Ascorbic Acid and Iron Supplement Treatment Improves Stem Cell-Mediated Cartilage Regeneration in a Minipig Model.

BACKGROUND: The transplantation of mesenchymal stem cells (MSCs) into cartilage defects has led to variable cartilage repair outcomes. Previous in vitro studies have shown that ascorbic acid and reduced iron independently can improve the chondrogenic differentiation of MSCs. However, the combined effect of ascorbic acid and iron supplementation on MSC differentiation has not been investigated. PURPOSE: To investigate the combined in vivo effects of ascorbic acid and a US Food and Drug Administration (FDA)-approved iron supplement on MSC-mediated cartilage repair in mature Göttingen minipigs. STUDY DESIGN: Controlled laboratory study. METHODS: We pretreated bone marrow-derived MSCs with ascorbic acid and the FDA-approved iron supplement ferumoxytol and then transplanted the MSCs into full-thickness cartilage defects in the distal femurs of Göttingen minipigs. Untreated cartilage defects served as negative controls. We evaluated the cartilage repair site with magnetic resonance imaging at 4 and 12 weeks after MSC implantation, followed by histological examination and immunofluorescence staining at 12 weeks. RESULTS: Ascorbic acid plus iron-pretreated MSCs demonstrated a significantly better MOCART (magnetic resonance observation of cartilage repair tissue) score (73.8 ± 15.5), better macroscopic cartilage regeneration score according to the International Cartilage Repair Society (8.6 ± 2.0), better Pineda score (2.9 ± 0.8), and larger amount of collagen type II (28,469 ± 21,313) compared with untreated controls (41.3 ± 2.5, 1.8 ± 2.9, 12.8 ± 1.9, and 905 ± 1326, respectively). The obtained scores were also better than scores previously reported in the same animal model for MSC implants without ascorbic acid. CONCLUSION: Pretreatment of MSCs with ascorbic acid and an FDA-approved iron supplement improved the chondrogenesis of MSCs and led to hyaline-like cartilage regeneration in the knee joints of minipigs. CLINICAL RELEVANCE: Ascorbic acid and iron supplements are immediately clinically applicable. Thus, these results, in principle, could be translated into clinical applications.

Coculturing of mesenchymal stem cells of different sources improved regenerative capability of osteochondral defect in the mature rabbit: An in vivo study.

PURPOSE: Choosing a therapeutic cell source for osteochondral repair remains a challenge. The present study investigated coculturing mesenchymal stem cells (MSCs) from different sources to provide an improved therapeutic cell option for osteochondral repair. METHODS: Dutch and Japanese white rabbits were used in this study, the first for isolating MSCs and the second for creating an osteochondral model in the medial femoral condyle. The 26 rabbit knees were divided randomly into four groups: control ( n = 6), bone marrow-derived MSCs (BMSCs) ( n = 7), synovial tissue MSCs (SMSCs) ( n = 7), and cocultured MSCs ( n = 6). Tissue repair was assessed using the Fortier scale, and colony-forming assay was performed. RESULTS: At different cell densities, cocultured and SMSCs formed larger colonies than BMSCs, indicating their high proliferative potential. After 2 months, complete filling of the defect with smooth surface regularity was detected in the cocultured MSC group, although there was no significant difference among the therapeutic groups macroscopically. Also, tissue repair was histologically better in the cocultured MSC group than in the control and SMSC groups, due to repair of the subchondral bone and coverage with hyaline cartilage. Additionally, toluidine blue and collagen-II staining intensity in the repaired tissue was better in the cocultured MSC group than in the remaining groups. CONCLUSION: Our results suggest that cocultured MSCs are a suitable option for the regeneration capability of osteochondral defects due to their enhanced osteochondrogenic potential.

Role of Mesenchymal Stem Cells Densities When Injected as Suspension in Joints with Osteochondral Defects.

OBJECTIVE: The aim of this study was to evaluate an intraarticular injection of different doses of autologous mesenchymal stem cells (MSCs) for improving repair of midterm osteochondral defect. DESIGN: At 4 weeks postoperative marrow stimulation model bilaterally (3 mm diameter; 4 mm depth) in the medial femoral condyle, autologous MSCs were injected into knee joint. Twenty-four Japanese rabbits aged 6 months were divided randomly into 4 groups ( n = 6 per group): the control group and and MSC groups including 0.125, 1.25, and 6.25 million MSCs. Repaired tissue was assessed macroscopically and histologically at 4 and 12 weeks after intraarticular injection of MSCs. RESULTS: At 12 weeks, there was no repair tissue in the control group. The gross appearance of the 1.25 and 6.25 million MSC groups revealed complete repair of the defect with white to pink tissue at 12 weeks. An osteochondral repair was histologically significantly better in the 1.25 and 6.25 million MSC groups than in the control and 0.125 million MSC groups at 4 and 12 weeks, due to presence of hyaline-like tissue in the deep layer at 4 weeks, and at 12 weeks hyaline cartilage formation at the periphery and fibrous tissue containing some chondrocytes in the deep layer of the center of the defect. Subchondral bone was restructured in the 1.25 and 6.25 million MSC groups, although it did not resemble the normal bone. CONCLUSION: An intraarticular injection of 1.25 or 6.25 million MSCs could promote the repair of subchondral bone, even in the case of midterm osteochondral defect.

Monitoring immune response after allogeneic transplantation of mesenchymal stem cells for osteochondral repair.

The aim of this study was to investigate the safety of using allogeneic magnetically labelled mesenchymal stem cells (m-MSCs) to ameliorate osteochondral repair, with immune surveillance using a mixed lymphocyte reaction (MLR) assay. Twenty knees of Japanese white rabbits were randomly divided into two groups: the control (autologous) group, where 2 × 105 autologous m-MSCs were transplanted into the defect site; the experimental (allogeneic) group, where 2 × 105  m-MSCs from Dutch rabbits were transplanted into the defect of Japanese white rabbits. The rabbits were then euthanized after 12 weeks. The repaired tissue was stained with toluidine blue stain in order to produce histological scoring on the Fortier scale. Splenocytes were used to evaluate anti-donor alloreactivity by a MLR assay using the carboxyfluorescein diacetate succimidyl ester (CFSE) labelling technique (CFSE-MLR). In both groups, complete repair of the subchondral bone covered by a layer of chondrogenic tissue was confirmed. Also, there was no histologically significant difference on the Fortier scale. Using the CFSE-MLR assay, CD4+ T-cells showed alloreactivity in each combination, while the stimulation index of CD4+ T-cells was not statistically different between the control and experimental groups. Allogeneic m-MSCs are a safe alternative source to autologous m-MSCs, upholding repair of an osteochondral defect for clinical application using universal donor MSCs through a one-stage surgical procedure. Copyright © 2017 John Wiley & Sons, Ltd.

Therapeutic Potential of Multilineage-Differentiating Stress-Enduring Cells for Osteochondral Repair in a Rat Model.

Multilineage-differentiating stress-enduring (Muse) cells are stage-specific embryonic antigen-3 (SSEA-3) positive cells existing in mesenchymal stem cell (MSC) populations. Muse cells have the pluripotency to differentiate into all germ layers as embryonic stem cells. In this study, we aimed to investigate the efficacy of Muse cell transplantation for osteochondral defect repair. Muse cells were isolated from human bone marrow MSCs. An osteochondral defect was created in the patellar groove of immunodeficient rats. After this, cell injection was performed, whereby rats were divided into 3 groups: the control group, the rats of which were given a PBS injection; the non-Muse group, which comprised 5 × 104 SSEA-3 negative non-Muse cells; and the Muse group, which comprised 5 × 104 SSEA-3 positive Muse cells. The white repaired tissue had a mostly smooth homogenous surface at 12 weeks after treatment in the Muse group, while no repair tissue was detected in the control and non-Muse groups. Histological assessments showed better repair at the cartilage defect sites in the Muse group compared to the other groups at 4 and 12 weeks after treatment. Muse cells could be a new promising cell source for the treatment of osteochondral defects.

Cell Magnetic Targeting System for Repair of Severe Chronic Osteochondral Defect in a Rabbit Model.

The aim of this study was to investigate a cell delivery system for repair of severe chronic osteochondral defects using magnetically labeled mesenchymal stem cells (m-MSCs), with the aid of an external magnetic device, through the accumulation of a small number of m-MSCs into a desired area and to detect the suitable number of autologous m-MSCs needed for repair of the defect. Twenty-six male Japanese white rabbits aged 6 months were used. An osteochondral defect was created bilaterally at the weight-bearing surface of the medial femoral condyle of the rabbits' knees (3 mm diameter; 4 mm depth). At 4 weeks after creation of the defect, autogenic transplantation of the m-MSCs into the defect area was performed, followed by 10-min exposure to an external magnetic device, where animals were divided into four groups: high (1 × 10(6) m-MSCs), medium (2 × 10(5) m-MSCs), low (4 × 10(4) m-MSCs), and control (PBS injection). At 4 and 12 weeks posttransplantation of m-MSCs, repaired tissue was assessed histologically using the Fortier score with toluidine blue staining. Transplantation of a low number of m-MSCs was not enough to improve osteogenesis and chondrogenesis, but the medium and high groups improved repair of the chronic defect with chondrogenic tissues and showed histologically significantly better results than the control and low groups. The use of a magnetic targeting system for delivering m-MSCs has the potential to overcome the clinical hurdles for repair of the severe chronic osteochondral defect. Furthermore, this system is predicted to produce good clinical outcomes for humans, not only to repair osteochondral defects but also to repair a variety of damaged tissues.

Combination therapy with intra-articular injection of mesenchymal stem cells and articulated joint distraction for repair of a chronic osteochondral defect in the rabbit.

The present study investigated intra-articular injection of bone-marrow-derived mesenchymal stem cells (MSCs) combined with articulated joint distraction as treatment for osteochondral defects. Large osteochondral defects were created in the weight-bearing area of the medial femoral condyle in rabbit knees. Four weeks after defect creation, rabbits were divided into six groups: control group, MSC group, distraction group, distraction + MSC group, temporary distraction group, and temporary distraction + MSC group. Groups with MSC received intra-articular injection of MSCs. Groups with distraction underwent articulated distraction arthroplasty. Groups with temporary distraction discontinued the distraction after 4 weeks. The rabbits were euthanized at 4, 8, and 12 weeks after treatment except temporary distraction groups which were euthanized at only 12 weeks. Histological scores in the distraction + MSC group were significantly better than in the control, MSC group or distraction group at 4 and 8 weeks, but showed no further improvement. At 12 weeks, the temporary distraction + MSC group showed the best results, demonstrating hyaline cartilage repair with regeneration of the osteochondral junction. In conclusion, joint distraction with intra-articular injection of MSCs promotes early cartilage repair, and compressive loading of the repair tissue after temporary distraction stimulates articular cartilage regeneration.

Repair mechanism of osteochondral defect promoted by bioengineered chondrocyte sheet.

Cell sheet engineering has developed as a remarkable method for cell transplantation. In the field of cartilage regeneration, several studies previously reported that cartilage defects could be regenerated by transplantation of a chondrocyte sheet using cell sheet engineering. However, it remains unclear how such a thin cell sheet could repair a deep cartilage defect. We, therefore, focused on the mechanism of cartilage repair using cell sheet engineering in this study. Chondrocyte sheets and synovial cell sheets were fabricated using cell sheet engineering, and these allogenic cell sheets were transplanted to cover an osteochondral defect in a rat model. Macroscopic and histological evaluation was performed at 4 and 12 weeks after transplantation. Analysis of the gene expression of each cell sheet and of the regenerated tissue at 1 week after transplantation was performed. In addition, green fluorescent protein (GFP) transgenic rats were used as donors (transplanted chondrocyte sheets) or recipients (osteochondral defect models) to identify the cell origin of regenerated cartilage. Cartilage repair was significantly better in the group implanted with a chondrocyte sheet than in that with a synovial cell sheet. The results of gene expression analysis suggest that the possible factor contributing to cartilage repair might be TGFß1. Cell tracking experiments using GFP transgenic rats showed that the regenerated cartilage was largely composed of cells derived from the transplanted chondrocyte sheets.