Christopher S. Adams

Inorganic phosphate induces apoptosis of osteoblast-like cells in culture.

The major goal of this investigation was to test the hypothesis that one of the major products of bone resorption, inorganic phosphate (Pi), activates osteoblast apoptosis. Osteoblast-like cells were isolated from explants of human bone. In monolayer culture, these cells showed an osteogenic phenotype. Thus, the cells exhibited raised alkaline phosphatase activity, expressed osteogenic messenger RNA transcripts, and formed biological mineral. When these cells were treated with 1-7 mmol/L Pi there was a dose- and time-dependent decrease in cell viability. Accordingly, after 48 h, 5 mmol/L Pi reduced the number of viable osteoblast-like cells by 25%; 7 mmol/L Pi reduced the number of cells by 60%. By 96 h, following treatment with 5 mmol/L Pi, the percentage of viable cells was 30%, whereas 7 mmol/L Pi caused an almost complete loss of osteoblast viability. Osteoblast death was blocked by treating the cells with phosphonoformic acid, an inhibitor of the plasma-membrane Na-Pi transporter. Using morphological and end-labeling procedures, we confirmed that cell death was through apoptosis. To probe the mechanism of cell death, osteoblast-like cells were probed with rhodamine 123, a dye that is responsive to the membrane potential. We noted that Pi-treated cells displayed a profound loss of mitochondrial membrane potential, suggesting that the anion activated the death program through the induction of a mitochondrial membrane permeability transition. We conclude that high levels of osteoblast apoptosis observed at sites of bone resorption may be linked to release of Pi from bone mineral.

Matrix Regulation of Skeletal Cell Apoptosis ROLE OF CALCIUM AND PHOSPHATE IONS

Previously, we noted that inorganic phosphate (Pi), a major component of bone extracellular matrix, induced osteoblast apoptosis (Meleti, Z., Shapiro, I. M., and Adams, C. S. (2000) Bone (NY) 27, 359–366). Since Ca2+ along with Pi is released from bone during the resorption process, we advanced the hypothesis that Ca2+ modulates Pi-mediated osteoblast apoptosis. To test this hypothesis, osteoblasts were incubated with both ions, and cell death was determined. We noted that a modest increase in the medium Ca2+ concentrations ([Ca2+] e ) of 0.1–1 mm caused a profound and rapid enhancement in Pi-dependent death of cultured osteoblasts. An elevation in [Ca2+] e alone had no effect on osteoblast viability, whereas Ca2+ channel blockers failed to inhibit killing of ion pair-treated cells. These results indicated that Pi-mediated cell death is not dependent on a sustained increase in the cytosolic Ca2+ concentration. Terminal dUTP nick-end labeling analysis and measurement of caspase-3 activity of the ion pair-treated cells suggested that death was apoptotic. Apoptosis was confirmed using caspase-3 and endonuclease inhibitors. The mitochondrial membrane potential and cytosolic Ca2+ status of the treated cells were evaluated. After incubation with [Ca2+ ] e and Pi, a decrease in mitochondrial fluorescence was noted, suggesting that the ions decreased the mitochondrial transmembrane potential. Subsequent to the fall in mitochondrial membrane potential, there was a transient elevation in the cytosolic Ca2+ concentration. Results of the study suggest that the ion pair conspire at the level of the plasma membrane to induce intracellular changes that result in loss of mitochondrial function. The subsequent increase in the cytosolic Ca2+ concentration may trigger downstream events that transduce osteoblast apoptosis.

Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism

In a previous investigation we showed that phosphate ions (Pi) induced apoptosis of terminally differentiated hypertrophic chondrocytes. To explore the mechanism by which Pi induces cell death, we asked the following two questions. First, can we prevent Pi-induced apoptosis by inhibiting plasma membrane Na-Pi cotransport? Second, which specific Na-Pi transporters are expressed in chondrocytes and are they developmentally regulated? Terminally differentiated hypertrophic chondrocytes were isolated from chick tibial cartilage and cell death was measured in the presence of 3-7 mmol/L Pi. To ascertain whether apoptosis was linked to a rise in cellular Pi loading, we examined the effect of phosphonoformic acid (PFA), a competitive inhibitor of Na-Pi cotransport on Pi-induced apoptosis in chondrocytes. We found that 1 mmol/L PFA blocked anion-induced cell death and prevented an increase in the cell Pi content. In a parallel study, we determined that the bisphosphonate, alendronate, also protected chondrocytes from death, albeit at a lower concentration than PFA. Using a DNA end-labeling procedure, we showed that the Pi-treated cells were apoptotic and, as might be predicted, the presence of PFA blocked induction of the death sequence. Next, we examined the expression of two Pi transporters in relation to chondrocyte maturation and anion treatment. We noted that there was expression of the constitutive transporter, Glvr-1, and a type II cotransporter in chick growth plate cells. Although these transport systems are active in terminally differentiated cells, it is probable that the initiation of apoptosis may require the induction of other Pi-transport systems. It is concluded that, at the mineralization front, cell death is linked directly to the elevation in environmental anion concentration and the concomitant rise in intracellular Pi levels.

The Transforming Growth Factor-β-inducible Matrix Protein βig-h3 Interacts with Fibronectin

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, βig-h3, is a highly conserved transforming growth factor-β-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length βig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on βig-h3-coated plates and developed actin stress fibers. Purified βig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the βig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of βig-h3 in the ECM.

RGD Peptides Immobilized on a Mechanically Deformable Surface Promote Osteoblast Differentiation

The major objective of this work was to attach bone cells to a deformable surface for the effective transmission of force. We functionalized a silastic membrane and treated it with 3‐aminopropyltriethoxysilane (APTS). A minimal RGD peptide was then covalently linked to the aminated surface. MC3T3‐E1 osteoblast‐like cells were cultured on the arginine‐glycine‐aspartic acid (RGD)‐treated membrane for 3–15 days and cell attachment and proliferation was evaluated. We observed that cells were immediately bound to the membrane and proliferated. After 8 days on the material surface, osteoblasts exhibited high levels of ALP staining, indicating that the cells were undergoing maturation. Alizarin red staining and Fourier transform infrared (FTIR) analysis showed that the mineral formed by the cells was a biological apatite. The second objective was to apply a mechanical force to cells cultured on the modified silicone membrane. Dynamic equibiaxial strain, 2% magnitude, and a 0.25‐Hz frequency were applied to bone cells for 2 h. Osteoblasts elicited increased phalloidin fluorescence, suggesting that there was reorganization of the cytoskeleton. Furthermore, the applied strain elicited increased expression of the αvβ3 integrin receptor. We concluded that the covalent binding of RGD peptides to a silicone membrane provides a compatible surface for the attachment and subsequent differentiation of osteoblasts. Moreover, the engineered surface transduces applied mechanical forces directly to the adherent cells via integrin receptors.

Effect of functional end groups of silane self assembled monolayer surfaces on apatite formation, fibronectin adsorption and osteoblast cell function

Bioactive glass (BG) can directly bond to living bone without fibrous tissue encapsulation. Key mechanistic steps of BGs activity are attributed to calcium phosphate formation, surface hydroxylation and fibronectin (FN) adsorption. In the present study, self‐assembled monolayers (SAMs) of alkanesilanes with different surface chemistry (OH, NH2 and COOH) were used as a model system to mimic BGs surface activity. Calcium phosphate (Ca‐P) was formed on SAMs by immersion in a solution that simulates the electrolyte content of physiological fluids. FN adsorption kinetics and monolayer coverage was determined on SAMs with or without Ca‐P coating. The surface roughness was also examined on these substrates before and after FN adsorption. The effects of FN‐adsorbed, Ca‐P‐coated SAMs on the function of MC3T3‐E1 were evaluated by cell growth, expression of alkaline phosphatase activity and actin cytoskeleton formation. We demonstrate that, although the FN monolayer coverage and the root mean square (rms) roughness are similar on OH and COOH terminated SAMs with or without Ca‐P coating, higher levels of ALP activity, more actin cytoskeleton formation and more cell growth are obtained on OH‐ and COOH‐terminated SAMs with Ca‐P coating. In addition, although the FN monolayer coverage is higher on Ca‐P‐coated NH2‐terminated SAMs and SiOx surfaces, higher levels of ALP activity and more cell growth are obtained on Ca‐P‐coated OH‐ and COOH‐terminated SAMs. Thus, with the same Ca‐P coatings, different surface functional groups have different effects on the function of osteoblastic cells. These findings represent new insights into the mechanism of bioactivity of BG and thereby may lead to designing superior constructs for bone grafting. Copyright

Bone Cell Autophagy Is Regulated by Environmental Factors

The goal of this investigation was to ascertain whether bone cells undergo autophagy and to determine if this process is regulated by environmental factors. We showed that osteocytes in both murine and human cortical bone display a punctuate distribution of microtubule-associated protein light chain 3, indicative of autophagy. In addition, we noted a basal level of autophagy in preosteocyte-like murine long bone-derived osteocytic (MLO)-A5 cells. Autophagy was upregulated following nutrient deprivation and hypoxic culture, stress conditions that osteocytes encounter in vivo. Furthermore, in response to calcium stress, the transcription factor hypoxia inducible factor 1 regulated MLO-A5 autophagy. Finally, we showed that the more differentiated MLO-Y4 osteocyte-like cells exhibited a significant basal autophagic flux. Based on these findings, we suggest that raising the level of autophagic flux is a mechanism by which differentiated bone cells survive in a stressful environment.

Tetracycline tethered to titanium inhibits colonization by Gram‐negative bacteria

As peri-prosthetic infection is one of the most devastating complications associated with implant placement, we have reasoned that such infection can be largely subverted by development of antibacterial implants. Our previous work demonstrated that covalent coupling of vancomycin to titanium alloy prevented colonization by the Gram-positive pathogens, Staphylococcus aureus and Staphylococcus epidermidis. Some orthopedic devices, including permanent prosthesis anchors, and most dental implants are transcutaneous or transmucosal and can be prone to colonization by Gram-negative pathogens. We report here the successful covalent coupling of the broad-spectrum antibiotic, tetracycline (TET), to titanium surfaces (Ti-TET) to retard Gram-negative colonization. Synthetic progress was followed by changes in water contact angle, while the presence of TET was confirmed by immunofluorescence. Ti-TET actively prevented colonization in the presence of bathing Escherichia coli, both by fluorescence microscopy and direct counting. Finally, the Ti-TET surface supported osteoblastic cell adhesion and proliferation over a 72-h period. Thus, this new surface offers a powerful means to protect transcutaneous implants from adhesion of Gram-negative pathogens, decreasing the need for replacement of this hardware.