Koji Otabe

Transcription factor Mohawk controls tenogenic differentiation of bone marrow mesenchymal stem cells in vitro and in vivo.

Mohawk homeobox (MKX) has been demonstrated as a tendon/ligament specific transcription factor. The aim of this study was to investigate the role of MKX in ligament/tenogenic differentiation of bone marrow derived mesenchymal stem cells (BMMSCs). Human BMMSCs were treated with 50 ng/ml BMP‐12 or transduced with MKX or scleraxis (SCX) adenoviral vector. Gene expression analysis was performed by quantitative reverse transcribed polymerase chain reaction (qRT‐PCR). Rat BMMSCs were seeded in a collagen scaffold and transplanted into a rat Achilles tendon defect model. Tenogenesis related gene expressions and histological features were analyzed. BMP‐12 induced tenogenesis in BMMSCs as indicated by increased COL1a1, TNXB, DCN and SCX mRNA, and MKX expression increased simultaneously. Rat BMMSCs enhanced defect repair and were still detectable 3 weeks after transplantation. Increased expressions of COL1a1, TNC and TNMD in vivo were also correlated with upregulated MKX. Adenoviral MKX promoted expression of COL1a1, TNXB, and TNMD in BMMSCs. This study demonstrated that MKX gene expression is enhanced during the tenogenic differentiation of BMMSCs in vitro and in vivo, and the adenoviral overexpression of MKX increases tendon extracellular matrix gene expression and protein production. Thus, MKX is a key factor for tenogenic differentiation of MSCs. 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1–8, 2015.

Transplantation of Achilles Tendon Treated With Bone Morphogenetic Protein 7 Promotes Meniscus Regeneration in a Rat Model of Massive Meniscal Defect

Objective This study was undertaken to examine whether bone morphogenetic protein 7 (BMP-7) induces ectopic cartilage formation in the rat tendon, and whether transplantation of tendon treated with BMP-7 promotes meniscal regeneration. Additionally, we analyzed the relative contributions of host and donor cells on the healing process after tendon transplantation in a rat model. Methods BMP-7 was injected in situ into the Achilles tendon of rats, and the histologic findings and gene profile were evaluated. Achilles tendon injected with 1 μg of BMP-7 was transplanted into a meniscal defect in rats. The regenerated meniscus and articular cartilage were evaluated at 4, 8, and 12 weeks. Achilles tendon from LacZ-transgenic rats was transplanted into the meniscal defect in wild-type rats, and vice versa. Results Injection of BMP-7 into the rat Achilles tendon induced the fibrochondrocyte differentiation of tendon cells and changed the collagen gene profile of tendon tissue to more closely approximate meniscal tissue. Transplantation of the rat Achilles tendon into a meniscal defect increased meniscal size. The rats that received the tendon treated with BMP-7 had a meniscus matrix that exhibited increased Safranin O and type II collagen staining, and showed a delay in articular cartilage degradation. Using LacZ-transgenic rats, we determined that the regeneration of the meniscus resulted from contribution from both donor and host cells. Conclusion Our findings indicate that BMP-7 induces ectopic cartilage formation in rat tendons. Transplantation of Achilles tendon treated with BMP-7 promotes meniscus regeneration and prevents cartilage degeneration in a rat model of massive meniscal defect. Native cells in the rat Achilles tendon contribute to meniscal regeneration.

Mid- to Long-term Results of Single-Bundle Versus Double-Bundle Anterior Cruciate Ligament Reconstruction: Randomized Controlled Trial

PURPOSE To evaluate the mid-to long-term results of a randomized controlled trial of single-bundle (SB) versus double-bundle (DB) anterior cruciate ligament (ACL) reconstruction using a semitendinosus tendon. METHODS Seventy-eight patients who underwent primary ACL reconstruction with an autologous semitendinosus tendon were prospectively randomized into 2 groups: SB reconstruction (n = 39) and DB reconstruction (n = 39). In both groups, grafts were fixed at 30° of flexion with a total tension of 80 N. The following evaluation methods were used: clinical examination, KT-1000 arthrometer (MEDmetric, San Diego, CA) measurement, muscle strength, Tegner activity score, Lysholm score, subjective rating scale regarding patient satisfaction and sports performance level, graft retear, contralateral ACL tear, and additional meniscus surgery. RESULTS Fifty-three patients (25 in SB group and 28 in DB group) who were followed up for a minimum of 3 years (mean, 69 months; range, 36 to 140 months) were evaluated. Preoperatively, there were no differences between the groups. Postoperatively, the Lachman and pivot-shift test results were better in the DB group (P = .024 and P < .0001, respectively). KT measurements were better in the DB group (mean, 1.4 mm v 2.7 mm; P = .0023). The Tegner score was also better in the DB group (P = .033). There were no significant differences in range of motion, muscle strength, Lysholm score, subjective rating scale, graft retear, and secondary meniscal tear. CONCLUSIONS In ACL reconstruction using the transtibial approach, DB reconstruction was significantly better than SB reconstruction regarding anterior and rotational stability during the 3- to 12-year follow-up. The results of KT measurements and the Lachman and pivot-shift tests were significantly better in the DB group, whereas there was no difference in the anterior drawer test results. The Tegner score was also better in the DB group; however, there were no differences in the other subjective findings. LEVEL OF EVIDENCE Level II, lesser-quality prospective randomized trial.

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. Methods For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. Results In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage. Conclusion Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.

Transcription Factor Mohawk and the Pathogenesis of Human Anterior Cruciate Ligament Degradation

OBJECTIVE To investigate the expression and function of Mohawk (MKX) in human adult anterior cruciate ligament (ACL) tissue and ligament cells from normal and osteoarthritis (OA)-affected knees. METHODS Knee joints were obtained at autopsy (within 24-48 hours postmortem) from 13 donors with normal knees (mean ± SD age 36.9 ± 11.0 years), 16 donors with knee OA (age 79.7 ± 11.4 years), and 8 aging donors without knee OA (age 76.9 ± 12.9 years). All cartilage surfaces were graded macroscopically. MKX expression was analyzed by immunohistochemistry and quantitative polymerase chain reaction. ACL-derived cells were used to study regulation of MKX expression by interleukin-1β (IL-1β). MKX was knocked down with small interfering RNA (siRNA) to analyze the function of MKX in extracellular matrix (ECM) production and differentiation in ACL-derived cells. RESULTS The expression of MKX was significantly decreased in ACL-derived cells from OA knees compared with normal knees. Consistent with this finding, immunohistochemistry analysis showed that MKX-positive cells were significantly reduced in ACL tissue from OA donors, in particular in cells located in disorientated fibers. In ACL-derived cells, IL-1β strongly suppressed MKX expression and reduced expression of the ligament ECM genes COL1A1 and TNXB. In contrast, SOX9, a chondrocyte master transcription factor, was up-regulated by IL-1β treatment. Importantly, knockdown of MKX expression with siRNA up-regulated SOX9 expression in ACL-derived cells, whereas the expression of COL1A1 and TNXB was reduced. CONCLUSION Reduced expression of MKX is a feature of degenerated ACL in OA-affected joints, and this may be mediated in part by IL-1β. MKX appears necessary to maintain the tissue-specific cellular differentiation status and ECM production in adult human tendons and ligaments.

Comparison of Gingiva, Dental Pulp, and Periodontal Ligament Cells From the Standpoint of Mesenchymal Stem Cell Properties.

The specific properties of mesenchymal stem cells (MSCs) in oral tissues still remain unknown though their existence has been previously reported. We collected gingiva, dental pulp, and periodontal ligament tissues from removed teeth and isolated MSCs. These MSCs were compared in terms of their yields per tooth, surface epitopes, and differentiation potentials by patient-matched analysis. For in vivo calcification analysis, rat gingival and dental pulp cells mounted on β-tricalcium phospateTCP were transplanted into the perivertebral muscle of rats for 6 weeks. Gingival cells and dental pulp cells showed higher yield per tooth than periodontal ligament cells (n=6, p<0.05). Yields of periodontal ligament cells were too low for further analysis. Gingival and dental pulp cells expressed MSC markers such as CD44, CD90, and CD166. Gingival and dental pulp cells obtained phenotypes of chondrocytes and adipocytes in vitro. Approximately 60% of the colonies of gingival cells and 40% of the colonies of dental pulp cells were positively stained with alizarin red in vitro, and both gingival and dental pulp cells were calcified in vivo. We clarified properties of MSCs derived from removed teeth. We could obtain a high yield of MSCs with osteogenic potential from gingiva and dental pulp. These results indicate that gingiva and dental pulp are putative cell sources for hard tissue regeneration.

Effect of Initial Graft Tension on Knee Stability and Graft Tension Pattern in Double-Bundle Anterior Cruciate Ligament Reconstruction

PURPOSE To determine the initial minimal tension for restoring knee stability during double-bundle anterior cruciate ligament (ACL) reconstruction in vivo. METHODS Patients who underwent primary double-bundle ACL reconstruction with an autologous semitendinosus tendon during 2012 were included. The bundles were fixed to a graft-tensioning system during surgery. Initial graft tensions were set to the following tensions per 6 mm in graft diameter: (1) 30 N, (2) 25 N, and (3) 20 N. Bundle tension was recorded during knee flexion-extension and in response to anterior or rotatory loads. In addition, anterior knee laxity was measured with the KT-1000 arthrometer (MEDmetric, San Diego, CA), and the pivot-shift test was evaluated. RESULTS Sixty patients were evaluated. The tension curves of both bundles among different initial tension settings were significantly different (P < .0001), with the tension in the 30-N setting being highest and that in the 20-N setting being lowest. The tension in both bundles showed reciprocal pattern during flexion-extension (P = .019). The tension of the posterolateral bundle graft was significantly lower than that of the anteromedial bundle graft in response to the anterior load at all settings (P = .0017, P = .0019, and P = .0021 at 30° in the 30-N, 25-N, and 20-N settings, respectively, and P < .0001 at 90° at all settings), whereas the tensions in both bundles in response to rotatory loads were equivalent. Two cases showed a grade 1 pivot shift in the 20-N setting, whereas no case showed a positive pivot shift in the other settings. KT measurements in the 30-N and 25-N settings showed no difference. CONCLUSIONS In double-bundle ACL reconstruction, initial tension could be set as low as 25 N; however, initial tension of 20 N is not recommended because it might result in residual pivot shift in some cases, although the pivot-shift difference was not significant. LEVEL OF EVIDENCE Level IV, therapeutic case series.

Evaluation of a behind-remnant approach for femoral tunnel creation in remnant-preserving double-bundle anterior cruciate ligament reconstruction — Comparison with a standard approach

PURPOSE To evaluate a novel approach for femoral tunnel creation, a behind-remnant approach, in remnant-preserving double-bundle anterior cruciate ligament (ACL) reconstruction through comparison with a standard approach. METHODS Sixty patients who underwent remnant-preserving double-bundle ACL reconstruction were included. Thirty patients with a standard approach were classified as the standard group, and 30 patients with a behind-remnant approach as the behind-remnant (BR) group. The anteromedial bundle (AMB) and posterolateral bundle (PLB) were provisionally fixed at 20° and 45° of flexion to a graft tensioning system during surgery. Bundle tension was recorded during knee flexion-extension and in response to anterior or rotatory loads. Femoral tunnel positions were then assessed using the quadrant method. RESULTS During flexion-extension, the BR group showed equivalent tension curves between AMB and PLB, while the standard group showed reciprocal tension curves. The tension on the PLB was lower than the AMB in response to anterior or rotatory loads in the BR group, while the AMB and PLB shared equivalent loads in the standard group. Tunnel position of the AMB in the BR group was lower and deeper, with smaller variances, than that in the standard group. Tunnel position of the PLB in the BR group was lower than that in the standard group. CONCLUSIONS In remnant-preserving double-bundle ACL reconstruction, a behind-remnant approach can be achieved without any removal of the remnant tissue, and could create a deeper and lower AMB tunnel and a lower PLB tunnel with higher reproducibility, showing equivalent tension curves between the AMB and PLB.

Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage

The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA.

Fibulin-3 in joint aging and osteoarthritis pathogenesis

The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA.