Particle-induced osteolysis is not accompanied by systemic remodeling but is reflected by systemic bone biomarkers.

Particle-induced osteolysis is caused by an imbalance in bone resorption and formation, often leading to loss of implant fixation. Bone remodeling biomarkers may be useful for identification of osteolysis and studying pathogenesis, but interpretation of biomarker data could be confounded if local osteolysis engenders systemic bone remodeling. Our goal was to determine if remote bone remodeling contributes to biomarker levels. Serum concentrations of eight biomarkers and bone remodeling rates at local (femur), contiguous (tibia), and remote (humerus and lumbar vertebra) sites were evaluated in a rat model of particle-induced osteolysis. Serum CTX-1, cathepsin K, PINP, and OPG were elevated and osteocalcin was suppressed in the osteolytic group, but RANKL, TRAP 5b, and sclerostin were not affected at the termination of the study at 12 weeks. The one marker tested longitudinally (CTX-1) was elevated by 3 weeks. We found increased bone resorption and decreased bone formation locally, subtle differences in contiguous sites, but no differences remotely at 12 weeks. Thus, the skeletal response to local particle challenge was not systemic, implying that the observed differences in serum biomarker levels reflect differences in local remodeling.

Pharmacologic augmentation of implant fixation in osteopenic bone.

Osteoporosis presents a challenge for successful implant fixation due to an impaired healing response. Preclinical studies have consistently reported reduced osseointegration capability in trabecular bone. Although clinical studies of implant success in dentistry have not found a negative effect due to osteoporosis, low bone mass is a significant risk factor for implant migration in orthopedics. Pharmacologic treatment options that limit bone resorption or upregulate formation have been studied preclinically. While, both treatment options improve implant fixation, direct comparisons to-date have found anti-catabolic more effective than anabolic treatments for establishing implant fixation, but combination approaches are better than either treatment alone. Clinically, anti-catabolic treatments, particularly bisphosphonates have been shown to increase the longevity of implants, while limited clinical evidence on the effects of anabolic treatment exists. Preclinical experiments are needed to determine the effects of osteoporosis and subsequent treatment on the long-term maintenance of fixation and recovery after bone loss.

Healing of rat femoral segmental defect with bone morphogenetic protein-2: a dose response study.

OBJECTIVE: Use of recombinant human bone morphogenetic protein-2 (rhBMP-2) is becoming a common clinical approach to enhance bone repair. There is little or no information in the literature on the dose of rhBMP-2 required for effective healing of critical-sized defects such as those associated with trauma. In this study, we used a segmental defect model to assess the dose response of rhBMP-2 using quantitative and qualitative endpoints. METHODS: Femoral defects in rats were replaced with absorbable collagen sponges carrying rhBMP-2 (0, 1, 5, 10 or 20 µg; N=5). At 4-weeks new bone formation was assessed using quantitative (radiography and microcomputed tomography) and qualitative (histology and backscattered-SEM) endpoints statistically compared. RESULTS: rhBMP-2 showed increased bridging in the gap. Quantitative evaluation presented a bi-phasic dose response curve. Histological assessment revealed that with rhBMP-2 the defect showed the presence of spongy bone with the trabeculae layered with active osteoblasts and osteoclasts. The density and compactness of the bone varied with the dose of rhBMP-2. CONCLUSIONS: Our findings revealed that all doses of rhBMP-2 result in new bone formation. However, there is an optimum dose of 12 µg of rhBMP-2 for bone repair in this model, above which and below which less stimulation of bone occurs.

Limitations of using micro-computed tomography to predict bone-implant contact and mechanical fixation.

Fixation of metallic implants to bone through osseointegration is important in orthopaedics and dentistry. Model systems for studying this phenomenon would benefit from a non-destructive imaging modality so that mechanical and morphological endpoints can more readily be examined in the same specimens. The purpose of this study was to assess the utility of an automated microcomputed tomography (µCT) program for predicting bone-implant contact (BIC) and mechanical fixation strength in a rat model. Femurs in which 1.5-mm-diameter titanium implants had been in place for 4 weeks were either embedded in polymethylmethacrylate (PMMA) for preparation of 1-mm-thick cross-sectional slabs (16 femurs: 32 slabs) or were used for mechanical implant pull-out testing (n= 18 femurs). All samples were scanned by µCT at 70 kVp with 16 µm voxels and assessed by the manufacturer's software for assessing 'osseointegration volume per total volume' (OV/TV). OV/TV measures bone volume per total volume (BV/TV) in a 3-voxel-thick ring that by default excludes the 3 voxels immediately adjacent to the implant to avoid metal-induced artefacts. The plastic-embedded samples were also analysed by backscatter scanning electron microscopy (bSEM) to provide a direct comparison of OV/TV with a well-accepted technique for BIC. In µCT images in which the implant was directly embedded within PMMA, there was a zone of elevated attenuation (>50% of the attenuation value used to segment bone from marrow) which extended 48 µm away from the implant surface. Comparison of the bSEM and µCT images showed high correlations for BV/TV measurements in areas not affected by metal-induced artefacts. In addition for bSEM images, we found that there were high correlations between peri-implant BV/TV within 12 µm of the implant surface and BIC (correlation coefficients ≥0.8, p < 0.05). OV/TV as measured on µCT images was not significantly correlated with BIC as measured on the corresponding bSEM images. However, OV/TV was significantly, but weakly, correlated with implant pull-out strength (r= 0.401, p= 0.049) and energy to failure (r= 0.435, p= 0.035). Thus, the need for the 48-µm-thick exclusion zone in the OV/TV program to avoid metal-induced artefacts with the scanner used in this study means that it is not possible to make bone measurements sufficiently close to the implant surface to obtain an accurate assessment of BIC. Current generation laboratory-based µCT scanners typically have voxel sizes of 6-8 µm or larger which will still not overcome this limitation. Thus, peri-implant bone measurements at these resolutions should only be used as a guide to predict implant fixation and should not be over-interpreted as a measurement of BIC. Newer generation laboratory-based µCT scanners have several improvements including better spatial resolution and X-ray sources and appear to have less severe metal-induced artefacts, but will need appropriate validation as they become available to researchers. Regardless of the µCT scanner being used, we recommend that detailed validation studies be performed for any study using metal implants because variation in the composition and geometry of the particular implants used may lead to different artefact patterns.

Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound.

Bone growth and repair are under the control of biochemical and mechanical signals. Low-intensity pulsed ultrasound (LIPUS) stimulation at 30mW/cm(2) is an established, widely used and FDA approved intervention for accelerating bone healing in fractures and non-unions. Although this LIPUS signal accelerates mineralization and bone regeneration, the actual intensity experienced by the cells at the target site might be lower, due to the possible attenuation caused by the overlying soft tissue. The aim of this study was to investigate whether LIPUS intensities below 30mW/cm(2) are able to provoke phenotypic responses in bone cells. Rat bone marrow stromal cells were cultured under defined conditions and the effect of 2, 15, 30mW/cm(2) and sham treatments were studied at early (cell activation), middle (differentiation into osteogenic cells) and late (biological mineralization) stages of osteogenic differentiation. We observed that not only 30mW/cm(2) but also 2 and 15mW/cm(2), modulated ERK1/2 and p38 intracellular signaling pathways as compared to the sham treatment. After 5 days with daily treatments of 2, 15 and 30mW/cm(2), alkaline phosphatase activity, an early indicator of osteoblast differentiation, increased by 79%, 147% and 209%, respectively, compared to sham, indicating that various intensities of LIPUS were able to initiate osteogenic differentiation. While all LIPUS treatments showed higher mineralization, interestingly, the highest increase of 225% was observed in cells treated with 2mW/cm(2). As the intensity increased to 15 and 30mW/cm(2), the increase in the level of mineralization dropped to 120% and 82%. Our data show that LIPUS intensities lower than the current clinical standard have a positive effect on osteogenic differentiation of rat bone marrow stromal cells. Although Exogen™ at 30mW/cm(2) continues to be effective and should be used as a clinical therapy for fracture healing, if confirmed in vivo, the increased mineralization at lower intensities might be the first step towards redefining the most effective LIPUS intensity for clinical use.

Additive enhancement of implant fixation following combined treatment with rhTGF-beta2 and rhBMP-2 in a canine model.

BACKGROUND: Gaps at the interface between implant and bone increase the risk of diminished implant fixation and eventual loosening. The purpose of the present study was to determine if combined use of recombinant human transforming growth factor-beta 2 (rhTGF-beta2) and bone morphogenetic protein 2 (rhBMP-2) led to greater implant fixation strength in the presence of interface gaps than the use of either growth factor alone. METHODS: Twenty-eight skeletally mature adult male dogs received one porous-coated titanium implant in the proximal part of each humerus, for a total of fifty-six implantation sites. Spacers were used to establish an initial 3-mm gap between the implant and the host bone at all fifty-six sites. Forty-two implants were coated with hydroxyapatite-tricalcium phosphate and were used in three growth-factor-treatment groups in which the implants placed in the left humerus were loaded with 12 microg of rhTGF-beta2 (Group 1, seven animals), 25 microg of rhBMP-2 (Group 2, seven animals), or 12 microg of rhTGF-beta2 combined with 25 microg of rhBMP-2 (Group 3, seven animals). In these animals, the twenty-one implants that were placed in the right humerus were loaded with buffer only to serve as contralateral controls. In Group 4 (seven animals), the implants were not coated with hydroxyapatite-tricalcium phosphate, the gap in the left humerus was lightly packed with autogenous bone graft, and the gap in the right humerus was left empty to serve as a contralateral control. All animals were killed at twenty-eight days. The primary end points included three mechanical variables: fixation strength, interface stiffness, and energy to failure. Secondary end points included bone ingrowth and bone volume and trabecular architecture in the gap and in a region located 2 mm medial to the implantation site. RESULTS: The hydroxyapatite-tricalcium phosphate coating had no effect on implant fixation, bone ingrowth, or bone formation in the 3-mm gap. Individual growth factor treatments led to 2.3 to 3.2-fold increases in fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.05). The combined growth factor treatment led to 5.7-fold increases in fixation strength and stiffness compared with the values for the contralateral controls (p < 0.01). Autogenous bone graft treatment was associated with 4.5 to 6.4-fold increases in implant fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.01). Compared with the relevant contralateral controls, energy to failure was increased 3.5-fold in association with TGF-beta2 alone (p < 0.05), 4.5-fold in association with TGF-beta2 combined with BMP-2 (p < 0.01), and 2.5-fold in association with autogenous bone-grafting. As much as 63% of the variance in the mechanical end points was associated with variance in bone volume and architecture in the 3-mm gap and in the region of interest located 2 mm medial to the implantation site (p < 0.01). CONCLUSIONS: In this animal model, the combined use of TGF-beta2 and BMP-2 led to more secure mechanical fixation of the implant than did the use of either growth factor alone and demonstrated results that were similar to those associated with the use of autogenous bone graft.

Saline irrigation does not affect bone formation or fixation strength of hydroxyapatite/tricalcium phosphate-coated implants in a rat model.

Intramembranous bone regeneration is critical to implant fixation. In cementless joint replacement (as opposed to cemented joint replacement), saline irrigation is not typically performed during surgery so that the osteogenic stimulus provided by the marrow is preserved. Several groups are now using the rat marrow ablation model to study intramembranous bone regeneration and implant fixation. In this model, the marrow contents are mechanically disrupted, and debris is often cleared by saline irrigation, a step that appears inconsistent with the clinical situation. Furthermore, in contrast to conventional wisdom, it has been reported that saline irrigation enhanced bone-implant contact and peri-implant bone formation in the rat model (Ishizaka et al. Bone 1996;19:589-594), although mechanical fixation of the implant was not investigated. Accordingly, the present study was performed to determine if saline irrigation leads to enhanced mechanical fixation of implants in the rat model. Forty-eight 400 to 450 g male rats were divided equally into two groups. The treatment group, in contrast to the control group, received saline irrigation in the ablated medullary canal prior to placement of hydroxyapatite/tricalcium phosphate-coated implants. Eight animals in each group were killed at 2, 4, or 8 weeks after implantation, at which time the specimens were analyzed by micro computed tomography to measure bone formation around the implant, followed by a mechanical pull-out test to measure the strength of fixation of the implant. As expected, there was increased fixation strength over time, but there were no significant differences in peri-implant bone volume, bone-implant contact, or implant fixation strength between the two groups. Thus, we found no effect of saline irrigation on bone formation or implant fixation strength in this study in which the implant had an osteoconductive coating.

A low-temperature biomimetic calcium phosphate surface enhances early implant fixation in a rat model.

The present study demonstrates increased early mechanical fixation of titanium implants coated with a new biomimetic apatite surface in a rat model. Male Sprague-Dawley rats received unilateral femoral medullary implants for periods of 1-4 weeks. The strength of fixation of the implant to the host bone increased more rapidly in the group receiving apatite-treated implants compared with the control group as evidenced by the apatite group's 21-fold greater fixation strength at 1 week (p = 0.009), 4-fold greater fixation strength at 2 weeks (p = 0.041), and 2-fold greater fixation strength at 4 weeks (p = 0.093) compared with the control. Fixation strength was correlated with bone-implant contact as determined from micro computed tomography assessment of the specimens (r2 = 0.338, p = 0.011 in the control group and r2 = 0.543, p < 0.001 in the apatite group). Furthermore, for a given amount of bone-implant contact, the fixation strength was higher in the apatite group than in the control group (p = 0.011), suggesting that the bone formed a stronger bond to the apatite coating than to the titanium. This difference in bonding strength accounted for the difference in mechanical behavior.

Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study.

The presence of osteogenic progenitors in human skeletal muscle is suggested by the formation of ectopic bone in clinical and experimental conditions, but their direct identification has not yet been demonstrated. The aims of this study were to identify osteogenic progenitor cells in human skeletal muscle tissue and to expand and characterize them in culture. Specimens of gracilis and semitendinosus muscle were obtained from young adults and digested to separate the connective tissue and satellite cell fractions. The cells were cultured and characterized morphologically and immunohistochemically using antibodies known to be reactive with primitive osteoprogenitor cells, pericytes, intermediate filaments, and endothelial cells. Alkaline phosphatase activity and osteocalcin gene expression were also determined. In the early stages of culture, the connective tissue cells obtained were highly positive for primitive osteoprogenitor cell and for pericyte markers. Alkaline phosphatase activity was detectable at early stages of culture and rose as a function of time, whereas primitive osteoprogenitor cell markers declined and osteocalcin mRNA expression became detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). It is shown that human skeletal muscle connective tissue contains osteogenic progenitor cells. Their identification as pericytes, perivascular cells with established osteogenic potential, suggests a cellular link between angiogenesis and bone formation in muscle tissue. These cells are easily cultured and expanded in vitro by standard techniques, providing an alternative source of osteogenic progenitor cells for possible cell-based therapeutic use in certain conditions.

Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo.

Amplification of multipotential stem cells, with or without ex vivo gene transfer, offers the potential for their use for beneficial repopulation of a host in which there is specific cellular deficiency or functional impairment. The aims of the current study were to immunoselect, genetically mark, and determine the fate of fibroblastic progenitor cells in vivo. A monoclonal antibody, HOP-26, which has high reactivity with a cell surface antigen present on human osteoprogenitors in bone marrow fibroblast populations, was used to select these cells by immunopanning. Following culture in 10% FCS in alphaMEM containing ascorbate-2-phosphate and dexamethasone the amplified cells expressed the osteoblast phenotype as determined by expression of osteocalcin protein determined immunohistochemically, and Type I collagen and osteocalcin mRNA expressions determined by RT-PCR analysis. The selected cells were genetically labeled using a murine leukemia virus (MuLV) encoding a reporter gene (lacZ) with a selective marker gene (neo(r)) using a triple transient transfection protocol. Transfected cells were implanted in CB17 scid/scid mice by local subcutaneous injection over the calvariae. Localization of the genetically marked cells within the calvarial tissues was detected by beta-galactosidase histochemistry and immunocytochemistry. Genetically marked cells were observed within the periosteal layer in close association with the osteoblast layer, covering mineralized bone surfaces and within bone osteoid at 5 and 7 days after injection. This study demonstrates the successful selection, expansion, and retroviral-marking of human osteoprogenitors and their migration and localization within calvariae of SCID mice following in vivo implantation. These basic studies indicate the migration of these cells to skeletal sites and support possibilities for future uses of human osteoprogenitors in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these conditions.

Osteogenic stem-cell characterization and development: potentials for cytotherapy.

BACKGROUND: Primitive progenitors of bone tissue exist postnatally and exhibit stem-cell characteristics, as shown by extensive renewal potential, and capacity to differentiate into all characteristic connective tissue types, including bone, cartilage, fat, fibrous tissue, muscle and hemopoietic stroma. METHODS: A wide variety of investigative techniques have been applied to characterize and assess differentiation of the normally non-cycling osteogenic stem cells. These include methods to assess in vitro and in vivo differentiation potentials, the production and use of Abs to identify surface markers, the expression of specific genes and, more recently, incorporation of marker genes (beta-galactosidase, green fluorescent protein) to study cell fate after implantation at tissue sites. RESULTS: Some antigenic cell-surface molecules reactive with MAbs generated by a number of laboratories have been identified. For cell-fate studies, retroviral insertion of beta-galactosidase or green fluorescent protein genes into human marrow stromal progenitors has been accomplished with high efficiency. The stromal cell phenotype and cellular functions in vitro are not significantly altered by these genetic modifications. In vivo transplantation in immunodeficient animals demonstrates migration and persistence of marrow stromal cells to skeletal and other tissue sites. DISCUSSION: None of the Abs generated against surface markers of early progenitors are absolutely lineage and cell-stage specific, but the respective Ags appear to participate in cell adhesion and cell-signalling mechanisms. These may be important in stem-cell activation and subsequent early osteogenic development. Studies of cell fate indicate feasibility for future uses in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these and other conditions.

Tissues formed during distraction osteogenesis in the rabbit are determined by the distraction rate: localization of the cells that express the mRNAs and the distribution of types I and II collagens.

An experimental model of leg lengthening was used to study the morphology of, the collagenous proteins present, and the collagen genes expressed in the regenerating tissue following 20% lengthening at four different distraction rates. At a distraction rate of 0.3 mm/day (8 weeks distraction), the regenerate consists of intramembranous bone and localized areas of fibrocartilage. At rates of 0.7 (4 weeks) and 1.3 mm/day (2 weeks), the bone that grows from the cut ends of the cortical bone is separated by fibrous tissue and cartilage is present. At 2.7 mm/day (1 week), only fibrous tissue and sparse bone are present. Type I collagen is present in the matrices around the cells expressing its mRNA and similarly, type II collagen is located around the chondrocytes. Type I collagen mRNA is expressed predominantly by the fibroblasts in the fibrous tissue, the bone surface cells and to a reduced extent by the osteocytes. Type II collagen mRNA is expressed by chondrocytes. The results suggest that osteoblasts and chondrocytes within the regenerate originate from the same pool of progenitor cells, and the differentiation of these cells and the expression of types I and II collagen genes are altered by different rates of distraction. These observations suggest that the optimal rate of distraction in the model is 0.7 mm/day.

Phenotypic and molecular heterogeneity in fibrodysplasia ossificans progressiva.

Fibrodysplasia (myositis) ossificans progressiva (FOP) is an extremely rare inherited disorder in which progressive ossification of major striated muscles, often following injury, is associated with abnormal skeletal patterning. Altered expression of bone morphogenetic proteins may be a contributory cause. To examine this hypothesis, we compared the patterns of expression of bone morphogenetic proteins (BMPs) mRNAs from lymphoblastoid cell lines from two small multigenerational families with autosomal dominant transmission of FOP. Although affected members of both families showed the characteristic phenotype of FOP, one family was more severely affected than the other. Expression of mRNAs for BMP-1, 2, 3, 5, and 6 mRNAs were not detected within the more severely affected family, but BMP-4 mRNA was expressed in affected but not unaffected members of this family. The results of linkage exclusion analysis using a highly polymorphic microsatellite marker near the BMP-4 gene were consistent with linkage of FOP and BMP-4 in this family. Within the less severely affected family, affected and unaffected members showed similar levels of mRNA expression of BMPs 1, 2, 4, and 5, and linkage of FOP to the BMP-4 gene was excluded. It is concluded that clinical, radiographic, and biochemical data in these two families with FOP establish clinical and molecular heterogeneity and also suggest the possibility of genetic heterogeneity.

Effects of interferon alpha on human osteoprogenitor cell growth and differentiation in vitro.

The specific effects of interferon alpha (IFNalpha), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNalpha on osteogenic development by investigating cell differentiation, colony formation (colony forming unit-fibroblastic, CFU-F), cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow osteoprogenitor cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNalpha (5-1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 microM), which are agents known to affect osteogenic differentiation. IFNalpha produced a significant dose-dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNalpha (50-1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU-F showed dose- and time-dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase-positive CFU-F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP-26), was reduced in IFNalpha-treated cultures. Northern blot analysis showed a significant dose-dependent up-regulation of BMP-2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNalpha significantly inhibited BMP-2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNalpha inhibits human osteoprogenitor cell proliferation, CFU- F formation, HOP-26 expression, and alkaline phosphatase specific activity and modulates BMP-2 gene expression. These results suggest a role for IFNalpha in local bone turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation.

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage.

Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERalpha), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERalpha mRNA, together with immunocytochemical analysis using a human ERalpha-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERalpha in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERalpha expression was detected in human bone marrow cultures treated with 1,25(OH)2D3 and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)2D3. However, in an in vitro model of human osteoclast formation, no ERalpha expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERalpha in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERalpha, but osteoclast maturation and bone resorption is associated with loss of ERalpha expression. This indicates that ERalpha expression and regulation may play a role in osteoclast formation.

Localization of estrogen receptor-alpha in human and rabbit skeletal tissues.

Estrogen is essential for the development and maintenance of optimal bone mass in women and men, and acts through activation of estrogen receptors (ER). We have examined the pathways of estrogen action on the skeleton by seeking to localize the "classical" estrogen receptor, ER alpha, to particular cells to test the hypotheses that 1) estrogen directly influences growth plate chondrocytes; and 2) estrogen has a principal action on bone tissue via osteoblasts. ER alpha messenger ribonucleic acid (mRNA) was localized by in situ hybridization in human specimens from five males (11-15 yr old), two females (9 and 11 yr old), and three growing rabbits. In all of the human material examined, ER alpha mRNA was consistently identified in chondrocytes. In all of the rabbit tissue studied, ER alpha mRNA was localized in chondrocytes of the growth plate and the subarticular epiphyseal growth center. ER alpha mRNA signals were readily observed in both active osteoblasts and lining cells on trabecular surfaces of all samples. No clear evidence of positive staining was detectable in osteoclasts or osteocytes in either species. The distribution of ER alpha mRNA coincided with immunolocalization of the ER protein in the human specimens. These data suggest a direct action of estrogen on growth plate chondrocytes that may affect longitudinal growth and subsequent fusion of the growth plate and also on osteoblasts to affect bone formation at trabecular sites.

Modulation of bone morphogenetic protein-2 and bone morphogenetic protein-4 gene expression in osteoblastic cell lines.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily and are crucial factors in the process of bone formation. Despite knowledge on their wide distribution and expression, however, there is very little information on the biological factors that affect gene transcription of these osteoinductive agents. To investigate this aspect of BMP gene regulation we have studied the effect of a number of factors known to affect osteogenic cells. Northern analysis showed modulation of the expression of BMP-2 and BMP-4 mRNAs in two human osteosarcoma cell lines, MG63 and Saos-2, by prostaglandin E2 (PGE2), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interferon-alpha (IFN-alpha), retinoic acid and 1,25(OH)2 vitamin D3. mRNA expressions of the normally used "housekeeping genes", glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin, were found to be susceptible to influence by some of the factors used. Hence, an oligo(dT)15-18 probe was used to reliably estimate the relative quantities of mRNA present for normalization of data. In general, all factors down-regulated mRNA expressions of BMP-2 and BMP-4 in MG63 cells. IL-6 completely abolished detectable expression of BMP-2 mRNA, which was also greatly reduced by IL-1beta, retinoic acid and 1,25(OH)2 vitamin D3. PGE2 had similar influences on BMP-2 and BMP-4 expressions, showing reductions to approximately 60% of normal. In Saos-2 cells only 1,25(OH)2 vitamin D3 had any great effect on BMP-2 expression, which was down-regulated to approximately 60% of control values. BMP-4 was down-regulated by IFN-alpha (approximately 60%) and IL-1beta (approximately 20%). We conclude that BMPs are subject to regulation by a variety of factors and that this is dependent on the stage of the cell in the osteogenic lineage. Furthermore, the use of GAPDH and beta-actin genes as "housekeeping genes" in expression-modulation studies must be treated with care.