Inflammation and Bone Metabolism in Rheumatoid Arthritis: Molecular Mechanisms of Joint Destruction and Pharmacological Treatments.

Rheumatoid arthritis (RA) is an inflammatory disease characterized by a variety of symptoms and pathologies often presenting with polyarthritis. The primary symptom in the initial stage is joint swelling due to synovitis. With disease progression, cartilage and bone are affected to cause joint deformities. Advanced osteoarticular destruction and deformation can cause irreversible physical disabilities. Physical disabilities not only deteriorate patients' quality of life but also have substantial medical economic effects on society. Therefore, prevention of the progression of osteoarticular destruction and deformation is an important task. Recent studies have progressively improved our understanding of the molecular mechanism by which synovitis caused by immune disorders results in activation of osteoclasts; activated osteoclasts in turn cause bone destruction and para-articular osteoporosis. In this paper, we review the mechanisms of bone metabolism under physiological and RA conditions, and we describe the effects of therapeutic intervention against RA on bone.

Mass spectrometric quantitation of AGEs and enzymatic crosslinks in human cancellous bone.

Advanced glycation end-products (AGEs) deteriorate bone strength. Among over 40 species identified in vivo, AGEs other than pentosidine were roughly estimated as total fluorescent AGEs (tfAGEs) due to technical difficulties. Using LC-QqTOF-MS, we established a system that enabled the quantitation of five AGEs (CML, CEL, MG-H1, CMA and pentosidine) as well as two mature and three immature enzymatic crosslinks. Human bone samples were collected from 149 patients who underwent total knee arthroplasty. Their clinical parameters were collected to investigate parameters that may be predictive of AGE accumulation. All the analytes were quantitated and showed significant linearity with high sensitivity and precision. The results showed that MG-H1 was the most abundant AGE, whereas pentosidine was 1/200-1/20-fold less abundant than the other four AGEs. The AGEs were significantly and strongly correlated with pentosidine, while showing moderate correlation with tfAGEs. Interestingly, multiple linear regression analysis revealed that gender contributed most to the accumulation of all the AGEs, followed by age, tartrate-resistant acid phosphatase-5b and HbA1c. Furthermore, the AGEs were negatively correlated with immature crosslinks. Mass spectrometric quantitation of AGEs and enzymatic crosslinks is crucial to a better understanding of ageing- and disease-related deterioration of bone strength.

Pre-operative hemoglobin level and use of sedative-hypnotics are independent risk factors for post-operative delirium following total knee arthroplasty.

BACKGROUND: Delirium is a well-known complication following surgery, especially with the increasing age of patients undergoing surgery. The increasing demands resulting from a prolonged healthy life expectancy has resulted in more arthroplasties despite their age and existing comorbidities. The purpose of this study is to explore the various risk factors that may contribute to delirium in unilateral and bilateral total knee arthroplasties in the elderly population. METHODS: 170 patients who underwent unilateral or bilateral total knee arthroplasties were analyzed retrospectively for delirium. Age, sex, comorbidities, use of sedative-hypnotics, peri-operative blood loss, pre- and post-operative laboratory blood test results were investigated and analyzed. RESULTS: The incidence of post-operative delirium was 6.5% (11 out of 170 patients) with a mean age of 79.5 (± 6.9) years, compared to 73.0 (± 9.0) years in the non-delirium group. Higher age, use of sedative-hypnotics, low pre-operative Hb and Ht, low post-operative Hb, Ht and BUN were observed in the delirium group. Multivariate logistic regression analysis identified that the use of sedative-hypnotics and pre-operative Hb level were independent risk factors for post-operative delirium after TKA. The odds ratios for the use of sedative-hypnotics and pre-operative Hb level were 4.6 and 0.53, respectively. Receiver operating characteristic curve analysis showed that pre-operative Hb of less than 11.1 g/dL was a predictor for the development of delirium, with a sensitivity of 54.6% and a specificity of 91.6%. CONCLUSION: Patients with a pre-operative Hb level of < 11.1 g/dL or those using sedative-hypnotics are associated with post-operative delirium. Peri-operative management and preventative measures are therefore needed to reduce the risks of post-operative delirium in such patients.

Enhancement of tendon-bone interface healing and graft maturation with cylindrical titanium-web (TW) in a miniature swine anterior cruciate ligament reconstruction model: histological and collagen-based analysis.

BACKGROUND: Tendon-bone interface healing and ligamentization of the graft in anterior cruciate ligament (ACL) reconstruction with autografts are important factors affecting treatment outcome. This study aimed to investigate the effectiveness of a cylindrical titanium-web (TW) in tendon-bone interface healing and graft maturation in ACL reconstruction. METHODS: Fourteen mature female CLAWN miniature swine underwent bilateral ACL reconstructions with patellar tendon (PT) autografts. In one limb, the TW/tendon complex was placed into the proximal side of the tibial tunnel. Only the graft was transplanted into the tunnel in the control limb. The proximal side of the graft was sutured into the stump of the native ACL and the distal end was stapled to the tibia. The animals were euthanized at 4 and 15 weeks postoperatively, for histological and biochemical analyses. RESULTS: Microscopic images in TW limbs showed that ingrowth of tendon-like tissue and mineralized bone tissue into the TW connected the bone and the tendon directly. In contrast, fibrous tissue intervened between the bone and tendon in the control limbs. The total amount of collagen cross-links (which defines the strength of collagen fibers) and the maturation of collagen cross-links in TW tendons were significantly higher (p < 0.05) than those of control limbs. There was no significant difference in the ratio of dihydroxy-lysinonorleucine to hydroxy-lysinonorleucine (an indicator of tissue specific collagen maturation) between TW tendons and that of the native PT. CONCLUSIONS: TW promoted the maturation and formation of collagen cross-links in the grafted tendon while maintaining the cross-links pattern of native tendon collagen, and enabled direct binding of tendon to bone.

The Regulation of Bone Metabolism and Disorders by Wnt Signaling.

Wnt, a secreted glycoprotein, has an approximate molecular weight of 40 kDa, and it is a cytokine involved in various biological phenomena including ontogeny, morphogenesis, carcinogenesis, and maintenance of stem cells. The Wnt signaling pathway can be classified into two main pathways: canonical and non-canonical. Of these, the canonical Wnt signaling pathway promotes osteogenesis. Sclerostin produced by osteocytes is an inhibitor of this pathway, thereby inhibiting osteogenesis. Recently, osteoporosis treatment using an anti-sclerostin therapy has been introduced. In this review, the basics of Wnt signaling, its role in bone metabolism and its involvement in skeletal disorders have been covered. Furthermore, the clinical significance and future scopes of Wnt signaling in osteoporosis, osteoarthritis, rheumatoid arthritis and neoplasia are discussed.

Positioning the femoral bone socket and the tibial bone tunnel using a rectangular retro-dilator in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to evaluate the positions of femoral bone sockets and tibial bone tunnels made with the rectangular retro-dilator (RRD), which we manufactured for anterior cruciate ligament reconstruction (ACLR) with a bone-patella tendon-bone (BPTB) graft which is fixed into the rectangular bone socket and tunnel made at anatomical ACL insertion sites. METHODS: 42 patients who had undergone ACLR with BPTB using the RRD were evaluated to assess bone socket and tunnel positions by the quadrant method and Magnussen classification using three-dimensional (3-D) CT. Intra-operative complications were also investigated in all patients. RESULTS: 3-D CT of the operated knee joints using the RRD showed that the bone socket and tunnel were placed in anatomical positions. In the quadrant method, the mean position of the femoral bone socket aperture was located at 22.0 ± 4.2% along the Blumensaat's line, and 37.4 ± 7.2% across the posterior condylar rim. The mean positions of the tibial bone tunnel aperture were 37.7 ± 5.2% and 46.1 ± 2.2% antero-posteriorly and medio-laterally, respectively. In addition, according to the Magnussen classification, 39 cases were evaluated as type 1, and almost all were located behind the lateral intercondylar ridge (also known as the resident's ridge). 3 cases were classified as type 2, which overlapped with the resident's ridge. A partial fracture of BPTB bone fragment was observed in 2 patients, but no serious complications including neurovascular injury were observed. CONCLUSION: The study indicates that the use of RRD achieves a safe anatomical reconstruction of the ACL.

The transcription factor mohawk homeobox regulates homeostasis of the periodontal ligament.

The periodontal ligament (PDL), which connects the teeth to the alveolar bone, is essential for periodontal tissue homeostasis. Although the significance of the PDL is recognized, molecular mechanisms underlying PDL function are not well known. We report that mohawk homeobox (Mkx), a tendon-specific transcription factor, regulates PDL homeostasis by preventing its degeneration. Mkx is expressed in the mouse PDL at the age of 10 weeks and expression remained at similar levels at 12 months. In Mkx-/- mice, age-dependent expansion of the PDL at the maxillary first molar (M1) furcation area was observed. Transmission electron microscopy (TEM) revealed that Mkx-/- mice presented collagen fibril degeneration in PDL with age, while the collagen fibril diameter gradually increased in Mkx+/+ mice. PDL cells lost their shape in Mkx-/- mice, suggesting changes in PDL properties. Microarray and quantitative polymerase chain reaction (qPCR) analyses of Mkx-/- PDL revealed an increase in osteogenic gene expression and no change in PDL- and inflammatory-related gene expression. Additionally, COL1A1 and COL1A2 were upregulated in Mkx-overexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis.

Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs.

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx(-/-) mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.

Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis.

Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx)(-/-) rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx(-/-) mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx(-/-) mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx(-/-) TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx(+/+) TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9 Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.

Gtf2ird1-Dependent Mohawk Expression Regulates Mechanosensing Properties of the Tendon.

Mechanoforces experienced by an organ are translated into biological information for cellular sensing and response. In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover. We show that Mohawk (Mkx), a tendon-specific transcription factor, is essential in mechanoresponsive tenogenesis through regulation of its downstream ECM genes such as type I collagens and proteoglycans such as fibromodulin both in vivo and in vitro Wild-type (WT) mice demonstrated an increase in collagen fiber diameter and density in response to physical treadmill exercise, whereas in Mkx(-/-) mice, tendons failed to respond to the same mechanical stimulation. Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation. Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.

[Analysis of Musculoskeletal Systems and Their Diseases. The role of extracellular matrix and its pathogenesis in musculoskeletal disorders].

Extracellular matrix is present in all organs, but its strength and character differ greatly from structure to structure. Organs in the musculoskeletal system require tissue specific strength, resistance to strains and elasticity. Extracellular matrix that forms the scaffold in tissues, is able to detect mechanical stress, and undergoes remodeling to dissipate the strain. Here we explain how excess mechanical and biochemical stresses disrupt normal matrix remodeling resulting in reduced strength/elasticity.

A systems approach and skeletal myogenesis.

Skeletal myogenesis depends on the strict regulation of the expression of various gene subsets. Therefore, the understanding of genome wide gene regulation is imperative for elucidation of skeletal myogenesis. In recent years, systems approach has contributed to the understanding of various biological processes. Our group recently revealed the critical genome network of skeletal myogenesis by using a novel systems approach combined with whole-mount in situ hybridization (WISH) database, high-throughput screening, and microarray analysis. In this paper, we introduce our systems approach for understanding the myogenesis regulatory network and describe the advantages of systems approach.