Mitsuo Nagase

Differential effects of different forms of hydroxyapatite and hydroxyapatite/tricalcium phosphate particulates on human monocyte/macrophages in vitro

A possible complication associated with the use of hydroxyapatite (HA) or HA/tricalcium phosphate (HA/TCP) coating on the surfaces of prosthetic devices used for dental and orthopedic implants is their potential to fragment and thus exist as wear debris. In contrast to the so-called osteoconductive properties of HA or HA/TCP coatings, in particulate form these materials may lead to an adverse pattern of cellular and tissue responses at the bone-implant interface. We have established an in vitro cell culture system to characterize the biologic and biochemical effects of various particulate materials. The present study demonstrates that the HA/TCP particles derived from different sintering temperatures exhibit differential effects on cultured human monocyte/macrophages (M/M). The HA/TCP particles dried at 110 degrees C were the most biologically active, stimulating significant release of interleukin 1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha, and prostaglandin E2 (PGE2), products implicated as important mediators of inflammation in diverse pathologic conditions. Other particles, sintered at either 900 or 1200 degrees C, did not stimulate production of cytokines or PGE2. HA/TCP particles from plasma-spray coatings also failed to release proinflammatory products. These results suggest that the biochemical and crystalline structural properties of particles markedly affects their capacity to modulate M/M function. This in vitro culture system should be useful in characterizing the specific physical and chemical properties of HA or HA/TCP particulates that are responsible for stimulating proinflammatory cell responses.

Toxicity of silica-containing calcium phosphate glasses demonstrated in mice

Suspensions of calcium phosphate glass containing various concentrations of silica (glass composition (moles): 100 Ca(PO3)2 to x SiO2,x = 0, 5, 10, 15 or 40) dispersed in normal saline were injected intraperitoneally into C57BL/6 mice to determine the mortality within 30 days. The mortality was 0/10, 3/10, 9/10, 10/10 and 10/10 at x = 0, 5, 10, 15 and 40 mol of silica, respectively. By means of inductively coupled plasma analysis, the amount of dissolved silica (Si4+) in water at 37 degrees C from the calcium phosphate glass depended on the amount of silica in the glasses. The mortality of mice was directly proportional to the silica content of the glass injected intraperitoneally. These results clearly show that the dissolved silica (Si4+) from the glass, monomeric or low molecular silicic anion, is highly toxic. The SiO2 component in biomaterials has toxic potential when dissolved in the body.

Sternocostoclavicular hyperostosis. A report of nineteen cases, with special reference to etiology and treatment.

The cases of nineteen patients with sternocostoclavicular hyperostosis were reviewed retrospectively. Of particular interest were the responses to antibiotics and prostaglandin inhibitors. The prostaglandin inhibitors relieved the pain within three to four weeks in sixteen of eighteen patients so treated. However, the inhibitors gradually became less effective in most patients. Oral antibiotics were more effective than the inhibitors in relieving the pain of eight of the eleven patients who were given antibiotics. Pustulosis palmaris and plantaris, commonly associated with sternocostoclavicular hyperostosis, diminished after antibiotic therapy, as did the chest pain in most patients. The similarities between the age and sex distributions and the responses to antibiotics of the patients with sternocostoclavicular hyperostosis and those with pustulosis suggest that these disorders have a common etiology, and that the pustulosis may be a so-called bacterid reaction and the hyperostosis, a manifestation of a systemic reaction to a focal infection.

The effect of crystallinity on hydroxyapatite‐induced production of reactive oxygen metabolites by polymorphonuclear leukocytes

To assess the role of crystallinity in biological response, we quantitated the generation of reactive oxygen metabolites in human polymorphonuclear leukocytes (PMN) with a chemiluminescence assay using three hydroxyapatite preparations with sintering temperatures of 1,200°C and 900°C and a drying temperature of 110°C on the basis of equal weights (1 mg/ml). These crystals have almost the same average diameters and similar average zeta potentials. The crystals prepared at higher temperatures have higher crystallinity, or larger domain sizes, which were calculated by X‐ray diffraction line broadening. The production of reactive oxygen metabolites by PMN in hydroxyapatite of 1,200°C was 10‐times that by PMN in hydroxyapatite of 900°C and more than 50‐times greater than that in hydroxyapatite of 110°C. A single linear correlation was observed for a plot of log (peak chemiluminescence levels) vs. a plot of log (domain sizes). These results clearly show that the maximal effect of crystallinity on hydroxyapatite‐induced production of reactive oxygen metabolites by human PMN was seen at higher crystallinity.

HLA phenotypes in patients who have osteosarcoma.

Human leukocyte antigen (HLA) phenotypes were studied in twenty Japanese patients who had typical osteosarcoma. The HLA-A11 phenotype was found in ten (50 per cent) of the twenty patients, compared with 16.6 per cent of 235 control subjects (chi square = 13.248; p less than 0.0005)--an odds ratio of 5.026. These data suggest that major histocompatibility complex-linked genes may determine susceptibility to osteosarcoma.

Effect of particle size on alumina-induced production of reactive oxygen metabolites by human leukocytes

To assess the role of crystal size in the biologic response, we quantitated the generation of reactive oxygen metabolites in human polymorphonuclear leukocytes (PMNs) or mononuclear leukocytes (MNs) with a chemiluminescence assay using 6 alpha-alumina preparations with average diameters of 0.6, 0.8, 3.2, 7.5, 28 and 68 microns on the basis of equal weights (1 mg/ml), and also on the basis of surface areas (100 cm2/ml). On an equal weight alumina particles induced PMN chemiluminescence in the following order of magnitude: 3.2 microns > 7.5 microns, 0.8 micron > 0.6 micron > 28 microns, 68 microns. The particulates induced MN chemiluminescence: 3.2 microns > 0.8 micron > 0.6 micron > 7.5 microns > 28 microns, 68 microns. On the basis of identical surface areas, alumina elicited PMN chemiluminescence: 3.2 microns > 7.5 microns, 0.8 micron > 0.6 micron, 28 microns, 68 microns. The particulates induced MN chemiluminescence: 3.2 microns > 0.8 micron > 0.6 micron, 7.5 microns > 28 microns, 68 microns. The maximal effect of particle size on the alumina-induced production of reactive oxygen metabolites by human leukocytes was seen at around 3 microns.

Human leukocyte antigen and aseptic loosening in Charnley total hip arthroplasty.

Human leukocyte antigen phenotypes and radiographic course were studied in 70 Japanese patients (90 hips) who had Charnley low friction arthroplasty. Of the 28 hips diagnosed as radiographically loose less than 15 years after surgery, seven hips (25.0%) were in patients who had a positive human leukocyte antigen A31(19). Of the 62 hips that were radiographically stable more than 15 years, two hips (3.2%) were in patients who had a positive human leukocyte antigen A31(19). Of the nine hips in patients who were positive to human leukocyte antigen A31(19), seven hips were diagnosed as radiographically loose (loosening rate was 77.8%) 15 years after surgery. On the other hand, of the 81 hips in the patients who were negative to human leukocyte antigen A31(19), only 21 hips were diagnosed as radiographically loose (loosening rate was 25.9%) 15 years after surgery. These data suggest that individual immune response to implant materials, linked to human leukocyte antigen, may participate in the process of aseptic loosening in Charnley low friction arthroplasty.

Analysis of muscle bioenergetic metabolism in rabbit leg lengthening.

The effect of lengthening on muscle metabolism was measured and correlated to the percent lengthening at early and late time points. Using the rabbit tibial lengthening model, the authors examined the effects of lengthening on the tibialis anterior muscle using phosphorus-31 magnetic resonance spectroscopy. Thirty-six rabbits were divided into five groups, four groups by percent lengthening (0%, 15%, 20%, and 25%), with each group divided into subgroups of early (end distraction) and late (12 weeks after end distraction), and the fifth group using the opposite untreated leg as control. Several parameters measuring metabolism of muscle using phosphorus-31 magnetic resonance spectroscopy analysis were compared. No changes occurred to 15% lengthening, but significant decreases were measured at 20% and 25% lengthening. After a 25% lengthening, the decreased metabolism persisted at 12 weeks after distraction, indicating the possibility of permanent damage. After 20% lengthening, the same parameters improved but never to normal levels. The authors conclude that lengthening to 15% is safe for muscle, but 20% to 25% lengthening may result in permanent metabolic damage. The current study also suggests that phosphorus-31 magnetic resonance spectroscopy may provide a viable clinical method for evaluating muscle damage during lengthening.

Priming effect of hydroxyapatite on the chemiluminescence response in human polymorphonuclear leukocytes

In order to determine whether hydroxyapatite modulates the response of polymorphonuclear leukocyte (PMN) to oxidative stimuli, human PMNs were incubated with a non‐activating concentration (1 or 10 μg/ml) of hydroxyapatite prior to stimulation with N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP; 0.1 or 1 μM), phorbol 12‐myristate 13‐acetate (PMA; 100 pg/ml), sodium fluoride (50 μM), zymosan (1 μg/ml), or the calcium ionophore A23187 (0.1 μM). Chemiluminescence was measured with an automatic microcomputer‐controlled luminescence analyzer at 37°C. Hydroxyapatite alone did not stimulate chemiluminescence at concentrations below 10 μg/ml. Levels 300–400% higher than ‘stimulus only’ controls without preincubation with hydroxyapatite have been recorded. This synergism between hydroxyapatite and subsequent stimuli reveals a new activity of hydroxyapatite and suggests that particulate material may prepare PMNs for an exaggerated inflammatory response to other phlogistic mediators. This is the first report demonstrating PMNs primed with particulate material.

Remodeling of large bone defects in the treatment of space-occupying lesions

SummaryCurettage without bone graft was performed in 17 patients with benign bone tumors and tumor-like lesions. New bone formation with uniformly increased radio-density appeared in serial plain radiographs within 3 months after the operation. The average period before full weight could be borne on the lower extremities was 14 weeks. Computed tomography revealed that the central part of the bone lesions persisted without bone formation. The thickening of cortical bone was predominant. These data indicate that enough mechanical strength for daily activity will be recovered by bone within 4 months after curettage without any filler, although remodeling continues for over a year. It is suggested that bone graft and implantation of “biomaterials” are not necessary in patients, especially younger ones, with benign bone tumors or tumorous conditions.