Bone Slices Research Articles

The effect of molecules in mother-of-pearl on the decrease in bone resorption through the inhibition of osteoclast cathepsin K

This study evaluates the effect of the mother-of-pearl (nacre) organic matrix on mammalian osteoclast activity and on cathepsin K protease. Rabbit osteoclasts were cultured on bovine cortical bone slices in the presence of water-soluble molecules extracted from nacre of the pearl oyster Pinctada margaritifera. Osteoclast resorption activity was determined by quantification of the resorption surface area on bovine bone slices. Papain and cathepsin K, B and L inhibition tests were performed in the presence of the nacre water-soluble extracts. The active crude extract was fractionated by dialysis and reversed-phase high-performance liquid chromatography before electrospray mass spectrometry analysis of inhibitory fractions. The water-soluble molecules extracted from nacre decreased bone resorption without jeopardizing osteoclast survival. The hydrolytic activity of cysteine proteinases was reduced when the enzymes were incubated with the nacre water-soluble molecules. Trending towards characterization of the molecules involved, it appears that cathepsin K inhibitors remain in different nacre water-soluble organic matrix subfractions, composed of low molecular weight molecules. Mollusk shell nacre contains molecules capable of reducing osteoclast bone resorption activity by inhibiting cathepsin K, giving a new facet of the bioactivity of nacre as bone biomaterial.

Increased resorptive activity and accompanying morphological alterations in osteoclasts derived from the oim/oim mouse model of osteogenesis imperfecta.

The current study addresses whether alterations in osteoclasts (OCs) derived from oim/oim mice, an established model of moderate-to-severe OI, are present. Bone marrow cells from oim/oim and wildtype (+/+) mice were cultured on bone slices in the presence of MCSF and RANKL and evaluated at days 0, 1, 2, 4, and 7. OCs were identified by tartrate-resistant acid phosphatase (TRAP) staining, and bone slice resorption pits were analyzed by reflection microscopy. Flow cytometry was used to examine CD51 (integrin alphaV) and CD61 (integrin beta3) markers. Confocal microscopy was used to assess changes in OC morphology and resorption. There was no difference between the OC precursors of the two genotypes in expression of CD51 and CD61 markers. At day 2, the bone slices seeded with oim/oim cells had a greater percentage of mononuclear cells associated with resorption pits compared to +/+ bone slices. At day 4, the diameter and area of oim/oim OCs were larger compared to the +/+ OCs, and the number of nuclei per OC was also greater for the oim/oim group. At day 7, the oim/oim OCs contained more F-actin rings compared to the +/+ OCs, and the number of OCs in the oim/oim group was greater compared to the +/+ group. The resorbed area of bone slices for the oim/oim group was also greater compared to the +/+ group at day 7. In conclusion, oim/oim mononuclear resorbing cells and OCs showed cellular changes and greater resorptive activity compared to +/+ cells, features that likely contribute to dysregulated bone remodeling in OI.

Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival

Osteoclasts are the sole cells possessing the ability to resorb calcified bone matrix. This occurs via secretion of hydrochloric acid mediated by the V-ATPase and the chloride channel ClC-7. Loss of acidification leads to osteopetrosis characterized by ablation of bone resorption and increased osteoclast numbers, indicating increased life span of the osteoclasts. To investigate the role of the inorganic phase of bone with respect to osteoclast life span, we used the V-ATPase inhibitor bafilomycin and the calcium uptake antagonist ryanodine on human osteoclasts cultured on calcified and decalcified bone slices. Bafilomycin inhibited bone resorption and increased osteoclast survival on calcified but not decalcified bones. Ryanodine attenuated calcium uptake and thereby augmented osteoclast survival on calcified bones. In summary, we found that acidification leading to calcium release from bone during resorption controls osteoclast survival, potentially explaining the increased numbers of osteoclasts in patients with osteopetrosis.

The dual specific Src/Abl kinase inhibitor AZD0530 inhibits the formation and activity of human osteoclasts

3602 Background: Gene knockout studies have demonstrated the critical importance of the non-receptor TK Src to osteoclast bone resorptive function. Deregulated Src TK activity is also reported as a hallmark of the invasive cancer cell. Bone metastatic cancer cells interact with and activate osteoclasts in a destructive cycle of bone degradation and stimulation of tumor cell growth. Therefore targeting Src activity would appear to be a rational therapeutic approach in treating metastatic bone disease. We have reported previously (AACR 2005) on the activity of the dual Src/Abl kinase inhibitor AZD0530 in inhibiting the bone resorptive activity of mature rabbit osteoclasts in a bone slice model. Here we tested the effect of AZD0530 in a human co-culture system, examining its activity on i) osteoclast formation by peripheral blood mononuclear cells (PBMCs) co-cultured with osteoblasts, and ii) osteoclastic bone resorption. Methods and Results: PBMC adhesion to osteoblasts and osteoblast morphology was not affected by AZD0530 (0.1–10 μM). However, AZD0530 inhibited the formation of multinucleated osteoclast-like cells dose dependently. PBMC-osteoblast co-cultures were then exposed to 1 μM AZD0530 for different time intervals. AZD0530 was most effective in inhibiting the formation of osteoclast-like cells when added at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial induction of osteoclast formation. Formation of actin rings, to which c-Src co-localizes, is a prerequisite for osteoclastic bone resorption. The effect of AZD0530 on formation of actin rings was analyzed using the co-culture system on cortical bone slices. AZD0530 prevented migration of osteoclast precursors to the bone surface, and the subsequent formation of actin rings. On withdrawal of the drug, this process was reversible. Conclusions: Our data suggest that Src activity is pivotal for the formation, migration and activity of osteoclasts. Data reported elsewhere suggest AZD0530 will also impact on tumor cells directly. AZD0530 is a promising new anti-cancer drug with potential to additionally treat metastatic bone disease through its inhibition of both osteoclast activity and tumor cell invasion into and within the bone micro-environment. No significant financial relationships to disclose.

Differential effects of selective oestrogen receptor modulators (SERMs) tamoxifen, ospemifene and raloxifene on human osteoclasts in vitro

Several selective oestrogen receptor modulators (SERMs) with oestrogen agonist effects in bone cells and without increased risk of breast and endometrial cancer have been developed. Here, we have investigated the effects of different types of SERMs on osteoclast differentiation, bone resorption and apoptosis in vitro. Human peripheral blood-derived CD14+ monocytes were cultured on bovine bone slices in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone for seven days. Also, CD14+ monocytes were co-cultured either with human SaOS-2 or MG-63 osteosarcoma cells, in the presence of parathyroid hormone. Osteoclast cultures were treated with different SERMs. TRACP+ multinucleated cells and C-terminal telopeptide of type I collagen were used as markers for osteoclast formation and bone resorption, respectively. In CD14+ monocyte cultures, tamoxifen directly inhibited human osteoclast formation and bone resorption, while raloxifene and ospemifene had no inhibitory effect. In the co-cultures either with SaOS-2 or MG-63 cells, ospemifene and raloxifene as well as tamoxifen inhibited osteoclast formation in a concentration-dependent manner. The inhibitory effect was associated with an increased production of osteoprotegerin. The anti-oestrogen ICI 182 780 completely reversed the effects of these SERMs. Tamoxifen had an oestrogen receptor dependent, direct, inhibitory effect on human osteoclast differentiation and bone resorption, whereas ospemifene and raloxifene required osteoblastic cells to achieve a similar inhibition. The effects of ospemifene and raloxifene were mediated by oestrogen receptors by a mechanism involving paracrine induction of osteoprotegerin in cultures with osteoblast derived osteosarcoma cells.

CD34 human hematopoietic progenitor cell line, MUTZ-3, differentiates into functional osteoclasts

CD14(+) monocyte cell lines can differentiate into an osteoclast (OC)-like lineage. However, the identification of human cell lines with stem cell characteristics, capable of differentiating into OCs, would provide a tool for the study of the molecular mechanisms regulating their commitment, differentiation, and function. Since the human acute myeloid leukemia cell line MUTZ-3 contains both CD34(+) stem cell and CD14(+) cell populations, we investigated the capacity of the stem/progenitor CD34(+) population to differentiate into functional OCs. Sorted MUTZ-3-CD34(+) and MUTZ-3-CD14(+) cells were cultured in presence of M-CSF, RANK-L, and TNF-alpha to generate OCs. Differentiation was evaluated by TRAP staining and RT-PCR, which assessed the expression of c-fms, RANK, MMP-9, CATK, TRAP, and CTR in -CD34(+)OC and -CD14(+)OC cells. Resorption pit formation was also evaluated. CD34, CD14, M-CSF-R, RANK, and CTR expression was assessed by FACS analysis. MUTZ-3-CD34(+) differentiated into OCs, displaying the full range of differentiation markers; MMP-9, CATK, TRAP, and RANK mRNA were detected from day 3 of culture, whereas CTR from day 12. Stimulated MUTZ-3-CD34(+) generated functional osteoclasts that formed extensive resorption lacunae on both mineralized surface and bone slices. Surprisingly, in both sorted populations we identified a population M-CSF-R(+)/RANK(+) that at the same time co-expressed CD14 and CD34. These findings demonstrate that MUTZ-3 cells constitute an invaluable model to study the expression pattern in different developmental stages of commitment and differentiation. Importantly, the data indicate that the CD14(+)CD34(+)M-CSF-R(+)RANK(+) population represents an intermediate stage of differentiation from CD34 precursors and monocytes to osteoclast.

Mechanical properties of femoral cortical bone following cemented hip replacement

Femoral bone remodeling following total hip replacement is a big concern and has never been examined mechanically. In this study, six goats underwent unilateral cemented hip hemiarthroplasty with polymethyl methacrylate (PMMA) bone cement. Nine months later animals were sacrificed, and the femoral cortical bone slices at different levels were analysed using microhardness testing and microcomputed tomography (micro-CT) scanning. Implanted femurs were compared to contralateral nonimplanted femurs. Extensive bone remodeling was demonstrated at both the proximal and middle levels, but not at the distal level. Compared with the nonimplanted side, significant decreases were found in the implanted femur in cortical bone area, bone mineral density, and cortical bone hardness at the proximal level, as well as in bone mineral density and bone hardness at the middle level. However, no significant difference was observed in either variable for the distal level. In addition, similar proximal-to-distal gradient changes were revealed both in cortical bone microhardness and bone mineral density. From the mechanical point of view, the results of the present study suggested that stress shielding is an important mechanical factor associated with bone adaptation following total hip replacement.

The in vitro effect of pH on osteoclasts and bone resorption in the cat: Implications for the pathogenesis of FORL

Dental disease due to osteoclast over‐activity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline osteoclastic resorptive lesions (FORL) involve extensive resorption of the tooth leaving it liable to root fracture and subsequent tooth loss. The aetio‐pathogenesis of FORL is not known. Recent work has shown that systemic acidosis causes increased osteoclast activation and that loci of infection or inflammation in cat mouth are likely to be acidotic. To investigate this, we generated osteoclasts from cat blood and found that they formed in large numbers (∼400) in cultures on bovine cortical bone slices. Acidosis caused an increase in the size of cells—in cultures maintained up to 14 days at basal pH 7.25, mean osteoclast area was 0.01 ± 0.003 mm2, whereas an 8.6‐fold increase was observed in cells cultured between 11 and 14 days at pH 7.15 (0.086 ± 0.004 mm2). Acidosis caused a modest increase in the number of osteoclasts. Exposure to pH 6.92 exhibited a 5‐fold increase in the area of bone slices covered by resorption lacunae (∼70% bone slice resorbed). In line with this finding, significant increases were observed in the expression of cathepsin K and proton pump enzymes (both approximately 3‐fold) that are key enzymes reflective of resorptive activity in osteoclasts. These results demonstrate that acidosis is a major regulator of osteoclast formation and functional activation in the cat, and suggest that local pH changes may play a significant role in the pathogenesis of FORL. J. Cell. Physiol. 213: 144–150, 2007. © 2007 Wiley‐Liss, Inc.

Icariin suppresses bone resorption activity of rabbit osteoclasts in vitro

The effect of icariin on the bone resorption activity of rabbit osteoclasts is assessed in vitro. Osteoclasts were isolated from Japanese white rabbits and cultured on plates with a sterilized bone slice in each well. After treatment with icariin at various concentrations, the bone resorption activity of osteoclasts was evaluated by examining pit areas, superoxide anion (·O 2 − ) generation, size and number of actin rings and intracellular calcium concentration [Ca2+]i. As revealed by these data, icariin elicited continuous decline of [Ca2+]i, making actin ring constricted and ·O2 − generation decreased. These events resulted in smaller and fewer pits which indicate suppressed bone resorption activity of rabbit osteoclasts by icariin.

Investigation of elemental distribution in human femoral head by PIXE and SRXRF microprobe

In order to study the distribution and possible degenerative processes inducing the loss of inorganic substances in bone and to provide a scientific basis for the prevention and therapy of osteoporosis, proton induced X-ray emission (PIXE) method is used for the determination of elemental concentrations in femoral heads from five autopsies and seven patients with femoral neck fractures. Synchrotron radiation X-ray fluorescence (SRXRF) microprobe analysis technique is used to scan a slice of the femoral head from its periphery to its center, via cartilage, compact and spongy zones. The specimen preparation and experiment procedure are described in detail. The results show that the concentrations of P, Ca, Fe, Cu, Sr in the control group are higher than those in the patient group, but the concentrations of S, K, Zn, Mn are not significantly different. The quantitative results of elemental distribution, such as Ca, P, K, Fe, Zn, Sr and Pb in bone slice tissue including cartilage, substantial compact and substantial spongy, are investigated. The data obtained show that the concentrations of Ca, P, K, (the major elements of bone composition), are obviously low in both spongy and cartilage zones in the patient group, but there are no remarkable differences in the compact zone. Combined with the correlations between P, K, Zn, Sr and Ca, the loss mechanism of minerals and the physiological functions of some metal elements in bone are also discussed.

In vitro study of the effects of copper ion on osteoclastic resorption in various dental mineralized tissues

To investigate the effects of copper ion on osteoclastic resorption in various dental mineralized tissues. Osteoclasts were separated from long-limb bones of neonatal rabbits, and cultured with de-activated human tooth slices and glass slices. The cells in the experiment group were treated with (1 x 10(-14))-1 x (10(-4)) mol/L copper + 10% (volume fraction) fetal calf serum (FCS) + alpha-MEM, while the cells in control group cells were grown in 10% FCS + alpha-MEM. Osteoclasts on glass slices were stained by TRAP staining. The absorption pits on tooth slices were observed by inverted phase contrast microscope. The resorbing activity was evaluated with the concentration of calcium in the supernatant liquid of osteoclasts. The ratio between the concentration of calcium in the experiment group and control group was termed as the resorption index. The isolated cells were multinuclear and TRAP positive in cytoplasma. Osteoclasts resorbed teeth slices first on the cementum and dentin. Compared with those on bone slices, the lacunae on the dental slices numbered less, with smaller volume and shallower in depth. Microscopy showed that the number and area of absorption pits formed on treated tissues were less than those on control tissues. The content of calcium in the supernatant liquid decreased in the concentrations of 1 x 10(-14) mol/L - 1 x 10(-4) mol/L copper, especially in the group of 1 x 10(-10) mol/L copper at 3rd day (P < 0.05) and 1 x 10(-4) mol/L, 1 x 10(-10) - 1 x 10(-12) mol/L copper at 7th day (P < 0.05). Their resorption index was lower than one. Extracellular copper ion can inhibit osteoclastic resorption on dental slices.

Non-enzymatic Glycation of Bone Collagen Modifies Osteoclastic Activity and Differentiation

Type I collagen, the major organic component of bone matrix, undergoes a series of post-translational modifications that occur with aging, such as the non-enzymatic glycation. This spontaneous reaction leads to the formation of advanced glycation end products (AGEs), which accumulate in bone tissue and affect its structural and mechanical properties. We have investigated the role of matrix AGEs on bone resorption mediated by mature osteoclasts and the effects of exogenous AGEs on osteoclastogenesis. Using in vitro resorption assays performed on control- and AGE-modified bone and ivory slices, we showed that the resorption process was markedly inhibited when mature osteoclasts were seeded on slices containing matrix pentosidine, a well characterized AGE. More specifically, the total area resorbed per slice, and the area degraded per resorption lacuna created by osteoclasts, were significantly decreased in AGE-containing slices. This inhibition of bone resorption was confirmed by a marked reduction of the release of type I collagen fragments generated by the collagenolytic enzymes secreted by osteoclasts in the culture medium of AGE-modified mineralized matrices. This effect is likely to result from decreased solubility of collagen molecules in the presence of AGEs, as documented by the reduction of pepsin-mediated digestion of AGE-containing collagen. We found that AGE-modified BSA totally inhibited osteoclastogenesis in vitro, most likely by impairing the commitment of osteoclast progenitors into pre-osteoclastic cells. Although the mechanisms remain unknown, AGEs might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs). These results suggest that AGEs decreased osteoclast-induced bone resorption, by altering not only the structural integrity of bone matrix proteins but also the osteoclastic differentiation process. We suggest that AGEs may play a role in the alterations of bone remodeling associated with aging and diabetes.

Effects of Carbonic Anhydrase Inhibitor Acetazolamide (AZ) on Osteoclasts and Bone Structure

We examined the changes of osteoclasts and bone structure in vivo and in vitro under the condition of low activity of bone resorption induced by acetazolamide (AZ). AZ is a specific inhibitor of carbonic anhydrase II (CA II), which is the enzyme indispensable for substantial proton generation by osteoclasts. In vivo study, we investigated histologically and histomorphometrically the changes of trabecular bone and osteoclasts at the cartilage-bone junction of tibiae in rats injected subcutaneously with AZ for 21 days. The area of trabecular bone of AZ treated group increased compared with control group. Moreover, AZ treatment decreased about 20% the number of osteoclasts at the cartilage-bone junction. In vitro study, we examined the effects of AZ on osteoclasts using culture system of rat bone marrow. The number of osteoclasts on the bone slice was suppressed by AZ of a concentration from 10-5 to 10-4 M in a doze-dependent manner. The number of osteoclasts cultured with 10-4 M AZ for 24 hours and with or without parathyroid hormone (PTH) on the bone slice and on the glass showed a significant decrease compared with the control group. Some osteoclasts treated with AZ showed morphological changes of apoptosis-like cell death, including shrinking of cytoplasm and fragmentation of nuclei. These results show that high-doze of AZ decreases bone resorption not only by inhibiting CA II but also by decreasing the number of osteoclasts via induction of cell death. This suggests that CA II plays an important role not only in proton formation but also in osteoclast survival.

Effect of Modified Pectin Molecules on the Growth of Bone Cells

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple pectins. MHRs were covalently attached to tissue culture polystyrene (TCPS) or glass. Uncoated substrata or bone slices were used as controls. Cell attachment, proliferation, and differentiation were investigated with fluorescence and confocal microscopy. Bone cells seem to prefer MHR-B coating to MHR-A coating. On MHR-A samples, the overall numbers as well as proportions of active osteoclasts were diminished compared to those on MHR-B, TCPS, or bone. Focal adhesions indicating attachment of the osteoblastic cells were detected on MHR-B and uncoated controls but not on MHR-A. These results demonstrate the possibility to modify surfaces with pectin nanocoatings.

Bortezomib Transiently Inhibits Osteoclast Resorptive Activity in Cell Culture Conditions Mimicking In Vivo Intermittent Treatment.

Multiple myeloma (MM) leads to high risk for bone pain and fractures. MM-induced bone disease is due to acute degradation of bone matrix by osteoclasts, and absence of repair by bone forming osteoblasts. It is currently treated with bisphosphonates, highly effective bone resorption inhibitors, which do not promote but rather inhibit bone formation and may cause renal damage and osteonecrosis of the jaw. Thus, it is important to reconsider the management of MM bone disease in long-term treatment. Recent preclinical studies reported that the proteasome inhibitor Bortezomib (V) used for the treatment of MM patients can stimulate bone formation, and that in MM patients treated with V, serum levels of bone formation markers are increased. The present study aims at investigating if V may inhibit osteoclast activity.Methods: Osteoclasts were differentiated from pure populations of blood derived CD14-positive monocytes cultured with M-CSF and RANKL for 6–7 days, and treated continuously with V at various concentrations. As prolonged inhibition of proteasome activity has been reported to be toxic for any cell type, and in vivo pharmacodynamic studies have shown V to be eliminated from the vascular compartment as soon as 30min after intravenous injection, displaying maximal inhibitory activity of the proteasome within 24 hours subsiding rapidly thereafter, V was also given intermittently, to mimick the in vivo situation. Osteoclast differentiation and activity were assessed by measuring Tartrate-Resistant Acid Phosphatase (TRACP) activity in the medium. Cell viability was determined with Celltiter Blue measuring metabolic activity. To extend our observations to the clinical situation, serum levels of CTX-I, a bone resorption marker, were measured during the 3 days following therapeutic V administration in a single patient.Results: A continuous treatment of cultures with V at 4 nM and higher concentrations proved to be highly toxic for differentiating osteoclasts but also monocytes. A 3-hour-pulse treatment with V followed by a 3-day culture in the absence of V, was not toxic neither to monocytes nor to osteoclasts, even at a concentration as high as 100 nM. This 3-hour pulse was however highly toxic for myeloma cells. Interestingly, a 3-hour pulse with 25 nM V induced a 50% inhibition of the resorptive activity of osteoclasts, as assessed by culturing them for 3 days on bone slices and measuring the formation of resorption pits. The release of TRACP in the medium was inhibited to a similar extent within the first 24 hours post-pulse, but tended to return to the control level during the next 2 days. This 3-hour pulse with 25 nM V inhibited strongly RANKL-induced translocation of NF-KB in the osteoclast nuclei, an event dependent on proteasome function and critical for osteoclastic activity. Serum CTX-I levels decreased during the first 48 hours after each V injection (n = 3), and tended to increase again after 72 hours suggesting a partial recovery of osteoclast activity between each administration.Conclusions: Our results suggest that Bortezomib temporarily inhibits osteoclast activity in vitro and in vivo. This effect is linked to RANKL-induced translocation of NF-KB in the osteoclast nuclei and proteasome function. Since recent reports suggested that formation of new bone requires at least a transient activity of osteoclasts transient inhibition of osteoclasts could be an advantage compared to the more persistent inhibition of osteoclast activity by bisphosphonate.

Hematopoietc Stem Cell Targeted Neonatal Gene Therapy Cures oc/oc Mice from Osteopetrosis.

Infantile malignant osteopeterosis (IMO) is a progressive, rare autosomal recessive disorder affecting osteoclast function. 50% of the affected children have a mutation in the Tcirg1 gene coding for one subunit of an osteoclast specific proton pump, OC116. The non-resorbed dense, sclerotic bones cause symptoms including pancytopenia and progressive visual loss and ultimately death. So far, the only curative treatment is hematopoietic stem cell (HSC) transplantation. The oc/oc mouse has a mutation in the gene homologous to Tcirg1 giving rise to similar symptoms as in patients leading to death of the mice at the age of 3–4 weeks. We have previously shown that the oc/oc mouse can be successfully treated with neonatal transplantation of normal HSC leading to prolonged survival and reversal of osteopetrosis (M. Johansson et al., Exp. Hematology 34;242, 2006). In the current study we set out to develop HSC directed gene therapy for osteopetrosis in the oc/oc mouse model. As the bone marrow compartment is severely reduced in the oc/oc mouse fetal liver (FL) cells depleted of Ter119+ erythroid cells were used as a source of hematopoietic stem cells. We first established that wild type Ter119 depleted FL cells marked with a GFP vector and transplanted to newborn oc/oc mice i.p. could correct the osteopetrotic phenotype just as was shown for fresh bone marrow cells previously. Subsequently, Ter119 depleted FL cells from oc/oc mice were transduced with a retroviral vector expressing OC116 and GFP. In vitro transduction efficiency was 60–85%. One-day-old oc/oc mice were irradiated (400cGy) and transplanted i.p. with the transduced FL cells (1–3.5x106). 7 out of 14 mice survived past the expected lifespan and had 8–53% GFP+ cells in the peripheral blood at 3, 6 and 12 weeks. Analysis of bone structure with X-ray and histopathology showed an improvement at 8 weeks and an almost normal structure at 18 weeks, indicating induction of osteoclast activity. In vitro culture of osteoclasts from bone marrow from transplanted animals on bovine bone slices showed GFP marked osteoclasts and bone resorption, albeit at lower levels than for wild type cells. In the oc/oc mouse there is a block in B-lymphopoiesis leading to a reduced number of B-lymphocytes in the peripheral blood. In treated mice a reversal of this deficiency was observed.In summary we have demonstrated that the osteoclast defect seen in oc/oc mice can be successfully corrected by neonatal transplantation of gene modified hematopoietic stem cells and that this can lead to long-term survival of treated mice. This represents a significant step towards the development of gene therapy for osteopetrosis.

Debridement of Cancellous Bone: A Comparison of Irrigation Methods

This study tests the bone debridement efficacy and damage to cancellous bone produced by different wound irrigation methods. Cancellous bone slices of bovine distal femurs (3 cm x 3 cm) were divided into eight test groups and scored with a saw in a latticed pattern. Four test groups were contaminated with 1.0 g rock dust and four were not. The specimens were then treated as follows: no treatment (control), bulb irrigation, brush-suction irrigation, or high-pressure pulsatile lavage (HPPL). Runoff from the irrigation was collected, filtered, lyophilized, and ashed to allow for quantitative determination of organic and inorganic material removed from the bone by each irrigation method. The bone samples were subjected to blinded grading on two five-point scales to assess: 1) macroscopic tissue damage and 2) amount of contaminant remaining following treatment. Significantly more (P < or = 0.05) mean organic material was removed from samples treated with HPPL (744.8 +/- 120.0 mg) than with bulb syringe (115.2 +/- 11.9 mg) or brush-suction irrigation (95.1 +/- 9.2 mg). Brush-suction irrigation removed statistically significantly more (P < or = 0.05) of the 1.0 g of initial inorganic contaminant (937.7 +/- 6.3 mg) than bulb syringe irrigation (866.2 +/- 30.1 mg), while HPPL (900.2 +/- 19.0 mg) did not. Past studies have shown HPPL to damage both soft tissue and bone structure. The tissue damage that HPPL produces has been accepted in the past in exchange for its presumed superiority in contaminant removal. In this study, HPPL damaged samples more than other irrigation methods by removing significantly more organic material from them. However, HPPL and bulb syringe removed a statistically similar amount of inorganic contaminant, while brush-suction irrigation removed a significantly greater amount of inorganic contaminant than bulb syringe. It is proposed that HPPL may drive some contaminants deeper into the tissue rather than removing them, rendering HPPL not only more deleterious to bone structure and healing, but also less efficacious at removing contaminant than brush-suction irrigation.

Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics

Gamma irradiation from Cobalt 60 sources has been used to terminally sterilize bone allografts for many years. Gamma radiation adversely affects the mechanical and biological properties of bone allografts by degrading the collagen in bone matrix. Specifically, gamma rays split polypeptide chains. In wet specimens irradiation causes release of free radicals via radiolysis of water molecules that induces cross-linking reactions in collagen molecules. These effects are dose dependent and give rise to a dose-dependent decrease in mechanical properties of allograft bone when gamma dose is increased above 25 kGy for cortical bone or 60 kGy for cancellous bone. But at doses between 0 and 25 kGy (standard dose), a clear relationship between gamma dose and mechanical properties has yet to be established. In addition, the effects of gamma radiation on graft remodelling have not been intensively investigated. There is evidence that the activity of osteoclasts is reduced when they are cultured onto irradiated bone slices, that peroxidation of marrow fat increases apoptosis of osteoblasts; and that bacterial products remain after irradiation and induce inflammatory bone resorption following macrophage activation. These effects need considerably more investigation to establish their relevance to clinical outcomes. International consensus on an optimum dose of radiation has not been achieved due to a wide range of confounding variables and individual decisions by tissue banks. This has resulted in the application of doses ranging from 15 to 35 kGy. Here, we provide a critical review on the effects of gamma irradiation on the mechanical and biological properties of allograft bone.

Hypoxia Induces Giant Osteoclast Formation and Extensive Bone Resorption in the Cat

Dental disease due to osteoclast (OC) overactivity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline odontoclastic resorptive lesions (FORL) involve extensive resorption of the tooth, leaving it liable to root fracture and subsequent loss. The etiopathogenesis of FORL remains unclear. Here, we explore the hypothesis that FORL is associated with hypoxia in the oral microenvironment, leading to increased OC activity. To investigate this, we developed a method of generating OCs from cat blood. Reducing O2 from 20% to 2% increased the mean area of OC eightfold from 0.01 to 0.08 mm2. In hypoxic cultures, very large OCs containing several hundred nuclei were evident (reaching a maximum size of approximately 14 mm2). Cultures exposed to 2% O2 exhibited an increase of approximately 13-fold in the area of bone slices covered by resorption lacunae. In line with this finding, there was a significant increase in cells differentiating under hypoxic conditions, reflected in increased expression of cathepsin K and proton pump enzymes. In conclusion, these results demonstrate that oxygen tension is a major regulator of OC formation in the cat. However, in this species, hypoxia induces the formation of "giant" OCs, which can be so large as to be visible with the naked eye and yet also actively resorb. This suggests that local hypoxia is likely to play a key role in the pathogenesis of FORL and other inflammatory conditions that are associated with bone resorption in cats.

Comprehensive Therapy in Osteoporosis Using a Single Drug: From ADFR to Strontium Ranelate

In vitro, strontium ranelate increases collagen and non-collagenic proteins synthesis by mature osteoblast enriched cells. The effects of strontium ranelate on bone formation were confirmed as the drug enhanced preosteoblastic cell replication. In the isolated rat osteoclast, a preincubation of bone slices with strontium ranelate induced a dose-dependent inhibition of the bone resorbing activity of treated rat osteoclast. Strontium ranelate dose-dependently inhibited preosteoclast differentiation. In a phase II dose ranging trial Strontium ranelate (500 mg, 1000 mg, 2000 mg/day) or placebo were given to 353 postmenopausal women with prevalent vertebral osteoporosis. At the conclusion of this 2-year study, the annual increase in lumbar BMD of the group receiving 2000 mg of strontium ranelate was + 7.3%, a significant increase in bone alkaline phosphatase, over a 6-month period and a significant decrease in N-telopeptide crosslinks throughout the 2-year period were seen. During the second year of treatment, the dose of 2000 mg was associated with a 44% reduction in the number of patients experiencing a new vertebral deformity. The primary analysis of the SOTI study, evaluating the effect of strontium ranelate 2000 mg on vertebral fracture rates, revealed a 41% reduction in the relative risk of patient experiencing a first new vertebral fracture with strontium ranelate throughout the 3-year study. The TROPOS study showed a significant reduction in the risk of experiencing a first non-vertebral fracture by 16% in the group treated with strontium ranelate throughout the 3-year study. A reduction in the risk of experiencing a hip fracture by 36% was also demonstrated in the patients at high risk of hip fracture (age > or =74 years and Femoral Neck T score < or = -2.4 according to NHANES normative value). All these results suggest that strontium ranelate is a new, effective and safe treatment of vertebral and non-vertebral osteoporosis, with a unique mode of action.