Variations in knee cartilage thickness: Fully automatic three-dimensional analysis of MRIs from five manufacturers.

BACKGROUND: Measurements of knee cartilage thickness derived from MR images are attractive biomarkers for osteoarthritis research. Although some cross-sectional multivendor studies exist, none have employed fully automatic three-dimensional MRI analysis. Our objective was to evaluate the variations in knee cartilage thickness measurements obtained using automated methods and MRI instruments from five different vendors. METHODS: The subjects were 10 healthy volunteers aged 22-60 years. MRI models with 3 Tesla strength from five different companies were used. Cartilage thickness was quantified fully automatically for seven regions. We hypothesized that "the MRI model influences cartilage thickness measurements." Inter-measurement error, defined as the absolute difference between the targeted and median thicknesses determined by the five MRI models, was analyzed using histograms. The factors generating the largest inter-measurement error were also examined. RESULTS: No exceptional trends attributable to a specific instrument model were observed, and the p-value from the Kruskal-Wallis test exceeded 0.05 in all seven regions. Therefore, the study hypothesis was rejected. Of the 350 measurements, the inter-measurement error was ≤0.05 mm in 53 %, ≤0.10 mm in 75 %, and ≤0.20 mm in 95 %. Analysis of the medial tibial cartilage, which had the largest inter-measurement error, revealed mis-extraction of synovial fluid as cartilage. CONCLUSIONS: The choice of MRI model did not influence cartilage thickness measurements. Overall, 95 % of the inter-measurement errors were within 0.20 mm. The greatest error resulted from mis-extracting synovial fluid as cartilage.

Association of AI-determined Kellgren-Lawrence grade with medial meniscus extrusion and cartilage thickness by AI-based 3D MRI analysis in early knee osteoarthritis.

The associations among Kellgren-Lawrence (KL) grade, medial meniscus extrusion (MME), and cartilage thickness in knee osteoarthritis (OA) remain insufficiently understood. Our aim was to determine these associations in early to moderate medial tibiofemoral knee OA. We included 469 subjects with no lateral OA from the Kanagawa Knee Study. KL grade was assessed using artificial intelligence (AI) software. The MME was measured by MRI, and the cartilage thickness was evaluated in 18 subregions of the medial femorotibial joint by another AI system. The median MME width was 1.4 mm in KL0, 1.5 mm in KL1, 2.4 mm in KL2, and 6.0 mm in KL3. Cartilage thinning in the medial femur occurred in the anterior central subregion in KL1, expanded inwardly in KL2, and further expanded in KL3. Cartilage thinning in the medial tibia occurred in the anterior and middle external subregions in KL1, expanded into the anterior and middle central subregions in KL2, and further expanded in KL3. The absolute correlation coefficient between MME width and cartilage thickness increased as the KL grade increased in some subregions. This study provides novel insights into the early stages of knee OA and potentially has implications for the development of early intervention strategies.

Morphological analysis of three-dimensional MR images of patellofemoral joints in asymptomatic subjects.

The existing methods for analyzing patellofemoral (PF) osteoarthritis (OA) are limited. Our purpose was to clarify the frequency, localization, and morphological progression of PFOA by observing three-dimensional (3D) magnetic resonance (MR) images from a cohort population. The subjects were 561 patients aged 30-79 years from the Kanagawa Knee Study who had not visited a hospital for more than three consecutive months for knee symptoms. MR images of the PF joints, separated into the medial and lateral types, were presented in order of the highest to lowest patella cartilage area ratios. Cartilage defects in the patella were detected in 37 subjects (6.6%). Medial lesions (4.6%) were significantly more frequent than lateral lesions (2.0%) (p < 0.01). For both medial and lateral lesions, the patellar cartilage defects were divided into confined and unconfined types. The 3D MR images of the PF joint showed that the patellar cartilage defect occurred along each ridge of the femoral trochlea. The 3D MR images revealed a 6.6% prevalence of patellar cartilage defects, higher in the medial than lateral regions. The 3D MR images can easily determine PF morphology and cartilage defect location, making them useful in understanding the pathophysiology and etiology of PFOA.

Autologous Synovial Mesenchymal Stem Cell Transplantation Suppresses Inflammation Caused by Synovial Harvesting and Promotes Healing in a Micro Minipig Repaired Meniscus Model.

PURPOSE: Allogeneic synovial mesenchymal stem cells (MSCs) effectively promote meniscus healing in micro minipigs. We investigated the effect of autologous synovial MSC transplantation on meniscus healing in a micro minipig model of meniscus repair showing synovitis after synovial harvesting. MATERIALS AND METHODS: Synovium was harvested from the left knee of the micro minipigs after arthrotomy and used to prepare synovial MSCs. The left medial meniscus in the avascular region was injured, repaired, and transplanted with synovial MSCs. First, synovitis was compared after 6 weeks in knees with and without synovial harvesting. Second, the repaired meniscus was compared for the autologous MSC group and the control group (in which synovium was harvested but MSCs were not transplanted) 4 weeks after transplantation. RESULTS: Synovitis was more severe in knees subjected to synovium harvesting than in knees not subjected to harvesting. Menisci treated with autologous MSCs showed no red granulation at the tear of the meniscus, but menisci not treated with MSCS showed red granulation. Macroscopic scores, inflammatory cell infiltration scores, and matrix scores assessed by toluidine blue staining were all significantly better in the autologous MSC group than in the control group without MSCs (n = 6). CONCLUSION: Autologous synovial MSC transplantation suppressed the inflammation caused by synovial harvesting in micro minipigs and promoted healing of the repaired meniscus.

Association between knee cartilage thickness determined by magnetic resonance imaging three-dimensional analysis and the International Cartilage Repair Society (ICRS) arthroscopic grade.

BACKGROUND: The purpose of this study was to retrospectively investigate whether the average cartilage thickness calculated by magnetic resonance imaging (MRI) three-dimensional (3D) analysis system was correlated with the International Cartilage Repair Society (ICRS) grade at each subregion, as a representative scoring for arthroscopic evaluation. METHODS: The subjects were 102 patients who underwent arthroscopy for meniscus repair or high tibial osteotomy for medial osteoarthritis of the knee. Cartilage lesions were arthroscopically quantified according to the ICRS grade at each subregion. Fluoroscopy was used to compare the subregions on arthroscopic evaluation with subregions on MRI. The average cartilage thickness at each subregion was also automatically calculated from MRI data using our 3D analysis system. The association between ICRS grade and the average cartilage thickness at 18 subregions in the medial femoral and medial tibial regions was evaluated using Spearman's rank correlation coefficient. RESULTS: Examination of the fluoroscopic images revealed that the posterior subregions in the medial femoral region did not match the position between arthroscopy and MRI; therefore, those three subregions were excluded. In the medial femoral region, the ICRS grade correlated moderately with cartilage thickness at five subregions and weakly at one subregion. In the medial tibial region, the ICRS grade correlated moderately with cartilage thickness at four subregions and weakly at one subregion, but it did not correlate at the other four subregions. CONCLUSION: The average cartilage thickness determined by MRI 3D analysis correlated with arthroscopic grade at 11 of 15 subregions in the medial femoral and tibial regions.

Comparison of adhesion of thawed and cultured synovial mesenchymal stem cells to the porcine meniscus and the relevance of cell surface microspikes.

BACKGROUND: Placement of a cultured synovial mesenchymal stem cell (MSC) suspension on a repaired meniscus for 10 min accelerated meniscus repair. Upon placement of the MSC suspension on the meniscus, microspikes projecting from the MSC surface trap meniscus fibers and promote MSC adhesion. Thawed cryopreserved MSCs are preferred materials for meniscus repair, as they can be transplanted without additional culture. However, the adhesion ability of thawed cryopreserved MSCs is unknown. Here, we compared the proportion of cultured versus thawed MSCs adhering to a porcine meniscus immediately and 10 min after placement. We also investigated the relationship between adhesion and the number of microspikes on the synovial MSCs. METHODS: Synovial MSCs were prepared from the knees of four donors with osteoarthritis. The "cultured MSCs" were thawed MSCs that were re-cultured and suspended in PBS for transplantation. A similarly prepared suspension was cryopreserved, thawed again, suspended in PBS, and used without further culture as the "thawed MSCs." MSCs with at least three microspikes in SEM images were defined as microspike-positive MSCs. Porcine meniscus surfaces were abraded, cut into a cylindrical shape, and treated with MSC suspension. Non-adherent cells were counted immediately and again 10 min after placement to calculate the adhesion proportion. RESULTS: The proportion of microspike-positive MSCs was significantly higher in thawed (53 ± 3%) than in cultured (28 ± 5%) MSC suspensions. MSC adhesion to the meniscus was significantly better for the thawed than for the cultured MSC suspensions immediately after placement on the meniscus, but no differences were detected after 10 min. The proportion of MSCs with microspikes in the cell suspension was significantly correlated with the proportion of adhered MSCs immediately after the placement, but not 10 min later. Addition of FBS to the cryopreservation medium promoted a concentration-dependent increase in the proportion of microspike-positive cells. CONCLUSIONS: Thawed MSCs adhered better than cultured MSCs immediately after placement, but adhesion was similar for both MSC preparations after 10 min. Immediately after placement, the presence of microspikes was associated with better adhesion of synovial MSCs to the meniscus.

Stress and motivation of cell processing operators: A pilot study of an online questionnaire survey.

Introduction: Cell processing operators (CPOs) are one of the most important human assets for the industrial development of regenerative therapy since cell processing is still carried out manually. However, investigating the psychological aspects of CPOs to take advantage of this important human asset has not been done. Thus, the objectives of the current study were, first, to investigate the attributes of CPOs, second, to identify the factors that give CPOs mental stress, and third, to identify the motivational factors of CPOs. Methods: An online questionnaire was conducted to investigate the attributes of participants, stresses, and motivations; the questionnaire included multiple-choice questions. A survey questionnaire was distributed to 64 operators in 31 CPFs, including universities, clinics, and pharmaceutical companies, from December 2021 to February 2022. Participants responded to the online survey via Microsoft Forms. Results: Approximately half of the CPOs had been employed for two years or less, and they were more likely to experience various stressful situations than CPOs with three or more years of experience. In particular, the wear of dust-free clothing and work in a unique environment of a clean room, where it is difficult to take breaks due to the nature of the closed space, were considered to be particularly stressful situations. However, these CPOs have high motivations based on their pride in being involved in cutting-edge medical care, which is highly expected by society, and in contributing to patient care. Conclusions: While stress with training and education programs for CPOs is an issue that can be resolved through in-house training, other issues that are difficult to resolve immediately, such as mechanization to relieve the stress of human manual operations, were identified. Continued efforts to analyze and alleviate the stress factors identified in this study are expected to improve the quality of CPOs' work and maintain the important human asset of CPOs.

Human synovial mesenchymal stem cells show time-dependent morphological changes and increased adhesion to degenerated porcine cartilage.

The possibility that mesenchymal stem cells (MSCs) can adhere to partial defects or degenerative areas in cartilage remains to be established. The purposes of the present study were to verify the adhesion of synovial MSCs to degenerated cartilage, the time course of that adhesion, and the morphological changes that MSCs might undergo during the adhesion process. The surface of pig cartilage was abraded, and a human synovial MSC suspension was placed on the abraded surface. The proportion/number of MSCs that adhered to the cartilage was quantified by counting non-adhered MSCs, measuring the fluorescence intensity of DiI-labeled MSCs, and scanning electron microscopy (SEM) observations. The presence of microspikes or pseudopodia on the MSCs that adhered to the cartilage was also evaluated. SEM confirmed the adhesion of synovial MSCs to degenerated cartilage. The three independent quantification methods confirmed increases in the proportion/number of adhered MSCs within 10 s of placement and over time up to 24 h. The MSCs that adhered at 10 s had a high proportion of microspikes, whereas those that adhered after 1 h had that of pseudopodia. MSCs showed time-dependent morphological changes and increased adhesion to degenerated cartilage after placement of the human synovial MSC suspension.

Cell membrane fluidity and ROS resistance define DMSO tolerance of cryopreserved synovial MSCs and HUVECs.

OBJECTIVES: Synovial mesenchymal stem cells (MSCs) have high freeze-thaw tolerance, whereas human umbilical vein endothelial cells (HUVECs) have low freezing tolerance. The differences in cell type-specific freeze-thaw tolerance and the mechanisms involved are unclear. This study thus aimed to identify the biological and physical factors involved in the differences in freeze-thaw tolerance between MSCs and HUVECs. MATERIALS AND METHODS: For biological analysis, MSC and HUVEC viability after freeze-thawing and alteration of gene expression in response to dimethyl sulfoxide (DMSO, a cryoprotectant) were quantitatively evaluated. For physical analysis, the cell membrane fluidity of MSCs and HUVECs before and after DMSO addition was assessed using a histogram for generalized polarization frequency. RESULTS: HUVECs showed lower live cell rates and higher gene expression alteration related to extracellular vesicles in response to DMSO than MSCs. Fluidity measurements revealed that the HUVEC membrane was highly fluidic and sensitive to DMSO compared to that of MSCs. Addition of CAY10566, an inhibitor of stearoyl-coA desaturase (SCD1) that produces highly fluidic desaturated fatty acids, decreased the fluidity of HUVECs and increased their tolerance to DMSO. The combination of CAY10566 and antioxidant glutathione (GSH) treatment improved HUVEC viability from 57 to 69%. Membrane fluidity alteration may thus contribute to pore-induced DMSO influx into the cytoplasm and reactive oxygen species production, leading to greater cytotoxicity in HUVECs, which have low antioxidant capacity. CONCLUSIONS: Differences in freeze-thaw tolerance originate from differences in the cell membranes with respect to fluidity and antioxidant capacity. These findings provide a basis for analyzing cell biology and membrane-physics to establish appropriate long-term preservation methods aimed at promoting transplantation therapies.

Three-dimensional MRI shows cartilage defect extension with no separation from the meniscus in women in their 70 s with knee osteoarthritis.

The positional relationship between cartilage defects and the meniscus is poorly understood for osteoarthritis of the knee. Our purpose was to clarify how cartilage defects extend and their association with the meniscus location during osteoarthritis progression. The subjects were women in their 70 s who were registered in the Kanagawa Knee Study. We obtained 3D MRI images of the tibial surfaces with menisci in subjects with cartilage area ratios < 0.95 and examined the morphological association between cartilage defects and the medial meniscus (MM) by viewing the defects according to the cartilage area ratio at the medial tibial region. Of the 561 Kanagawa Knee Study subjects, 45 were included in the analyses, and 11 had a cartilage area ratio < 0.95 at the medial tibia. Significant differences were observed in the localization of cartilage defects among 9 subregions, with cartilage defects occurring predominantly in the middle external subregion. The inner margin of the MM contacted the cartilage defect in 7 knees and crossed the cartilage defect in 4 knees but was never found separated from the cartilage defect. The cartilage defects occurred from the middle external subregion and extended to the surrounding area without separating from the inner margin of the MM.Trial registration UMIN, UMIN000032826; 1 September 2018.

Yields of mesenchymal stromal cells from synovial fluid reflect those from synovium in patients with rheumatoid arthritis.

The yield of primary synovial mesenchymal stromal cells (MSCs) from synovium of patients with rheumatoid arthritis (RA) is highly variable, but cell transplantation therapy with autologous synovial MSCs requires accurate prediction of the synovial MSC yield per synovium weight. Here, we determined whether the yield of synovial fluid MSCs might predict the ultimate yield of primary MSCs from the synovium of RA knees. Synovial fluid and synovium were harvested during total knee arthroplasty from the knee joints of 10 patients with RA. Synovial fluid (1.5 mL) was diluted fourfold and plated equally into six 60 cm2 dishes. Nucleated cells from digested synovium were similarly plated at 1 × 104 cells in 6 dishes. All dishes were cultured for 14 days and analyzed for MSC yields and properties, including in vitro chondrogenesis. The cultured synovial cell number was correlated with the cultured synovial fluid cell number (n = 10, R2 = 0.64, p < 0.01). Synovial fluid cells formed cell colonies and showed MSC-like surface epitopes and multi-differentiation potential. However, the cartilage pellet weight indicated a greater chondrogenic potential of the synovial MSCs (n = 8). The primary MSC yields from synovial fluid and synovium were correlated, indicating that the synovial fluid MSC yield can predict the ultimate synovial MSC yield.

Medial Tibial Osteophyte Width Strongly Reflects Medial Meniscus Extrusion Distance and Medial Joint Space Width Moderately Reflects Cartilage Thickness in Knee Radiographs.

BACKGROUND: The presence of medial tibial osteophytes on knee radiographs suggests cartilage wear, but may be associated with medial meniscus extrusion (MME). The joint space width of the medial compartment consists anatomically of cartilage and the medial meniscus, but which is most responsible for joint space narrowing remains unclear. Magnetic resonance imaging (MRI) reveals MME and cartilage thickness. PURPOSES: To determine which radiographic medial tibial osteophyte width correlates better with cartilage thickness or MME distance and which radiographic medial joint space width correlates better with cartilage thickness or MME distance. STUDY TYPE: Cross-sectional. POPULATION: Total of 527 subjects, 253 females and 274 males, aged 30-79 years, included in the Kanagawa Knee Study. FIELD STRENGTH/SEQUENCE: 3 T/fat-suppressed spoiled gradient echo and proton density weighted. ASSESSMENT: The medial tibial osteophyte width and "the minimum joint space width at the medial compartment" (mJSW) were measured from plain radiographs. The cartilage region was automatically extracted from MRI data using software. The medial femoral and tibial cartilage regions were each divided into nine subregions, and the average thickness of the cartilage was determined in each region and subregion. MME was manually measured by two orthopedic surgeons using MRI coronal section images. STATISTICAL TESTS: Pearson's correlation coefficient and their comparison, with P < 0.05 considered statistically significant. RESULTS: The absolute values of the correlation coefficients were 0.33 at maximum between osteophyte width and cartilage thickness and 0.76 between osteophyte width and MME; the value was significantly higher with MME than with cartilage thickness (P < 0.001). The absolute values of the correlation coefficients were 0.50 at maximum between mJSW and cartilage thickness and 0.16 between mJSW and MME; the value was significantly higher with cartilage thickness than with MME (P < 0.001). DATA CONCLUSION: The medial tibial osteophyte width strongly reflected MME and the medial joint space width moderately reflected cartilage thickness. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 3.

Biomechanical analysis of a centralization procedure for extruded lateral meniscus after meniscectomy in porcine knee joints.

The recently developed arthroscopic centralization for lateral meniscal extrusion has obtained satisfactory short-term clinical and radiological results and improves the meniscus biomechanical properties. However, the effectiveness of treatment for meniscus extrusion after partial meniscectomy still requires elucidation. This study investigated the effect of centralization with modifications from a mechanical viewpoint. Porcine knee joints (N = 6) were set in a universal tester under the following conditions: (1) Intact; (2) Meniscectomy: Inner half of the posterior half meniscus was removed; (3) Extrusion: Posterior meniscus was dislocated laterally by transecting the posterior root and the meniscotibial ligament; (4) Centralization-1: Centralization procedure using one anchor; (5) Centralization-2: Centralization procedure using two anchors; and (6) Centralization-ad: Centralization with capsular advancement using two anchors. Load distributions and contact pressure in the meniscus and tibial cartilage were evaluated with an axial compressive force of 200 N. After meniscectomy, the tibial cartilage load increased and that of the medial margin of the posterior part of the meniscus decreased. When the meniscus was extruded, the load was concentrated only on the tibial cartilage. Centralization-1 increased the load on the meniscus, while Centralization-2 further increased the meniscus load but decreased the tibial cartilage load. Centralization-ad further decreased the load on the tibial plateau. The average contact pressure of the tibial cartilage was significantly higher in the Extrusion group than in the Intact group or the Centralization-ad group. From a biomechanical viewpoint, centralization with capsular advancement was the most effective of the tested procedures for treatment for an extruded meniscus after partial meniscectomy.

Difference in the joint space of the medial knee compartment between full extension and Rosenberg weight-bearing radiographs.

OBJECTIVES: Radiographs are the most widespread imaging tool for diagnosing osteoarthritis (OA) of the knee. Our purpose was to determine which of the two factors, medial meniscus extrusion (MME) or cartilage thickness, had a greater effect on the difference in the minimum joint space width (mJSW) at the medial compartment between the extension anteroposterior view (extension view) and the 45° flexion posteroanterior view (Rosenberg view). METHODS: The subjects were 546 participants (more than 50 females and 50 males in their 30 s, 40 s, 50 s, 60 s, and 70 s) in the Kanagawa Knee Study. The mJSW at the medial compartment was measured from both the extension and the Rosenberg views, and the "mJSW difference" was defined as the mJSW in the Rosenberg view subtracted from the mJSW in the extension view. The cartilage region was automatically extracted from MRI data and constructed in three dimensions. The medial region of the femorotibial joint cartilage was divided into 18 subregions, and the cartilage thickness in each subregion was determined. The MME was also measured from MRI data. RESULTS: The mJSW difference and cartilage thickness were significantly correlated at 4 subregions, with 0.248 as the highest absolute value of the correlation coefficient. The mJSW difference and MME were also significantly correlated, with a significantly higher correlation coefficient (0.547) than for the mJSW difference and cartilage thickness. CONCLUSIONS: The MME had a greater effect than cartilage thickness on the difference between the mJSW at the medial compartment in the extension view and in the Rosenberg view. KEY POINTS: • The difference in the width at the medial compartment of the knee between the extension and the flexion radiographic views was more affected by medial meniscus extrusion than by cartilage thickness.

Second-look arthroscopy after meniscus repair and synovial mesenchymal stem cell transplantation to treat degenerative flaps and radial tears of the medial meniscus: A case report.

BACKGROUND: The purpose of this study was to compare arthroscopic findings of a degenerative flap and radial tear of the medial meniscus (MM) before and one year after treatment by meniscus repair and synovial mesenchymal stem cell (MSC) transplantation. METHODS: Patients with a degenerative flap and radial MM tear that would generally be treated by meniscectomy were included. The patients ranged in age from 45 to 62 years and all underwent meniscus repair and synovium harvest at time 0. The digested synovium was cultured with autologous serum for 12 days, and an average of 4 × 107 MSCs were transplanted at two weeks. A second-look arthroscopy was performed at 52 weeks (n = 6). The average duration of symptoms was 24 months. For flap tears, arthroscopic findings were quantified in terms of the presence, stability, and smoothness of the meniscus at each zone and area. The Lysholm score was evaluated throughout the 52 week follow-up. RESULTS: Four patients with MM flap tears showed deficiencies in the central area at the posterior junctional zone before treatment, but this zone was completely restored to a stable and smooth condition in two patients and partially restored in the other two patients. The arthroscopy score for a flap tear at the central area of the posterior junctional zone was 0.3 ± 0.5 before treatment and 4.3 ± 2.1 after treatment. The score was significantly higher after treatment (p < 0.05, n = 4). The original radial MM tears in two patients were healed one year after treatment. Lysholm scores were significantly higher at 4 and 52 weeks after treatment than before treatment (n = 6). CONCLUSIONS: Arthroscopic findings for a degenerative flap and radial tear of the MM were improved at the central area of the posterior junctional zone one year after meniscus repair and MSC transplantation.

Ultrasound-Guided Harvesting of Synovium for Regenerative Medicine of Cartilage and Meniscus Using Synovial Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC) therapy for cartilage or meniscus pathologies, including osteoarthritis, requires the easy and safe collection of MSC source materials. Synovial MSCs are attractive cell sources for joint pathology because of their high proliferative and chondrogenic potential in vitro and in vivo. We developed an ultrasound-guided harvesting procedure for synovium for the regenerative medicine of cartilage and meniscus. A ∼1-cm skin incision is made at the proximal side of the patellae, and a forceps is inserted under ultrasound guidance of the suprapatellar pouch to grasp the synovium. Here, several synovium samples were retrieved and transported sterilely for culture at the cell-processing facility. After a 14-day culture of the nucleated cells, crystal violet confirmed colony formation. Cell growth was enough for MSC therapy of joint pathology (0.89 ± 0.06 × 106 cells/dish). No adverse events occurred during synovium harvesting. A key advantage of this procedure is its minimal invasiveness, as synovium is harvested from a 1-cm skin incision in the knee joint. A disadvantage is the possible risk of hemostasis, as arresting bleeding at the synovial harvest site is difficult, even though the suprapatellar pouch contains no major vessels.

Transplantation of human autologous synovial mesenchymal stem cells with trisomy 7 into the knee joint and 5 years of follow-up.

Mesenchymal stem cells (MSCs) can show trisomy 7; however, the safety of these cells has not been fully investigated. The purposes of this study were to determine the ratio of patients whose synovial MSCs were transplanted clinically, to intensively investigate MSCs with trisomy 7 from a safety perspective, and to follow up the patients for 5 years after transplantation. Synovial MSCs at passage 0 were transplanted into a knee for degenerative meniscus tears in 10 patients, and the patients were checked at 5 years. The synovial MSCs were evaluated at passages 0 to 15 by G-bands and digital karyotyping, and trisomy 7 was found in 3 of 10 patients. In those three patients, 5% to 10% of the synovial MSCs showed trisomy 7. The mRNA expressions of representative oncogenes and genes on chromosome 7 did not differ between MSCs with and without trisomy 7. Whole-genome sequencing and DNA methylation analysis showed similar results for MSCs with and without trisomy 7. Transplantation of human synovial MSCs with trisomy 7 into eight mouse knees did not result in tumor formation under the skin or in the knees after 8 weeks in any mouse, whereas transplanted HT1080 cells formed tumors. In vitro chondrogenic potentials were similar between MSCs with and without trisomy 7. Five-year follow-ups revealed no serious adverse events in all 10 human patients, including 3 who had received MSCs with trisomy 7. Overall, our findings indicated that synovial MSCs with trisomy 7 were comparable with MSCs without trisomy 7 from a safety perspective.

Optimal Pore Size of Honeycomb Polylactic Acid Films for In Vitro Cartilage Formation by Synovial Mesenchymal Stem Cells.

BACKGROUND: Tissue engineering of cartilage requires the selection of an appropriate artificial scaffold. Polylactic acid (PLA) honeycomb films are expected to be highly biodegradable and cell adhesive due to their high porosity. The purpose of this study was to determine the optimal pore size of honeycomb PLA films for in vitro cartilage formation using synovial mesenchymal stem cells (MSCs). METHODS: Suspensions of human synovial MSCs were plated on PLA films with different pore sizes (no pores, or with 5 µm or 20 µm pores) and then observed by scanning electron microscopy. The numbers of cells remaining in the film and passing through the film were quantified. One day after plating, the medium was switched to chondrogenic induction medium, and the films were time-lapse imaged and observed histologically. RESULTS: The 5 µm pore film showed MSCs with pseudopodia that extended between several pores, while the 20 µm pore film showed MSC bodies submerged into the pores. The number of adhered MSCs was significantly lower for the film without pores, while the number of MSCs that passed through the film was significantly higher for the 20 µm pore film. MSCs that were induced to form cartilage peeled off as a sheet from the poreless film after one day. MSCs formed thicker cartilage at two weeks when growing on the 5 µm pore films than on the 20 µm pore films. CONCLUSIONS: Honeycomb PLA films with 5 µm pores were suitable for in vitro cartilage formation by synovial MSCs.

Alterations in cartilage quantification before and after injections of mesenchymal stem cells into osteoarthritic knees.

Several studies have reported improvement in knee pain following mesenchymal stem cell (MSC) injections for knee osteoarthritis (OA). We developed a novel 3D magnetic resonance imaging (MRI) analysis software program that provides "projected cartilage area ratios" for automatic detection of changes in cartilage amounts. The primary objective of this prospective interventional study was to compare alterations in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral posteromedial region between 30 weeks before and 30 weeks after synovial MSC injections. Secondary objectives were to assess the clinical scores and safety of MSC injections. Patients with OA who complained of knee pain underwent autologous synovial MSC injections into the knee at time 0 and again 15 weeks later. MRI examinations were performed at - 30, - 15, - 1, and 30 weeks. Patients showing < 3% decreases in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral the posteromedial region from - 30 weeks to - 15 weeks were excluded from the study. The Lysholm Knee Score, Knee Injury and Osteoarthritis Outcome Scale (KOOS), and Numerical Rating Scale (NRS) scores were evaluated at - 30, - 15, - 5, - 2, 0, 5, 10, 15, 20, 25, and 30 weeks. Five patients were excluded because 3D MRI analysis showed no cartilage loss at - 15 weeks. Ultimately, eight OA patients underwent MSC injections. The projected cartilage area ratio significantly decreased by 0.07 in the 30 weeks before MSC injections (p = 0.01), but no further decreases occurred in the 30 weeks after MSC injections. The projected cartilage area ratio at the femoral posteromedial region showed a significant difference between 30 weeks before and 30 weeks after MSC injections. The Lysholm Knee Score, KOOS, and NRS values improved significantly after the injections. MSC injection could not be ruled out as the cause of two adverse events: transient knee pain and itching in both hands. Fully automatic 3D MRI analysis showed that synovial MSC injections suppressed cartilage loss in patients with progressive OA.Trial registration: Intraarticular injections of synovial stem cells for osteoarthritis of the knee (Number UMIN 000026732). Date of registration; June 1, 2017. https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000029967 .

Thawed cryopreserved synovial mesenchymal stem cells show comparable effects to cultured cells in the inhibition of osteoarthritis progression in rats.

Intra-articular injections of mesenchymal stem cells (MSCs) can inhibit the progression of osteoarthritis (OA). Previous reports have used cultured MSCs, but the ability to use thawed cryopreserved MSC stocks would be highly advantageous. Our purpose was to elucidate whether thawed cryopreserved MSCs show comparable inhibitory effects on OA progression in rats to those obtained with cultured MSCs. Cultured rat synovial MSCs or thawed MSCs were compared for in vitro viability and properties. The inhibitory effect of thawed MSCs on OA progression was evaluated by injecting cryopreservation fluid and thawed MSCs in meniscectomized rats. Cartilage degeneration was assessed using gross finding and histological scores. Cultured MSCs were then injected into one knee and thawed MSCs into the contralateral knee of the same individual to compare their effects. Cultured MSCs and MSCs thawed after cryopreservation had comparable in vitro colony formation and chondrogenic potentials. In the rat OA model, the gross finding and histological scores were significantly lower in the thawed MSC group than in the cryopreservation fluid group at 8 weeks. Finally, cartilage degeneration did not differ significantly after injection of cultured and thawed MSCs. In conclusion, thawed MSCs showed comparable inhibitory effects on OA progression to cultured MSCs.