Paradoxical effects of cyclosporin A on collagen arthritis in rats.

The effect of the immunosuppressive agent cyclosporin A (CS-A) on collagen arthritis in Sprague-Dawley rats is investigated. A 14-d course of CS-A treatment at doses of 15 mg/kg per day or more, begun on the same day as type II collagen immunization, suppressed the development of arthritis as well as humoral and delayed-type hypersensitivity (DTH) skin test responses to type II collagen, possibly by interfering with helper T cells. Additional studies demonstrated that CS-A treatment only during the induction phase of immunity proved to be successful. When CS-A treatment was started only during the immediately preclinical phase of arthritis or after the disease onset, a significant enhancement of the disease was obtained in a dose-dependent manner. This enhancement was accompanied by an augmentation of DTH skin reactions, while antibody responses were either suppressed or unaffected. These results appear to be attributable at least in part to a suppressive effect of CS-A on a population of suppressor T cells, thus resulting in a T cell-mediated helper effect. It is therefore reasonable to assume that the paradoxical effects of CS-A on collagen arthritis in rats might be caused by an altering of the sensitive balance of the two regulatory subpopulations of T cells. It is also possible that cell-mediated immune responses may play an important role in influencing the course of the disease.

Pathogenetic difference between collagen arthritis and adjuvant arthritis.

Daily treatment with cyclosporin at a dose of 25 mg/kg for 14 d gave complete suppression of the development of collagen arthritis and adjuvant arthritis in Sprague-Dawley rats during an observation period of 45 d. To study whether the immunologic unresponsiveness produced by cyclosporin is antigen specific, we rechallenged the cyclosporin-protected rats with either type II collagen or complete Freund's adjuvant (CFA) after discontinuation of cyclosporin treatment. Type II collagen-immunized, cyclosporin-protected rats did not develop arthritis in response to reimmunization with type II collagen, but, they did develop arthritis in response to a subsequent injection of CFA. Similarly, CFA-injected, cyclosporin-protected rats showed a suppressed arthritogenic reaction in response to reinjection of CFA, whereas their response to a subsequent immunization with type II collagen was unaffected. On the other hand, the rats that were treated with cyclosporin without any prior antigenic challenge could develop arthritis in response to a subsequent injection of CFA or type II collagen after cessation of cyclosporin treatment. These results indicate that specific immunologic unresponsiveness can be induced by cyclosporin in the two experimental models of polyarthritis, collagen arthritis and adjuvant arthritis, and that there is no cross-reactivity between type II collagen and the mycobacterial cell wall components. The results further indicate that immunity to type II collagen plays a critical role in the pathogenesis of collagen arthritis but that its pathogenetic role in adjuvant arthritis is insignificant.

Changes in the WOMAC, EuroQol and Japanese lifestyle measurements among patients undergoing total hip arthroplasty.

OBJECTIVE: To assess changes in the health outcomes of Japanese patients before and after total hip arthroplasty (THA), and to assess the impact of THA on commonly performed postures or body positions requiring deep flexion of the hip joint such as the use of Japanese squat toilets. METHODS: Consecutive patients undergoing primary THA between July 2003 and July 2004 were eligible for the study. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the EuroQol 5D (EQ-5D) were administered at the preoperative period and two postoperative periods of 6 weeks and 6 months. The patients were also asked to rate three items regarding common activities of daily living in Japan such as squatting. Changes in scores were examined using effect size and proportion at the floor and ceiling. RESULTS: Four-hundred and fifty-one patients completed both pre- and post-THA surveys. Significant improvements in pain and physical function as measured by WOMAC and EQ-5D were evident within 6 weeks. Changes in WOMAC and EQ-5D subscale scores and scores for each item from the three time periods were highly significant (P=0.000). The effect size was 1.56 for WOMAC pain and 1.38 for physical function at 6 months. In contrast, two items (Japanese toilet and seiza) became significantly worse at the 6-week postoperative period (P=0.000) and returned to preoperative levels by the 6-month postoperative period. CONCLUSION: These results highlight the importance of evaluating culturally sensitive physical functions in addition to conventional measurements for the health outcomes of THA patients.

Effectiveness of autologous fibrin tissue adhesive in reducing postoperative blood loss during total hip arthroplasty: a prospective randomised study of 100 cases.

PURPOSE: To evaluate the effectiveness of autologous fibrin tissue adhesive (auto-FTA) in reducing blood loss during cementless total hip arthroplasty (THA). METHODS: From September 2000 to August 2001, 100 patients who predonated 400 ml of autologous blood were randomised to undergo either standard treatment with auto-FTA (auto-FTA group) or standard treatment alone (control group). The volume of postoperative blood loss and the decrease in haemoglobin level were measured. All patients were followed up for 3 years to evaluate the rate of bone ingrowth and heterotopic ossification. RESULTS: The mean postoperative blood loss was 580 ml (standard deviation [SD], 240 ml) in the auto-FTA group and 810 ml (SD, 341 ml) in the control group; the difference was significant (230 ml, p<0.001). The decrease in haemoglobin concentration was 17 g/l (SD, 11 g/l) in the auto-FTA group and 22 g/l (SD, 12 g/l) in the control group. The difference was significant (5 g/l, p=0.03). The percentage of total blood loss of >1200 ml in any single patient was significantly lower in the auto-FTA group (4%) than in the control group (20%) [p=0.01]. CONCLUSION: Auto-FTA is a safe and effective means of reducing perioperative blood loss in THA.

Tumor necrosis factor-alpha cooperates with receptor activator of nuclear factor kappaB ligand in generation of osteoclasts in stromal cell-depleted rat bone marrow cell culture.

A member of the tumor necrosis factor (TNF) family, receptor activator of nuclear factor kappaB ligand (RANKL; also known as ODF, OPGL, and TRANCE), plays critical roles in osteoclast differentiation and activation in the presence of macrophage colony-stimulating factor (M-CSF). Recently, TNF-alpha has also been shown to induce the formation of multinucleated osteoclast-like cells (MNCs) in the presence of M-CSF from mouse macrophages. We demonstrated that mononuclear preosteoclast-like cells (POCs) were formed in the presence of conditioned medium of osteoblastic cells in a rat bone marrow culture depleted of stromal cells. Using this culture system, in this study we examined whether TNF-alpha affects differentiation into POCs from hematopoietic progenitor cells. Human TNF-alpha (hTNF-alpha) markedly stimulated the formation of POCs. Moreover, a concentration as low as 0.005 ng/mL of hTNF-alpha increased the level of mRNA for calcitonin receptor (CTR) and cathepsin-K of POCs. The POCs induced by hTNF-alpha formed MNCs, which showed dentine-resorbing activity after coculture with primary osteoblasts. Stimulation was observed after 24 h of treatment with hTNF-alpha only on day 1 or day 2 of the culture. After 24 h of hTNF-alpha treatment, expression of the receptor activator of nuclear factor kappaB (RANK) mRNA was markedly increased. The addition of soluble RANKL (sRANKL) to the preformed POCs efficiently induced MNCs. Interestingly, treatment of bone marrow cells with hTNF-alpha and sRANKL synergistically augmented the formation of MNCs. This formation was abolished by the addition of human osteoprotegerin (hOPG). These results suggest that cooperation of TNF-alpha and RANKL is important for osteoclastogenesis.

Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and indefinite survival.

We have tested the effects of FK506 (FK), a new immunosuppressive agent, on a rat limb allograft model. Histoincompatible BN limb allografts were rejected in untreated F344 hosts within 11 +/- 1 days (mean +/- SD) after operation. A single injection of 2 mg/kg, 10 mg/kg, or 50 mg/kg of FK on the day of limb transplantation (day 0) significantly prolonged graft survival in a dose-dependent manner--i.e., mean limb survival times (MST) based on gross signs of skin rejection were 16 +/- 3 days, 51 +/- 6 days, or 104 +/- 17 days, respectively (P less than 0.01). Delayed treatment with a single injection of 10 mg/kg of FK at when early signs of rejection were visible (day 7 or day 10) reversed the ongoing rejection. The MSTs in these groups were comparable to that of those treated with the same dosage of FK on day 0. The FK-induced unresponsiveness toward limb allografts was donor-specific because limb-allografted. FK-protected rats could not accept the skin grafts from a third-party donor. In the next set of experiments, rats were given a single administration of 10 mg/kg of FK on the day of limb allograft, followed by intermittent injections of 3 mg/kg of FK once a week. This regimen produced complete graft survival for more than 200 days, though Pneumocystis carinii pneumonia occurred in most of the recipients. These results represent the unique effects of FK in preventing or reversing the graft rejection and in inducing indefinite survival in this animal model of composite tissue allografts.

Collagen arthritis in rats: the importance of humoral immunity in the initiation of the disease and perpetuation of the disease by suppressor T cells.

Arthritis could be passively transferred with a serum concentrate from collagen arthritic rats to nude rats and cyclosporin-treated, type II collagen-tolerant rats. These findings suggest that collagen arthritis could be inducible by humoral immunity alone in the absence of cellular immunity to type II collagen or functional T cells. In addition, passive arthritis induced by anticollagen antibody is a mild, transient disease from which the animals normally recover and the rats that have recovered from passive arthritis are resistant to develop a second phase of arthritis following a second administration of anticollagen antibody or the subsequent challenge with type II collagen. However, when a serum concentrate was transferred while cyclosporin was administered continuously, transferred arthritis in cyclosporin-treated, type II collagen-tolerant rats lasted as long as cyclosporin treatment and arthritis was significantly enhanced compared to those of naive recipients. Further, enhancement and prolongation of passively transferred arthritis in nude rats was observed. Furthermore, treatment with cyclophosphamide reversed acquired resistance to collagen arthritis subsequent to recovery from passive arthritis. These findings suggest that suppressor T cells might, at least in part, affect the clinical course of collagen arthritis and reverse acquired resistance to arthritis.

Administration of growth hormone modulates the gene expression of basic fibroblast growth factor in rat costal cartilage, both in vivo and in vitro.

We examined the effect of growth hormone on local growth factor mRNA expression in male Sprague-Dawley rats. Repetitive systemic administration of growth hormone (0.4 IU every 4 h) increased the expression of IGF-I mRNA up to 2.8-fold in costal cartilage tissue compared with controls. Basic FGF (bFGF) mRNA expression gradually increased up to 15.5-fold compared with pre-injection samples, where the mRNA expression was 5.3-times greater than vehicle-injected controls. TGF-beta mRNA showed little changes. Moreover, one microgram/ml of growth hormone enhanced the expression of bFGF mRNA in costal chondrocytes in culture. We conclude that growth hormone increased the local expression of bFGF, as well as that of IGF-I, in cartilage, and suggest that bFGF is directly regulated by growth hormone within a local area.

Induction of abundant osteoclast-like multinucleated giant cells in adjuvant arthritic rats with accompanying disordered high bone turnover.

The development of an in vivo system for investigating osteoclast differentiation is important because molecular events occurring in vivo can be observed during the differentiation of the authentic osteoclasts. In adjuvant arthritic rats, an experimental model of human rheumatoid arthritis, extensive bone resorption is observed in the distal diaphysis of the tibia. In the area of extensive bone resorption, it is always accompanied with clusters of numerous multinucleated giant cells (MGCs) as well as bone-resorbing osteoclasts. Here we characterized the morphological properties of these MGCs with the use of enzymehistochemical and immunohistochemical techniques. Extensive destruction but also a marked formation of the inner and outer bone surfaces were the predominant features in the tibiae of such arthritic rats 4 weeks after the adjuvant injection. Numerous MGCs were frequently clustered in the bone marrow spaces located apart from the bone matrices. Although the MGCs lacked ruffled borders, these cells were rich in mitochondria and vacuoles. These multinucleated cells revealed a positive reaction for tartrate-resistant acid phosphatase but a negative reaction for non-specific esterase staining. Most of these MGCs expressed the Kat 1-antigen, an immunological marker specifically expressed on the cell surface of rat osteoclasts. In a dentin resorption experiment using a cluster of MGCs excised from the bone marrow tissues of the tibial distal diaphyses of rats with adjuvant arthritis, many resorption lacunae were formed on dentin slices after a 3-day culture. These results suggest that the majority of the MGCs are osteoclasts but not macrophage polykaryons.

Evaluation of blood flow within the subchondral bone of the femoral head: use of the laser speckle method at surgery for osteonecrosis.

The laser speckle method is a new form of tissue flowmetry that can analyze the interference pattern that appears when tissue is illuminated with a laser beam. During surgery for 100 cases of osteonecrosis of the femoral head, we measured the blood flow within the subchondral bone using this method. We compared the flow maps (two-dimensional distribution of the microcirculation) obtained this way with the necrotic area estimated by the preoperative magnetic resonance images and with the collapse seen during surgery. The laser speckle method was able to distinguish between the ischemic areas and the normal areas in 92 femoral heads, including five hips for which neither the magnetic resonance images nor the collapse observed during surgery demonstrated a distinct margin surrounding the necrotic area. We concluded that the laser speckle method is useful for defining the margin around a necrotic area.

Assessment of subchondral bone blood flow in the rabbit femoral condyle using the laser speckle method.

The laser speckle method is a new form of flowmetry that can obtain a two-dimensional distribution of blood flow in tissue. This method is a noncontact, simple, and rapid technique that may aid in the diagnosis of osteonecrosis. We investigated whether the subchondral bone blood flow within the femoral condyles of rabbits could be measured by the laser speckle method. The hydrogen washout method was chosen as a comparison technique because of its ability to allow repetitive measurements of blood flow in various conditions in one rabbit and because of its reliability, which already has been established. We simultaneously measured the bone blood flow in 20 femoral condyles of 10 rabbits with the laser speckle and hydrogen washout methods and found a significant correlation between the blood flow levels with use of these two methods. For the clinical application of the laser speckle method, we also investigated the influence of cartilage thickness on the measurements and the depth in the bone to which blood flow could be measured with this method. A cartilage thickness of 0.2 mm did not influence the measurement of the bone blood flow, and the depth in the bone to which the laser speckle method could be used was approximately 2 mm.

Inhibition of endochondral ossification during fracture repair in experimental hypothyroid rats.

Using a rat fracture model, we investigated the effects of a decrease in serum levels of thyroid hormone on the fracture-repair process. Rats were divided into the following groups: (a) controls, (b) those treated with methimazole for the duration of the experiment, and (c) those treated with methimazole and L-thyroxine, receiving both for the same duration. Three weeks after the initiation of pharmacologic treatment, closed femoral fractures were produced. The formation of cartilage tissue in the fracture callus in all rats was not obviously different on day 7 after fracture. In the rats treated with methimazole, differentiation from proliferating to hypertrophic chondrocytes in the fracture callus was less advanced and vascular invasion was clearly inhibited on day 12. Gene expression of alkaline phosphatase and osteocalcin in the callus was significantly lower in these rats than in the controls on days 10, 12, and 14. The mechanical properties of the fracture callus were also significantly weaker in these animals than in the controls on day 21, resulting in impaired fracture repair. These results demonstrate that hypothyroidism inhibits endochondral ossification, resulting in an impaired fracture-repair process. L-thyroxine replacement in the rats treated with methimazole caused the impaired repair process to revert to normal. These results indicate that thyroid hormone is one of the critical systemic factors for fracture repair.

Changes in growth-plate morphology associated with rejection of rat-limb allografts.

Histological and electron microscopic studies were performed to demonstrate the changes in the morphology of the growth plate that occur in allografts obtained from the limbs of growing rats. A genetically defined model was used in which the right hindlimbs of Lewis rats were orthotopically transplanted into Fischer-344 recipient rats. These strains are matched for major histocompatibility antigens but mismatched for minor histocompatibility antigens. The disparity at the minor histocompatibility complex between the Lewis donors and the Fischer recipients creates a weak histocompatibility barrier to transplantation. Lewis-to-Lewis syngeneic limb grafts were used as controls. The proximal parts of the transplanted tibiae were excised during acute rejection of the allograft on days 1, 5, 8, 11, 14, and 28 postoperatively. During rejection, a widened zone of calcified cartilage in the growth plate was observed at eleven days; this zone increased progressively thereafter. The number of chondroclasts in the primary spongiosa of the metaphysis had decreased significantly at eleven days, and chondroclasts had disappeared completely at fourteen days, in association with mononuclear cell infiltration. Electron microscopic examination revealed inactive morphology in some chondroclasts at eight days, and the number of inactive chondroclasts had increased significantly on day 11. At fourteen days, there were no viable chondroclasts in the primary spongiosa, and only remnants of degenerated chondroclasts were present. These findings suggest that the chondroclasts were early targets of rejection and their loss resulted in the cessation of resorption of the calcified cartilage. However, the proliferation and maturation of chondrocytes in the growth plate and the calcification of the matrix continued, despite progression of rejection in the metaphysis. Thus, survival of the chondrocytes and rejection of the chondroclasts apparently led to the formation of a widened calcifying zone in the growth plate.

Diagnostic criteria for non-traumatic osteonecrosis of the femoral head. A multicentre study.

Six major and seven minor diagnostic criteria have been developed by the Japanese Investigation Committee for osteonecrosis of the femoral head (ONFH). We have carried out a multicentre study to clarify these. We studied prospectively 277 hips in 222 patients, from six hospitals, who had ONFH and other hip pathology and from whom histological material was available. We identified five criteria with high specificity: 1) collapse of the femoral head without narrowing of the joint space or acetabular abnormality on radiographs, including the crescent sign; 2) demarcating sclerosis in the femoral head without narrowing or acetabular abnormality; 3) a 'cold-in-hot' appearance on the bone scan; 4) a low-intensity band on T1-weighted images (band pattern); and 5) evidence of trabecular and marrow necrosis on histological examination. With any combination of two of these criteria, the sensitivity and specificity of the diagnosis were 91% and 99%, respectively.

Prolonged limb allograft survival with short-term treatment with FK-506 in rats.

The effect of the new immunosuppressive agent FK-506 on rat limb allograft was investigated across the BN-to-F344 histocompatibility barrier. A 14-day course of FK-treatment at doses of 1 mg/kg per day or more, begun on the day of operation, significantly increased the period of graft survival. Additional studies demonstrated that single treatment with FK only on the day of operation prolonged the graft survival in a dose-dependent manner. These results stress the profound immunosuppressive effect of FK and suggest the possible future application of composite tissue allograft in humans.

Limb allografts in rats immunosuppressed with cyclosporine: as a whole-joint allograft.

We performed limb allografts in three inbred rat strains immunosuppressed with cyclosporine. As controls, 10 autografts and 10 isografts exhibited an excellent result. In minor-mismatched allografts (Lewis to Fischer, n = 45), with the use of cyclosporine, the grafted limbs survived and the articular cartilage retained normal architecture and cell viability 52 weeks after grafting, but without cyclosporine treatment severe degeneration and destruction of articular cartilage were shown by 16 weeks after operation. In major-mismatched allografts (Brown Norway to Fischer, n = 35), the articular cartilage of cyclosporine-treated animals maintained normal architecture and cell viability 52 weeks after operation despite the gross appearance of skin rejection, while that of non-cyclosporine-treated animals was degenerated and destroyed by 6 weeks. These results suggest the possibility of whole-joint allografts in humans with the use of cyclosporine, as well as other organ transplantations.

Experimental osteochondritis dissecans--the role of cartilage canals in chondral fractures of young rabbits.

Skeletal immature rabbits were used to study the pathogenesis of Osteochondritis Dissecans (OCD). Both histological studies and a radiographical examination were utilized after sagittal and coronal surgical chondral fractures were made in the femoral condyles cartilage. Serial microangiographies were performed in rabbits between 0 and 84 days after the chondral fractures were made. Analyses of the histology and microradiography findings suggest in either a coronal or sagittal direction, that avascular lesions like an experimental OCD occur as a sequence of chondral injury. A fracture in a wide pedicle of a stable cartilaginous flap with abundant cartilage canals heals in the usual way. However, a fracture in an unstable fragment with a small isthmus devoid of cartilage canals and of nutritious vessels, probably doesn't heal completely and a fragment closely resembling OCD is instead formed. An experimental OCD depends on the slender hinge of the flap and on the lack of stability in a rabbit's non-ossified epiphyseal cartilage. The damage to the cartilage canals and the rupture of vessels in the canals by a chondral fracture and the disturbance in the revascularization in the healing process by abnormal mechanical forces are thus most likely considered to be the main factor for OCD production.

[Two cases of Turner's syndrome with spondyloepiphyseal dysplasia like bone appearance].

We report two cases of mosaic karyotype (45XO/46XiXq) Turner's syndrome with unique bone appearance. The cases were 44 and 34 year-old women and latter was complicated by Hashimoto's thyroiditis (hypothyroidism). Following the systemic bone surveys, we found the patients showed not only osteoporotic bone change and short stature, but also spondyloepiphyseal dysplasia (SED) like bone appearance (thinness of vertebral bodies, irregularity of vertebral end-plates, shortness of femoral necks, Coxa valga, Coxa magna and hypoplasia of acetabula). Those findings can not be explained by degenerative bone changes like osteoporosis, rather are suggestive the sequelae of malgrowth of the bone system in Turner's syndrome.

Osteoconductivity of thermal-sprayed silver-containing hydroxyapatite coating in the rat tibia.

A silver-containing hydroxyapatite (Ag-HA) coating has been developed using thermal spraying technology. We evaluated the osteoconductivity of this coating on titanium (Ti) implants in rat tibiae in relation to bacterial infection in joint replacement. At 12 weeks, the mean affinity indices of bone formation of a Ti, an HA, a 3%Ag-HA and a 50%Ag-HA coating were 97.3%, 84.9%, 81.0% and 40.5%, respectively. The mean affinity indices of bone contact of these four coatings were 18.8%, 83.7%, 77.2% and 40.5%, respectively. The indices of bone formation and bone contact around the implant of the 3%Ag-HA coating were similar to those of the HA coating, and no significant differences were found between them (bone formation, p = 0.99; bone contact, p = 0.957). However, inhibition of bone formation was observed with the 50%Ag-HA coating. These results indicate that the 3%Ag-HA coating has low toxicity and good osteoconductivity, and that the effect of silver toxicity on osteoconductivity depends on the dose.