Disc in flames: Roles of TNF-α and IL-1ß in intervertebral disc degeneration.

The intervertebral disc is an important mechanical structure that allows range of motion of the spinal column. Degeneration of the intervertebral disc--incited by aging, traumatic insult, genetic predisposition, or other factors--is often defined by functional and structural changes in the tissue, including excessive breakdown of the extracellular matrix, increased disc cell senescence and death, as well as compromised biomechanical function of the tissue. Intervertebral disc degeneration is strongly correlated with low back pain, which is a highly prevalent and costly condition, significantly contributing to loss in productivity and health care costs. Disc degeneration is a chronic, progressive condition, and current therapies are limited and often focused on symptomatic pain relief rather than curtailing the progression of the disease. Inflammatory processes exacerbated by cytokines tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are believed to be key mediators of disc degeneration and low back pain. In this review, we describe the contributions of TNF-α and IL-1ß to changes seen during disc degeneration at both cellular and tissue level, as well as new evidence suggesting a link between infection of the spine and low back pain, and the emerging therapeutic modalities aimed at combating these processes.

Molecular engineering of an orthopaedic implant: from bench to bedside.

The use of metallic implants has revolutionised the practice of orthopaedic surgery. While the safety and biocompatibility of these devices are excellent, a small percentage becomes infected. These infections are due to the formation of a biofilm that harbours bacteria encased in a complex extracellular matrix. The matrix serves as a barrier to immune surveillance as well as limiting the biocidal effects of systemic and local antibiotics. The objective of the review is to describe a novel approach to controlling implant infection using an antibiotic that is linked to titanium through a self-assembled monolayer of siloxy amines. We show that the hybrid-engineered surface is stable, biocompatible and resists colonisation by bacterial species most commonly associated with implant-related infections. Studies with rodent bone infection models suggest that the engineered titanium surface prevents bone infection. Results of a very recent investigation utilising a sheep model of infection indicate that the titanium-tethered antibiotic controls infection without compromising bone formation and remodelling. From all of these perspectives, the tethered antibiotic holds promise of providing a novel and practical approach to reducing implant-associated infections.

TonEBP regulates the hyperosmotic expression of aquaporin 1 and 5 in the intervertebral disc.

The central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes.

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca2+ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro-osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Infection of normal human epithelial cells by Epstein-Barr virus.

Primary cultures of epithelial cells were grown from the tonsils and adenoids of patients with diseases not related to Epstein-Barr virus. The cells could not be infected by Epstein-Barr virus. Fluorescein-labeled Epstein-Barr virus and a cytofluorograph were then used to show that the epithelial cells do not have detectable receptors for the virus. However, implantation with Epstein-Barr virus receptors gave the cells the ability to bind the labeled virus. One to 5 percent of receptor-implanted cells exposed to the transforming B95-8 substrain of the virus expressed Epstein-Barr nuclear antigen. The early and viral capsid Epstein-Barr virus-determined antigens were not detected in the virus-infected cultures. The results show that normal human epithelial cells from the nasopharynx become susceptible to infection by Epstein-Barr virus when the membrane barrier resulting from the lack of viral receptors is overcome by receptor implantation.

Initiation of endochondral calcification is related to changes in the redox state of hypertrophic chondrocytes.

The level of pyridine nucleotides (NADH and NAD+) in the mineralizing growth plate of the chick was ascertained by high-resolution scanning microfluorimetry and biochemical analysis. Scanning electron microscopy and light microscopy were used to relate the concentrations of NADH and NAD+ to stages of chondrocyte maturation. A dramatic increase was found in the relative concentration of reduced pyridine nucleotides in the hypertrophic zone. On either side of this zone, in proliferating and calcifying cartilage, there was a decrease in NADH fluorescence, and the NADH/NAD+ ratio was depressed. The finding that NADH accumulated in the tissue zone associated with the earliest deposition of bone mineral supports the hypothesis that a change in the redox state initiates tissue mineralization.

The Impact of Incorporating Antimicrobials into Implant Surfaces.

With the increase in numbers of joint replacements, spinal surgeries, and dental implantations, there is an urgent need to combat implant-associated infection. In addition to stringent sterile techniques, an efficacious way to prevent this destructive complication is to create new implants with antimicrobial properties. Specifically, these implants must be active in the dental implant environment where the implant is bathed in the glycoprotein-rich salivary fluids that enhance bacterial adhesion, and propagation, and biofilm formation. However, in designing an antimicrobial surface, a balance must be struck between antimicrobial activity and the need for the implant to interact with the bone environment. Three types of surfaces have been designed to combat biofilm formation, while attempting to maintain osseous interactions: 1) structured surfaces where topography, usually at the nanoscale, decreases bacterial adhesion sufficiently to retard establishment of infection; 2) surfaces that actively elute antimicrobials to avert bacterial adhesion and promote killing; and 3) surfaces containing permanently bonded agents that generate antimicrobial surfaces that prevent long-term bacterial adhesion. Both topographical and elution surfaces exhibit varying, albeit limited, antimicrobial activity in vitro. With respect to covalent coupling, we present studies on the ability of the permanent antimicrobial surfaces to kill organisms while fostering osseointegration. All approaches have significant drawbacks with respect to stability and efficacy, but the permanent surfaces may have an edge in creating a long-term antibacterial environment.

RGDS peptides immobilized on titanium alloy stimulate bone cell attachment, differentiation and confer resistance to apoptosis.

A major cause of implant failure in skeletal tissues is failure of osseointegration, often due to lack of adhesion of cells to the titanium (Ti) alloy interface. Since arginine-glycine-aspartic acid (RGD)-containing peptides have been shown to regulate osteoblast adhesion, we tested the hypothesis that, bound to a Ti surface, these peptides would promote osteoblasts differentiation, while at the same time inhibit apoptosis. RGDS and RGES (control) peptides were covalently linked to Ti discs using an APTS linker. While the grafting of both RGDS and RGES significantly increased Ti surface roughness, contact angle analysis showed that APTS significantly increased the surface hydrophobicity; when the peptides were tethered to Ti, this was reduced. To evaluate attachment, MC3T3-E1 osteoblast cells were grown on these discs. Significantly more cells attached to the Ti-grafted RGDS then the Ti-grafted RGES control. Furthermore, expression of the osteoblasts phenotype was significantly enhanced on the Ti-grafted RGDS surface. When cells attached to the Ti-grafted RGDS were challenged with staurosporine, an apoptogen, there was significant inhibition of apoptosis; in contrast, osteoblasts adherent to the Ti-grafted RGES were killed. It is concluded that RGD-containing peptides covalently bonded to Ti promotes osteoblasts attachment and survival with minimal changes to the surface of the alloy. Therefore, such modifications to Ti would have the potential to promote osseointegration in vivo.

Nucleation and growth of calcium phosphate on amine-, carboxyl- and hydroxyl-silane self-assembled monolayers.

Upon implantation, calcium phosphate (Ca-P) surfaces form on materials that are bone bioactive. In this study, the evolving surface characteristics associated with calcium phosphate precipitation are modeled using self-assembled monolayers (SAMs), in a one-step nucleation process. SAMs were used to create amine (-NH2), carboxyl (-COOH) and hydroxyl (-OH) functionalized surfaces by grafting 3-aminopropyltriethoxysilane, 3-triethoxysilylpropyl succinic anhydride and glycidoxypropyl tri-methoxysilane, respectively, onto oxidized silicon wafers. The SAM surfaces were characterized using ellipsometry to establish the presence of grafted molecules. On the surfaces incubated in simulated physiological fluids for 7 days, the thickness of Ca-P layer grew slowly over the first few hours, increasing strongly between 1 and 5 days and then slowed down again. FTIR showed the dependence of calcium phosphate morphology on the type of surface groups, with stronger P-O bands seen on the OH-terminated surface. SEM analysis showed dispersed Ca-P precipitates on the -COOH and -OH terminated surfaces after 1 day immersion. After 7 days, all SAM surfaces were covered with uniformly dispersed and denser Ca-P precipitates. The underlying Ca-P layer showed cracks on the -NH2-terminated surface. Rutherford backscattering spectrometry (RBS) data analysis confirmed that Ca/P ratio is in excellent agreement with the theoretical value of 1.67 for hydroxyapatite. X-ray diffraction (XRD) analysis also showed evidence of apatite formation on all the surfaces, with stronger evidence on the -OH-terminated surface. Highly porous Ca-P precipitates were observed on the SAM surfaces portrayed by the AFM scans with nanoscale RMS roughness. Thus, using highly controlled surface chemistry, under physiological conditions, in vitro, this study demonstrates that a hydroxylated surface enhances Ca-P nucleation and growth relative to other surfaces, thereby supporting the concept of its beneficial effect on bone tissue formation and growth.

Epstein-Barr virus co-reconstituted with Sendai virus envelopes infects Epstein-Barr virus-receptor negative cells.

Epstein-Barr virus (EBV) was co-reconstituted with Sendai virus envelopes. The reconstituted "hybrid' virus could bind and penetrate into EBV-receptor negative cells. Using this approach, T-cell-derived human and mouse leukemia cells, human T-lymphocytes and mouse spleen cells were successfully infected as judged by the induction of EBV-determined antigens and stimulation of DNA synthesis. The T-cell-derived human leukemia line Molt-4, that can absorb EBV but without virus penetration, could be also infected by the reconstituted EBV.

Infection of a murine T-cell line with a retrovirus carrying c-myc decreases the interleukin-2 cell dependence by a nonautocrine mechanism.

A murine interleukin-2 (IL-2)-dependent T-cell line, CTLL-2, was infected with a retrovirus carrying the mouse c-myc cDNA and the Tn-5 neogene. Transduced cells were selected in the presence of Geneticin sulfate (G418); these cells were shown to express both the endogenous and the transduced c-myc genes. The IL-2 requirement of these cells was then found to be significantly reduced. The cells did not express IL-2 mRNA nor did they produce an activity mitogenic for CTLL-2 cells. This suggests that the reduction of IL-2 dependence observed following retroviral transduction of c-myc is caused by a nonautocrine mechanism.

Adenine, guanine, and inosine nucleotides of chick growth cartilage: relationship between energy status and the mineralization process.

The major aim of this investigation was to measure the nucleotide content of the developing chick epiphysis and to relate changes in nucleotide levels to chondrocyte maturation and the development of mineralization. Using a cryostat, sections of cartilage were isolated from the proximal head of the tibial growth cartilage, care being taken to preserve the metabolic integrity of the tissue. Sections were identified microscopically, pooled, and the nucleotide and nucleoside content of each sample determined by HPLC. Procedures used for the study were shown to minimize degradation of nucleotides. Their effectiveness was assessed through an evaluation of the rapid freezing technique and by examination of the effects of apatite on the recovery of endogenous and added nucleotides. Analysis of nucleotide levels in the growth cartilage indicated that chondrocytes undergo a profound change in energy metabolism during development and maturation. Thus, in the premineralized resting and proliferative zones, ATP and, to a lesser extent, GTP values were high, suggesting that the chondrocytes obtained metabolic energy through both glycolytic and mitochondrial oxidative processes. In the hypertrophic zone and in calcified cartilage, there was a profound decrease in the ATP concentration and a corresponding fall in the energy charge and the ATP/ADP ratios. The nucleotide levels in this zone indicated that there was increased reliance on nonoxidative metabolism. Measurement of nucleoside levels in premineralized cartilage suggested that there was little resynthesis of nucleotides through the salvage pathway. These observed changes in nucleotide values are consistent with earlier observations concerning chondrocyte redox and the low pO2 tension of the hypertrophic zone.2+off

The phosphatidylinositol-glycolipid anchor on alkaline phosphatase facilitates mineralization initiation in vitro.

Alkaline phosphatase (AP) is required for the proper mineralization of cartilage and bone. The enzyme is localized to the outer surface of cells through a phosphatidylinositol-glycolipid anchor, which is covalently attached to the carboxyl terminus of the protein. In calcifying cartilage, AP-rich matrix vesicles (MVs) are released into the matrix from chondrocytes, and apatite formation is initiated within and around these particles. In this paper we examine the role of the AP glycolipid anchor using an in vitro mineralization assay system. AP was purified to homogeneity, and the purified enzyme was used to drive mineral formation in vitro with and without the anchor. Mineral formation was initiated through phosphate release from beta-glycerol phosphate (beta-GP). The amount of PO4(-3) released was similar whether the anchor was present or absent. However, SEM and X-ray microanalysis revealed that the mineral produced by anchored AP was indistinguishable from that produced by MVs and that both of those minerals were more apatite-like than mineral formed by soluble AP or through spontaneous precipitation. Taken together, the data suggest that in addition to providing PO4(-3) to drive mineralization, AP influences the nature of the mineral formed. Further, AP containing its glycolipid anchor produces mineral comparable with that formed by tissue-derived MVs. Thus, in the absence of extracellular matrix, MV mineralization in vitro can be emulated by glycolipid-anchor containing AP.

Retinoic acid induces a shift in the energetic state of hypertrophic chondrocytes.

In the epiphyseal growth plate, chondrocyte maturation is accompanied by dramatic alterations in energy metabolism. To explore the relationship between these two events, we used retinoic acid (RA) to promote chondrocyte maturation in culture. The specific question that was addressed was, does RA treatment of cultured chondrocytes in vitro induce a change in energy status similar to that seen in hypertrophic chondrocytes in vivo. Maturing chondrocytes isolated from the cephalic region of day 18 chick embryo sterna were allowed to grow for 7-14 days in monolayer until confluent and then treated with 10-300 nM RA. Immature chondrocytes from the caudal region of sternum were grown in parallel and served as control cells for the study. We found that in maturing cephalic cell cultures, RA had a rapid and profound effect on oxidative metabolism. The retinoid caused a reduction in the energy charge ratio (ECR) and the ATP/ADP ratio and a sharp decrease in cell ATP levels. Maximum inhibition was observed when the RA concentration was 10-35 nM. Compared with the adenine nucleotides, creatine phosphate levels were decreased to a lesser extent by RA, although there was substantial inhibition of creatine kinase activity. We expected to find a compensatory elevation in glycolytic activities; however, the lactate levels in the medium of the treated cells indicated that anaerobic glycolysis was depressed. In contrast to the cephalic chondrocytes, when caudal cell cultures were treated with RA, lactate formation was stimulated and there were minimal effects on oxidative metabolism. To determine the mechanism of inhibition of glycolysis, we measured the activity of pyruvate kinase in RA-treated cephalic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide dismutase and catalase activities in the growth cartilage: relationship between oxidoreductase activity and chondrocyte maturation.

Superoxide dismutase (SOD) and catalase are enzymes that protect cells from radical attack. Catalase disproportionates hydrogen peroxide, and SOD is an oxidoreductase that serves to dismutate the superoxide anion. The objective of this communication was to measure the activity of these disproportionating enzymes in the chick tibial growth cartilage and to relate enzyme activity to chondrocyte maturation and tissue calcification. Analytic techniques were optimized for the measurement of both enzymes; particular care was taken to ensure that the values obtained were due to SOD and catalase, not to the presence of other oxidases or contaminants. Catalase and SOD had similar profiles of activity in cartilage. For both enzymes, the highest levels of activity were observed in premineralized cartilage; as chondrocytes matured there was a progressive decrease in the activity of SOD and catalase. Comparison of chondrocyte SOD activity with nonmineralizing tissues indicated that the activity of cultured cartilage cells was low. We also measured the SOD activity of avascular chondrodystrophic cartilage and found it to be less than that of proliferating cartilage. When cartilage was electrofocused, three SOD isozymes were detected. The pI of the major isozyme corresponded to the copper-zinc isoform. We suggest that the observed changes in enzymatic activity are dependent on a number of cartilage-specific factors that include the vascular supply, the local production of oxygen radicals by chondrocytes, and the oxidative state of the tissue.

Further observations on programmed cell death in the epiphyseal growth plate: comparison of normal and dyschondroplastic epiphyses.

The objective of the investigation was to provide information on apoptosis in the normal epiphysis and to assess apoptosis in the plate of the dyschondroplastic chick. Apoptosis was evaluated using two terminal deoxynucleotide transferase end-labeling procedures, DNA fragmentation and nuclear morphology. We found that there was a minimal level of apoptosis in the dyschondroplastic cartilage. In the tibial dyschondroplastic (TD) lesion itself, only about 3% of cells are positive in the articular and proliferative regions; 11% of prehypertrophic chondrocytes are stained by the end-labeling procedure, and most of the cells are localized around vascular channels at the calcifying front. This finding suggests that dyschondroplasia is linked to impairment of apoptosis, and as a result the tissue contains immature cells that have outlived their normal life span. In contrast, in the normal plate, we noted that when the proliferative period was complete, the cells became terminal transferase positive; in addition, chondrocytes in the normal plate exhibited DNA fragmentation. Semiquantitative analysis of stained chondrocytes in the growth plate indicate that in the proliferative zone 15.5% of cells are terminal deoxynucleotidyl transferase (TUNEL) positive; in contrast, 44% of postmitotic chondrocytes are stained by the TUNEL procedure. The presence of a sharp border between the pre- and postmitotic zones suggests that the stimulus for apoptosis is maturation dependent and reflects local metabolic control. We also examined apoptosis in metaphyseal osteoblasts. We found that adjacent to the epiphysis, many osteoblasts were undergoing apoptosis. In more mature sites in the metaphysis, there was less cell death, indicating that osteoblast apoptosis was delayed and cells were completing their normal life cycle. Although terminal transferase end-labeled cells were not seen in articular cartilage, we noted that fibroblasts, in the perichondrial ligament surrounding the articular as well as the epiphyseal regions of the plate, were undergoing apoptosis. Apoptosis at this site may be related to lateral expansion of the cartilages, reflect a high cell turnover rate at the junction between the tissues, and result from paracrine signals received from the underlying cartilage.

End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes.

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.

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 alpha(v)beta3 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.

Developmental regulation of creatine kinase activity in cells of the epiphyseal growth cartilage.

During the process of endochondral bone formation, the maturing chondrocyte exhibits profound changes in energy metabolism. To explore the mechanism of energy conservation in cartilage we examined the expression of creatine kinase, an enzyme that catalyzes the formation of ATP in tissues under oxygen stress. Measurement of creatine kinase activity and cytochemical assessment of enzyme distribution clearly showed that the level of enzyme activity was related to chondrocyte maturation. Thus, as the cells hypertrophied, there was a progressive increase in creatine kinase activity. Similarly, an elevation in creatine kinase activity was noted in chondrocyte cultures as the cells assumed an hypertrophic state. When cartilage calcification was disturbed by rickets, there was a decrease in enzyme activity in the hypertrophic region. Studies were performed to examine the creatine kinase isozyme profile of cells of the epiphysis. In resting and proliferating cartilage, the isoform was MM. In hypertrophic cartilage, the predominant isoforms were MB and BB. In terms of the creatine phosphate content, the highest values were seen in the proliferative region; lower amounts were present in hypertrophic and resting cartilage; and no creatine phosphate was detected in calcified cartilage. These data suggest that turnover of creatine phosphate is greatest in the mineralized region of the epiphysis. The results of these investigations point to creatine kinase as being under developmental control. The activity of the enzyme in cartilage cells should serve as a marker of developmental events associated with chondrocyte proliferation, hypertrophy, and mineralization.