The association of the medial meniscus covering ratio with knee cartilage thickness at 15 medial compartment subregions as found by three-dimensional analysis using MRI.

Purpose: The correlation of cartilage thickness measured by three-dimensional (3D) magnetic resonance imaging (MRI) and the medial meniscal coverage ratio (MMCR), which presented pathology of the medial meniscus extrusion (MME) in 3D MRI, has not yet been elucidated. The study's purpose was to retrospectively verify whether the average cartilage thickness calculated by the automatic MRI 3D analysis system for each subregion was correlated with MMCR. Methods: A total of 60 patients underwent medial meniscus repair or high tibial osteotomy to treat their medial knee osteoarthritis. Cartilage thickness and MMCR were automatically calculated using 3D MRI software. The MMCR was defined as the ratio of the area covered by the meniscus within the medial tibial cartilage area to the total medial tibial cartilage area. The association between MMCR and the average cartilage thickness at 15 subregions in the medial femoral condyle (MFC) and medial tibial plateau (MTP) was evaluated using Spearman's rank correlation coefficient. Results: Kellgren-Lawrence grade exhibited a negative correlation with MMCR and a positive correlation with MME width. Cartilage thickness in the MTP had a moderately positive correlation with MMCR at four subregions and a weakly positive correlation at another subregion. Cartilage thickness in the MFC showed a moderately positive correlation with MMCR at five subregions and a weakly positive correlation at one subregion. Conclusions: Cartilage thickness calculated by automatic MRI 3D analysis system had a positive correlation with MMCR for all subregions of the anterior and middle subregions in the MFC and for five regions of nine subregions of the anterior and middle subregions in the MTP. Level of evidence: Level II, cross-sectional study (diagnosis).

Evaluation of cleaning methods for change-over after the processing of cell products to avoid cross-contamination risk.

Introduction: Cell-processing facilities face the risk of environmental bacteria contaminating biosafety cabinets during processing, and manual handling of autologous cell products can result in contamination. We propose a risk- and evidence-based cleaning method for cross-contamination, emphasizing proteins and DNA. Methods: The transition and residual risks of the culture medium were assessed by measuring both wet and dried media using fluorescence intensity. Residual proteins and DNA in dried culture medium containing HT-1080 cells were analyzed following ultraviolet (UV) irradiation, wiping, and disinfectant treatment. Results: Wet conditions showed a higher transition to distilled water (DW), whereas dry conditions led to higher residual amounts on SUS304 plates. Various cleaning methods for residual culture medium were examined, including benzalkonium chloride with a corrosion inhibitor (BKC + I) and DW wiping, which demonstrated significantly lower residual protein and DNA compared to other methods. Furthermore, these cleaning methods were tested for residual medium containing cells, with BKC + I and DW wiping resulting in an undetectable number of cells. However, in some instances, proteins and DNA remained. Conclusions: The study compared cleaning methods for proteins and DNA in cell products, revealing their advantages and disadvantages. Peracetic acid (PAA) proved effective for nucleic acids but not proteins, while UV irradiation was ineffective against both proteins and DNA. Wiping emerged as the most effective method, even though traceability remained challenging. However, wiping with ETH was not effective as it caused protein immobilization. Understanding the characteristics of these cleaning methods is crucial for developing effective contamination control strategies.

Cartilage thickness in the medial compartment of the knee joint evaluated by MRI three-dimensional analysis correlates with weight-bearing line ratio and joint line convergence angle.

PURPOSE: The correlation between cartilage thickness evaluated by 3D MRI and knee alignment has not been elucidated. The study's purpose was to retrospectively verify whether average cartilage thickness calculated by the automatic MRI 3D analysis system for each subregion was correlated with knee alignment. METHODS: A total of 53 patients underwent medial meniscus repair or high tibial osteotomy for medial knee osteoarthritis. Cartilage thickness was automatically calculated using 3D MRI software. Knee alignment, weight-bearing line ratio (WBLR), joint line convergence angle (JLCA), medial proximal tibial angle (MPTA), and lateral distal femoral angle (LDFA) were calculated on a weight-bearing long-leg radiograph using digital planning software. The association between knee alignment and the average cartilage thickness at 18 subregions in the medial femoral condyle (MFC) and medial tibial plateau (MTP) was evaluated using Spearman's rank correlation coefficient. RESULTS: Cartilage thickness of the MFC had moderately positive correlations with WBLR at four subregions and weak correlations at two subregions. Cartilage thickness of the MTP had moderately positive correlations with WBLR at four subregions. Cartilage thickness of the MFC had moderately negative correlations with JLCA at six subregions. Cartilage thickness of the MTP had moderately negative correlations with JLCA at four subregions and a weak correlation at one subregion. Conversely, cartilage thickness had weak correlations with MPTA or LDFA. CONCLUSIONS: In knees with pathological conditions in the medial compartment, cartilage thicknesses by 3D MRI had positive correlations with WBLR and JLCA at almost all subregions of the MFC and at the anterior-middle and central-external of the MTP. Treatment strategies should be considered in light of these regions. LEVEL OF EVIDENCE: Cross-sectional study (diagnosis); Level of evidence, 2.

Presoaking Grafts in Vancomycin Does Not Impair Graft-Bone Healing in a Rat Anterior Cruciate Ligament Reconstruction Model.

BACKGROUND: The vancomycin presoaking technique (wherein grafts are treated with a vancomycin solution [VS] for anterior cruciate ligament reconstruction [ACLR]) reduces the infection rate after ACLR. However, the effects of this technique on graft-bone healing have not been fully elucidated. PURPOSE: To investigate the effects of vancomycin presoaking on graft-bone healing in a rat ACLR model. STUDY DESIGN: Controlled laboratory study. METHODS: Long flexor digitorum longus tendons were obtained from 9 Wistar rats, and each was randomly allocated to the normal saline (NS) or VS groups. The grafts were immersed in sterile saline for 30 minutes in the NS group and in a 5-mg/mL VS in the VS group. The presence of time-zero graft bacterial contamination was confirmed, and the grafts were incubated in Fluidised Thioglycollate Broth for 2 weeks. ACLR was performed on the right knees of 65 male Wistar rats using the flexor digitorum longus tendons. Each graft was similarly treated. Biomechanical testing, micro-computed tomography, and histological evaluations were performed 4 and 12 weeks postoperatively. RESULTS: The VS group showed significantly reduced graft contamination at time zero (P = .02). The mean maximum loads to failure were 13.7 ± 8.2 N and 11.6 ± 4.8 N in the NS and VS groups, respectively, at 4 weeks (P = .95); and 23.2 ± 13.2 N and 30.4 ± 18.0 N in the NS and VS groups, respectively, at 12 weeks (P = .35). Regarding micro-computed tomography, the mean bone tunnel volumes were 3.76 ± 0.48 mm3 and 4.40 ± 0.58 mm3 in the NS and VS groups, respectively, at 4 weeks (P = .41); and 3.51 ± 0.38 mm3 and 3.67 ± 0.35 mm3 in the NS and VS groups, respectively, at 12 weeks (P = .54). Histological semiquantitative examination revealed no clear between-group differences at any time point. CONCLUSION: Presoaking grafts in vancomycin in a rat ACLR model demonstrated no discernible adverse effects on short- and midterm biomechanical, radiological, and histological investigations. CLINICAL RELEVANCE: The findings provide guidance for surgeons when considering this technique.

Operator-derived particles and falling bacteria in biosafety cabinets.

Introduction: To ensure the sterility of cell products that cannot undergo conventional sterilization processes, it is imperative to establish and maintain a clean room environment, regulated through environmental monitoring, including particle counts. Nevertheless, the impact of particles generated by operators as potential contaminants remains uncertain. Thus, in this study, we conducted an accelerated test to assess the correlation between particles generated by operators and airborne bacteria, utilizing biosafety cabinets within a typical laboratory setting. These biosafety cabinets create a controlled environment with air conditioning and high-efficiency particulate air (HEPA) filters, offering fundamental data relevant to cell production. Materials and methods: We conducted a simulation followed by real-time experiments involving human operations to explore the quantity of particles, particle sizes, and the percentage of bacteria within these particles. This investigation focused on conditions with heightened particle generation from operators within a biosafety cabinet. The experiment was conducted on operators wearing textile and non-woven dustless clothing within biosafety cabinets. It entailed tapping the upper arms for a duration of 2 min. Results: Observations under biosafety cabinet-off conditions revealed the presence of various particles and falling bacteria in textile clothing. In contrast, no particles or falling bacteria were detected in operators wearing dustless clothing within biosafety cabinets. Notably, a correlation between 5 µm particles and colony-forming units in textile clothing was identified through this analysis. The ratio of falling bacteria to the total number of particles within the biosafety cabinet was 0.8 ± 0.5 % for textile clothing, while it was significantly lower at 0.04 ± 0.2 % for dustless clothing. Conclusion: This study demonstrated that the number of particles and falling bacteria varied depending on the type of clothing and that quantitative data could be used to identify risks and provide basic data for operator education and evidence-based control methods in aseptic manufacturing areas. Although, this study aims to serve as an accelerated test operating under worst-case conditions, the results need to make sure the study range in general research.

Noninvasive total counting of cultured cells using a home-use scanner with a pattern sheet.

The inherent variability in cell culture techniques hinders their reproducibility. To address this issue, we introduce a comprehensive cell observation device. This new approach enhances the features of existing home-use scanners by implementing a pattern sheet. Compared with fluorescent staining, our method over- or underestimated the cell count by a mere 5%. The proposed technique showcased a strong correlation with conventional methodologies, displaying R2 values of 0.91 and 0.99 compared with the standard chamber and fluorescence methods, respectively. Simulations of microscopic observations indicated the potential to estimate accurately the total cell count using just 20 fields of view. Our proposed cell-counting device offers a straightforward, noninvasive means of measuring the number of cultured cells. By harnessing the power of deep learning, this device ensures data integrity, thereby making it an attractive option for future cell culture research.

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.

Volatile organic compounds and ionic substances contamination in cell processing facilities during rest period; a preliminary assessment of exposure to cell processing operators.

Introduction: Cell processing operators (CPOs) use a variety of disinfectants that vaporize in the workspace environment. These disinfectants can induce allergic reactions in CPOs, due to their long working hours at cell processing facilities (CPFs). Ionic substances such as CH3COO- generated from peracetic acid, nitrogen oxides (NOx) and sulfur oxides (SOx) from outdoor environment are also known to pollute air. Therefore, our objective was to assess the air quality in CPFs and detect volatile organic compounds (VOCs) from disinfectants and building materials, and airborne ionic substances from outdoor air. Methods: Sampling was conducted at three CPFs: two located in medical institutions and one located at a different institution. Air samples were collected using a flow pump. Ion chromatographic analysis of the anionic and cationic compounds was performed. For VOC analysis, a thermal desorption analyzer coupled with capillary gas chromatograph and flame ionization detector was used. Results: Analysis of the ionic substances showed that Cl-, NOx, and SOx, which were detected in large amounts in the outdoor air, were relatively less in the CPFs. Ethanol was detected as the main component in the VOC analysis. Toluene was detected at all sampling points. As compared to the other environments, air in the incubator contained larger amounts of VOCs, that included siloxane, tetradecane, and aromatics. Conclusions: No VOCs or ionic substances of immediate concern to the health of the CPOs were detected during the non-operating period. However, new clinical trials of cell products are currently underway in Japan, and a variety of new cell products are expected to be approved. With an increase in cell processing, health risks to CPOs that have not been considered previously, may become apparent. We should continue to prepare for the future expansion of the industry using a scientific approach to collect various pieces of information and make it publicly available to build a database.

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.

Effect of disinfectants and manual wiping for processing the cell product changeover in a biosafety cabinet.

Introduction: The process of cell product changeover poses a high risk of cross-contamination. Hence, it is essential to minimize cross-contamination while processing cell products. Following its use, the surface of a biosafety cabinet is commonly disinfected by ethanol spray and manual wiping methods. However, the effectiveness of this protocol and the optimal disinfectant have not yet been evaluated. Here, we assessed the effect of various disinfectants and manual wiping methods on bacterial removal during cell processing. Methods: The hard surface carrier test was performed to evaluate the disinfectant efficacy of benzalkonium chloride with a corrosion inhibitor (BKC + I), ethanol (ETH), peracetic acid (PAA), and wiping against Bacillus subtilis endospores. Distilled water (DW) was used as the control. A pressure sensor was employed to investigate the differences in loading under dry and wet conditions. The pre-spray for wiping was monitored by eight operators using a paper that turns black when wet. Chemical properties, including residual floating proteins, and mechanical properties, such as viscosity and coefficient of friction, were examined. Results: In total, 2.02 ± 0.21-Log and 3.00 ± 0.46-Log reductions from 6-Log CFU of B. subtilis endospores were observed for BKC + I and PAA, respectively, following treatment for 5 min. Meanwhile, wiping resulted in a 0.70 ± 0.12-Log reduction under dry conditions. Under wet conditions, DW and BKC + I showed 3.20 ± 0.17-Log and 3.92 ± 0.46-Log reductions, whereas ETH caused a 1.59 ± 0.26-Log reduction. Analysis of the pressure sensor suggested that the force was not transmitted under dry conditions. Evaluation of the amount of spray by eight operators showed differences and bias in the spraying area. While ETH had the lowest ratio in the protein floating and collection assays, it exhibited the highest viscosity. BKC + I had the highest friction coefficient under 4.0-6.3 mm/s; however, that of BKC + I decreased and became similar to the friction coefficient of ETH under 39.8-63.1 mm/s. Conclusions: DW and BKC + I are effective for inducing a 3-Log reduction in bacterial abundance. Moreover, the combination of optimal wet conditions and disinfectants is essential for effective wiping in specific environments containing high-protein human sera and tissues. Given that some raw materials processed in cell products contain high protein levels, our findings suggest that a complete changeover of biosafety cabinets is necessary in terms of both cleaning and disinfection.

Cross-contamination risk and decontamination during changeover after cell-product processing.

Introduction: During changeover in cell-product processing, it is essential to minimize cross-contamination risks. These risks differ depending on the patient from whom the cells were derived. Human error during manual cell-product processing increases the contamination risk in biosafety cabinets. Here, we evaluate the risk of cross-contamination during manual cell-processing to develop an evidence-based changeover method for biosafety cabinets. Methods: Contaminant coverage was analyzed during simulated medium preparation, cell seeding, and waste liquid decanting by seven operators, classified by skill. Environmental bacteria were surveyed at four participating facilities. Finally, we assessed the effect of conventional UV irradiation in biosafety cabinets on bacteria and fungi that pose a cross-contamination risk. Results: Under simulated conditions, scattered contamination occurred via droplets falling onto the surface from heights of 30 cm, and from bubbles rupturing at this height. Visible traces of contaminants were distributed up to 50 cm from the point of droplet impact, or from the location of the pipette tip when the bubble ruptured. In several facilities, we detected Bacillus subtilis, of which the associated endospores are highly resistant to disinfection. Irradiation at 50 mJ/cm2 effectively eliminated Bacillus subtilis vegetative cells and Aspergillus brasiliensis, which is highly resistant to UV. Bacillus subtilis endospores were eliminated at 100 mJ/cm2. Conclusions: Under these simulated optimal conditions, UV irradiation successfully prevents cross-contamination. Therefore, following cell-product processing, monitoring the UV dose in the biosafety cabinet during cell changeover represents a promising method for reducing cross-contamination.

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.

Safety of using cultured cells with trisomy 7 in cell therapy for treating osteoarthritis.

Cell therapy is a promising alternative treatment approach currently under study for osteoarthritis (OA), the most common chronic musculoskeletal disease. However, the mesenchymal stem cells (MSCs) used in cell therapy to treat OA are usually expanded in vitro to obtain sufficient numbers for transplantation, and their safety has not been fully assessed from multiple perspectives. Analysis of karyotypic abnormalities, in particular, is important to ensure the safety of cells; however, chromosomal mutations may also occur during the cell-expansion process. In addition, there have been many reports showing chromosome abnormalities, mainly trisomy 7, in the cartilage and synovium of patients with OA as well as in normal tissues. The suitability of cells with these karyotypic abnormalities as cells for cell therapy has not been evaluated. Recently, we assessed the safety of using cells with trisomy 7 from the osteoarthritic joint of a patient for transplantation, and we followed up with the patient for 5 years. This study showed analysis for copy number variant and whole-genome sequencing, compared with blood DNA from the same patient. We did not find any abnormalities in the genes regardless of trisomy 7. No side effects were observed for at least 5 years in the human clinical study. This suggests that the transplantation of cultured cells with trisomy 7 isolated from an osteoarthritic joint and transplanted into the osteoarthritic joints of the same person is not expected to cause serious adverse events. However, it is unclear what problems may arise in the case of allogeneic transplantation. Different types of risks will also exist depending on other transplantation routes, such as localization to the knee-joint only or circulation inflow and lung entrapment. In addition, since the cause of trisomy 7 occurrence remains unclear, it is necessary to clarify the mechanism of trisomy 7 in OA to perform cell therapy for OA patients in a safe manner.

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.

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.

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.

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.

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.