Toshiyuki Morito

Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect

IntroductionCurrent cell therapy for cartilage regeneration requires invasive procedures, periosteal coverage and scaffold use. We have developed a novel transplantation method with synovial mesenchymal stem cells (MSCs) to adhere to the cartilage defect.MethodsFor ex vivo analysis in rabbits, the cartilage defect was faced upward, filled with synovial MSC suspension, and held stationary for 2.5 to 15 minutes. The number of attached cells was examined. For in vivo analysis in rabbits, an autologous synovial MSC suspension was placed on the cartilage defect, and the position was maintained for 10 minutes to adhere the cells to the defect. For the control, either the same cell suspension was injected intra-articularly or the defects were left empty. The three groups were compared macroscopically and histologically. For ex vivo analysis in humans, in addition to the similar experiment in rabbits, the expression and effects of neutralizing antibodies for adhesion molecules were examined.ResultsEx vivo analysis in rabbits demonstrated that the number of attached cells increased in a time-dependent manner, and more than 60% of cells attached within 10 minutes. The in vivo study showed that a large number of transplanted synovial MSCs attached to the defect at 1 day, and the cartilage defect improved at 24 weeks. The histological score was consistently better than the scores of the two control groups (same cell suspension injected intra-articularly or defects left empty) at 4, 12, and 24 weeks. Ex vivo analysis in humans provided similar results to those in rabbits. Intercellular adhesion molecule 1-positive cells increased between 1 minute and 10 minutes, and neutralizing antibodies for intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and activated leukocyte-cell adhesion molecule inhibited the attachment.ConclusionPlacing MSC suspension on the cartilage defect for 10 minutes resulted in adherence of >60% of synovial MSCs to the defect, and promoted cartilage regeneration. This adherent method makes it possible to adhere MSCs with low invasion, without periosteal coverage, and without a scaffold.

Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans

OBJECTIVE The existence of mesenchymal stem cells (MSCs) in SF was previously reported. However, the behaviour and properties of MSCs derived from SF have not been fully elucidated. METHODS Human SFs were obtained from 19 knee joints with anterior cruciate ligament injury around the time of reconstruction surgery, and from three healthy volunteers. SF was plated, cultured and examined for colony-forming number, in vitro differentiation, surface epitopes and gene profiles. Also, rabbit synovium-MSCs were injected into the knee joint in a rabbit partial anterior cruciate ligament defect model, and the injected cells were traced. RESULTS SF-MSCs from IA ligament injury patients were 100 times more in number than those from healthy volunteers. Total colony number was positively correlated with post-injury period. No significant differences were observed among the cells derived from SF around the time of the surgery in relation to surface epitopes and differentiation potentials. Cluster analysis of gene profiles demonstrated that SF-MSCs were more similar to synovium MSCs than bone marrow MSCs. In rabbit experiments, the MSCs injected into the knee in which IA ligament was partially defective were observed more on the defected area than on the intact area of the ligament at 24 h. CONCLUSION We demonstrated that SF-MSCs, similar to synovium MSCs, increased in number after IA ligament injury and surgery without marked alteration of the properties.

Analysis of the chondrogenic potential of human synovial stem cells according to harvest site and culture parameters in knees with medial compartment osteoarthritis

OBJECTIVE Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage regeneration because of their high chondrogenic ability. In this study, we examined the synovium of patients with medial compartment knee osteoarthritis (OA) to determine the proportion of MSCs in relation to cellular compartmentalization, and to identify the culture parameters that could affect the chondrogenic potential of synovial MSCs. METHODS Human synovium was collected from 4 different harvest sites in the knees of patients with medial compartment OA. Each synovial tissue sample was divided into 2 parts, one for histologic assessment and the other for analysis of the cell size, surface epitopes, and chondrogenic potential of colony-forming cells in vitro. RESULTS The numbers of alpha-smooth muscle actin-positive vessels and CD31+ endothelial cells were higher in the medial outer region than in the other regions of OA synovial tissue. The numbers of these cells correlated with the number of colony-forming cells. In parallel with increasing duration of the preculture period, the size of the cells increased, while the chondrogenic potential decreased, and this was correlated with expression of CD90. CONCLUSION Medial compartment knee OA demonstrates variability in the distribution of vessels, which results in a varying distribution of MSCs. The preculture period should be utilized to assess both the potential for expansion and the chondrogenic potential of MSCs.

Autologous synovial fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in tissue culture system

Synovial fluid from osteoarthritic knee contains mesenchymal stem cells (MSCs). One of the possible reservoirs of MSCs in synovial fluid is synovial tissue, and synovial fluid may induce mobilization of MSCs into synovial fluid in osteoarthritis patients. Here, we investigated whether synovial fluid expanded synovial MSCs in a tissue culture system. Human synovium and synovial fluid were obtained from osteoarthritis patients during total knee arthroplasties. In the tissue culture system, autologous synovial fluid expanded synovial cells statistically higher than αMEM + FBS, and the addition of TGFβ3 to αMEM + FBS increased expansion to a similar level in all 11 donors. The addition of decorin or anti‐TGFβ neutralizing antibody to synovial fluid partially inhibited synovial cell expansion. In cell culture assay, synovial fluid proliferated synovial cells fewer than αMEM + FBS. The expanded synovial cells in synovial fluid retained multipotentiality and showed surface markers similar to those of MSCs. We demonstrated that autologous synovial fluid enhanced expansion of MSCs in tissue culture of synovium from osteoarthritis patients by promoting cell migration. This effect was partially affected by TGFβ.

Periodic knee injections of BMP-7 delay cartilage degeneration induced by excessive running in rats

Strenuous running of rats enhances mechanical stress on the knee, thereby inducing degeneration of articular cartilage. Bone morphogenetic protein‐7 (BMP‐7) has an inhibitory effect on cartilage degeneration, suggesting its usefulness for human osteoarthritis patients. However, its mode of administration should be investigated. We examined whether weekly knee injections of BMP‐7 delayed the progression of cartilage degeneration. Wistar rats were forced to run 30 km in 6 weeks on a rodent treadmill, and BMP‐7 was injected weekly into the knee. Macroscopically and histologically, this strenuous running regimen induced cartilage degeneration. Weekly injections of 250 ng BMP‐7 delayed the progression of cartilage degeneration. Immunohistochemically, in the control knee, type II collagen expression decreased, while BMP‐7 expression in chondrocytes slightly increased. Interestingly, weekly injection of BMP‐7 increased BMP‐7 expression even 9 days after the final injection. Disulfate disaccharide keratan sulfate in serum transiently increased in the control group, while it remained at a low level in the BMP‐7 group. Weekly BMP‐7 injection increased BMP‐7 expression in chondrocytes and its effect seemed to last more than 7 days. The effect of BMP‐7 could be monitored by serum keratan sulfate concentration. Periodical injections of BMP‐7 delayed progression of cartilage degeneration induced by excessive running in rats.

Revision anterior cruciate ligament reconstruction by double-bundle technique using multi-strand semitendinosus tendon.

PURPOSE The purpose of the study was to compare the outcome of revision anterior cruciate ligament (ACL) reconstruction by the double-bundle (DB) technique using multi-strand semitendinosus tendon with that of primary reconstruction by use of the same technique. METHODS The study included 21 patients who underwent revision ACL reconstruction (mean follow-up, 40 months) with the semitendinosus tendon DB technique between 1995 and 2006 and 86 unilateral primary DB ACL reconstructions (mean follow-up, 33 months) between 2000 and 2004. The outcome of both groups was compared based on differences between operated and unoperated limbs and modified International Knee Documentation Committee grades. Both the overall and sports-related subjective scores were evaluated between the 2 groups. RESULTS The KT measurements (MEDmetric, San Diego, CA) averaged 1.7 mm (SD, 1.8 mm) in the revision group and 1.5 mm (SD, 1.6 mm) in the primary group. There was no significant difference in KT measurements between the 2 groups. The Lachman test was negative in 83% of revision cases and 87% of primary cases; the anterior drawer test was negative in 83% and 91%, respectively, and the pivot-shift test was negative in 78% and 90%, respectively. There was a tendency for a positive pivot-shift test in the revision group being higher. The Lysholm score and subjective recovery score were significantly lower in the revision group. CONCLUSIONS The semitendinosus tendon DB revision procedure provided range of motion and anterior stability comparable to those after primary DB surgery and a comparable return to athletic activities. However, the patients tended to have positive pivot-shift test results. The revision cases were also inferior in terms of the general evaluation of recovery of knee condition. The outcome scores were lower overall in the revision group. LEVEL OF EVIDENCE Level IV, therapeutic case series.

Human sclera maintains common characteristics with cartilage throughout evolution.

Background The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. Methodology/Principal Findings We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-βs and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human ‘sclera’-derived cells can be considered chondrocytes when cultured ex vivo. Conclusions/Significance Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia.

High-flex Posterior Cruciate-Retaining vs Posterior Cruciate-Substituting Designs in Simultaneous Bilateral Total Knee Arthroplasty: A Prospective, Randomized Study

The superiority between the posterior cruciate-retaining and the posterior cruciate-substituting designs still remains controversial. We performed a prospective, randomized control study for evaluation of the superiority of these designs. This study investigated 58 knees in 29 patients with simultaneous bilateral total knee arthroplasty, in which the high-flex CR design was randomly implanted in one knee and the high-flex PS design was implanted in the other knee. The follow-up duration averaged 5.0 years, with a minimum duration of 3 years. Postoperatively, Knee Score and pain points in Knee Score resulted in no significant differences between the 2 designs. However, postoperative arc of range of motion, patient satisfaction, and posterior knee pain at passive flexion in the PS design were significantly superior to that of the CR design.

Hip Acetabular Dysplasia and Joint Laxity of Female Anterior Cruciate Ligament–Injured Patients

Background: It has been noted that some female anterior cruciate ligament–injured patients have complaints of both coxalgia and joint laxity. Hypothesis: Female anterior cruciate ligament–injured patients tend to have both acetabular dysplasia and generalized joint laxity. Study Design: Cohort study (prevalence); Level of evidence, 2. Methods: Hip radiographs of 100 female anterior cruciate ligament–injured patients and 40 female athletes without any hip joint complaints or history of anterior cruciate ligament injury were evaluated by measuring their center-edge angle (CEA). In addition, generalized joint laxity tests using 8 items were performed for anterior cruciate ligament–injured patients. Anterior-posterior (A-P) tibiofemoral translation of the uninjured knee was measured using a KT-1000 knee arthrometer to evaluate joint laxity under anesthesia before anterior cruciate ligament reconstruction. Results: The average (± standard deviation) CEA of female anterior cruciate ligament–injured patients was 25.5° ± 5.3° (uninjured side) and 25.8° ± 4.8° (injured side), and that of the control group was 28.2° ± 4.2° (right side) and 29.2° ± 5.7° (left side), both P < .05. Among the 100 patients with anterior cruciate ligament tears, both the generalized joint laxity score and A-P tibiofemoral translation of the group with acetabular dysplasia (CEA of <25°, n = 37) were significantly greater than that of the normal group (CEA of ≥25°, n = 63). There was a negative correlation between the CEA of female anterior cruciate ligament–injured patients and both the generalized joint laxity score and A-P tibiofemoral translation. Conclusion: The CEA of female anterior cruciate ligament–injured patients was significantly smaller than that of the control group. Statstical analysis showed a moderate negative correlation between the CEA and generalized joint laxity score. Female athletes with an anterior cruciate ligament injury had an increased prevalence of acetabular dysplasia and generalized joint laxity.

Doppler ultrasonography-aided early diagnosis of venous thromboembolism after total knee arthroplasty.

OBJECTIVES Postoperative deep venous thrombosis (DVT) is usually asymptomatic but can result in a fatal pulmonary embolism (PE). To assess the ability of transcranial Doppler (TCD) ultrasound apparatus to detect venous emboli in patients who had undergone total knee arthroplasty (TKA). METHODS Forty-eight patients undergoing TKA were examined postoperatively by using compression ultrasonography, computed tomographic angiography, and TCD ultrasonography that detected high-intensity transient signals (HITS) in femoral veins. An original scoring system based on both the number of HITS and the locations of DVT was tested for its accuracy in predicting PE development. RESULTS Twenty-three of the 48 patients had DVT postoperatively, and 8 had an asymptomatic PE. The sensitivity and specificity of the HITS assessment alone in identifying PE development were 75% and 92.5%, respectively. The scoring system, however, had a sensitivity of 100% and a specificity of 85% and the area under the receiver operating characteristic (ROC) curve (AUC) was 0.96. CONCLUSIONS Application of a scoring system based on the detection of both DVT and HITS may be an effective and efficient method of screening for PE after knee arthroplasty.