Published papers

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.

Background: Although various reconstruction techniques are available for anterior cruciate ligament (ACL) injuries, a long recovery time is required before patients return to sports activities, as the reconstructed ACL requires time to regain strength. To date, several studies have reported use of mesenchymal stem cells in orthopaedic surgery; however, no studies have used adipose-derived stem cell (ADSC) sheets in ACL reconstruction (ACLR). Hypothesis: ADSC sheet transplantation can improve biomechanical strength of the autograft used in ACLR. Study Design: Controlled laboratory study. Methods: A total of 68 healthy Japanese white rabbits underwent unilateral ACLR with a semitendinosus tendon autograft after random enrollment into a control group (no sheet; n = 34) and a sheet group (ADSC sheet; n = 34). At 2, 4, 8, 16, and 24 weeks after surgery, rabbits in each group were sacrificed to evaluate tendon-bone healing using histological staining, micro–computed tomography, and biomechanical testing. At 24 weeks, scanning transmission electron microscopy of the graft midsubstance was performed. Results: The ultimate failure load for the control and sheet groups, respectively, was as follows: 17.2 ± 5.5 versus 37.3 ± 10.3 (P =.01) at 2 weeks, 28.6 ± 1.9 versus 47.4 ± 10.4 (P =.003) at 4 weeks, 53.0 ± 14.3 versus 48.1 ± 9.3 (P =.59) at 8 weeks, 66.2 ± 9.3 versus 95.2 ± 43.1 (P =.24) at 16 weeks, and 66.7 ± 27.3 versus 85.3 ± 29.5 (P =.39) at 24 weeks. The histological score was also significantly higher in the sheet group compared with the control group at early stages up to 8 weeks. On micro–computed tomography, relative to the control group, the bone tunnel area was significantly narrower in the sheet group at 4 weeks, and the bone volume/tissue volume of the tendon-bone interface was significantly greater at 24 weeks. Scanning transmission electron microscopy at 24 weeks indicated that the mean collagen fiber diameter in the midsubstance was significantly greater, as was the occupation ratio of collagen fibers per field of view, in the sheet group. Conclusion: ADSC sheets improved biomechanical strength, prevented bone tunnel enlargement, and promoted tendon-bone interface healing and graft midsubstance healing in an in vivo rabbit model. Clinical Relevance: ADSC sheets may be useful for early tendon-bone healing and graft maturation in ACLR.

Currently, implants are utilized clinically for bone transplant procedures. However, if infectious osteomyelitis occurs at implant sites, removal of bacteria can be challenging. Moreover, altered blood flow at peri-implant infectious sites can create an anaerobic environment, making it more difficult to treat infection with antibiotics. Thus, it would be beneficial if implants could be modified to exhibit antibacterial activity, even in anaerobic conditions. Here, we show antibacterial activity of silver ions coated on titanium rods, even against the anaerobic bacteria Porphyromonas gingivalis (P. gingivalis), both in vitro and in vivo. Specifically, we implanted silver-coated or control uncoated titanium rods along with P. gingivalis in mouse femoral bone BM cavities and observed significantly inhibited P. gingivalis infection with silver-coated compared with non-coated rods, based on in vivo bio-imaging. Osteonecrosis by infectious osteomyelitis and elevation of the inflammatory factors C-reactive protein and IL-6 promoted by P. gingivalis s were also significantly reduced in the presence of silver-coated rods. Overall, our study indicates that silver ion coating of an implant represents a therapeutic option to prevent associated infection, even in anaerobic conditions or against anaerobic bacteria.

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.

Ossification of the posterior longitudinal ligament (OPLL), a disease characterized by the ectopic ossification of a spinal ligament, promotes neurological disorders associated with spinal canal stenosis. While blocking ectopic ossification is mandatory to prevent OPLL development and progression, the mechanisms underlying the condition remain unknown. Here we show that expression of hydroxyacid oxidase 1 (Hao1), a gene identified in a previous genome-wide association study (GWAS) as an OPLL-associated candidate gene, specifically and significantly decreased in fibroblasts during osteoblast differentiation. We then newly established Hao1-deficient mice by generating Hao1-flox mice and crossing them with CAG-Cre mice to yield global Hao1-knockout (CAG-Cre/Hao1flox/flox; Hao1 KO) animals. Hao1 KO mice were born normally and exhibited no obvious phenotypes, including growth retardation. Moreover, Hao1 KO mice did not exhibit ectopic ossification or calcification. However, urinary levels of some metabolites of the tricarboxylic acid (TCA) cycle were significantly lower in Hao1 KO compared to control mice based on comprehensive metabolomic analysis. Our data indicate that Hao1 loss does not promote ectopic ossification, but rather that Hao1 functions to regulate the TCA cycle in vivo.

Introduction: In women, the female athlete triad, marked by low energy availability, functional hypothalamic amenorrhea and osteoporosis, is a recognized risk for stress fractures. Stress injuries also occur in men, but by contrast risks and mechanisms underlying them are less characterized. Materials and methods: 5 week-old wild-type male mice were fed ad libitum (ad) or subjected to 60% food restriction (FR) for five weeks. In both groups, some mice were allowed access to an exercise wheel in cages to allow voluntary wheel running (ex) and/or treated with active vitamin D analogues. Mice were sacrificed and analyzed at 10 weeks of age. Result: Male FR mice exhibited significantly reduced testicle weight, serum testosterone levels and bone mass. Such bone losses in FR male mice were enhanced by exercise. Histological analysis revealed that both bone-resorbing and -forming activities were significantly reduced in FR or FR plus exercise (FR + ex) mice, mimicking a state of low bone turnover. Significantly reduced bone mass in FR or FR + ex male mice was significantly rescued by treatment with active vitamin D analogues, with significant restoration of osteoblastic activities. Serum levels of insulin-like growth factor I (IGF-I), which is critical for bone remodeling, were significantly lower in FR versus control male mice. Conclusions: Low energy availability puts men at risk for stress injuries as well, and low energy availability is upstream of gonadal dysfunction and osteoporosis in males. Active vitamin D analogues could serve as therapeutic or preventive options for stress injuries in men.

Invasive dental treatment such as tooth extraction following treatment with strong anti-bone resorptive agents, including bisphosphonates and denosumab, reportedly promotes osteonecrosis of the jaw (ONJ) at the extraction site, but strategies to prevent ONJ remain unclear. Here we show that in mice, administration of either active vitamin D analogues, antibiotics or anti-inflammatory agents can prevent ONJ development induced by tooth extraction during treatment with the bisphosphonate zoledronate. Specifically, tooth extraction during treatment with zoledronate induced osteonecrosis in mice, but administration of either 1,25(OH)2D3 or ED71, both active vitamin D analogues, significantly antagonized osteonecrosis development, even under continuous zoledronate treatment. 1,25(OH)2D3 or ED71 administration also significantly inhibited osteocyte apoptosis induced by tooth extraction and bisphosphonate treatment. Administration of either active vitamin D analogue significantly inhibited elevation of serum inflammatory cytokine levels in mice in response to injection of lipopolysaccharide, an infection mimetic. Furthermore, administration of either anti-inflammatory or antibiotic reagents significantly blocked ONJ development following tooth extraction and zoledronate treatment. These findings suggest that administration of active vitamin D, anti-inflammatory agents or antibiotics could prevent ONJ development induced by tooth extraction in patients treated with zoledronate.

Auto-inflammatory syndromes are rare diseases characterized by arthritis and joint destruction, symptoms similar to but distinct from rheumatoid arthritis (RA). Therapeutic targets have not been well characterized for auto-inflammatory syndromes, although the E3 ligase Synoviolin was previously shown to be a novel therapeutic target for RA. Here, we show that Synoviolin loss has little impact on a model of auto-inflammatory diseases. We previously established such a model, the hIL-1 cTg mouse, in which IL-1 signaling was constitutively activated, and animals exhibit symptoms recapitulating auto-inflammatory syndromes such as major joint dominant arthritis. Here, we crossed hIL-1 cTg with Synoviolin flox'd mice to yield hIL-1 cTg/Synoviolin cKO mice. Synoviolin gene expression was ablated in adult hIL-1 cTg/Synoviolin cKO mice by injection of pIpC to activate Mx1 promoter-driven Cre recombinase. However, symptoms seen in hIL-1 cTg mice such as arthritis and joint destruction were not alleviated by targeting Synoviolin, ruling out Synoviolin as a therapeutic target for auto-inflammatory disease. Our results indicate that although similar, RA and auto-inflammatory diseases are different diseases, and treatment strategies should differ accordingly.

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.

Changes in bone metabolism occur in mothers during pregnancy or lactation that may decrease bone mass and result in fragility fractures after partum. However, use of drugs during pregnancy or lactation to counteract these effects is often prohibited or strongly discouraged. Therefore, approaches to protect mothers from fragility fractures have not been established. Here we show that bone mineral density was significantly lower in female mice after partum than in age-matched female mice without partum. We also show that temporary administration of the bisphosphonate alendronate, either just before or just after pregnancy, to female mice was protective against bone loss due to pregnancy or lactation and had no adverse effects on offspring, such as growth retardation. Furthermore, we show that alendronate administration to female mice during lactation was effective in increasing bone mass in mothers without promoting bone abnormalities or growth retardation in offspring. Calcium levels in milk from female mice administered alendronate during lactation were equivalent to those in milk from mothers not treated with alendronate. Overall, we propose that alendronate administration to mothers could prevent bone loss and fragility fractures during pregnancy and lactation.

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.