Usefulness of osteoprotegerin in assessing responses to neridronate treatment in Paget's disease of bone

Osteoprotegerin (OPG) is a protein that inhibits of osteoclastogenesis. The aim this study was to evaluate the response of serum OPG levels to neridronate treatment in patients with Paget's disease of bone resistant to previous therapy. Nine patients (4 men) affected by active Paget’s disease of bone (6 polyostotic, 3 monostotic) not responsive to clodronate were studied. Serum OPG, osteocalcin, total and bone isoenzyme of alkaline phosphatase (AP and BAP, respectively), and urinary deoxypyridinoline (DPD) were measured before and 5 months after neridronate treatment (100 mg/day, i.v. for two days). A scintigraphic activity index (SAI) was also calculated before treatment. Mean baseline OPG levels were within normal values and were not significantly different 5 months after neridronate treatment. In contrast, there were significant reductions in AP (41.9%, p<0.02) and BAP (38.8%, p<0.04). Serum OPG levels correlated with DPD (r=0.925) and SAI (r=0.689). Although OPG is an important regulator of bone metabolism, in our series of already treated patients it was not a sensitive marker for diagnosing Paget's disease and for monitoring the response to pharmacological treatment, whereas AP and BAP confirmed their clinical usefulness. This preliminary study requires confirmation by a study with a larger population.     

Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D3.

In this study we assessed whether osteogenic cells respond in a differential manner to changes in surface roughness depending on their maturation state. Previous studies using MG63 osteoblast-like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhibited and differentiation (osteocalcin production) was stimulated by culture on titanium (Ti) surfaces of increasing roughness. This effect was further enhanced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we examined the response of three additional cell lines at three different maturation states: fetal rat calvarial (FRC) cells (a mixture of multipotent mesenchymal cells, osteoprogenitor cells, and early committed osteoblasts), OCT-1 cells (well-differentiated secretory osteoblast-like cells isolated from calvaria), and MLO-Y4 cells (osteocyte-like cells). Both OCT-1 and MLO-Y4 cells were derived from transgenic mice transformed with the SV40 large T-antigen driven by the osteocalcin promoter. Cells were cultured on Ti disks with three different average surface roughnesses (Ra): PT, 0.5 microm; SLA, 4.1 microm; and TPS, 4.9 microm. When cultures reached confluence on plastic, vehicle or 10(-7) M or 10(-8) M 1,25(OH)2D3 was added for 24 h to all of the cultures. At harvest, cell number, alkaline phosphatase-specific activity, and production of osteocalcin, transforming growth factor beta1 (TGF-beta1) and prostaglandin E2 (PGE2) were measured. Cell behavior was sensitive to surface roughness and depended on the maturation state of the cell line. Fetal rat calvarial (FRC) cell number and alkaline phosphatase-specific activity were decreased, whereas production of osteocalcin, TGF-beta1, and PGE2 were increased with increasing surface roughness. Addition of 1,25(OH)2D3 to the cultures further augmented the effect of roughness for all parameters in a dose-dependent manner; only TGF-beta1 production on plastic and PT was unaffected by 1,25(OH)2D3. OCT-1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE2, osteocalcin, and TGF-beta1 were increased on SLA and TPS. Response to 1,25(OH)2D3 varied with the parameter being measured. Addition of the hormone to the cultures had no effect on cell number or TGF-beta1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE2 was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO-Y4 cultures, cell number was decreased on SLA and TPS; alkaline phosphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF-beta1, and PGE2 were increased on SLA and TPS. Although 1,25(OH)2D3 had no effect on cell number, alkaline phosphatase, or production of TGF-beta1 or PGE2 on any surface, the production of osteocalcin was stimulated by 1,25(OH)2D3 on SLA and TPS. These results indicate that surface roughness promotes osteogenic differentiation of less mature cells, enhancing their responsiveness to 1,25(OH)2D3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roughness.     

Tissue reactions adjacent to well-fixed hydroxyapatite-coated acetabular cups. Histopathology of ten specimens retrieved at reoperation after 0.3 to 5.8 years

Ten acetabular cups coated with hydroxyapatite (HA) had originally been inserted in five primary and five revision total hip replacements. The thickness of the HA was 155 +/- 35 microm. The cups, which were well-fixed, were retrieved, with their adherent tissue, at reoperation after 0.3 to 5.8 years because of infection (five hips), wear of polyethylene (three hips), and instability (two hips). Undecalcified sections showed a direct contact between bone and osteoid-like tissue which had formed directly onto the HA coating. The area within the threads and their mirror images, as well as the implant-tissue interfaces consisted of similar amounts of bone and soft tissue. Degradation of HA was seen in all hips. The mean thickness of the remaining HA coating was 97 microm (95% CI 94 to 101). The metal interface comprised 66% HA. The HA-tissue interface contained more bone than soft tissue (p = 0.001), whereas the metal-tissue interface included more soft tissue than bone (p = 0.019). Soft tissue at the implant interface and poor replacement of HA by bone may interfere with long-term fixation.     

Expression of RANKL and OPG correlates with age-related bone loss in male C57BL/6 mice.

Osteoblasts regulate the recruitment and activity of osteoclasts through expression of RANKL and osteoprotegerin (OPG). To determine whether expression of RANKL and OPG change with age and how these changes relate to the bone loss of aging, we measured bone mass and cancellous volume, and expression of RANKL, OPG, alkaline phosphatase (AP), osteocalcin (OC), and alpha I collagen (COLL) in whole bone and osteoblast-like cells in culture using 6-week- (young), 6-month- (adult), and 24-month-old (old) mice. Cancellous volume decreased by 20% from young to adult and by 52% from adult to old. RANKL mRNA levels in whole bone were 2.1-fold and 4.4-fold higher in adult and old mice, respectively, compared with young mice, whereas OPG mRNA levels decreased with age slightly. RANKL expression was negatively (r = -0.99) and OPG was positively (r = 0.92) correlated with cancellous bone volume. Expression of RANKL was higher and OPG lower in cells from older animals early in culture (day 7). With cell maturation, RANKL mRNA levels in cells from young and adult mice increased, whereas levels in cells from old animals decreased. By 21 and 28 days of culture, no differences were found in RANKL mRNA in osteoblast-like cells among different age groups. We conclude that expression of RANKL and OPG change with age in whole bone and in cultured osteoblast-like cells. These changes favor increased osteoclast over osteoblast activity, and may explain, in part, the imbalance in bone formation and resorption associated with aging.     

Skeletal changes in osteoprotegerin and receptor activator of nuclear factor-kappab ligand mRNA levels in primary hyperparathyroidism: effect of parathyroidectomy and association with bone metabolism

The effect of parathyroid hormone (PTH) on the production of osteoprotegerin (OPG) and ligand of receptor activator of NF-kappaB (RANKL) in human bone is incompletely understood. Most in vitro studies indicate that PTH decreases OPG and increases RANKL production. In primary hyperparathyroidism (PHPT), hypersecretion of PTH leads to enhanced bone resorption and formation with increased risk of fracture. Decreasing PTH levels by surgery normalizes bone metabolism, but the effects on skeletal OPG and RANKL production are unknown. In this study, 24 patients referred to our clinic for evaluation, and treatment of PHPT were included. A transiliac bone biopsy was done before (n = 24) and 12 months after parathyroidectomy (PTX) (n = 21). Biopsies were frozen in liquid nitrogen and RNA extracted using Trizol. A competitive RT-PCR assay for RANKL and OPG mRNA using artificial cDNA standards was developed and used for quantification. Results were normalized for GAPDH mRNA content. Before surgery, the RANKL/GAPDH gene expression ratio showed positive correlations with serum osteocalcin (r = 0.42, P < 0.05) and urinary NTX (r = 0.43, P < 0.05). The OPG/GAPDH mRNA levels in iliac bone before surgery correlated with serum osteocalcin (r = 0.52, P < 0.01), but not with bone resorption markers. The mRNA ratio of RANKL/OPG decreased significantly (P < 0.05) after surgery. In conclusion, RANKL and OPG gene expression within the human bone microenvironment are influenced by PTH, as the ratio RANKL/OPG decreased upon PTX. In addition, locally produced RANKL appears to affect bone turnover in the hyperparathyroid state.     

HA particles can be released from well-fixed HA-coated stems: histopathology of biopsies from 20 hips 2-8 years after implantation

20 hip arthroplasties with a Landos Corail Ti6Al4V stem entirely plasma-sprayed with a 155+/-35 microm thick HA coating were reoperated on after median 6 (2-8) years because of polyethylene wear (10), acetabular loosening (7), instability (2), or infection (1). We took biopsies from the proximal femurs adjacent to the well-fixed stems. Undecalcifled sections were prepared and examined with a light microscope. The biopsies contained median 5 (1.3-16 ) mm metal interface with 54% HA, 32% bone, and 14% soft tissue. The median thickness of the remaining HA coating was 137 (6-380) microm, and the HA-tissue interface included 89% bone and 11% soft tissue. All HA coatings showed partial degradation and replacement by soft tissue, osteoid-like tissue, or bone. 6 hips had tissue ingrowth between HA and metal consistent with delamination. 14 hips showed bone resorptive areas containing some HA particles and large amounts of polyethylene and metal particles, partly internalized in multinucleated giant cells and macrophages. Bone resorption was associated with metal and polyethylene particles, but not with HA particles. The HA coatings were undermined, resulting in release of large flakes of HA with free access to the articulation. We believe this mechanism may be responsible for third-body wear.     

Influence of experimental conditions on osteoblast activity in human primary bone cell cultures

Primary bone cell cultures were derived from human bone explants. Cellular activity was characterized by the alkaline phosphatase (AP) activity, osteocalcin, and type I and III collagen secretions in the supernatant. The determination of bone cell activity was performed in three different wells. No significant difference was noted between wells: the coefficient of variation was 8.0 +/- 2.9% for AP activity, 18.3 +/- 1.9% for osteocalcin secretion, and 22.5 +/- 14.3% for collagen. The AP activity and osteocalcin secretion significantly decreased with the number of passages: they were the highest after the first passage. Between each subject, the coefficient of variation was 85% for AP activity and osteocalcin secretion and 63 and 57% for type I and III collagen secretion, respectively. The AP activity did not differ with the age or sex of the donor. In contrast, osteocalcin secretion was significantly lower in females than in males. In males, osteocalcin significantly decreased with the age of the donor (r = -0.61; p less than 0.05). Cellular activity did not depend on the site of the biopsy. When bone explants from one donor were cultured in two different petri dishes, the activity of cells was similar in both dishes, except in one case. Primary cell cultures derived from human bone explants are the only model providing untransformed osteoblastlike cells of human origin. Because of the experimental conditions, some factors may have influenced the cellular activity and they must be taken into account to validate further in vitro studies.     

Comparative study of human cancellous bone remodeling to titanium and hydroxyapatite-coated implants

The human cancellous bone response was compared in weight-bearing porous hydroxyapatite (HA) and titanium-coated implants placed in the distal medial femoral condyles of consenting staged bilateral knee patients. The Institutional Review Board approved study quantified the amount of bone ingrowth, the mineral apposition rate, and the bone mineral content. Results showed that the osteoconductive HA coating increased the amount of bone ingrowth by 8% (P=.018). The HA coating did not effect the mineral apposition rate of the bone but had an 8% lower bone mineral content at the implant interface (P=.042). The influence of HA coatings on human cancellous bone appears highly focal along the coating surface. Gaps of 50–500 μm filled with fibrous connective tissue were observed along the porous-coated surfaces of both implant types suggesting that HA coatings still require precision placement adjacent to human cancellous bone.     

A comparison of bone remodelling around hydroxyapatite-coated, porous-coated and grit-blasted hip replacements retrieved at post-mortem

We investigated the implant-bone interface around one design of femoral stem, proximally coated with either a plasma-sprayed porous coating (plain porous) or a hydroxyapatite porous coating (porous HA), or which had been grit-blasted (Interlok). Of 165 patients implanted with a Bimetric hip hemiarthroplasty (Biomet, Bridgend, UK) specimens were retrieved from 58 at post-mortem. We estimated ingrowth and attachment of bone to the surface of the implant in 21 of these, eight plain porous, seven porous HA and six Interlok, using image analysis and light morphometric techniques. The amount of HA coating was also quantified. There was significantly more ingrowth (p = 0.012) and attachment of bone (p < 0.05) to the porous HA surface (mean bone ingrowth 29.093 +/- 2.019%; mean bone attachment 37.287 +/- 2.489%) than to the plain porous surface (mean bone ingrowth 21.762 +/- 2.068%; mean bone attachment 18.9411 +/- 1.971%). There was no significant difference in attachment between the plain porous and Interlok surfaces. Bone grew more evenly over the surface of the HA coating whereas on the porous surface, bone ingrowth and attachment occurred more on the distal and medial parts of the coated surface. No significant differences in the volume of HA were found with the passage of time. This study shows that HA coating increases the amount of ingrowth and attachment of bone and leads to a more even distribution of bone over the surface of the implant. This may have implications in reducing stress shielding and limiting osteolysis induced by wear particles.     

Expression of bone resorption genes in osteoarthritis and in osteoporosis

Cathepsin K and MMP-9 are considered to be the most abundant proteases in osteoclasts. TRAP is a marker for osteoclasts, and there is increasing evidence of its proteolytic role in bone resorption. RANKL is a recently discovered regulator of osteoclast maturation and activity and induces expression of many genes. This study compared cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin gene expression in the proximal femur of patients with osteoarthritis with that of patients with femoral neck fracture. Fifty-six patients undergoing arthroplasty because of osteoarthritis or femoral neck fracture were included in the study. Total mRNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femur. Real-time RT-PCR was used to quantify CTSK (cathepsin K), MMP-9 (matrix metalloproteinase 9), ACP5 (TRAP), TNFSF11 (RANKL), TNFRSF11B (OPG), and BGLAP (osteocalcin) mRNAs. The levels of mRNAs coding for MMP-9 and osteocalcin indicated higher expression in the osteoarthritic group (P = 0.011, P = 0.001, respectively), whereas RANKL expression and the ratio RANKL/OPG were both significantly lower in the osteoarthritic group than in the fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the osteoarthritic group. Ratios of all three proteolytic enzymes relative to formation marker osteocalcin were higher in the fracture group. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin and the association between their mRNA levels pointed to higher bone resorption and bone formation in osteoarthritis, differences in balance between them, and differences in regulation of bone resorption in osteoarthritic and osteoporotic bone.     

In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: immunosuppressive and osteogenic potential of FK506 in vivo.

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagle's minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of beta-glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.     

The influence of crystallinity of the hydroxyapatite coating on the fixation of implants. Mechanical and histomorphometric results.

We inserted two hydroxyapatite (HA)-coated implants with crystallinities of either 50% (HA-50%) or 75% (HA-75%) bilaterally into the medial femoral condyles of the knees of 16 dogs. The implants were allocated to two groups with implantation periods of 16 and 32 weeks. They were weight-bearing and subjected to controlled micromovement of 250 microm during each gait cycle. After 16 weeks, mechanical fixation of the HA-50% implants was increased threefold as compared with the HA-75% implants. After 32 weeks there was no difference between HA-50% and HA-75%. Fixation of HA-75% increased from 16 to 32 weeks whereas that of HA-50% was unchanged. HA-50% implants had 100% more bone ingrowth than HA-75% implants after 16 weeks. More HA coating was removed on HA-50% implants compared with HA-75% implants after both 16 and 32 weeks. No further loss of the HA coating was shown from 16 to 32 weeks. Our study suggests that the crystallinity of the HA coating is an important factor in its bioactivity and resorption during weight-bearing conditions. Our findings suggest two phases of coating resorption, an initial rapid loss, followed by a slow loss. Resorbed HA coating was partly replaced by bone ingrowth, suggesting that implant fixation will be durable.     

Osteoprotegerin and its correlations with new markers of bone formation and bone resorption in kidney transplant recipients

Osteoprotegerin (OPG), a natural decoy receptor for osteoclast differentiation factor, is produced by osteoblasts in response to PTH. OPG and its ligand RANKL constitute a complex mediator system involved in the regulation of bone resorption, probably playing an important role in the homeostasis of bone turnover. At present, little is known about the effects of OPG on uremic bone. Successful kidney transplantation reverses many abnormalities of bone metabolism; however, the improvement is often incomplete. The aim of the study was to assess OPG and RANKL concentrations in long-term kidney allograft recipients and their correlations with biochemical markers of bone resorption and formation. The present studies on 48 kidney transplant recipients and 25 healthy volunteers included concentrations of parathormone, osteocalcin, bone-specific alkaline phosphatase, serum CrossLaps, calcidiol, calcitriol, ICTP, PICP, tartrate-resistant acid phosphatase, beta2 microglobulin, IGF-1, IFGBP-1, IGFBP-3, OPG, and RANKL using commercially available kits for measurements. Among kidney transplant recipients OPG and RANKL did not differ between transplant patients and healthy volunteers, whereas other markers of bone formation and resorption were significantly higher in the former group. OPD was related to age, time on dialysis prior transplantation, urea, platelet count, CSA dose, azathioprine dose, 25(OH)D(3), TRAP, IGF-1, IGFBP-3, whereas RANKL was related to leukocyte count, CSA concentration and dose, urine DPD, and beta2 microglobulin content. In healthy volunteers OPG correlated only with CrossLaps, whereas RANKL correlated only with osteocalcin and TRAP. Correlations between OPG, IGF system components, and some markers of bone metabolism may indicate the role of OPG/RANKL system in the pathogenesis of bone metabolism disturbances following renal transplantation.     

Formation of bone-like mineralized matrix by periodontal ligament cells in vivo: a morphological study in rats

Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey’s fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.     

Hyaluronan increases RANKL expression in bone marrow stromal cells through CD44.

HA activates CD44 to stimulate RANKL expression in bone marrow stromal cells. HA stimulation of RANKL is blocked by anti-CD44 antibody and is absent in cells from CD44(-/-) mice. CD44(-/-) mice exhibit thicker cortical bone and a smaller medullary cavity, but indices of bone resorption are not affected. INTRODUCTION: Hyaluronan (HA), the major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, functions in part through its receptor, CD44, to stimulate a series of intracellular signaling events that lead to cell migration, adhesion, and activation. To determine whether HA activation of CD44 influences RANKL and osteoprotegerin (OPG) expression and whether CD44 is functionally important in bone metabolism, we studied whole bone and bone marrow stromal cells (BMSCs) from wildtype and CD44(-/-) mice. MATERIALS AND METHODS: BMSCs from wildtype and CD44(-/-) mice at 7 weeks of age were cultured and treated with either HA or anti-CD44 antibody. The levels of mRNA of RANKL, OPG, CD44, alkaline phosphatase (ALP), osteocalcin (OC), and alphaI collagen (COLL) were determined by quantitative real-time RT-PCR. Levels of RANKL and CD44 protein were measured by immunoblotting, and expression of CD44 in whole bone was determined by immunohistochemical staining. Double immunofluorescence staining and confocal microscopy were used to study colocalization of Cbfa1, CD44, and HA. Tibias were imaged using muCT, and cancellous and cortical parameters were measured. Osteoblast and osteoclast surface in the distal femoral metaphysis and osteoclast on the endocortical surface at the tibio-fibular junction were measured using quantitative histomorphometry. Differences were analyzed using ANOVA and the Newman-Keuls test. RESULTS: Addition of HA dose-dependently increased RANKL mRNA (3.6-fold) and protein (3-fold) levels in BMSCs. Stimulation of RANKL by HA could be blocked with anti-CD44 antibody. Treatment of cells with HA or anti-CD44 antibody had no significant effect on OPG mRNA levels. Both CD44 and HA localized on the plasma membrane in cells expressing Cbfa1. HA localization on the cell membrane disappeared when cells were preincubated with anti-CD44 antibody. Compared with control mice, cortical bone of CD44(-/-) was thicker, and medullary area was smaller at both 7 and 17 weeks, but at 7 weeks, indices of bone resorption were normal. At 17 weeks of age, tibial mass of CD44(-/-) mice was higher than control mice. CD44(-/-) animals expressed less RANKL in whole bone (-30%) and in BMSCs (-50%). Cells from CD44(-/-) animals failed to respond to either HA or CD44 antibody treatment. CONCLUSIONS: HA can increase RANKL expression in BMSCs through CD44.     

Bone tissue engineering using novel interconnected porous hydroxyapatite ceramics combined with marrow mesenchymal cells: quantitative and three-dimensional image analysis

We developed fully opened interconnected porous calcium hydroxyapatite ceramics having two different pore sizes. One has pores with an average size of 150 microm in diameter, an average 40-microm interconnecting pore diameter, and 75% porosity (HA150). The other has pores with an average size of 300 microm in diameter, an average 60-100-microm interconnecting pore diameter, and 75% porosity (HA300). Because of its smaller pore diameter, HA150 has greater mechanical strength than that of HA300. These ceramics were combined with rat marrow mesenchymal cells and cultured for 2 weeks in the presence of dexamethasone. The cultured ceramics were then implanted into subcutaneous sites in syngeneic rats and harvested 2-8 weeks after implantation. All the implants showed bone formation inside the pore areas as evidenced by decalcified histological sections and microcomputed tomography images, which enabled three-dimensional analysis of the newly formed bone and calculation of the bone volume in the implants. The bone volume increased over time. At 8 weeks after implantation, extensive bone volume was detected not only in the surface pore areas but also in the center pore areas of the implants. A high degree of alkaline phosphatase activity with a peak at 2 weeks and a high level of osteocalcin with a gradual increase over time were detected in the implants. The levels of these biochemical parameters were higher in HA150 than in HA300. The results indicate that a combination of HA150 and mesenchymal cells could be used as an excellent bone graft substitute because of its mechanical properties and capability of inducing bone formation.     

Bone ingrowths to press-fit and loose-fit implants: comparisons between titanium and hydroxyapatite

The aim of this study was to investigate whether the coating of titanium (Ti) implants with hydroxyapatite (HA) might create a better fixation when titanium implants are implanted into a gap. In each of 16 rats, the medullary cavity of both femurs was entered by an awl from the trochanteric area. With steel burrs it was successively reamed to a diameter of 1.5 mm. In a random manner the proximal part of the cavity in half of the bones was reamed once again to a diameter of 2.0 mm. Nails with a diameter of 1.5 mm and a length of 34 mm were then inserted into the medullary cavity of these bones with press fit at the distal half and a gap to the bone in the proximal half. In the remaining bones the whole medullary canal was reamed to a diameter of 2.0 mm, and nails with a diameter of 2.0 mm and a length of 34 mm were introduced. In all cases, either a pure Ti nail or a Ti nail entirely plasma sprayed with HA was used in a random manner. The surface roughness of the pure Ti was characterized by Ra 2.6 microm and Rt 22 microm. Ra of HA was 7.5 microm and Rt 52 microm. At sacrifice after 16 weeks, both femurs were dissected free from soft tissues and then immersed in fixative. A specimen slice of about 5 mm in thickness was prepared from the subtrochanteric region with a water-cooled band saw. Sample preparation for undecalcified tissue followed the internal guidelines at the laboratories of the Department of Biomaterials/Handicap Research. Generally, bone contact to the nails with HA coating was more predictable than was bone contact to the Ti nails. But due to rather large variations in bone contact between the samples, statistical analyses revealed non-significant differences between the 4 groups (p = 0.083). There were no significant differences between Ti and HA coated nails of 2.0 mm (p = 0.633), nor between Ti and HA coated nails of 1.5 mm (p = 0.924). The pooled values for the 2.0 mm nails showed significantly higher bone bonding contact than the pooled values of the 1.5 mm nails (p = 0.011). Our results, then, indicate that bone bonding contact to implants with a loose fit insertion is less predictable than in press fit insertion, and HA coating seemed to be more predictable than pure Ti. However, due to large variations between the samples, the differences did not reach significant levels.     

Glucocorticoid decreases circulating osteoprotegerin (OPG): possible mechanism for glucocorticoid induced osteoporosis.

BACKGROUND: Osteoporosis is a well known side-effect of long-term treatment with glucocorticoids. However, the precise mechanism of this disorder is unclear. Recently, osteoprotegerin (OPG) [osteoclastogenesis inhibitory factor (OCIF)] has been identified as a novel cytokine, which inhibits differentiation and activation of osteoclast. In the present study, in order to clarify the roles of OPG in the development of glucocorticoid-induced osteoporosis, we measured circulating OPG before and after glucocorticoid therapy. METHODS: The study subjects were 12 patients (five males, seven females, 53.4 +/- 4.8 [SE] years) with various renal diseases that required glucocorticoid therapy. All patients received glucocorticoids for the first time. Treatment was initiated at an average dose of 32.5 +/- 3.0 mg per day. Serum OPG was measured using enzyme-linked immunosorbent assay (ELISA). Laboratory data, markers of bone metabolism and circulating OPG were compared before and after treatment for 4 weeks. RESULTS: Serum OPG prior to glucocorticoid therapy was positively and independently correlated with serum creatinine. Serum OPG decreased significantly (P: < 0.0001) from 1.03 +/- 0.14 to 0.77 +/- 0.12 ng/ml after short-term administration of glucocorticoids. Furthermore, serum osteocalcin as a marker of bone formation was also reduced markedly (P: = 0.001). On the other hand, there were no remarkable changes in serum calcium, total alkaline phosphatases, creatinine and intact parathyroid hormone in response to glucocorticoid administration. CONCLUSION: These findings indicate that short-term administration of glucocorticoids significantly suppresses serum OPG and osteocalcin. It might participate in the development of glucocorticoid-induced osteoporosis via an enhancement of bone resorption and suppression of bone formation.     

Osteoprotegerin and bone mineral density in hemodiafiltration patients

A newly identified cytokine, osteoprotegerin (OPG) appears to be involved in the regulation of bone remodeling. In vitro studies suggest that OPG, a soluble member of the TNF receptor family of proteins, inhibits osteoclastogenesis by interrupting the intercellular signaling between osteoblastic stromal cells and osteoclast progenitors. As patients with chronic renal failure (CRF) often have renal osteodystrophy (ROD), we investigated the role of osteoprotegerin (OPG) in ROD, and investigated whether there was any relationship between serum OPG, intact parathyroid (PTH) (iPTH), vitamin D, and trabecular bone. Serum OPG combined with iPTH might be a useful tool in the noninvasive diagnosis of ROD, at least in cases in which the range of PTH values compromises reliable diagnosis. Thirty-six patients on maintenance hemodiafiltration (HDF) and a control group of 36 age and sex matched healthy subjects with no known metabolic bone disease were studied. The following assays were made on serum: iPTH, osteocalcin (BGP), bone alkaline phosphatase, 25(OH)-cholecalciferol, calcium, phosphate, OPG, IGF-1, estradiol, and free testosterone. Serum Ca++, P, B-ALP, BGP, IGF-1, iPTH, and OPG levels were significantly higher in HDF patients than in controls, while DXA measurements and quantitative ultrasound (QUS) parameters were significantly lower. On grouping patients according to their mean OPG levels, we observed significantly lower serum IGF-1, vitamin D3 concentrations, and lumbar spine and hip bone mineral density in the high OPG groups. No correlation was found between OPG and bone turnover markers, whereas a negative correlation was found between serum OPG and IGF-1 levels (r=-0.64, p=0.032). Serum iPTH concentrations were positively correlated with bone alkaline phosphatase (B-ALP) (r=0.69, p=0.038) and BGP (r=0.92, p<0.001). The findings made suggest that an increase in OPG levels may be a compensatory response to elevated bone loss. The low bone mineral density (BMD) levels found in the high OPG group might have been due to the significant decrease in serum IGF-1 and vitamin D3 observed. In conclusion, the findings made in the present study demonstrate that increased OPG in hemodiafiltration patients is only partly due to decreased renal clearance. As it may partly reflect a compensatory response to increased bone loss, this parameter might be helpful in the identification of patients with a marked reduction in trabecular BMD.