金子 陽介

BACKGROUND: Chronic inflammation and impaired immune microenvironment are key barriers to effective cartilage regeneration. Herein, we developed an integrated, injectable thermosensitive hydrogel system termed CS@Gel-Mg. This system modularly combines exosome-loaded chitosan microspheres (CM@Exo)​ embedded within a Mg2+-coordinated poly(N-isopropylacrylamide)-based ionic liquid hydrogel (Gel-Mg), aiming to modulate local immune response and promote cartilage repair. METHODS: Hydroxypropyl-modified chitosan microspheres were synthesized via emulsion crosslinking and effectively loaded with bioactive exosomes. The Gel matrix was fabricated using an ionic liquid monomer (IL-C4) copolymerized with NIPAM and N-vinylimidazole, followed by Mg2+ complexation. Physicochemical properties, including morphology (SEM/TEM), surface chemistry (XPS/FTIR), and thermoresponsive behavior (DSC/TGA), were systematically characterized. The effects of CS@Gel-Mg on immune and cartilage regeneration were evaluated in vivo and in vitro using Western blot, qPCR, histological staining, flow cytometry, gait analysis, and asymmetric weight-bearing analysis. RESULTS: In vitro, the system demonstrated excellent cytocompatibility, promoted ATDC5 cell proliferation and migration, and inhibited apoptosis. The hydrogel also reprogrammed macrophage polarization from M1 to M2 phenotype, as confirmed by qPCR and flow cytometry. In vivo, using a rat full-thickness cartilage defect model, CS@Gel-Mg exhibited improved weight-bearing function, reduced inflammatory infiltration, and superior cartilage histological scores compared to control groups. Mechanistically, immunofluorescence and Western blot analyses suggested enhanced expression of SOX9, COL2A1 and COL1A1, along with suppression of inflammatory cytokines. Furthermore, SOX9 knockdown in vivo diminished the therapeutic effect, confirming its regulatory role in the immuno-chondrogenic axis. CONCLUSION: This exosome-integrated thermoresponsive hydrogel provides a multifunctional platform for immune modulation and cartilage regeneration. Our findings highlight the potential of CS@Gel-Mg as a promising therapeutic strategy for osteochondral repair.

OBJECTIVES: Whether to resurface the patella in total knee arthroplasty for patients with rheumatoid arthritis remains controversial. This study evaluated long-term clinical and radiographic outcomes after total knee arthroplasty without patellar resurfacing. METHODS: Of 100 knees in 74 patients who underwent total knee arthroplasty without patellar resurfacing, 64 knees in 49 patients were available for follow-up (mean, 13.6 years). Clinical evaluations included the presence of anterior knee pain (AKP) at final follow-up, Knee Society Score, Knee Society Functional Score, and Kujala score. Plain radiographic evaluations assessed patellar thinning and its relationship with clinical outcomes. We also compared outcomes between remission and non-remission groups based on Disease Activity Score in 28 joints using C-reactive protein at final follow-up. RESULTS: The incidence of AKP was 9.3%. At follow-up, Knee Society Scores and Functional Scores had improved significantly (P < .05). Although patellar thinning progressed, it did not appear to affect clinical outcomes or incidence of AKP. Disease activity did not differ significantly between groups or by presence of AKP. CONCLUSIONS: Long-term outcomes of total knee arthroplasty without patellar resurfacing in rheumatoid arthritis were favourable, indicating that this approach is acceptable.

INTRODUCTION: Tendinopathy, a disease that causes inflammation and pain and limits patients' activities of daily living, is considered particularly important to treat during the acute inflammatory phase to prevent the transition to chronic degeneration. Recently, platelet-rich plasma (PRP) has been used to treat tendinopathy; however, it is not clear whether platelets themselves, which are the active component of PRP, could be effective in treating tendinopathy. MATERIALS AND METHODS: We made rat Achilles tendinopathy models by incision of the calcaneal attachment and administrated platelet-like cells derived from adipose-derived mesenchymal stem cells (ASCL-PLCs) to the injury site and investigated the anti-inflammatory effect. RESULTS: ASCL-PLCs significantly inhibits the inflammatory cytokine expression and inflammatory cell infiltration in acute tendonitis in a rat Achilles tendon injury model in vivo. Interestingly, we observed no xeno-reaction when human-derived ASCL-PLCs were administered to wild-type rats in vivo. Moreover, IL-6 expression and phosphorylation seen in NIH3T3 fibroblasts treated with IL-6 plus soluble IL-6 receptor were both significantly suppressed by ASCL-PLCs in vitro. CONCLUSION: ASCL-PLC has advantages over existing PRP therapies, including the ability to be cryopreserved after quality checks, and homogeneous populations of ASCL-PLCs can be prepared in large quantity. We conclude that in the future ASCL-PLCs may serve as an allogeneic transplant effective to treat tendinopathy.

INTRODUCTION: With ongoing societal aging, the demand for regenerative medicine has increased. Recently, reports demonstrating the efficacy of knee regenerative therapies have increased, although substantial proportions of these studies involved relatively younger populations. However, in Japan, where our hospital is located, it is common for patients well beyond the WHO-defined elderly age of 65 years to present with a desire for regenerative therapy. This retrospective cohort study investigated the utility of autologous protein solution (APS) knee joint injections by comparing their effects in patients aged 80 years and older and those younger than 80 years. METHODS: From July 2021 to August 2023, we administered single APS injections into the knee joints of patients diagnosed with osteoarthritis. Clinical assessment was conducted at baseline and at one and six months post-injection. Forty-one patients (44 knees) who completed all scheduled assessments were included in this analysis. Subjects were stratified into group E (≥80 years old) or group Y (<80 years old). Clinical evaluations included assessments of joint range of motion, inflammatory biomarkers (C-reactive protein), radiographic Kellgren-Lawrence classification, MRI findings including bone marrow edema, and standardized patient-reported outcome measures (Knee Injury and Osteoarthritis Outcome Score {KOOS} and Knee Society Score {KSS}), with KSS subdomain scores normalized to a 100-point scale. RESULTS: No distinct intergroup differences were observed for knee range of motion, blood test parameters, or MRI findings. Group E had the highest scores on the KOOS activities of daily living and quality of life subscales at one month post-APS administration; both subscale scores decreased at six months post-administration, whereas these subscale scores consistently increased over time in group Y. Compared with the findings in prior research reporting the minimum clinically important difference after platelet-rich plasma administration, the findings at six months after APS administration were superior for all KOOS subscales, excluding sports, in both groups. Concerning KSS scores, most subscale scores consistently increased throughout the follow-up period in both groups, whereas the expectation subscale score tended to decrease over time, likely reflecting the evolution of expectations after surgery. CONCLUSIONS: Intra-articular APS injections demonstrated clinical efficacy in patients aged 80 years and older. When the analysis was limited to those who continued follow‑up at both one and six months after treatment, a tendency toward a shorter duration of effect was still observed, indicating the need for careful patient selection.

INTRODUCTION: Hallux hyperdorsiflexion and stiffness are typically associated with hallux rigidus. Although the underlying cause remains unclear, this case appears to have arisen in a patient undergoing treatment for schizophrenia, highlighting the importance of individualized and carefully tailored treatment approaches to achieve optimal patient outcomes and maintain high-quality care. CASE REPORT: A 71-year-old Asian woman undergoing treatment for schizophrenia presented with severe hyperdorsiflexion and stiffness of the right hallux, causing intense pain, which was particularly noticeable during gait and daily activities. Initially, the patient underwent rehabilitation to restore normal active motion of the right extensor hallucis longus (EHL). However, despite continuous rehabilitation, the stiffness persisted. Consequently, the patient underwent surgery involving dissection, lengthening of the distal EHL, and partial transposition of the proximal EHL. Postoperatively, the patient regained active hallux movement and showed significantly improved gait. CONCLUSION: Surgical treatment should be considered as an alternative option if symptoms persist for long in patients with hallux hyperdorsiflexion and stiffness. However, this approach is technically demanding and requires careful consideration of the strategy for successful recovery and optimal patient outcomes.

BACKGROUND: In total knee arthroplasty (TKA), cementless fixation is initially weaker than cement fixation. This study aimed to examine whether filling the tibial peg holes with bone improves initial fixation strength in cementless TKA. METHODS: This prospective, comparative study examined 88 joints in 66 patients randomized to the bone filling (48 joints) or conventional group (no bone filling; 40 joints). All patients underwent TKA with the NexGen® trabecular metal modular tibial component. In the bone filling group, resected cancellous bone was filled into the peg holes before insertion of the tibial component. We performed clinical and plain radiographic evaluations after the operation and measured bone mineral density (BMD) at five sites below the component at 1, 3, 6, and 12 months postoperatively. RESULTS: Operative time and clinical evaluations were not significantly different. Plain radiography showed significant longitudinal thickening of the trabecula below the peg (P<0.05) and decreased occurrence of reactive lines (P=0.07) in the bone filling group compared with the conventional group. BMD was significantly higher in the bone filling group in the medial region below the peg at 1, 3, and 6 months and in the central region at 1 and 3 months (all P<0.05). CONCLUSIONS: When using the NexGen trabecular metal modular tibial component, concurrent peg hole bone filling increases the initial component fixation strength. Possible effects on long-term stabilization warrant further study.

Rheumatoid arthritis (RA) is a disease characterized by chronic joint inflammation, pain and joint destruction, leading to alteration in activities of daily living, yet pathological mechanisms underlying the condition are not fully clarified. To date, various therapeutic agents have been developed as RA therapy including DMARDs and/or biological agents that target inflammatory cytokines or inhibit JAK. Here we asked whether inhibiting signal transducer and activator of transcription 3 (Stat3) activity would antagonize RA. Stat3 forms dimers when activated and undergoes nuclear translocalization; thus we screened approximately 4.9 million small compounds as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening. We identified 15 as strong candidates as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening from those compounds. Four of the 15 significantly inhibited expression of IL-6 and RANKL, both of which are direct targets of Stat3, induced by IL-6. Among four, one compound, F0648-0027, significantly inhibited arthritis development without apparent adverse effects in vivo in collagen-induced arthritis model mice. F0648-0027 also significantly blocked Stat3 phosphorylation and nuclear localization following IL-6 stimulation of fibroblasts. These data suggest that Stat3 is a target for collagen-induced arthritis in mice, and that F0648-0027 could serve as a therapeutic reagent against comparable conditions in humans.

This paper describes up-scalable microfiber-shaped tissues for macroscale tendon tissue reconstruction in vitro. C3H10T1/2 cells were encapsulated in a calcium alginate hydrogel microfiber that was fabricated via a double coaxial microfluidic device. The C3H10T1/2 cells gradually merged to construct the microfiber-shaped tendon-like tissue. Our microfiber-shaped tendon-like tissues were alive and maintained their microfiber-shaped morphology over 600 days. Immunostaining and real-time quantitative polymerase chain reaction analyses showed that our fabricated microfiber-shaped tendon-like tissue properly expressed tenomodulin and the orientation of the filaments of actin, which are one of the characteristics of tendon tissue in vivo. Furthermore, a macroscale tendon tissue assembly with ∼1 cm in length and ∼200 µm in thickness was successfully constructed by bundling the microfiber-shaped tendon-like tissues together. This feature enabled us to fabricate a macroscale tendon tissue with uniform cell distribution. We believe that our fabricated microfiber-shaped tendon-like tissue would be a suitable strategy to reconstruct tendon tissue in vitro for the treatments of tendon-related injuries.

INTRODUCTION: Osteonecrosis of the jaw (ONJ) occurring after invasive dental treatment often adversely affects patients' activities of daily living. Long-term administration of strong anti-bone resorptive agents such as bisphosphonates prior to invasive dental treatment is considered an ONJ risk factor; however, pathological mechanisms underlying ONJ development remain unclear. MATERIALS AND METHODS: We developed an ONJ mouse model in which a tooth is extracted during treatment with the bisphosphonate zoledronate. RESULTS: We observed induction of apoptosis in osteocytes, resulting in formation of empty lacunae in jaw bones at sites of tooth extraction but not in other bones of the same mice. We also observed elevated levels of inflammatory cytokines such as TNFα, IL-6 and IL-1 in jaw bone at the extraction site relative to other sites in zoledronate-treated mice. We also report that treatment in vitro with either zoledronate or an extract from Porphyromonas gingivalis, an oral bacteria, promotes expression of inflammatory cytokines in osteoclast progenitor cells. We demonstrate that gene-targeting of either TNFα, IL-6 or IL-1 or treatment with etanercept, a TNFα inhibitor, or a neutralizing antibody against IL-6 can antagonize ONJ development caused by combined tooth extraction and zoledronate treatment. CONCLUSIONS: Taken together, the cytokine storm induced by invasive dental treatment under bisphosphonate treatment promotes ONJ development due to elevated levels of inflammatory cytokine-producing cells. Our work identifies novel targets potentially useful to prevent ONJ.

Estrogen deficiency can be caused by ovarian dysfunction in females. Mechanisms underlying osteoporosis in this condition have been characterized in animal models, such as ovariectomized mice and rats, although it remains unclear how hypothalamic dysfunction promotes osteoporosis. Here, we show that administration of a gonadotropin-releasing hormone antagonist (GnRHa) significantly decreases uterine weight, a manifestation of hypothalamic dysfunction, and promotes both cortical and trabecular bone loss in female mice in vivo. We also report that osteoclast number significantly increased in mice administered GnRHa, and that the transcription factor hypoxia inducible factor 1 alpha (HIF1α) accumulated in those osteoclasts. We previously reported that treatment of mice with the active vitamin D analogue ED71, also known as eldecalcitol, inhibited HIF1α accumulation in osteoclasts. We show here that in mice, co-administration of ED71 with GnRHa significantly rescued the reduced cortical and trabecular bone mass promoted by GnRHa administration alone. GnRHa-dependent HIF1α accumulation in osteoclasts was also blocked by co-administration of ED71. We conclude that hypothalamic dysfunction promotes HIF1α accumulation in osteoclasts and likely results in reduced bone mass. We conclude that treatment with ED71 could serve as a therapeutic option to counter osteoporotic conditions in humans.

BACKGROUND: Knot tying technique is an extremely important basic skill for all surgeons. Clinically, knot slippage or suture breakage will lead to wound complications. Although some previous studies described the knot-tying technique of medical students or trainees, little information had been reported on the knot-tying technique of instructors. The objective of the preset study was to assess surgeons' manual knot tying techniques and to investigate the differences of tensile strength in knot tying technique between surgical instructors and trainees. METHODS: A total of 48 orthopaedic surgeons (postgraduate year: PGY 2-18) participated. Surgeons were requested to tie surgical knots manually using same suture material. They were divided into two groups based on each career; instructors and trainees. Although four open conventional knots with four throws were chosen and done with self-selected methods, knot tying practice to have the appropriate square knots was done as education only for trainees before the actual trial. The knots were placed over a 30 cm long custom made smooth polished surface with two cylindrical rods. All knots were tested for tensile strength using a tensiometer. The surgical loops were loaded until the knot slipped or the suture broke. The tensile strength of each individual knot was defined as the force (N) required to result in knot failure. Simultaneously, knot failure was evaluated based on knot slippage or suture rupture. In terms of tensile strength or knot failure, statistical comparison was performed between groups using two-tailed Mann-Whitney U test or Fisher exact probability test, respectively. RESULTS: Twenty-four instructors (PGY6-PGY18) and 24 trainees (PGY2-PGY5) were enrolled. Tensile strength was significantly greater in trainees (83.0 ± 27.7 N) than in instructors (49.9 ± 34.4 N, P = 0.0246). The ratio of slippage was significantly larger in instructors than in trainees (P < 0.001). Knot slippage (31.8 ± 17.7 N) was significantly worse than suture rupture (89.9 ± 22.2 N, P < 0.001) in tensile strength. CONCLUSIONS: Mean tensile strength of knots done by trainees after practice was judged to be greater than that done by instructors in the present study. Clinically, knot slippage can lead to wound dehiscence, compared to suture rupture.

Low energy availability in female athletes often causes hypothalamic amenorrhea and osteoporosis, in turn promoting stress fractures. Mechanisms underlying these conditions remain unclear. Here we show that model mice subjected to food restriction (FR) or FR-plus-voluntary running exercise exhibit significantly reduced bone mineral density, cortical bone parameters and uterine weight than do control mice, and that these parameters worsen in the FR-plus-exercise group. Relative to controls, FR and FR-plus-exercise groups showed significantly lower mineral apposition rate and osteoclast number and significantly reduced serum insulin-like growth factor-1 (IGF1) levels. Outcomes were rescued by ED71 or 1.25(OH)2D3 treatment. Thus, we conclude that administration of active vitamin D analogues represents a possible treatment to prevent these conditions.

Vitamin D deficiency is a recognized risk factor for sarcopenia development, but mechanisms underlying this outcome are unclear. Here, we show that low vitamin D status worsens immobilization-induced muscle atrophy in mice. Mice globally lacking vitamin D receptor (VDR) exhibited more severe muscle atrophy following limb immobilization than controls. Moreover, immobilization-induced muscle atrophy was worse in neural crest-specific than in skeletal muscle-specific VDR-deficient mice. Tnfα expression was significantly higher in immobilized muscle of VDR-deficient relative to control mice, and was significantly elevated in neural crest-specific but not muscle-specific VDR-deficient mice. Furthermore, muscle atrophy induced by limb immobilization in low vitamin D mice was significantly inhibited in Tnfα-deficient mice. We conclude that vitamin D antagonizes immobilization-induced muscle atrophy via VDR expressed in neural crest-derived cells.

The number of osteoarthritis patients is increasing with the rise in the number of elderly people in developed countries. Osteoarthritis, which causes joint pain and deformity leading to loss of activities of daily living, is often treated surgically. Here we show that mechanical stress promotes accumulation of reactive oxygen species (ROS) in chondrocytes in vivo, resulting in chondrocyte apoptosis and leading to osteoarthritis development in a rat model. We demonstrate that mechanical stress induces ROS accumulation and inflammatory cytokine expression in cultured chondrocytes in vitro and that both are inhibited by treatment with the anti-oxidant N-acetyl cysteine (NAC). In vivo, osteoarthritis development in a rat osteoarthritis model was also significantly inhibited by oral administration of NAC. MMP13 expression and down-regulation of type II collagen in chondrocytes, both of which indicate osteoarthritis, as well as chondrocyte apoptosis in osteoarthritis rats were inhibited by NAC. Interestingly, osteoarthritis development in sham-operated control sides, likely due to disruption of normal weight-bearing activity on the control side, was also significantly inhibited by NAC. We conclude that osteoarthritis development in rats is significantly antagonized by oral NAC administration. Currently, no oral medication is available to prevent osteoarthritis development. Our work suggests that NAC may represent such a reagent and serve as osteoarthritis treatment.

CTLA4-Ig (cytotoxic T-lymphocyte antigen 4-immunoglobulin; Abatacept) is a biologic drug for rheumatoid arthritis. CTLA4 binds to the CD80/86 complex of antigen-presenting cells and blocks the activation of T cells. Although previous reports showed that CTLA4-Ig directly inhibited osteoclast differentiation, the whole inhibitory mechanism of CTLA4-Ig for osteoclast differentiation is unclear. Bone marrow macrophages (BMMs) from WT mice were cultured with M-CSF and RANKL with or without the recombinant mouse chimera CTLA4-Ig. Intracellular calcium oscillations of BMMs with RANKL were detected by staining with calcium indicator fura-2 immediately after administration of CTLA4-Ig or after one day of treatment. Calcium oscillations were analyzed using Fc receptor gamma- (FcRγ-) deficient BMMs. CTLA4-Ig inhibited osteoclast differentiation and reduced the expression of the nuclear factor of activated T cells NFATc1 in BMMs in vitro. Calcium oscillations in BMMs were suppressed by CTLA4-Ig both immediately after administration and after one day of treatment. CTLA4-Ig did not affect osteoclastogenesis and did not cause remarkable changes in calcium oscillations in FcRγ-deficient BMMs. Finally, to analyze the effect of CTLA4-Ig in vivo, we used an LPS-induced osteolysis model. CTLA4-Ig suppressed LPS-induced bone resorption in WT mice, not in FcRγ-deficient mice. In conclusion, CTLA4-Ig inhibits intracellular calcium oscillations depending on FcRγ and downregulates NFATc1 expression in BMMs. © 2019 American Society for Bone and Mineral Research.

Insulin-like growth factor-I (IGF-I) is a peptide with diverse functions, among them regulation of embryonic development and bone homeostasis. Serum IGF-I levels decline in the elderly; however, IGF-I function in adults has not been clearly defined. Here, we show that IGF-I is required to maintain muscle mass in adults. We crossed Igf-I flox'd and Mx1 Cre mice to yield Mx1 Cre/Igf-Iflox/flox (IGF-I cKO) mice, and deleted Igf-I in adult mice by polyIpolyC injection. We demonstrate that, although serum IGF-I levels significantly decreased after polyIpolyC injection relative to (Igf-Iflox/flox) controls, serum glucose levels were unchanged. However, muscle mass decreased significantly after IGF-I down-regulation, while bone mass remained the same. In IGF-I cKO muscle, expression of anabolic factors such as Eif4e and p70S6K significantly decreased, while expression of catabolic factors MuRF1 and Atrogin-1 was normal and down-regulated, respectively, suggesting that observed muscle mass reduction was due to perturbed muscle metabolism. Our data demonstrate a specific role for IGF-I in maintaining muscle homeostasis in adults.

Auto-inflammatory syndrome, a condition clinically distinct from rheumatoid arthritis, is characterized by systemic inflammation in tissues such as major joints, skin, and internal organs. Autonomous innate-immune activation is thought to promote this inflammation, but underlying pathological mechanisms have not been clarified nor are treatment strategies established. Here, we newly established a mouse model in which IL-1 signaling is conditionally activated in adult mice (hIL-1 cTg) and observed phenotypes similar to those seen in auto-inflammatory syndrome patients. In serum of hIL-1 cTg mice, IL-6 and IL-17 levels significantly increased, and signal transducer and activator of transcription 3 (Stat3) was activated in joints. When we crossed hIL-1 cTg with either IL-6- or IL-17-deficient mice or with Stat3 conditional knockout mice, phenotypes seen in hIL-1 cTg mice were significantly ameliorated. Thus, IL-6, IL-17 and Stat3 all represent potential therapeutic targets for this syndrome.

Rheumatoid arthritis (RA) is a multi-factorial disease characterized by chronic inflammation and destruction of multiple joints. To date, various biologic treatments for RA such as anti-tumor necrosis factor alpha antibodies have been developed; however, mechanisms underlying RA development remain unclear and targeted therapy for this condition has not been established. Here, we provide evidence that signal transducer and activator of transcription 3 (Stat3) promotes inflammation and joint erosion in a mouse model of arthritis. Stat3 global KO mice show early embryonic lethality; thus, we generated viable Stat3 conditional knockout adult mice and found that they were significantly resistant to collagen-induced arthritis (CIA), the most common RA model, compared with controls. We then used an in vitro culture system to screen ninety-six existing drugs to select Stat3 inhibitors and selected five candidate inhibitors. Among them, three significantly inhibited development of arthritis and joint erosion in CIA wild-type mice. These findings suggest that Stat3 inhibitors may serve as promising drugs for RA therapy.