Effects of hydroxyapatite particulate debris on the production of cytokines and proteases in human fibroblasts.

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.

Injury to the popliteal artery and its anatomic location in total knee arthroplasty.

Injury to the popliteal artery during total knee arthroplasty (TKA) is a devastating complication. Although infrequent, these injuries can result in the need for further surgery, including revascularization or possibly even amputation. Several mechanisms are capable of producing direct trauma to the popliteal artery, including the use of posterior ret ractors. We investigated the proximity of the popliteal artery to the tibial joint surface during TKA to identify crucial steps in the procedure at which the artery was at highest risk for injury. TKA was performed on cadaveric specimens, and serial intraoperative arteriograms were taken throughout the procedure, demonstrating the potential for arterial injury by the instrumentation. Additionally, 50 transverse magnetic resonance imaging scans of unrelated knees were analyzed for the position of the popliteal artery relative to the midline of the tibial plateau as well as at a level 5 to 10 mm below this, at the site of a typical resection during TKA. All of the arteriograms showed the artery to be a lateral structure at the joint line. Additionally a posterior retractor placed the artery at risk when it was placed in a position lateral to the posterior cruciate ligament or when it was injudiciously inserted more than 1 cm into the soft tissues. Hyperextension of the knee, which might occur during preparation of the patella, produced dramatic tenting of the artery over the posterior joint line. These results demonstrate that the popliteal artery is at significant risk during TKA, particularly if posterior retractors are placed in a position lateral to the midline of the joint. Both hyperflexion and especially hyperextension produced severe deformities and kinking of the artery and would particularly jeopardize an artery with atherosclerosis. Our findings suggest that the popliteal artery may be at least risk during TKA if posterior retractors are placed medial to the midline of the tibial plateau and if care is taken to avoid extremes of both flexion and extension.

Bone marrow cells produce soluble factors that inhibit osteoclast activity.

Cytokines that stimulate bone resorption are produced by cells found in bone marrow. However, marrow cells produce multiple factors, some of which may be inhibitors of osteoclast differentiation or activity. Thus, it is not possible to predict a priori whether the mixture of factors produced by marrow cells will have a net stimulatory or inhibitory effect on bone resorption. In this study, we showed that the net effect of whole marrow is to inhibit osteoclast activity induced by parathyroid hormone. Fractionation of the marrow revealed that the inhibitory activity was in the marrow fluid. However, conditioned media obtained from marrow cell cultures also inhibited osteoclast activity. Thus, it is likely that the inhibitory factors are produced in vivo by cells residing in the marrow. These inhibitory factors may represent a physiological regulatory process that plays an important role in maintaining the balance between bone resorption and formation. Because we have previously shown that interleukin-6 is one of the cytokines that parathyroid hormone induces in osteoblastic cells to stimulate osteoclast activity, one potential mechanism by which the marrow-derived inhibitory factors might act is by preventing this production of interleukin-6. However, we found that the marrow cell-conditioned media do not inhibit the production or activity of interleukin-6. Thus, the inhibitory factors appear to block osteoclast activity through a mechanism that does not involve interleukin-6. Taken together, these results demonstrate the importance of factors that inhibit bone resorption and emphasize that the presence of cytokines that stimulate bone resorption in conditions such as osteoporosis and orthopaedic implant loosening should be interpreted with caution unless evidence exists demonstrating their functional importance.

Measurement and removal of adherent endotoxin from titanium particles and implant surfaces.

Aseptic loosening is thought to be due primarily to osteolysis induced by cytokines and prostaglandins that are produced in response to implant-derived wear particles. Because endotoxin has many of the same effects as have been reported for wear particles, we hypothesized that adherent endotoxin may be responsible for the biological responses induced by wear particles. We demonstrated the presence of significant levels of adherent endotoxin on commonly used preparations of titanium particles as well as on titanium and titanium-alloy implant surfaces. In contrast, supernatants obtained by centrifugation of particle suspensions contained approximately 1% as much endotoxin as did the particles. Therefore, it is erroneous to assume that particles do not contain endotoxin on the basis of data that it cannot be detected in their supernatants or filtrates. These results emphasize the importance of considering the potential role of adherent endotoxin when examining the in vitro effects of wear particles and the in vivo performance of orthopaedic implants. We also developed a protocol that removed more than 99.94% of the adherent endotoxin from the titanium particles without detectably affecting their size or shape. The removal of adherent endotoxin will allow comparison of the biological responses induced by particles with or without adherent endotoxin.

A PHEX gene mutation is responsible for adult-onset vitamin D-resistant hypophosphatemic osteomalacia: evidence that the disorder is not a distinct entity from X-linked hypophosphatemic rickets.

Previous investigators described a kindred with an X-linked dominant form of phosphate wasting in which affected children did not have radiographic evidence of rickets, whereas older individuals were progressively disabled by severe bowing. They proposed that this kindred suffered from a distinct disorder that they referred to as adult-onset vitamin D-resistant hypophosphatemic osteomalacia (AVDRR). We recently identified a gene, PHEX, that is responsible for the disorder X-linked hypophosphatemic rickets. To determine whether AVDRR is a distinct form of phosphate wasting, we searched for PHEX mutations in affected members of the original AVDRR kindred. We found that affected individuals have a missense mutation in PHEX exon 16 that results in an amino acid change from leucine to proline in residue 555. Clinical evaluation of individuals from this family indicates that some of these individuals display classic features of X-linked hypophosphatemic rickets, and we were unable to verify progressive bowing in adults. In light of the variability in the clinical spectrum of X-linked hypophosphatemic rickets and the presence of a PHEX mutation in affected members of this kindred, we conclude that there is only one form of X-linked dominant phosphate wasting.

Heterogeneity of human bone.

Matched samples of bone from the lumbar spine and tibia were obtained at autopsy from three adult males who had no known evidence of metabolic bone disease at the time of their demise. The soluble noncollagenous bone proteins were quantitatively extracted from these samples and assayed for the relative content of two bone-associated proteins, osteocalcin and osteonectin. When compared to trabecular bone, cortical bone had higher levels of osteocalcin and much lower levels of osteonectin. When concentration is expressed per gram of dried bone, the osteocalcin excess in cortical bone ranged from 30- to 32-fold, and the osteonectin excess in trabecular bone ranged from 21- to 47-fold. These differences were significant (P less than 0.01) using analysis of variance. We conclude that the human skeleton is not homogeneous with regard to these biochemical markers and that cortical and trabecular bone are biochemically quite distinct. This implies that these two types of bone may be subject to distinct regulatory mechanisms and that global assessments of skeletal function and bone quality based upon soluble markers should be applied with caution. The data also imply that a differential assessment of skeletal performance may be possible using biochemical serum markers.