title: Effects of hydroxyapatite particulate debris on the production of cytokines and proteases in human fibroblasts.
abstract: 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 hydroxyapatite/beta-tricalciumphosphate (HA/TCP) on cellular proliferation, cytokine gene expression and protein secretion, protease synthesis, and gelatinolytic activity were investigated in human fibroblasts. HA and HA/TCP 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 microm, 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 time-course 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-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), 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 vol%. 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 HA/TCP. HA particles also tripled the secretion of IL-1beta at 0.00021 vol%, and doubled TNF-alpha secretion at 0.021 vol%. 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. Stromelysin secretion into the culture medium was decreased by cobalt chromium, but increased by titanium, HA, and HA/TCP. All of the particles including HA increased the gelatinolytic activity of the fibroblasts. These findings demonstrate that HA and HA/TCP particles are capable of stimulating the expression and secretion of cytokines and proteases that enhance bone resorption, and suggest that particulate debris from implants using these coatings may also increase osteolysis and loosening.
authors.affiliation: Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee, USA. jninomi@ibm.net
authors.forename: J T
authors.initials: JT
authors.lastname: Ninomiya
authors.forename: J A
authors.initials: JA
authors.lastname: Struve
authors.forename: C T
authors.initials: CT
authors.lastname: Stelloh
authors.forename: J M
authors.initials: JM
authors.lastname: Toth
authors.forename: K E
authors.initials: KE
authors.lastname: Crosby
mesh_terms: D001672:Biocompatible Materials / Q000633:toxicity*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D017364:Collagenases / Q000032:analysis / Q000378:metabolism*; D016207:Cytokines / Q000032:analysis / Q000235:genetics*; D017886:Durapatite / Q000633:toxicity*; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000032:analysis / Q000235:genetics; D015850:Interleukin-6 / Q000032:analysis / Q000235:genetics; D019278:Matrix Metalloproteinase 3 / Q000032:analysis / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D011475:Prosthesis Failure; D012333:RNA, Messenger / Q000032:analysis; D012867:Skin / Q000166:cytology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics
chemical_list: D001672:Biocompatible Materials; D016207:Cytokines; D007375:Interleukin-1; D015850:Interleukin-6; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D017886:Durapatite; D017364:Collagenases; D019278:Matrix Metalloproteinase 3