Analysis of proteins released from osteoarthritic cartilage by compressive loading.

In osteoarthritis (OA), synovial pathology may be induced by proteins released from degenerated cartilage. This study was conducted to identify the proteins released from OA cartilage. OA cartilage was obtained from OA knees at macroscopically preserved areas (PRES) and degenerated areas (DEG), while control cartilage (CONT) was collected from non-arthritic knees. Released proteins were obtained from these cartilage samples by repeatedly applying compressive loading, which simulated loading on cartilage in vivo. The released proteins were analyzed comprehensively by antibody array analyses and a quantitative proteomic analysis. For several proteins, the exact amounts released were determined by Luminex assays. The amount of active TGF-ß that was released was determined by an assay using genetically-engineered HEK cells. The results of the antibody array and proteomic analyses revealed that various biologically active proteins are released from OA cartilage, particularly from DEG, by loading. The Luminex assay confirmed that several alarmins, complement proteins C3a and C5a, and several angiogenic proteins including FGF-1, FGF-2 and VEGF-A were released in greater amounts from DEG than from CONT. The HEK cell assay indicated that active TGF-ß was released from DEG at biologically significant levels. These findings may be helpful in understanding the pathology of OA.

Systemic inflammatory responses after orthopedic surgery in patients with rheumatoid arthritis treated with tofacitinib.

OBJECTIVE: To investigate the acute phase response to surgical stress in patients with rheumatoid arthritis (RA) treated with tofacitinib, a Janus kinase (JAK) inhibitor. METHODS: A retrospective matched pair analysis of 34 patients treated with tofacitinib and 34 patients treated with conventional disease-modifying anti-rheumatic drugs (csDMARDs) was performed. Patients were matched for age, sex, and type of surgery; body temperature, C-reactive protein (CRP) level, and white blood cell (WBC) count, neutrophil count, and lymphocyte count were compared between the tofacitinib and csDMARDs groups within 2 weeks after orthopedic surgery. Postoperative complications within 90 days were also assessed. RESULTS: No surgical site infection or delayed wound healing was observed in the tofacitinib group; whereas, one case of superficial infection was noted in the csDMARDs group. A similar postoperative increase in body temperature and CRP level was observed in both the groups. Postoperatively, the tofacitinib group showed an increase in WBC and neutrophils counts and a decrease in lymphocyte count, unlike the csDMARDs group. In contrast to two patients (2.6%) in the csDMARDs group, seven patients (20.6%) in the tofacitinib group had lymphocyte counts below 500 cells/µL within 2 weeks postoperatively. CONCLUSION: Tofacitinib did not suppress postoperative increase in body temperature and CRP level. Because of the postoperative decrease in lymphocyte count in patients treated with tofacitinib, the timing for resuming tofacitinib treatment after surgery should be carefully considered. Key Points • This study is the first to report the complications and systemic inflammatory responses after orthopedic surgery in patients treated with tofacitinib in comparison with matched pairs treated with conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) • While tofacitinib does not suppress postoperative increase in body temperature and CRP level, the postoperative decrease in lymphocyte count in patients treated with tofacitinib is significant compared with patients treated with csDMARDs • Attention should be paid to a reduced lymphocyte count when to resume tofacitinib after surgery.

Fracture of ossified Achilles tendons: A review of cases.

Fracture of an ossification of the Achilles tendon (OAT) is a rare entity, and its etiology, pathology, and treatment remain unclear. We reviewed and scrutinized 18 cases (16 articles) of the fracture of an OAT. The most common etiologies of the ossifications include previous surgery and trauma. The fractures often occur without any trigger or with minimal trigger. The long, > 5 cm, ossification in the body of the Achilles tendon may have a higher risk of fracture. The OAT itself is often asymptomatic; however, its fracture causes severe local pain, swelling, and weakness of plantar flexion, which forces patients to undergo aggressive treatments. Regarding the treatments of the fractures, nonoperative treatment by immobilizing ankle joint could be an option for elderly patients. However, because it often cannot produce satisfactory results in younger patients, surgical treatment is typically recommended. Excision of the fractured mass and repairing the tendon is applicable if the remnant is enough. If there is a defect after the excision, reconstruction with autologous grafts or adjacent tendon transfer is performed. Gastrocnemius fascia turndown flap, hamstring tendon and tensor fascia lata are used as autologous grafts, whereas peroneus brevis and flexor hallucis longus tendons are used for the tendon transfer. If the fracture of an OAT is treated properly, the functional result will be satisfactory.

The attenuation of insulin-like growth factor signaling may be responsible for relative reduction in matrix synthesis in degenerated areas of osteoarthritic cartilage.

BACKGROUND: In osteoarthritis (OA), cartilage matrix is lost gradually despite enhanced matrix synthesis by chondrocytes. This paradox may be explained, at least partly, by reduced chondrocyte anabolism in degenerated area of OA cartilage. However, to date, it is not known why chondrocyte anabolism is suppressed in those areas. METHODS: Cartilage was obtained from control knees and end-stage OA knees in macroscopically preserved areas and degenerated areas, and gene expression was analyzed in respective regions of cartilage using laser capture microdissection and qPCR. For the cartilage protein analysis, cartilage was obtained from preserved areas and degenerated areas of OA knees in pairs, and proteins were extracted using urea buffer. Protein concentrations were determined by Luminex and compared between the areas. Cartilage explants prepared from preserved areas and degenerated areas of OA knees were cultured in the presence or absence of an AKT inhibitor, and the gene expression was evaluated by qPCR. Finally, the expression of SP1 was evaluated in OA and control cartilage, and the significance of Sp1 on the expression of IGF1R and IRS1 was investigated in experiments using primary cultured chondrocytes. RESULTS: Within OA cartilage, the expression of IGF-1, IGF-2, IGF1R and IRS1 was reduced in degenerated areas compared to preserved areas, while the expression of all six IGF-binding protein genes examined was enhanced in the former areas. Consistent results were obtained by a protein analysis. In explant culture, the inhibition of AKT signaling abrogated the abundant matrix gene expression in the preserved areas over the degenerated areas, indicating that suppressed matrix synthesis in degenerated areas may be ascribed, at least partly, to attenuated IGF signaling. Within OA cartilage, the expression of Sp1 was considerably reduced in severely degenerated areas compared to preserved areas, which correlated well with the expression of IGF1R and IRS1. In experiments using primary cultured chondrocytes, the expression of IGF1R and IRS1 was enhanced by the induction of Sp1 expression and reduced by the suppression of Sp1 expression. CONCLUSIONS: The results of this study suggest that attenuated IGF signaling may be responsible, at least partly, for the reduced matrix synthesis in degenerated areas of OA cartilage.

Time Course of Quality of Life Improvement Between Resection Arthroplasty and Metatarsophalangeal Joint-Preserving Forefoot Arthroplasty for Rheumatoid Arthritis.

BACKGROUND: Resection arthroplasty has long been a major treatment option for forefoot deformity caused by rheumatoid arthritis (RA). However, metatarsophalangeal (MTP) joint-preserving surgery is now surpassing classic resection arthroplasty. This study was performed to compare the postoperative results of these 2 operative methods. METHODS: Fifty-one toes of 40 patients with RA who underwent resection arthroplasty (resection group) or MTP joint-preserving arthroplasty (preservation group) from 2014 to 2017 for forefoot deformity were followed up for >1 year and were retrospectively analyzed. In the preservation group, open reduction of joint dislocation was performed if needed, and the deformity was corrected by metatarsal shortening osteotomy. The mean follow-up period was 21 months. The Japanese Society for Surgery of the Foot (JSSF) scales (objective outcome measures), the Self-Administered Foot Evaluation Questionnaire (SAFE-Q) (subjective outcome measure), and radiographic indices were compared between the groups. The resection group and preservation group comprised 15 toes of 11 patients and 36 toes of 29 patients, respectively. RESULTS: There were no significant differences in the preoperative radiographic indices, JSSF scales, or SAFE-Q results between the 2 groups. The preservation group showed better JSSF scores at the last follow-up (median hallux scale, 89 vs 74; median lesser scale, 87 vs 79). In the preservation group, the SAFE-Q scores gradually improved with time until 12 months postoperatively. In the resection group, the scores decreased 3 months postoperatively and then improved and reached a plateau 6 months postoperatively. At 12 months postoperatively, there was no significant difference in the SAFE-Q scores between the 2 groups. CONCLUSIONS: MTP joint-preserving arthroplasty resulted in superior objective scores to resection arthroplasty in patients with RA forefoot deformity. Although the subjective scores did not differ between the groups at the last follow-up, the time course of postoperative quality of life improvement was different between the 2 surgeries. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Factors affecting bone union after distal shortening oblique osteotomy of the lesser metatarsals.

Objectives: There have been few reports on factors affecting bone union after metatarsal osteotomies. The purpose of this study was to clarify the factors affecting bone union after distal shortening oblique osteotomy of the lesser metatarsals.Methods: Patients who underwent distal shortening oblique osteotomy of the lesser metatarsals were retrospectively investigated. Failure to achieve bone union at 6 months after surgery was defined as delayed union. Background characteristics and radiographic measurements were compared between patients with and those without delayed union, and factors affecting bone union were assessed using multivariate analysis.Results: Among 204 toes in 58 patients evaluated in this study, delayed union occurred in 28%. In multivariate analysis, corticosteroid use (odds ratio (OR), 3.68; 95% confidence interval (CI), 1.65-8.16; p< .01), larger preoperative overlap between the metatarsal and the proximal phalanx (OR, 1.11 (per 1 mm increase); 95% CI, 1.02-1.21; p= .02), and larger gap at the osteotomy site (OR, 3.02 (per 1 mm increase); 95% CI, 1.76-5.16; p< .01) were identified as independent risk factors of delayed union.Conclusion: The identified risk factors of delayed union after distal shortening metatarsal osteotomies were corticosteroid use, preoperative overlap between the metatarsal and the proximal phalanx, and a gap at the osteotomy site.

Successful treatment of a fracture of a huge Achilles tendon ossification with autologous hamstring tendon graft and gastrocnemius fascia flap: a case report.

BACKGROUND: Fracture of an ossified Achilles tendon is a rare entity, and no standard treatment has been established. This is the first report to describe the use of a hamstring tendon graft and gastrocnemius fascia flap for Achilles tendon reconstruction. CASE PRESENTATION: We present the case of a 50-year-old woman with fracture of an ossified Achilles tendon. She presented to our clinic with acute right hindfoot pain, which started suddenly while going up the stairs. Plain radiography and magnetic resonance imaging revealed a massive ossification on the right Achilles tendon extending over 14 cm in length; the ossification was fractured at 5 cm proximal to the calcaneus insertion. Surgical treatment included removal of the ossified tendon and reconstruction with an autologous hamstring tendon graft and gastrocnemius fascia flap. One year after surgery, she was able to walk with little pain or discomfort and to stand on her right tiptoe. CONCLUSION: Our novel surgical procedure may be useful in the treatment of fractured ossified Achilles tendons and large Achilles tendon defects.

Intracellular and extracellular ATP coordinately regulate the inverse correlation between osteoclast survival and bone resorption.

Osteoclasts, highly differentiated bone-resorbing cells of hematopoietic origin, have two conflicting tendencies: a lower capacity to survive and a higher capacity to execute energy-consuming activities such as bone resorption. Here, we report that when compared with their precursors, mature mitochondria-rich osteoclasts have lower levels of intracellular ATP, which is associated with receptor activator of nuclear factor κ-B ligand (RANKL)-induced Bcl-x(L) down-regulation. Severe ATP depletion, caused by disrupting mitochondrial transcription factor A (Tfam) gene, leads to increased bone-resorbing activity despite accelerated apoptosis. Although AMP-activated protein kinase (AMPK) activation by ATP depletion is not involved in the regulation of osteoclast function, the release of ATP from intracellular stores negatively regulates bone-resorbing activity through an autocrine/paracrine feedback loop by altering cytoskeletal structures. Furthermore, osteoclasts derived from aged mice exhibit reduced mitochondrial DNA (mtDNA) and intracellular ATP levels with increased bone-resorbing activity, implicating the possible involvement of age-related mitochondrial dysfunction in osteoporosis. Thus, our study provides evidence for a mechanism underlying the control of cellular functions by reciprocal changes in intracellular and extracellular ATP, which regulate the negative correlation between osteoclast survival and bone resorption.

Class IA phosphatidylinositol 3-kinase regulates osteoclastic bone resorption through protein kinase B-mediated vesicle transport.

Class IA phosphatidylinositol 3-kinases (PI3Ks) are activated by growth factor receptors and regulate a wide range of cellular processes. In osteoclasts, they are activated downstream of α(v) ß(3) integrin and colony-stimulating factor-1 receptor (c-Fms), which are involved in the regulation of bone-resorbing activity. The physiological relevance of the in vitro studies using PI3K inhibitors has been of limited value, because they inhibit all classes of PI3K. Here, we show that the osteoclast-specific deletion of the p85 genes encoding the regulatory subunit of the class IA PI3K results in an osteopetrotic phenotype caused by a defect in the bone-resorbing activity of osteoclasts. Class IA PI3K is required for the ruffled border formation and vesicular transport, but not for the formation of the sealing zone. p85α/ß doubly deficient osteoclasts had a defect in macrophage colony-stimulating factor (M-CSF)-induced protein kinase B (Akt) activation and the introduction of constitutively active Akt recovered the bone-resorbing activity. Thus, the class IA PI3K-Akt pathway regulates the cellular machinery crucial for osteoclastic bone resorption, and may provide a molecular basis for therapeutic strategies against bone diseases.

Distinguishing the proapoptotic and antiresorptive functions of risedronate in murine osteoclasts: role of the Akt pathway and the ERK/Bim axis.

OBJECTIVE: Nitrogen-containing bisphosphonates are one of the most successful therapeutics for osteoporosis. The aim of this study was to elucidate the functional mechanism of one of the typical nitrogen-containing bisphosphonates, risedronate. METHODS: Osteoclasts generated from murine bone marrow macrophages were treated with risedronate in vitro, and its effects on apoptosis and bone-resorbing activity were examined. The mechanism of action of risedronate was examined by gene induction of constitutively active Akt-1 and constitutively active MEK-1, and by gene deletion of Bim. Bim(-/-) mice, in which osteoclasts were resistant to apoptosis, were treated with risedronate and analyzed radiographically, biochemically, and histologically. RESULTS: Risedronate induced osteoclast apoptosis through the mitochondria-dependent pathway with an increased expression of Bim, and the proapoptotic effect of risedronate was suppressed by Bim deletion and constitutively active MEK-1 introduction. In contrast, the risedronate-induced suppression of bone resorption was completely reversed by inducing constitutively active Akt-1, but not by Bim deletion or constitutively active MEK-1 introduction. These results suggested that apoptosis and bone-resorbing activity of osteoclasts were regulated through the ERK/Bim axis and the Akt pathway, respectively, both of which were suppressed by risedronate. Although osteoclast apoptosis in response to risedronate administration was suppressed in the Bim(-/-) mice, risedronate treatment increased bone mineral density in Bim(-/-) mice at a level equivalent to that in wild-type mice. CONCLUSION: Our findings indicate that the antiresorptive effect of risedronate in vivo is mainly mediated by the suppression of the bone-resorbing activity of osteoclasts and not by the induction of osteoclast apoptosis.

Antiapoptotic molecule Bcl-2 is essential for the anabolic activity of parathyroid hormone in bone.

The antiapoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Since Bcl-2(-/-) mice die before six weeks of age on account of renal failure and cannot be compared to adult wild-type mice, we generated Bcl-2(-/-)Bim(+/-) mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2(+/-)Bim(+/-) littermates. Bcl-2(-/-)Bim(+/-) mice grew normally, but exhibited decreased bone mass compared to Bcl-2(+/-)Bim(+/-) mice, mainly due to impaired osteoblast function. Interestingly, the anabolic effect of parathyroid hormone (PTH) was not observed in Bcl-2(-/-)Bim(+/-) mice. This data demonstrates that Bcl-2 is indispensable for the anabolic activity of PTH in bone.

Anti-apoptotic molecule Bcl-2 regulates the differentiation, activation, and survival of both osteoblasts and osteoclasts.

The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2(-/-) mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2(-/-) mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2(-/-) mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2(-/-)Bim(+/-) mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2(+/-)Bim(+/-) littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2(-/-) mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts.

The antiapoptotic protein Bcl-xL negatively regulates the bone-resorbing activity of osteoclasts in mice.

The B cell lymphoma 2 (Bcl-2) family member Bcl-xL has a well-characterized antiapoptotic function in lymphoid cells. However, its functions in other cells--including osteoclasts, which are of hematopoietic origin--and other cellular processes remain unknown. Here we report an unexpected function of Bcl-xL in attenuating the bone-resorbing activity of osteoclasts in mice. To investigate the role of Bcl-xL in osteoclasts, we generated mice with osteoclast-specific conditional deletion of Bcl-x (referred to herein as Bcl-x cKO mice) by mating Bcl-xfl/fl mice with mice in which the gene encoding the Cre recombinase has been knocked into the cathepsin K locus and specifically expressed in mature osteoclasts. Although the Bcl-x cKO mice grew normally with no apparent morphological abnormalities, they developed substantial osteopenia at 1 year of age, which was caused by increased bone resorption. Bcl-x deficiency increased the bone-resorbing activity of osteoclasts despite their high susceptibility to apoptosis, whereas Bcl-xL overexpression produced the opposite effect. In addition, Bcl-x cKO osteoclasts displayed increased c-Src activity, which was linked to increased levels of vitronectin and fibronectin expression. These results suggest that Bcl-xL attenuates osteoclastic bone-resorbing activity through the decreased production of ECM proteins, such as vitronectin and fibronectin, and thus provide evidence for what we believe to be a novel cellular function of Bcl-xL.

Potential involvement of p53 in ischemia/reperfusion-induced osteonecrosis.

Idiopathic osteonecrosis of the femoral head (ION) is a devastating pathological condition of unknown etiology. In this study, we developed a simple murine model of osteonecrosis and investigated the underlying molecular mechanisms. In this model, the central portion of the tails of male C57BL/6 mice were tightly ligated to produce ischemic regions at sites distal to the ligatures. The occlusive ligatures were maintained for the indicated periods and then removed to induce reperfusion. The tails were histologically examined, and gene expression was analyzed by PCR array. The effect of p53 expression on osteocytes apoptosis was examined using preosteocytic MLO-A5 cells. In addition, the expression of p53 was analyzed in the femoral head samples obtained from hip osteoarthritis (OA) patients and ION patients. Caudal vertebrae distal to the ligatures (distal region) exhibited histological changes mimicking those observed in ION. Expression of p53 was increased in the distal region, and overexpression of p53 induced apoptosis in MLO-A5 cells. Treatment with a p53 inhibitor suppressed osteocyte apoptosis in the distal region. Strong p53 immunostaining was observed in osteocytes, vascular endothelial cells, and bone marrow cells in the femoral heads from ION patients but not from OA patients. Ischemia/reperfusion of the caudal vertebrae is a useful murine model of osteonecrosis, mimicking the histological changes found in ION. Using this model, we found the possible involvement of p53 in the osteocyte apoptosis observed in ION. Therapeutics targeting p53 might be a useful approach to ameliorating or even preventing osteonecrosis in ION patients.

Pivotal role of Bcl-2 family proteins in the regulation of chondrocyte apoptosis.

During endochondral ossification, chondrocytes undergo hypertrophic differentiation and die by apoptosis. The level of inorganic phosphate (P(i)) elevates at the site of cartilage mineralization, and when chondrocytes were treated with P(i), they underwent rapid apoptosis. Gene silencing of the proapoptotic Bcl-2 homology 3-only molecule bnip3 significantly suppressed P(i)-induced apoptosis. Conversely, overexpression of Bcl-xL suppressed, and its knockdown promoted, the apoptosis of chondrocytes. Bnip3 was associated with Bcl-xL in chondrocytes stimulated with P(i). Bcl-xL was expressed uniformly in the growth plate chondrocytes, whereas Bnip3 expression was exclusively localized in the hypertrophic chondrocytes. Finally, we generated chondrocyte-specific bcl-x knock-out mice using the Cre-loxP recombination system, and we provided evidence that the hypertrophic chondrocyte layer was shortened in those mice because of an increased apoptosis of prehypertrophic and hypertrophic chondrocytes, with the mice afflicted with dwarfism as a result. These results suggest the pivotal role of Bcl-2 family members in the regulation of chondrocyte apoptosis.