The Effects Of Substance P On The Fibroblasts From Hip Periprosthetic Membrane

Aseptic loosening of the implant following total joint arthroplasty remains a major long-term complication that results from periprosthetic osteolysis.Resorption of bone is associated with a chronic inflammatory response to implant wear debris accumulating at the prosthetic interface.The inflammatory response manifests as a granulomatous membrane between the implant and bone that is infiltrated with macrophages,fibroblasts,and giant cells.Chiba et al. studied interfacial tissues from failed cementless total hip replacements and found greater levels of tumor necrosis factor-alpha (TNF-a), interleukin (IL-1) and IL-6 in hips with osteolysis. Increased expression of IL-1, IL-6,and TNF-a has been reported in the macrophages and fibroblasts in the pseudomembrane in patients with hip arthroplasties. Particles from different prosthetic materials activate macrophages and induce secretion of proinflammatory cytokines such as IL-1, IL-6, prostaglandin E2 and TNF-a. These inflammatory cytokines in the tissue surrounding failed implants have been implicated as factors in bone resorption. It has been shown that the expression of RANKL is elevated in the tissues surrounding failed prostheses, Fibroblasts in the periprosthetic membrane express RANKL. In response to wear particles, fibroblasts express RANKL and stimulate osteoclast formation.Macrophages compose -15% of the cells in the inflammatory membrane. the macrophage has been extensively studied as a mediator of osteolysis. In contrast, there is much more limited information regarding the role of other cells, especially fibroblasts, which compose -70% of the cells in the periprosthetic membrane. Recent information suggests that synovial fibroblasts present in the periprosthetic membrane are important targets of wear debris during osteolysis.The nervous system has been implicated in the etiology and pathogenesis of joint diseases. M. Ahmed has shown that the interface membrane in aseptic loose cemented hip prostheses is supplied by sensory nerve fibres immunoreactive to SP. SP-immunoreactive nerve fibers have also been detected in the pseudocapsular tissues.Therefore we hypothesized that fibroblasts may play an important role in periprosthetic osteolysis, and that SP might stimulate secretion of cytokines in fibroblasts in the periprosthetic membrane and cause increase in cytokines production by titanium-stimulated fibroblasts, thus contributing to periprosthetic osteolysis. We compared levels of substance P (SP) in pseudosynovial fluid from patients with aseptic loosening after THA with those in synovial fluid from patients undergoing primary THA for osteoarthritis (control). SP levels were measured using an enzyme immunoassay. We found that SP levels were significantly higher in pseudosynovial fluid of loose artificial joints than in synovial fluid controls .We isolated fibroblasts from periprosthetic membrane at the time of revision hip arthroplasty performed due to aseptic loosening. Fibroblasts were incubated for 24 hours in the presence of various concentrations of SP, and the levels of IL-1βand TNF-αin the media were determined using ELISA kit.the levels of IL-1βand TNF-a in the media increased in a time- and concentration-dependent manner.Fibroblasts were stimulated with titanium, or remained unstimulated, and PGE2 and IL-6 assays were performed using ELISA kit after 24 h . PGE2 and IL-6 secretion by unstimulated fibroblasts have been significantly increased in the presence of SP or titamium particles. Moreover SP caused significant increase in PGE2 and IL-6 production by titanium-stimulated fibroblasts.Fibroblasts were examined by real time RT-PCR and enzyme-linked immunosorbent assay (ELISA) for expression of the receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin(OPG), cyclooxygenase(COX)-1, and COX-2. Experiments were performed in the presence and absence of Ti particles ,SP and NS-398 (a selective COX-2 inhibitor).Ti particles or SP stimulated RANKL gene and protein expression in fibroblasts from periprosthetic membrane. Ti particles or SP induced COX-2 mRNA, whereas NS-398 inhibited RANKL production, suggesting a COX-2-mediated event. Moreover, SP enhanced COX-2 and RANKL expression by Ti particles-stimulated fibroblasts.SP levels were significantly higher in pseudosynovial fluid of loose artificial joints than in synovial fluid controls. SP stimulated production of IL-1β, TNF-α, PGE2, IL-6 and RANKL in fibroblasts from hip periprosthetic membrane. Moreover, SP enhanced PGE2, IL-6 and RANKL expression by Ti particles-stimulated fibroblasts. These cytokines play an important role in the development of periprosthetic osteolysis and implant loosening, Therefore SP might be implicated in interface inflammation, bone resorption and subsequent aseptic loosening of joint implants.