Acid attack and cathepsin K in bone resorption around total hip replacement prosthesis.

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid- and cathepsin K-driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.     

Induction of monocyte chemotaxis in devascularized rabbit bone

Summary Temporary impairment of blood supply has been suggested to cause bone remodeling. The degradation of cells and matrix and the attraction of resorbing cells were examined in this study. Bone specimens of rabbits were stored in vitro for 2–20 days. At the end of this aging process the probes were tested for their chemotactic activity toward autologous leukocytes in a diffusion chamber. Both supernatant from the aged bone specimens and ground bone particles exhibited significant chemotactic activity that was specifically attracting monocytes. It is suggested that soluble bone matrix proteins or degeneration products liberated during ischemic damage to cortical bone initiate the resorptive process.This work was supported by the Swiss National Science Foundation, grant no. 3.857.0.83, and by the Studienstiftung des deutschen Volkes     

Osteoclastic acidification pathways during bone resorption

Osteoclasts resorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclast and bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases. This study summarizes the characterization and role of each type of ion transport and defines the main biochemical mechanisms involved in the dissolution of bone mineral during bone resorption. The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar H+-adenosine triphosphatase (ATPase) coupled with Cl- conductance localized to the ruffled membrane. Carbonic anhydrase II (CAII) provides the proton source for extracellular acidification by H+-ATPase and the HCO3- source for the HCO3-/Cl- exchanger. Whereas some transporters are responsible for the bone resorption process, others are essential for pH regulation in the osteoclast. The HCO3-/Cl- exchanger, in association with CAII, is the major transporter for maintenance of normal intracellular pH. An Na+/H+ antiporter may also contribute to the recovery of intracellular pH during early osteoclast activation. Once this mechanism has been rendered inoperative, another conductive pathway translocates the protons and modulates cytoplasmic pH. Inward-rectifying K+ channels may also be involved by compensating for the external acidification due to H+ transport. These different effects of transport processes, either on bone resorption or pH homeostasis, increase the number of possible sites for pharmacological intervention in the treatment of metabolic bone diseases.     

The relative contribution of cysteine proteinases and matrix metalloproteinases to the resorption process in osteoclasts derived from long bone and scapula

It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.     

A possible role of bone morphogenetic protein (BMP) in the regulation of bone remodeling

Bone morphogenetic protein (BMP) from an osteosarcoma powder was found to be solubilized in weak acid (pH 2.6–3.0), but not in acidic solutions of a lower or higher pH. This indicates that the solubility of BMP strictly depends on the hydrogen ion concentration of the solution. Ectopic osteogenesis induced by the osteosarcoma powder was greatly enhanced by treating the powder with acid solutions below pH 3.0. The acid-treated preparations were neutralized before bioassay. Acid treatment resulted in almost a five-fold increase in the amount of new bone formation. This finding suggests that a high concentration of hydrogen ion is important in regulating the activity of BMP. Lyophilized cultured cells of the osteosarcoma preserved the osteogenic activity even after incubation in acidic and neutral buffers at 37°C for six days. This observation suggests that BMP is stable to endogenous enzymes such as lysosomal or proteolytic enzymes. Based on these results, the role possibly played by BMP in the regulation of bone remodeling is discussed.     

A back-scattered electron microscopy (BSEM) study of the tight apposition between bone and hydroxyapatite coating

We performed a back-scattered electron microscopy analysis of the interface between newly formed bone and hydroxyapatite coating, in an experimental rabbit model. Twenty cylinders made of Ti6A14V and coated with hydroxyapatite at different crystallinity were implanted in the distal femural canal and retrieved at 4, 8, 26 an 34 weeks. Crystallinity of the coating varied from 90% to 60% and thickness varied between 50 and 100 μm. Osteocytes were detectable a few micrometers in proximity of the coating. They produced new bone which was so tightly apposed to the coating that high magnification BSEM did not resolve any discontinuity at the interface. This was not observed in uncoated implants. Degradation of the hydroxyapatite coating is not a simple hydrolytic process because newly formed bone is remodelled in areas were a tight apposition with hydroxyapatite is present. The coatint itself is likely to be attacked by the resorptive action of multinucleated giant cells and osteoclasts. In conclusion, response to coated samples is morphologically characterized by tight apposition with bone. The substitution of areas of the coating by newly formed bone is possible. Received: 28 April 2000/Accepted: 2 June 2000     

Acidification enhances peritoneal macrophage phagocytic activity

BACKGROUND: Laparoscopic surgery is currently used in an array of diverse clinical situations, including cases with potential bacterial contamination. Previous studies have shown that CO2 insufflation during laparoscopic procedures modulates the immune response due to the acidification of the peritoneum. In the present study, we investigated whether exposure of macrophages to an acidic environment, such as that produced by CO2 insufflation, could affect phagocytosis, which is the fundamental process for bacterial clearance. MATERIALS AND METHODS: A murine peritoneal macrophage line (J774) was pre-incubated at pH levels of 6.0 or 7.4 for 3 h at 37 degrees C and returned to neutral pH (7.4). Phagocytosis was evaluated by incubation with fluorescein isothiocyanate-conjugated IgG-opsonized bacterial particles, IgG-opsonized fluorescent latex beads, and non-opsonized fluorescent latex beads at 37 degrees C, pH 7.4. The intensity of the internalized signal was measured by using a fluorometer. RESULTS: Pre-incubation of macrophages at a pH of 6.0 resulted in a significant increase of phagocytic activity of opsonized particles. However, it did not change the uptake of non-opsonized particles. This effect was due to the internalization process since there were no differences in foreign particle binding of cells exposed to acidic or neutral pH levels. CONCLUSIONS: This study demonstrates that environmental acidification increases the phagocytosis of opsonized particles by macrophages. These results suggest that CO2 insufflation during laparoscopic surgery may be beneficial for the clearance of pathogens, particularly in cases where there is a high risk of potential intra-abdominal infections.     

Resorptive response of rhBMP2 simulating infection in an anterior lumbar interbody fusion with a femoral ring

RhBMP-2 has been available for general use since July of 2002. No literature regarding its use with femoral ring allografts exists. This case demonstrates how a resorptive effect of rhBMP-2 with a femoral ring allograft on host bone at the interbody fusion site can easily be confused with infection. Spine surgeons should be aware of this effect to avoid unnecessary antibiotic use or additional surgery.     

The relationship between stress shielding and bone resorption around total hip stems and the effects of flexible materials.

Bone resorption around hip stems is a disturbing phenomenon, although its clinical significance and its eventual effects on replacement longevity are as yet uncertain. The relationship between implant flexibility and the extent of bone loss, frequently established in clinical patient series and animal experiments, does suggest that the changes in bone morphology are an effect of stress shielding and a subsequent adaptive remodeling process. This relationship was investigated using strain-adaptive bone-remodeling theory in combination with finite element models to simulate the bone remodeling process. The effects of stem material flexibility, bone flexibility, and bone reactivity on the process and its eventual outcome were studied. Stem flexibility was also related to proximal implant/bone interface stresses. The results sustain the hypothesis that the resorptive processes are an effect of bone adaptation to stress shielding. The effects of stem flexibility are confirmed by the simulation analysis. It was also established that individual differences in bone reactivity and mechanical bone quality (density and stiffness) may account for the individual variations found in patients and animal experiments. Flexible stems reduce stress shielding and bone resorption. However, they increase proximal interface stresses. Hence, the cure against bone resorption they represent may develop into increased loosening rates because of interface debonding and micromotion. The methods presented in this paper can be used to establish optimal stem-design characteristics or check the adequacy of designs in preclinical testing procedures.     

The fate of onlay membranous bone grafts in different facial recipient sites

The fate of onlay bone grafts was studied in the growing rabbit facial skeleton with regard to the depository and resorptive characteristics of bone surfaces. The nasal snout was chosen as the depository field and the vestibular surface of the mandibular ramus as the resorptive field. The membranous bone graft applied on a depository surface maintained its volume and morphology significantly better than the graft placed on a resorptive field. There was no evident difference between the fate of full-thickness and split-thickness membranous bone grafts in the different facial recipient beds. The authors suggest that the reconstructive procedures to improve the facial skeleton contour in growing children should be planned keeping in mind the depository or resorptive characteristics of the facial recipient site.     

Membrane-bound carbonic anhydrases in osteoclasts

Osteoclasts are multinucleated bone-resorbing cells that use multiple pH regulation mechanisms to create an acidic pH in the resorption lacuna. Carbonic anhydrase II and vacuolar H(+)-ATPases produce and transport protons, while chloride channels provide a Cl(-) flux into the resorption site. These activities are required for inorganic matrix dissolution that precedes enzymatic removal of organic bone matrix. In other cell types it has become evident that carbonic anhydrase isoenzymes interact with AE proteins to form transport metabolons that regulate intracellular pH. Membrane-bound carbonic anhydrase isoenzymes may also compensate for the lack of cytoplasmic carbonic anhydrase II. Therefore, our goal was to explore the expression of membrane-bound carbonic anhydrase (CA) isoenzymes CA IV, CA IX, CA XII and CA XIV in bone-resorbing osteoclasts. Immunohistochemistry and confocal microscopy showed expression of CA IV, CA XII and CA XIV in cultured rat and human osteoclasts. To confirm these results, RT-PCR was used. Immunohistochemistry revealed distinct staining patterns for CA IV, CA XII and CA XIV in rat trabecular bone specimens. A plasma membrane staining was observed in bone lining cells with the CA XII antibody while osteoclast plasma membranes were stained with CA IV and CA XIV antibodies. Confocal microscopy of cultured human osteoclasts showed a punctated intracellular CA IV staining and a perinuclear CA XIV staining while no CA IX or CA XII staining was observed. To evaluate the physiological role of membrane-bound CAs in osteoclasts, we used PCS, a novel membrane-impermeable CA inhibitor. Increased osteoclast number and bone resorption activity was observed in rat osteoclast cultures exposed to a low concentration of PCS while higher concentrations affected cell survival. PCS treatment also disturbed intracellular acidification in osteoclasts, as determined by live cell microscopy. In conclusion, our data shows that membrane-bound carbonic anhydrase isoenzymes CA IV and CA XIV are expressed both at mRNA and protein levels in osteoclasts in vivo and in vitro. In addition, the inhibitor experiments provide novel evidence to support the hypothesis that intracellular pH regulation in osteoclasts may indeed involve transport metabolons.     

Acute effect of the carbon dioxide laser on the epicerebral microcirculation. Experimental study by fluorescein angiography

The effect of laser radiation on the central nervous system has been studied in cases of clinical and experimental tumors. However, no report yet exists on the effects of laser radiation on the cerebral microcirculation in vivo. Cerebral fluorescein angiography permits observations of small vessels that are not possible by conventional angiography. In this study, disturbance in the epicerebral microcirculation after carbon dioxide laser radiation was localized. On fluorescein angiograms, a circular zone of nonfilling of fluorescein dye around the site of impact, 1 to 1.5 mm in diameter, was seen throughout from the arterial to later venous phase. Around the nonfilling area, thrombus formation in small vessles and extravasation of the dye were demonstrated. Such extravasation of the fluorescein remained after the dye had faded from the venules and veins. Microscopically, coagulation necrosis was observed to coincide with the area of nonfillin of fluorescein dye in the fluorescein angiograms. In areas surrounding this, edema, dilatation or rupture of the capillaries, and thrombus formation in the arterioles were observed. Such areas coincided with those of extravasation of the fluorescein dye.     

Polymethylmethacrylate-induced release of bone-resorbing factors

A pseudomembranous structure that has the histological characteristics of a foreign-body-like reaction invariably develops at the bone-cement interface in the proximity of resorption of bone around aseptically loosened cemented prostheses. This study was an attempt to implicate polymethylmethacrylate in this resorptive process. Unfractionated peripheral-blood mononuclear cells (consisting of lymphocytes and monocytes) and surface-adherent cells (monocyte-enriched) were prepared from control subjects who did and did not have clinical evidence of osteoarthrosis and from patients who had osteoarthrosis and were having a revision for failure of a cemented hip or knee implant. Cells were cultured for varying periods in the presence and absence of nonpolymerized methacrylate (one to two-micrometer spherules), pulverized polymerized material, or culture chambers that were pre-coated with polymerized cement. Conditioned media that were derived from both methacrylate-stimulated cell populations were shown to contain specific bone-resorbing mediators (interleukin-1, tumor necrosis factor, or prostaglandin E2) and to directly affect bone resorption in 45Ca-labeled murine limb-bone assays.     

The fate of onlay membranous bone grafts in different facial recipient sites

Summary The fate of onlay bone grafts was studied in the growing rabbit facial skeleton with regard to the depository and resorptive characteristics of bone surfaces. The nasal snout was chosen as the depository field and the vestibular surface of the mandibular ramus as the resorptive field. The membranous bone graft applied on a depository surface maintained its volume and morphology significantly better than the graft placed on a resorptive field. There was no evident difference between the fate of full-thickness and split-thickness membranous bone grafts in the different facial recipient beds. The authors suggest that the reconstructive procedures to improve the facial skeleton contour in growing children should be planned keeping in mind the depository or resorptive characteristics of the facial recipient site.     

Acidosis Inhibits Bone Formation by Osteoblasts In Vitro by Preventing Mineralization

The negative effect of acidosis on the skeleton has been known for almost a century. Bone mineral serves an important pathophysiologic role as a reserve of hydroxyl ions to buffer systemic protons if the kidneys and lungs are unable to maintain acid-base balance within narrow physiologic limits. Extracellular hydrogen ions are now thought to be the primary activation signal for osteoclastic bone resorption, and osteoclasts are very sensitive to small changes in pH within the pathophysiologic range. Herein, we investigated the effects of acidosis on osteoblast function by using mineralized bone nodule-forming primary osteoblast cultures. Osteoblasts harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone. pH was manipulated by addition of 5 to 30 mmol/L HCl and monitored by blood gas analyzer. Abundant, matrix-containing mineralized nodules formed in osteoblast cultures at pH 7.4, but acidification progressively reduced mineralization of bone nodules, with complete abolition at pH 6.9. Osteoblast proliferation and collagen synthesis, assessed by 3H-thymidine and 3H-proline incorporation, respectively, were unaffected by pH in the range 7.4 to 6.9; no effect of acidification on collagen ultrastructure and organization was evident. The apoptosis rate of osteoblasts, assessed by the enrichment of nucleosomes in cell lysates, was also unaffected by pH within this range. However, osteoblast alkaline phosphatase activity, which peaked strongly near pH 7.4, was reduced eight-fold at pH 6.9. Reducing pH to 6.9 also downregulated messenger ribonucleic acid (mRNA) for alkaline phosphatase, but upregulated mRNA for matrix Gla protein, an inhibitor of mineralization. The same pH reduction is associated with two-and four-fold increases in Ca2+ and PO4(3-) solubility for hydroxyapatite, respectively. Our results show that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response. Thus, in uncorrected acidosis, the deposition of alkaline mineral in bone by osteoblasts is reduced, and osteoclast resorptive activity is increased in order to maximize the availability of hydroxyl ions in solution to buffer protons.     

磨损微粒诱导细胞凋亡与无菌性松动的研究进展

无菌性松动是关节置换术后最常见的远期并发症之一,限制了关节假体的使用寿命。近年来人们对假体周围界膜组织内的巨噬细胞、成骨细胞、破骨细胞及成纤维细胞作了大量的研究发现,人工关节置换产生的磨损颗粒在与界膜组织周围的细胞接触或被吞噬后可诱导假体周围界膜的慢性炎症反应并引起细胞凋亡,最终导致假体周围骨溶解以及无菌性松动的发生,提示细胞凋亡在无菌性松动中起着重要作用,细胞凋亡有可能成为无菌性松动新的治疗方法。本文就磨损颗粒引起细胞凋亡与无菌松动之间关系做一综述。     

Microcracks and Osteoclast Resorption Activity In Vitro

During bone remodeling osteoclasts resorb bone, thus removing material, e.g., damaged by microcracks, which arises as a result of physiological loading and could reduce bone strength. Such a process needs targeted bone resorption exactly at damaged sites. Osteocytic signaling plays a key role in this process, but it is not excluded that osteoclasts per se may possess toposensitivity to recognize and resorb damaged bone since it has been shown that resorption spaces are associated with microcracks. To address this question, we used an in vitro setup of a pure osteoclast culture and mineralized substrates with artificially introduced microcracks and microscratches. Histomorphometric analyses and statistical evaluation clearly showed that these defects had no effect on osteoclast resorption behavior. Osteoclasts did not resorb along microcracks, even when resorption started right beside these damages. Furthermore, quantification of resorption on three different mineralized substrates, cortical bone, bleached bone (bone after partial removal of the organic matrix), and dentin, revealed lowest resorption on bone, significantly higher resorption on bleached bone, and highest resorption on dentin. The difference between native and bleached bone may be interpreted as an inhibitory impact of the organic matrix. However, the collagen-based matrix could not be the responsible part as resorption was highest on dentin, which contains collagen. It seems that osteocytic proteins, stored in bone but not present in dentin, affect osteoclastic action. This demonstrates that osteoclasts per se do not possess a toposensitivity to remove microcracks but may be influenced by components of the organic bone matrix.     

Kinetics of dissolution of calcium hydroxyapatite powder. III: pH and sample conditioning effects

Summary The kinetics of dissolution of synthetic hydroxyapatite powder (HAP) were studied at 37°C and constant pH in the pH range 3.7–6.9 by continuously recording proton uptake and calcium release. The effect of sample conditioning was carefully investigated. The powder previously equilibrated in saturated solutions shows an initial dissolution rate higher than the one obtained when dry powder directly added to the dissolution solution is used. This effect is interpreted by considering surface state differences. As previously shown, dry powder contains important amounts of calcium and phosphate ions adsorbed onto apatite surface, ions which are desorbed during equilibration. It is assumed that the initial presence of these ions slows the dissolution rate during the first stage of the process by the formation of a permselective layer. Except for these adsorption phenomena which are less important for human enamel powder (HEP) having a lower specific surface area, it is shown that in spite of structural, morphological, and purity differences, the general dissolution behavior of HAP is quite similar to that of HEP, previously studied, and for which a quantitative model has been proposed. The dissolution rates are stirring dependent in a large range of stirring speeds and are proportional to [H⁺]^0.64. Moreover, it is shown that in the whole range of studied pH, a calcium accumulation process occurs at the interface during the first minutes of the acidic attack. It is concluded that in our experimental conditions, the dissolution process is limited by the diffusion of calcium and/or phosphate ions in the interface. The calcium-rich interface constitutes a layer of low permeability in which strong interactions considerably reduce the diffusion of calcium and/or phosphate ions released during the attack and thus considerably slows the dissolution process.     

Purification,composition, and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix

Summary Fractionation of the EDTA-soluble, noncollagenous proteins of the organic matrix of chicken bone by Sephadex G-100 molecular sieving has revealed that the majority of the organic phosphorus is present in two fractions, from one of which a homogeneous phosphoprotein has been isolated. The purified phosphoprotein has an apparent molecular weight of 12,000 and contains bothO-phosphoserine andO-phosphothreonine.³¹P-NMR spectroscopy demonstrates that all of the organic phosphorus exists in the form of phosphomonoesters which have an average pK₂ of 6.8. The phosphoprotein is highly acidic due to its high content of dicarboxylic acids in addition to the presence of organic phosphorus. The characteristic amino acid composition of the phosphoprotein establishes its noncollagenous nature and highlights the differences among bone, dentin, and enamel phosphoproteins. The absence ofγ-carboxyglutamic acid distinguishes it from osteocalcin, the noncollagenousγ-carboxyglutamic acid-containing peptide of bone matrix.