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Background: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing., Hypothesis: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair., Study Design: Controlled laboratory study., Methods: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro-computed tomography, and mechanical analysis., Results: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro-computed tomography of the subchondral bone., Conclusion: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs., Clinical Relevance: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by the National Research Foundation of Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) program through the Critical Analytics for Manufacturing Personalized–Medicine (CAMP) Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology (SMART). H.J. and J.H. have a US patent on microfluidic devices (“Multi-Dimensional Double Spiral Device and Methods of Use Thereof”; patent pending No. US20220088588A1 [2019]). J.H. has a US patent on microfluidic devices (“Micro-Fluidic Devices and Uses Thereof”; patent No. 9,789,485 [2017]). AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Purpose: To compare the efficacy of mesenchymal stem cell (MSC) exosomes with hyaluronic acid (HA) against HA alone for functional cartilage regeneration in a rabbit osteochondral defect model., Methods: Critical-size osteochondral defects (4.5-mm diameter and 1.5-mm depth) were created on the trochlear grooves in the knees of 18 rabbits and were randomly allocated to 2 treatment groups: (1) exosomes and HA combination and (2) HA alone. Three 1-mL injections of either exosomes and HA or HA alone were administered intra-articularly immediately after surgery and thereafter at 7 and 14 days after surgery. At 6 and 12 weeks, gross evaluation, histologic and immunohistochemical analysis, and scoring were performed. The functional biomechanical competence of the repaired cartilage also was evaluated., Results: Compared with defects treated with HA, defects treated with exosomes and HA showed significant improvements in macroscopic scores (P = .032; P = .001) and histologic scores (P = .005; P < .001) at 6 and 12 weeks, respectively. Defects treated with exosomes and HA also demonstrated improvements in mechanical properties compared with HA-treated defects, with significantly greater Young's moduli (P < .05) and stiffness (P < .05) at 6 and 12 weeks. By 12 weeks, the newly-repaired tissues in defects treated with exosomes and HA composed mainly of hyaline cartilage that are mechanically and structurally superior to that of HA-treated defects and demonstrated mechanical properties that approximated that of adjacent native cartilage (P > .05). In contrast, HA-treated defects showed some repair at 6 weeks, but this was not sustained, as evidenced by significant deterioration of histologic scores (P = .002) and a plateau in mechanical properties from 6 to 12 weeks., Conclusions: This study shows that the combination of MSC exosomes and HA administered at a clinically acceptable frequency of 3 intra-articular injections can promote sustained and functional cartilage repair in a rabbit post-traumatic cartilage defect model, when compared with HA alone., Clinical Relevance: Human MSC exosomes and HA administered in combination promote functional cartilage repair and may represent a promising cell-free therapy for cartilage repair in patients., (Copyright © 2020 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Background: Articular cartilage has a zonal architecture and biphasic mechanical properties. The recapitulation of surface lubrication properties with high compressibility of the deeper layers of articular cartilage during regeneration is essential in achieving long-term cartilage integrity. Current clinical approaches for cartilage repair, especially with the use of mesenchymal stem cells (MSCs), have yet to restore the hierarchically organized architecture of articular cartilage., Hypothesis: MSCs predifferentiated on surfaces with specific nanotopographic patterns can provide phenotypically stable and defined chondrogenic cells and, when delivered as a bilayered stratified construct at the cartilage defect site, will facilitate the formation of functionally superior cartilage tissue in vivo., Study Design: Controlled laboratory study., Methods: MSCs were subjected to chondrogenic differentiation on specific nanopatterned surfaces. The phenotype of the differentiated cells was assessed by the expression of cartilage markers. The ability of the 2-dimensional nanopattern-generated chondrogenic cells to retain their phenotypic characteristics after removal from the patterned surface was tested by subjecting the enzymatically harvested cells to 3-dimensional fibrin hydrogel culture. The in vivo efficacy in cartilage repair was demonstrated in an osteochondral rabbit defect model. Repair by bilayered construct with specific nanopattern predifferentiated cells was compared with implantation with cell-free fibrin hydrogel, undifferentiated MSCs, and mixed-phenotype nanopattern predifferentiated MSCs. Cartilage repair was evaluated at 12 weeks after implantation., Results: Three weeks of predifferentiation on 2-dimensional nanotopographic patterns was able to generate phenotypically stable chondrogenic cells. Implantation of nanopatterned differentiated MSCs as stratified bilayered hydrogel constructs improved the repair quality of cartilage defects, as indicated by histological scoring, mechanical properties, and polarized microscopy analysis., Conclusion: Our results indicate that with an appropriate period of differentiation, 2-dimensional nanotopographic patterns can be employed to generate phenotypically stable chondrogenic cells, which, when implanted as stratified bilayered hydrogel constructs, were able to form functionally superior cartilage tissue., Clinical Relevance: Our approach provides a relatively straightforward method of obtaining large quantities of zone-specific chondrocytes from MSCs to engineer a stratified cartilage construct that could recapitulate the zonal architecture of hyaline cartilage, and it represents a significant improvement in current MSC-based cartilage regeneration.

Background: The use of bone marrow-derived mesenchymal stem cells (BMSCs) in cartilage repair procedures circumvents some of the limitations of autologous chondrocyte implantation (ACI), but long-term outcomes for this newer procedure are lacking. The authors previously reported comparable outcomes for the 2 procedures at 2-year follow-up., Purpose/hypothesis: The purpose was to compare the long-term clinical outcomes of ACI versus BMSCs. It was hypothesized that there would be no significant difference between the groups in terms of patient-reported outcome scores and safety outcomes at 10-year follow-up., Study Design: Cohort study; Level of evidence, 2., Methods: Seventy-two patients who underwent either ACI or BMSC implantation-matched in terms of age and lesion site- were followed up to a median of at least 10 years. Patients were assessed with the 36-item Short Form Health Survey (SF-36), the International Knee Documentation Committee knee evaluation form, the Lysholm Knee Score, and the Tegner Activity Scale. In addition, information was obtained regarding any additional surgical procedures as well as safety data, with particular attention to infection and tumor formation., Results: There was an improvement in all patient-reported outcomes scores apart from the Mental Component Summary of the SF-36 after cartilage repair surgery. There was no significant difference in any of the patient-reported outcomes between cohorts at any time point. Six and 5 patients in the ACI and BMSC groups, respectively, underwent subsequent surgical procedures, including 1 total knee replacement in the BMSC group. None of the patients in either group developed any deep infection or tumor within the follow-up period., Conclusion: BMSC implantation used for the treatment of chondral defects of the knee appears to result in equivalent clinical outcomes to first-generation ACI at up to 10 years, with no apparent increased tumor formation risk.

Objective: To compile and analyze the published studies on cell therapy for type 2 diabetes mellitus (T2DM) to obtain a better insight into management of T2DM that involved stem cell therapy., Methods: We searched all published studies in Pubmed/Medline, and Cochrane library, using keywords: ‘stem cell’ AND ‘therapy’ AND ‘diabetes type 2’. Inclusion criteria: original articles on the use of stem cells in humans with T2DM. Exclusion criteria: articles in the non-English literature, studies on T2DM complications that did not assess both adverse events and any of the common diabetes study outcomes. Data collection: type of study, number of cases, and all data that were related to outcome and adverse events. Data were analyzed descriptively to conclude the possible cause of adverse reactions, and which protocols gave a satisfactory outcome., Results: We collected 25 original articles, out of which 17 studies did not have controls and were classified as case reports, while there were 8 studies that were controlled clinical trials. Most studies used autologous bone marrow mononuclear cells (BM-MNCs) or autologous or allogeneic mesenchymal stem cells (MSCs) from various sources. Adverse events were mild and mostly intervention related. Efficacy of autologous BM-MNCs that were given via interventional route was comparable to Wharton jelly or umbilical cord MSCs that were given via intravenous (IV), Intra muscular (IM), or subcutaneous (SC) route., Conclusion: Further controlled studies that compare BM-MNCs to BM-MSCs or WJ-MSCs or UCSCs are recommended to prove their comparable efficacy. In addition, studies that compare various routes of administration (IV, IM or SC) versus the more invasive interventional routes are needed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Purpose: To analyze the results of the use of intra-articular cultured autologous bone marrow-derived mesenchymal stem cell (MSC) injections in conjunction with microfracture and medial opening-wedge high tibial osteotomy (HTO)., Methods: Fifty-six knees in 56 patients with unicompartmental osteoarthritic knees and genu varum were randomly allocated to the cell-recipient group (n = 28) or control group (n = 28). Patients who had a joint line congruity angle of more than 2°, malalignment of the knee from femoral causes, a fixed flexion deformity, or age older than 55 years were excluded. All patients underwent HTO and microfracture. The cell-recipient group received intra-articular injection of cultured MSCs with hyaluronic acid 3 weeks after surgery, whereas the control group only received hyaluronic acid. The primary outcome measure was the International Knee Documentation Committee (IKDC) score at intervals of 6 months, 1 year, and 2 years postoperatively. Secondary outcome measures were Tegner and Lysholm clinical scores and 1-year postoperative Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scores., Results: The median age of the patients was 51 years, with a mean body mass index of 23.85. Both treatment arms achieved improvements in Tegner, Lysholm, and IKDC scores. After adjustment for age, baseline scores, and time of evaluation, the cell-recipient group showed significantly better scores. The effect of treatment showed an added improvement of 7.65 (95% confidence interval [CI], 3.04 to 12.26; P = .001) for IKDC scores, 7.61 (95% CI, 1.44 to 13.79; P = .016) for Lysholm scores, and 0.64 (95% CI, 0.10 to 1.19; P = .021) for Tegner scores. Magnetic resonance imaging scans performed 1 year after surgical intervention showed significantly better MOCART scores for the cell-recipient group. The age-adjusted mean difference in MOCART score was 19.6 (95% CI, 10.5 to 28.6; P < .001)., Conclusions: Intra-articular injection of cultured MSCs is effective in improving both short-term clinical and MOCART outcomes in patients undergoing HTO and microfracture for varus knees with cartilage defects., Level of Evidence: Level II, randomized controlled trial., (Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Background: First-generation autologous chondrocyte implantation has limitations, and introducing new effective cell sources can improve cartilage repair., Purpose: This study was conducted to compare the clinical outcomes of patients treated with first-generation autologous chondrocyte implantation to patients treated with autologous bone marrow-derived mesenchymal stem cells (BMSCs)., Study Design: Cohort study; Level of evidence, 3., Methods: Seventy-two matched (lesion site and age) patients underwent cartilage repair using chondrocytes (n = 36) or BMSCs (n = 36). Clinical outcomes were measured before operation and 3, 6, 9, 12, 18, and 24 months after operation using the International Cartilage Repair Society (ICRS) Cartilage Injury Evaluation Package, which included questions from the Short-Form Health Survey, International Knee Documentation Committee (IKDC) subjective knee evaluation form, Lysholm knee scale, and Tegner activity level scale., Results: There was significant improvement in the patients' quality of life (physical and mental components of the Short Form-36 questionnaire included in the ICRS package) after cartilage repair in both groups (autologous chondrocyte implantation and BMSCs). However, there was no difference between the BMSC and the autologous chondrocyte implantation group in terms of clinical outcomes except for Physical Role Functioning, with a greater improvement over time in the BMSC group (P = .044 for interaction effect). The IKDC subjective knee evaluation (P = .861), Lysholm (P = .627), and Tegner (P = .200) scores did not show any significant difference between groups over time. However, in general, men showed significantly better improvements than women. Patients younger than 45 years of age scored significantly better than patients older than 45 years in the autologous chondrocyte implantation group, but age did not make a difference in outcomes in the BMSC group., Conclusion: Using BMSCs in cartilage repair is as effective as chondrocytes for articular cartilage repair. In addition, it required 1 less knee surgery, reduced costs, and minimized donor-site morbidity.

Current techniques in biological resurfacing of cartilage defects require an open arthrotomy or arthroscopy and involve the direct transplantation of isolated cells and/or scaffolds or whole tissue grafts with chondrogenic potential onto the cartilage defect. Our study investigates the possibility of direct intra-articular injection of mesenchymal stem cells suspended in hyaluronic acid (HA) as an alternative to the much more invasive methods currently available. A partial-thickness (without penetration of the subchondral bone) cartilage defect was created in the medial femoral condyle of an adult minipig. Mesenchymal stem cells from the iliac crest marrow of the same pig harvested in a separate procedure and suspended in 2 milliliters of hylan G-F 20 (Synvisc) were injected intra-articularly after the creation of the defect. This was followed by two more injections of hylan G-F 20 (HA) at weekly intervals. Either saline or HA was injected into the knees of the controls. The pigs were sacrificed at 6 and 12 weeks for morphological and histological analysis. The cell-treated groups showed improved cartilage healing both histologically and morphologically at 6 and 12 weeks compared with both controls. The use of intra-articular injections of mesenchymal stem cells suspended in HA is a viable option for treating large cartilage defects. This would be further explored in clinical trials.

Background: A repaired tendon needs to be protected for weeks until it has accrued enough strength to handle physiological loads. Tissue-engineering techniques have shown promise in the treatment of tendon and ligament defects. The present study tested the hypothesis that bone marrow-derived mesenchymal stem cells can accelerate tendon-healing after primary repair of a tendon injury in a rabbit model., Methods: Fifty-seven New Zealand White rabbits were used as the experimental animals, and seven others were used as the source of bone marrow-derived mesenchymal stem cells. The injury model was a sharp complete transection through the midsubstance of the Achilles tendon. The transected tendon was immediately repaired with use of a modified Kessler suture and a running epitendinous suture. Both limbs were used, and each side was randomized to receive either bone marrow-derived mesenchymal stem cells in a fibrin carrier or fibrin carrier alone (control). Postoperatively, the rabbits were not immobilized. Specimens were harvested at one, three, six, and twelve weeks for analysis, which included evaluation of gross morphology (sixty-two specimens), cell tracing (twelve specimens), histological assessment (forty specimens), immunohistochemistry studies (thirty specimens), morphometric analysis (forty specimens), and mechanical testing (sixty-two specimens)., Results: There were no differences between the two groups with regard to the gross morphology of the tendons. The fibrin had degraded by three weeks. Cell tracing showed that labeled bone marrow-derived mesenchymal stem cells remained viable and present in the intratendinous region for at least six weeks, becoming more diffuse at later time-periods. At three weeks, collagen fibers appeared more organized and there were better morphometric nuclear parameters in the treatment group (p < 0.05). At six and twelve weeks, there were no differences between the groups with regard to morphometric nuclear parameters. Biomechanical testing showed improved modulus in the treatment group as compared with the control group at three weeks (p < 0.05) but not at subsequent time-periods., Conclusions: Intratendinous cell therapy with bone marrow-derived mesenchymal stem cells following primary tendon repair can improve histological and biomechanical parameters in the early stages of tendon-healing.

Osteoporosis might be due to defects in mesenchymal stem cells (MSCs) that lead to reduced proliferation and osteoblast differentiation. We hypothesized that transplantation of MSCs into sites at risk for developing osteoporotic bone could improve bone structure and biomechanics. The aim of this study was to establish an osteoporosis rabbit model by ovariectomy (OVX), characterize the autologous MSCs from the OVX rabbits, and transplant the autologous MSCs into the OVX rabbits. MSCs harvested from bone marrow of normal and OVX rabbits were culture expanded and differentiated in osteogenic medium. Phenotypes were evaluated by collagen I immunostaining, von Kossa staining, and quantitative assays of bone-specific alkaline phosphatase (B-ALP) and osteocalcin (OCN). MSCs were transfected with green fluorescence protein (GFP) and implanted in the gluteus muscle to trace their fate in vivo. Cultured autologous MSCs from OVX rabbits were constructed in calcium alginate gels and then transplanted in the distal femurs. At 4 and 8 weeks after implantation, histomorphometrical and biomechanical analyses were performed on the samples. MSCs from OVX rabbits displayed higher B-ALP activity, but had similar OCN levels as compared to those from sham rabbits. After 8 weeks of implantation, more bone apposition was found in the MSC-alginate-treated group. Histomorphometry indicated increased trabecular thickness. Histology also illustrated improved microstructures with newly formed osteoids and enhanced trabecular thickness. In addition, biomechanical testing revealed stronger stiffness in the MSC-alginate treatment group. Therefore, this study implies that transplantation of MSCs can help to strengthen osteoporotic bone in rabbits.

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter x 5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host-tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site.

This study evaluates the ability of MSCs isolated from different origins--bone marrow, periosteum, or fat--to treat partial growth arrest in immature (6-week-old) New Zealand White rabbits. Up to 50% of the medial half of the proximal physis of the tibia was excised in these New Zealand White rabbits. Three weeks later, the bony bridge was excised, and fibrin glue with and without MSCs were transferred into the physeal defect of different rabbits. Contralateral tibias, without undergoing operation, served as self-control. Four groups of rabbits were involved in the study. Each group was injected separately with bone marrow-derived MSCs (group I), periosteum-derived MSCs (group II), fat-derived MSCs (group III), and fibrin glue alone (control, group IV). The rabbits were killed 8 and 16 weeks postoperatively. Clinical, radiological, and histological analyses were subsequently performed. Similar proliferative rates for three MSCs were demonstrated on days 4, 7, and 11 of primary culture. However, MSCs derived from bone-marrow and periosteum appeared to be more homogeneous than that from fat. All MSCs demonstrated chondrogenic and osteogenic differentiation potentials in vitro. The tibias in groups I and II showed significant correction of varus angulation at 16 weeks. However, the varus angulation in group III remained significantly obvious when compared with group I (p < 0.05). The length discrepancies between operated and normal tibiae in groups I, II, and III were significantly corrected compared with control (p < 0.01). In conclusion, bone-marrow and periosteum yielded more homogenous MSCs than fat, providing better correction of physeal arrest in rabbits. The source of MSCs itself could influence the success in the treatment of growth arrest.

Purpose: To study the effect of coating tendon grafts with mesenchymal stem cells (MSCs) on the rate and quality of graft osteointegration in anterior cruciate ligament (ACL) reconstruction., Type of Study: Animal model., Methods: Bilateral ACL reconstructions using hamstring tendon autografts were performed on 48 adult rabbits. Grafts were coated with MSCs in a fibrin glue carrier in one limb, and fibrin glue only in the other. Assessment was done at 2, 4, and 8 weeks. Histologic analysis was carried out using standard and immunohistochemical stains. Biomechanical testing of force and stiffness during loading to ultimate failure was performed., Results: Control reconstructions showed mature scar tissue with some Sharpey's-like fibers spanning the tendon-bone interface at 8 weeks. The MSC-enhanced reconstructions had large areas of cartilage cells at the tendon-bone junction at 2 weeks. By 8 weeks, a mature zone of cartilage was seen gradually blending from bone into the tendon grafts. This zone stained strongly for type II collagen and showed histologic characteristics similar to normal rabbit ACL insertions. Biomechanically, there was no statistical difference between limbs at 2 and 4 weeks. At 8 weeks, the MSC-enhanced grafts had significantly higher failure load and stiffness., Conclusions: Coating of tendon grafts with MSCs results in healing by an intervening zone of cartilage resembling the chondral enthesis of normal ACL insertions rather than collagen fibers and scar tissue. MSC-enhanced ACL reconstructions perform significantly better than controls on biomechanical testing., Clinical Relevance: Enhancement of tendon graft osteointegration with MSCs is a novel method offering the potential for more physiologic and biomechanically stronger ligament reconstructions.

Management of chondral lesions in osteochondritis dissecans remains a challenge. This study investigated the efficacy of periosteal graft, osteochondroidal autograft, autologous chondrocyte and mesenchymal stem cell transplants in the treatment of chondral lesions in animal models. Full-thickness articular cartilage defects were created in the weight-bearing surface of the medial femoral condyle in 20-week-old NZW rabbits. A total of 56 knees were randomly divided into four groups as follows: group 1, transfer of cultured chondrocytes; group 2, transfer of cultured mesenchymal stem cells; group 3, repair by periosteal graft; and group 4, mosaicplasty. All of the contralateral knees served as control. Gross, histologic, and biomechanical examinations at 36 weeks after the operation showed that the cultured chondrocytes and mesenchymal stem cells had comparable enhancing effects on the repair of chondral defects in advanced osteochondritis dissecans, whereas mosaicplasty did well initially and periosteal graft did less favorably., (Copyright 2004 Lippincott Williams and Wilkins)

To investigate the feasibility of using chitin, a natural polymer, as a scaffold to repair large growth plate defects (50% of physis) in immature rabbits, mesenchymal stem cells (MSCs) were harvested from periosteum. The compatibilities of chitin with the MSCs were investigated in vitro using immunohistochemistry and fluorescence confocal microscopy. The results showed high compatibilities of chitin. An experimental model of growth arrest was created by excising the medial half of the proximal growth plate of the tibia in 6-week-old New Zealand White rabbits. The physeal defect after excision of the bony bridge was transplanted either with no interposition (group 1), chitin alone (group 2), or chitin with MSCs (group 3). In groups 2 and 3, both angulatory deformities and length discrepancies of the tibia were corrected. The differences between group 3 and group 1 were greater than those between group 2 and group 1.