marrow cells in serum-containing medium nonadherent cells were removed. Further cultivation was carried out in either serum-containing or serum-free conditions, with or without feeder cells (irradiated bone marrow cells). This approach revealed differences between animal species in initial MSF growth requirements. In serum-containing conditions, mouse MSF precursor cells (colony-forming units-fibroblast, CFU-Fs) were shown to be feeder cell dependent: MSF colonies were formed only in the presence of feeder cells. Guinea pig CFU-Fs were partially feeder cell dependent, whereas human CFU-Fs were feeder cell independent. In serum-free conditions, CFU-Fs of all three species were feeder cell dependent. The difference between the growth requirements for mouse and human MSFs was not caused by serum origin or concentration, feeder cell origin, or differences in the preparation of marrow cell suspensions. Received: 9 November 1995 / Accepted: 8 March 1996     

Expression of Bone Sialoprotein in Human Lung Cancer

Lung cancer belongs to the group of malignant lesions that specifically select bone as secondary implantation site. The molecular bases for this property, defined as osteotropism, is still largely unknown. The recent demonstration that human breast cancer cells express and attach to bone sialoprotein (BSP), a sulfated phosphoprotein rich in bone and other mineralized tissues, could provide a clue to elucidating bone metastases formation. BSP contains the integrin binding peptide Arg-Gly-Asp (RGD), as well as non-RGD cell attachment domain. Using an immunoperoxidase technique and a specific polyclonal antibody directed against a BSP synthetic peptide, we examined the expression of BSP in 48 lung lesions including 25 squamous carcinoma, 21 adenocarcinoma, and 2 bronchioloalveolar cancers, as well as 38 human ovarian carcinoma that constitute a group of generally nonosteotropic cancers. BSP was not specifically detected in normal lung tissue with the exception of cartilage associated with bronchi. Most of the adenocarcinoma (74%) and all squamous carcinoma of the lung examined exhibited detectable levels of BSP. Staining was mainly cytoplasmic and membrane associated. The two bronchioloalveolar lung cancers examined did not show detectable amounts of BSP. When microcalcifications were observed in pulmonary malignant lesions, they were usually associated with cancer cells expressing BSP. Only 21% of the ovarian cancers examined contained malignant cells with 2+ or 3+ positivity for BSP. We further demonstrated that in 8 of 10 additional lung cancers, BSP was detected at the mRNA level. Our observation is the first demonstration that BSP is expressed in non-small cell lung carcinoma. Lung cancer cells are now the second type of osteotropic malignant cells described to express BSP. Added to the observation that BSP expression is not frequent in ovarian carcinoma, a low osteotropic cancer, our study supports our hypothesis that BSP could play a role in determining the affinity of cancer cells to bone. Received: 30 August 1996 / Accepted: 23 April 1997     

Whole-Cell Membrane Currents from Human Osteoblast-like Cells

Bone cells share common responses to external stimuli with most other cells. Among these are changes in membrane ion channel activity, although at present, relatively little is known about their nature or significance in human bone cells. Using the whole-cell configuration of the patch-clamp technique, we have revealed two types of membrane current in MG63 human osteoblast-like cells. With a potassium-based dialysis solution and a holding potential of −40 mV, voltage commands to more negative potentials elicited an inward current. This current showed little inactivation with time during the command pulse and exhibited some characteristics of an inwardly rectifying K current, including sensitivity to external K and Ba. The second type of current was outward, activated by depolarizing pulses from −40 mV. This current was transient in nature, activating in the first 50 ms of the pulse and then showing rapid inactivation to reach a steady-state level after 4 to 5 seconds. The transient outward current was sensitive to block by TEA, CTX, and to a lesser extent, Ba. These data suggest that a large proportion of this outward current is carried by K ions through channels that may be sensitive to the internal Ca ion concentration. The transient outward current was enhanced by setting the holding potential at −100 mV, and greatly inactivated by setting it at 0 mV. Increased understanding of the significance of these membrane currents may allow development and use of agents to modulate their action and therefore influence bone cell behavior in disease states such as osteoporosis. Received: 13 March 1997 / Accepted: 7 August 1997     

Effects of Pregnancy and Lactation on Trabecular Bone and Marrow Adipocytes in Rats

Changes in the structure and metabolism of trabecular bone and marrow adipocytes in rats during pregnancy and the early stage of postpartum were evaluated by investigating bone mineral density (BMD) and bone and fat histomorphometry. Forty-nine female virgin Sprague-Dawley rats aged 200 days were mated and divided into seven groups: (1) beginning controls; (2) antepartum-on-day-7; (3) antepartum-on-day-21; (4) nonlactating on the fourth postpartum day; (5) nonlactating on the sixth postpartum day; (6) nonlactating on the eighth postpartum day; (7) lactating on the eight postpartum day. The significant decreases occurred in the trabecular bone at the end of pregnancy and lactation, and the bone formation increases and the bone structure is almost recovered in nonlactating rats within 6 days of postpartum. The percent adipocyte volume, adipocyte number, and unit adipocyte volume significantly decreased during postpartum whether lactating or nonlactating, and they significantly showed negative correlation with the osteoid volume values. The serum triglyceride value and body weight of the seven groups correlated significantly with the unit adipocyte volume value (r = 0.49, P= 0.004; r = 0.58, P= 0.0005, respectively). We concluded that bone resorption and formation are regulated separately during late pregnancy and lactation and that the recovery of BMD from lactation appears to rely on an acceleration of bone formation. Furthermore, the metabolism of the marrow adipocyte may be correlated with bone formation rates, serum triglyceride value, and body weight during pregnancy and early stage of puerperium. Received: 5 October 1999 / Accepted: 27 April 2000 / Online publication: 27 July 2000     

Effects of Indomethacin Administration on Bone Turnover and Bone Mass in Adjuvant-Induced Arthritis in Rats

We examined the bone turnover and bone mass in adjuvant-induced arthritis in rats and assessed the effects of indomethacin in this model. One hundred ten SD rats, 6 weeks of age, were assigned to 11 groups and injected with adjuvant or solvent in the right foot. Adjuvant-injected rats were orally administered indomethacin at doses of 0 (vehicle), 0.1 (low), 0.5 (medium), and 1.5 (high) mg/kg body weight from the start (day 0). Animals were sacrificed on days 0, 14 (acute phase), and 28 (chronic phase). In the arthritic-control group, serum osteocalcin level and bone mineral content of the fourth lumbar body (L4) and the femur were significantly reduced on day 14. Serum alkaline-phosphatase was increased on day 28. Trabecular bone volume of L4 was decreased on day 14, and the value was further decreased on day 28. Bone formation rate (BFR/BS) was significantly reduced on day 14, and then osteoclast number (Oc.N/BS) increased on day 28. Indomethacin treatment dose-dependently prevented increases in paw volume and osteoclast number. In the high dose group, these indices were maintained at the same level with those in the normal group. However, indomethacin treatments were not able to maintain the parameters of bone formation such as serum osteocalcin and BFR/BS values, and the trabecular bone mass decrease was only partially prevented. These data clearly indicated both reduced bone formation and increased bone resorption as the causes of bone loss in adjuvant-induced arthritis in rats. Increased bone resorption seemed to be due to the increased activity of prostaglandins, but bone formation defect would be related to other factors in this animal model. Received: 13 January 1996 / Accepted: 3 May 1996     

Effects of Bone CS-Proteoglycans, DS-Decorin, and DS-Biglycan on Hydroxyapatite Formation in a Gelatin Gel

The small leucine-rich bone proteoglycans, biglycan and decorin, can be purified by chromatography on hydroxyapatite columns, demonstrating their potential affinities for bone apatite. To determine their effects on in vitro apatite formation and growth, a mixture of the chondroitin-sulfate (CS) bone proteoglycans, or purified fractions of the dermatan sulfate (DS) containing proteoglycans, DS-decorin and DS-biglycan obtained from skin and articular cartilage, respectively, were analyzed in a gelatin gel diffusion system in which apatite formation occurs in the absence of proteins in a 3.5 day period. Low concentrations of the bone CS-proteoglycan mixture and low DS-biglycan concentrations (5–25 μg/ml) increased apatite formation relative to proteoglycan-free controls at 3.5 days. The CS-proteoglycan mixture was less effective at 50 μg/ml than at 10 μg/ml. DS-biglycan was similarly most effective at 5–25 μg/ml. At 5 days, when apatite growth and proliferation were assessed, 10 and 50 μg/ml of both CS-bone proteoglycan and DS-biglycan increased mineral yields. DS-decorin, in contrast, had no significant effect on mineral accumulation at any of these concentrations. In seeded growth experiments, 1 and 10 μg/ml CS-proteoglycan and 10 and 50 μg/ml DS-biglycan were significant effective inhibitors of mineral accretion, whereas DS-decorin showed no tendency to inhibit seeded growth. Using molar extinction coefficients to determine concentrations, the binding of DS-biglycan and DS-decorin to apatite (specific surface 54 m²/g) was determined using a Langmuir adsorption isotherm model. DS-biglycan had a greater affinity for apatite than DS-decorin (0.285 ml/μmol versus 0.0098 ml/μmol). DS-biglycan binding was more specific with fewer binding sites (3.5 μmol/m² compared with 18.2 μmol/m² for DS-decorin). Data suggest that of the small proteoglycans, biglycan may play a more significant role than decorin in the regulation of mineralization. Received: 10 June 1996 / Accepted: 25 April 1997     

Inhibitory and stimulatory effects of prostaglandins on osteoclast differentiation

The effect of prostaglandins (PGs) on osteoclast differentiation, an important point of control for bone resorption, is poorly understood. After an initial differentiation phase that lasts at least 4 days, murine monocytes, cocultured with UMR106 osteoblastic cells (in the presence of 1,25-dihydroxyvitamin D₃) give rise to tartrate-resistant acid phosphatase (TRAP) positive osteoclast-like cells that are capable of lacunar bone resorption. PGE₂ strongly inhibits TRAP expression and bone resorption in these cocultures. To examine further the cellular mechanisms associated with this inhibitory effect, we added PGE₂ to monocyte/UMR106 cocultures at specific times before, during, and after this initial 4-day differentiation period. To determine whether this PGE₂ inhibition was dependent on the type of stromal cell supporting osteoclast differentiation, we also added PGE₂ to cocultures of monocytes with ST2 preadipocytic cells. Inhibition of bone resorption was greatly reduced when the addition of PGE₂ to monocyte/UMR106 cocultures was delayed until the fourth day of incubation; when delayed until the seventh day, inhibition did not occur. PGE₂ inhibition of bone resorption was concentration-dependent and at 10⁻⁶ M was also mediated by PGE₁ and PGF_2α. In contrast to its effects on monocyte/UMR106 cocultures, PGE₂ stimulated bone resorption in monocyte/ST2 cocultures. Both ST2 cells and UMR106 cells were shown to express functional receptors for PGE₂. These results show that PGs strongly influence the differentiation of osteoclast precursors and that this effect is dependent not only on the type and dose of PG administered, but also on the nature of the bone-derived stromal cell supporting this process. Received: 12 October 1995 / Accepted: 1 April 1996