Puzas Je

Increased Levels of Tumor Necrosis Factor-α and Interleukin-6 Protein and Messenger RNA in Human Peripheral Blood Monocytes due to Titanium Particles*

Cytokines produced by macrophages in the periprosthetic membranes surrounding joint replacements have been implicated as causal agents in osteolysis and prosthetic loosening. The present study characterizes the response of human peripheral blood monocytes to titanium particles. Monocytes were obtained from volunteers and blood that had been donated to the American Red Cross and were cultured in the presence of titanium particles (one to three micrometers in diameter). There were consistent dose-dependent increases in the production of TNF-&agr; (tumor necrosis factor-&agr;) and IL-6 (interleukin-6) protein, with the greatest stimulation generally observed with a concentration of 6 x 105 to 6 x 106 particles of titanium per milliliter. The level of TNF-&agr; was the greatest (fifty to 1000 times greater than the control level) after eight hours of exposure to titanium particles; the level of IL-6 was two to five times greater than the control level after sixteen hours of exposure. These increases were similar to those observed after stimulation with lipopolysaccharide and depended on de novo synthesis rather than on release from intracellular stores. The production of TNF-&agr; was inhibited in a dose-dependent manner by the translational inhibitor cycloheximide and the transcriptional inhibitor actinomycin D, indicating the requirement for both mRNA (messenger RNA) and protein synthesis for the induction of cytokine synthesis by titanium particles. Although the increase in the levels of cytokine mRNA in response to titanium was rapid (thirty to ninety minutes), the increase in the level of TNF-&agr; mRNA preceded that of IL-6 mRNA. The level of TNF-&agr; mRNA was the greatest at ninety minutes and the level of IL-6 mRNA was the greatest at three hours. After stimulation with titanium particles, the level of TNF-&agr; mRNA was increased as much as fivefold and the level of IL-6 mRNA, as much as twelvefold. CLINICAL RELEVANCE: Awareness of the importance of wear debris particles in cytokine-induced bone resorption has resulted in improvements in the designs of implants and in operative techniques to reduce wear of components. The present study further elucidates the biological mechanisms involved in periprosthetic osteolysis. Titanium-stimulated biosynthesis of the cytokines TNF-&agr; and IL-6, which are both potent stimulators of bone resorption, requires increases in the synthesis of both mRNA and protein by monocytes. An understanding of the complex mechanisms of the induction of cytokine synthesis by particles of wear debris will facilitate the design of pharmacological agents to control periprosthetic bone resorption. These agents, in combination with other efforts to reduce the generation of wear debris, may improve the longevity of orthopaedic implants.

Pathologic bone formation.

The literature on pathologic bone formation is reviewed first on the basis of clinical syndromes and second in relation to newer knowledge of the origin of the bone-forming cells and regulatory molecules. Pathologic bone formation can be categorized into three groups based on the initiating stimulus: trauma, tumors, and idiopathic causes. In the trauma category, the formation of ectopic bone is concerned with major and minor traumatic incidents, surgery, burns, and other causes. In the tumor category, direct and reactive pathologic bone formation is discussed with an emphasis on the different neoplasms capable of ectopic bone formation. The category of idiopathic causes involves the formation of pathologic bone following neurologic injury and in systemic ossification disorders. The origin of the bone-forming cells in all forms of pathologic bone has not been unequivocally determined. However, there is evidence suggesting that these cells may arise from osteogenic stromal elements. Potent bone formation growth-regulating factors have been recently identified, and these molecules must also participate in the formation of pathologic bone. Increased understanding of the processes that control pathologic bone formation will lead to better methods of preventing and treating disorders of ossification.

Transforming Growth Factor Beta: An Autocrine Regulator of Chondrocytes

Transforming growth factor beta (TGF-beta) is a ubiquitous regulator of cellular growth and differentiation. The present study investigated the effects of TGF-beta on chick growth plate chondrocyte proliferation, matrix synthesis, and alkaline phosphatase activity in short term cultures. TGF-beta markedly stimulated DNA synthesis in a dose-dependent manner, while collagen synthesis and cellular and matrix vesicle alkaline phosphatase activity were inhibited. Biologic effects of TGF-beta were correlated with binding to specific receptors, and both high and low affinity receptors were identified. Countercurrent centrifugal elutriation was used to fractionate growth plate chondrocytes to obtain populations of cells in different stages of maturation (effectively from different zones of the growth plate). TGF-beta showed increasing mitogenicity with increasing cellular maturation in the growth plate, with maximal stimulation in the proliferating and early hypertrophic cells. The smallest cells expressed only the high affinity receptor, while with hypertrophy there was increasing expression of the low affinity receptor and a progressive increase in the number of both receptors per cell. Furthermore, the dose-response curves for TGF-beta-stimulated DNA synthesis were not biphasic in the smaller cells, but became progressively more biphasic with cellular hypertrophy and expression of the low affinity receptor. Finally, TGF-beta activity was identified in partially purified chondrocyte conditioned medium by specific bioassay, indicating TGF-beta production by growth plate chondrocytes. The data suggests a potentially important autocrine function for TGF-beta in modulating chondrocyte proliferation and matrix synthesis in endochondral calcification.

Modulation of the Production of Cytokines in Titanium-Stimulated Human Peripheral Blood Monocytes by Pharmacological Agents. The Role of cAMP-Mediated Signaling Mechanisms*†

Cytokines secreted by activated macrophages play a role in the development of osteolysis adjacent to prosthetic joints. To determine whether the synthesis of cytokines can be inhibited by pharmacological agents, we studied the role of the cAMP-protein kinase A signal transduction pathway in the synthesis of interleukin-6 and tumor necrosis factor-&agr; and examined the effect of potential pharmacological regulators of this pathway in human peripheral blood monocytes stimulated with titanium particles. Dibutyryl cAMP enhanced the synthesis of interleukin-6 by titanium-stimulated monocytes and resulted in a marked increase (maximum, seventyfold) in the synthesis of interleukin-6 even in the absence of titanium particles. However, the active analogs (agonists) of cAMP, dibutyryl cAMP and Sp cAMP, inhibited the production of tumor necrosis factor-&agr; by titanium-stimulated monocytes (the maximum effects resulted in complete inhibition), while the cAMP antagonist, Rp cAMP, enhanced the production of tumor necrosis factor-&agr;. Additional agents that alter the intracellular levels of cAMP were examined for their effects on the synthesis of cytokines. Prostaglandins E1 and E2 were potent inhibitors of the synthesis of tumor necrosis factor-&agr; but stimulated the synthesis of interleukin-6. In contrast, indomethacin enhanced the stimulatory effects of titanium particles on tumor necrosis factor-&;, resulting in a more than threefold increase in the maximum levels of tumor necrosis factor-&agr;. Phosphodiesterase inhibitors, such as isobutyryl methylxanthine and pentoxifylline, which increase intracellular levels of cAMP, caused a decrease in the production of tumor necrosis factor-&agr; and an increase in the production of interleukin-6. In contrast, the fluoroquinolone antibiotic ciprofloxacin, which is also a phosphodiesterase inhibitor, caused a dose-dependent inhibition of the synthesis of both tumor necrosis factor-&agr; and interleukin-6 by titanium-stimulated monocytes, suggesting that ciprofloxacin suppresses the synthesis of interleukin-6 through a mechanism that is independent of cAMP. CLINICAL RELEVANCE: Pharmacological agents, in combination with efforts at reducing the generation of wear debris, may lead to novel therapeutic strategies to prevent the loosening of implants. Alteration of the synthesis of cytokines in response to pharmacological manipulation of the cAMP-protein kinase A signaling pathway suggests that these events are regulated by normal physiological mechanisms in titanium-stimulated human peripheral blood monocytes rather than by an irreversible toxic response. The differential response of tumor necrosis factor-&agr; and interleukin-6 to agents that alter intracellular levels of cAMP indicates that these two cytokines are independently regulated. Thus, pharmacological strategies to inhibit the release of cytokines may need to involve multiple agents to inhibit diverse regulatory pathways. Basic-science studies identifying the pathways involved in titanium-mediated synthesis of cytokines are essential for the efficient design and selection of inhibitory agents.

Effect of transforming growth factor-β on DNA synthesis by growth plate chondrocytes: Modulation by factors present in serum

SummaryChondrocytes in the growth plate undergo rapid proliferation during the process of enchondral ossification. The factors that regulate this proliferative activity have not been defined although several local autocrine or paracrine growth factors have recently been discovered in cartilage. Transforming growth factor-β1 (TGF-β) is an important regulator of cell metabolism and growth and is present in cartilage. The present study was designed to examine the influence of TGF-β on DNA synthesis in chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% FBS, and after 24 hours in monolayer culture, were treated with TGF-β caused a biphasic dose-dependent increase in thymidine incorporation. The effect was greatly increased in serum-containing cultures where a maximal 20-fold increase in thymidine incorporation occurred compared with the 21/2-fold maximal increase obtained in serum-free cultures. The effect was present by 12 hours and maximal between 0.3 and 1.0 ng/ml of TGF-β. Higher concentrations of TGF-β were less stimulatory. The serum enhancement was dependent upon the simultaneous presence of TGF-β and serum factors and was abolished when chondrocytes were plated and exposed to TGF-β in medium containing dialyzed FBS (12–14 kD MW membrane). The nature of the uptake and incorporation of thymidine into DNA by individual chondrocytes appeared to be the same in both TGF-β-treated and control cultures as the apparent Kms were similar. The present study indicates that TGF-β increases DNA synthesis by growth plate chondrocytes. The effect is enhanced by factors present in serum.

Pretreatment of bone with osteoclasts affects phenotypic expression of osteoblast-like cells.

Implant surface morphology regulates osteoblast phenotypic expression. Osteoblast sensitivity to non‐biologic surfaces suggests that native bone surface features may also affect osteoblast response. To test this, MG63 osteoblast‐like cells were grown for 7 days on bovine cortical bone wafers pretreated with rat bone marrow osteoclasts for 0, 10 or 20 days. Response to osteoclast‐treated surfaces was compared to the response of MG63 cells to titanium surfaces with smooth and rough microtopographies. Cell number, differentiation (alkaline phosphatase activity and osteocalcin levels), and local factors (PGE2 and TGF‐β1) were measured in confluent cultures. Compared to culture on plastic, cell number was reduced on all three types of bone wafers; this effect was dose‐dependent with increasing resorption of the surface. Alkaline phosphatase specific activity was increased (P ⩽ 0.05) on all three surfaces compared with plastic, but this increase was not dependent on resorption time, indicating this parameter was sensitive to the surface (bovine bone vs. plastic) but not to osteoclast‐resorption. There was a direct correlation between the area of the bone surface resorbed and the amount of osteocalcin, TGF‐β1 and PGE2 (R2 = 0.8025, 0.8689, 0.8896, respectively). With 20 days of osteoclast pretreatment, there was a 20‐fold increase in osteocalcin over plastic and a 7‐fold increase over cultures on untreated bone wafers. Similar increases were found for TGF‐β1 and PGE2. Thus, surface changes resulting from osteoclast pretreatment have a strong effect on osteoblast phenotypic expression, and suggest that microtopography may play a role.

Targeted Deletion of Minpp1 Provides New Insight into the Activity of Multiple Inositol Polyphosphate Phosphatase In Vivo

ABSTRACT Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1,Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed inMinpp1-deficient mice. Biochemical analyses demonstrate that InsP5 and InsP6 are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates inMinpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP5 and InsP6. These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP5 and InsP6 is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.

Preliminary findings on the effects of sex hormones on bone metabolism in anorexia nervosa

We performed tetracycline double-labeled bone biopsies and serial bone densitometry on four patients with anorexia nervosa to determine the effects of various conditions of sex hormone replacement and body weight status. We found negligible bone mineral apposition rate in two patients with anorexia nervosa who were taking estrogen and progestin pills while weighing only 79% of normal. However, bone mineral apposition rate was normal in two patients with anorexia nervosa who took no sex hormones and remained amenorrheic while weighing 87% of normal. Histomorphometric analysis revealed a decrease in intracortical trabecular area by 57% and an increase in active osteoblastic surface area by 300%, comparing the subjects who were taking hormone replacement to those who were not. There was no direct evidence of increased resorption in any subject. Six months after biopsy, the two subjects taking sex hormones decreased lumbar bone mineral density by 1.9%, while the two taking no sex hormones remained amenorrheic, but increased bone mineral density by 1.3%. Therefore, we propose that sex hormone therapy in the presence of persistently low body weight might be of limited benefit for bone mineral apposition and density.

The Effect of Bone Cell Stimulatory Factors Can be Measured with Thymidine Incorporation Only Under Specific Conditions

SummaryThis study examines the efficacy of using radiolabeled thymidine as a measure of bone cell DNA synthesis. The effect of a bone-active peptide on cell proliferation is assessed under different labeling conditions and it is shown that the apparent stimulation in DNA synthesis is due to an increase in participating cells and not to labeling artifacts. In another series of experiments we demonstrate how the use of carrier-free thymidine can cause erroneous results. From these data it is shown that only 10–28% of the cells in culture are participating in DNA synthesis. Therefore, under specific conditions, a 100% stimulation in thymidine incorporation by a mitogenic factor can be caused by as little as a 10% increase in the number of DNA synthesizing cells.

Countercurrent centrifugal elutriation. High-resolution method for the separation of growth-plate chondrocytes.

Countercurrent centrifugal elutriation was used to separate, on the basis of size, isolated growth-plate chondrocytes in chicks. The mean cellular volume, activity of alkaline phosphatase, and synthesis of type-X collagen increased progressively in each of seven successive fractions. Slices of tissues that contained either proliferating or hypertrophic chondrocytes were also removed by manual dissection from the superficial and deep regions of the growth plate. These cells demonstrated differences in size and biochemistry that were similar to those observed in chondrocytes that were separated by elutriation. These differences included increased synthesis of proteoglycan and collagen in the larger chondrocytes. Radiolabeled hypertrophic chondrocytes were mixed with unlabeled resting and proliferating chondrocytes, and then were separated by elutriation. The radioactivity was recovered in fractions that contained the largest cells, confirming that differences in the sizes of the cells can be used to effect a zonal separation by centrifugal elutriation.