Cytokines and proteases are secreted by fibroblasts in response to particulate wear debris, and these proteins are felt to play an important role in the development of osteolysis and implant loosening. Although metallic and polyethlyene debris have been studied extensively, little is known about the cellular responses to hydroxyapatite, despite the wide clinical use of these materials. Therefore, the effects of hydroxyapatite (HA) and hydroxyapatitelp-tricalciumphosphate (HARCP) on cellular proliferation, cytokine gene expression and protein secretion, protease synthesis, and gelatinolytic activity were investigated in human fibroblasts.HA and HMTCP particles were synthesized, and their effects were compared to the responses elicited by titanium and cobalt chromium. Sample characterization by scanning electron microscopy and Coulter Counter demonstrated that the materials had a mean particle size of less than 10 pm, and all of the particles were compared using the same concentration ranges.Aliquots of particle suspensions were added to human fibroblasts maintained in tissue culture, and dose-response and timecourse experiments were performed. Effects of the particles on fibroblast proliferation were assessed, and alterations in cytokine levels were determined by specific enzyme linked immunosorbent assays (ELISA). Cytokines that were evaluated included interleukin-1 (IL-1 p), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a), all of which have been demonstrated to enhance bone resorption and are associated with osteolysis and implant loosening. Gene expression was determined using Northern blot analysis with cytokine-specific probes, while secretion of the proteases collagenase and stromelysin was determined by Western blot analysis.Functional gelatinolytic assay was assessed using zymogram gels.The particles were evaluated in a concentration range from 0.000021 to 0.021 ~01%. All of the particles produced increases in cellular proliferation up to 0.0021 vol%, with the largest increases being seen at 0.021 vol% with HA/TCP and titanium. At the highest concentration, both cobalt chromium and HA samples decreased cellular proliferation relative to lower doses, possibly representing cytotoxicity.Hydroxyapatite particles yielded a 30-fold increase in interleukin-6 secretion compared to unstimulated controls, which was also greater than three times the levels produced by cobalt chromium, titanium, or HAITCP. HA particles also tripled the secretion of IL-Ip at 0.00021 vol%, and doubled TNF-a secretion at 0.021 ~01%. Addition of conditioned media prepared by incubation of the particles in culture medium in the absence of cells did not alter the secretion of any of the cytokines. Northern blot analysis using IL-6 probes also demonstrated strong increases with HA compared to the other materials, suggesting that the action of the HA particles was at the level of transcription. Secretion of the protease collagenase was increased by all of the samples including HA when compared to unstimulated controls. Stro...

Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystal deposition diseases are a group of heterogeneous arthritides which are a significant source of morbidity in the elderly. Both crystals induced mitogenesis and metalloproteinase (MP) synthesis and secretion by fibroblasts and chondrocytes which may promote degradation of intra-articular tissue. We have previously shown that phosphocitrate (PC), an inhibitor of hydroxyapatite crystallization, specifically blocks BCP crystal-induced mitogenesis in 3T3 cells. This led us to examine the effect of PC on BCP and CPPD crystal induction of MP synthesis in human fibroblasts. PC (10(-3) to 10(-4) M) specifically inhibited the crystal-induced collagenase and stromelysin mRNA accumulation while having no effect on epidermal growth factor-induced or basal levels of mRNA for both enzymes. Western blots (collagenase) of conditioned media confirmed that PC blocked crystal-induced proteinase secretion as well. Moreover, PC (10(-3) M) also blocked the crystal induction of c-fos and c-jun. Since FOS and JUN proteins form a transacting activator (AP-1) for expression of collagenase and stromelysin genes, PC may block the synthesis of both enzymes by inhibiting the transcription of c-fos and c-jun.

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.

Synovial fluid basic calcium phosphate crystals (BCP) are often found in severely degenerated joints. Crystalline BCP is a growth factor stimulating fibroblast mitogenesis and acting as a competence factor similar to platelet-derived growth factor. In human fibroblasts (HF), the synthesis of collagenase and stromelysin is coordinately induced after stimulation with a variety of cytokines and growth factors. We sought to determine whether BCP, like other growth factors, might induce proteases that would damage articular tissue. Northern blot analysis of mRNA for collagenase and stromelysin in HF stimulated with BCP was performed. Secreted enzymes were analyzed by immunoblot using a monoclonal antibody to collagenase and by immunoprecipitation using a polyclonal antibody to stromelysin. Stromelysin activity was confirmed using casein substrate gels. A significant, dose-dependent accumulation of collagenase and stromelysin message was evident after 4 h and continued for at least 24 h in BCP-stimulated cultures. Forty-nine and 54 kD proteins immunoreacting with collagenase antibody were identified in the conditioned media (CM) from BCP-stimulated cultures while 50 and 55 kD proteins were identified by immunoprecipitation with stromelysin antibody. Collagenase activity was increased significantly in the CM from BCP treated cells; casein substrate gels showed casein degrading bands at molecular weights consistent with stromelysin. BCP stimulates coordinate induction of collagenase and stromelysin which may mediate the joint destruction associated with these crystals.

Objective. To investigate BCP (basic calcium phosphate) crystal-stimulated mitogenesis and collagenase gene transcription in primary cultures of porcine chondrocytes.Methods. The role of protein kinases in BCP crystal4imulated DNA synthesis was investigated using thymidine incorporation, kinase inhibitors, and protein kinase C (PKC) assays. Northern blot analysis was used to determine the levels of collagenase c-fos and c-jun message.Results. BCP crystals stimulated chondrocyte proliferation in a PKC-dependent manner. Increased levels of collagenase message were preceded by an increased accumulation of c-fos, but not c-jun.Conclusion. BCP crystals could contribute to the abnormal chondrocyte proliferation and collagenase secretion observed in some rheumatic diseases.Intraarticular deposition of basic calcium phosphate (BCP) crystals (hydroxyapatite, octacalcium phosphate, and tricalcium phosphate) and/or calcium pyrophosphate dihydrate (CPPD) crystals is associated with a number of destructive arthropathies involving cartilage degeneration (1,2). Collagenase activity has been demonstrated in the synovial fluid of patients with BCP crystal deposition disease (3,4), although Dieppe et a1 (5) were unable to show an increase in collagenase activity in synovial fluids from a series of patients with crystal deposition disease of the shoulder ("idiopathic destructive disease of the shoulder"). Both CPPD crystals (6,7) and BCP crystals (6)(7)(8)(9) are commonly found in osteoarthritic cartilage and have occasionally been observed within chondrocytes (6,9).It has previously been demonstrated that cultured human rheumatoid synovial cells or normal canine synovial cells will endocytose BCP or CPPD crystals (10). Crystal endocytosis was followed by the release of prostaglandins and, in addition, collagenase and neutral protease activity (11,12). It has also been demonstrated that BCP crystals are mitogenic in cultures of human foreskin fibroblasts, canine synovial fibroblasts (13), and murine fibroblasts (14). Crystalstimulated mitogenesis has been postulated to be a contributing factor in the synovial hyperplasia associated with BCP or CPPD crystal deposition diseases (1 3,15).Earlier studies demonstrated that the time to peak BCP crystal-stimulated thymidine uptake in fibroblasts was delayed by 2-3 hours compared with the peak uptake time using 10% serum (13). In addition, solubilization of the crystals was necessary in order for mitogenesis to proceed (16). In fibroblasts, BCP crystals acted similarly to a competence growth factor (17), since insulin-like growth factor 1 (IGF-1) was required in order to elicit a full mitogenic response (18). BCP crystals also induced accumulation of the competence genes c-fos and c-myc, in a protein kinase C (PKCwependent manner, in BALBk-3T3 mouse fibroblasts (14).The proliferative effects of BCP crystals on