The safety and efficacy of magnetic targeting using autologous mesenchymal stem cells for cartilage repair.

PURPOSE: A new cell delivery system using magnetic force, termed magnetic targeting, was developed for the accumulation of locally injected cells in a lesion. The aim of this study was to assess the safety and efficacy of mesenchymal stem cell (MSC) magnetic targeting in patients with a focal articular cartilage defect in the knee. METHODS: MSC magnetic targeting for five patients was approved by the Ministry of Health Labour and Welfare of Japan. Autologous bone marrow MSCs were cultured and subsequently magnetized with ferucarbotran. The 1.0-T compact magnet was attached to a suitable position around the knee joint to allow the magnetic force to be as perpendicular to the surface of the lesion as possible. Then 1 × 107 MSCs were injected into the knee joint. The magnet was maintained in the same position for 10 min after the MSC injection. The primary endpoint was the occurrence of any adverse events. The secondary endpoints were efficacy assessed by magnetic resonance imaging (MRI) T2 mapping and clinical outcomes using the International Knee Documentation Committee (IKDC) Subjective Knee Evaluation and the Knee Injury and Osteoarthritis Outcome Score (KOOS). RESULTS: No serious adverse events were observed during the treatment or in the follow-up period. Swelling of the treated knee joint was observed from the day after surgery in three of the five patients. The swelling resolved within 2 weeks in two patients. MRI showed that the cartilage defect areas were almost completely filled with cartilage-like tissue. MOCART scores were significantly higher 48 weeks postoperatively than preoperatively (74.8 ± 10.8 vs 27.0 ± 16.8, p = 0.042). Arthroscopy in three patients showed complete coverage of their cartilage defects. Clinical outcome scores were significantly better 48 weeks postoperatively than preoperatively for the IKDC Subjective Knee Evaluation (74.8 ± 17.7 vs 46.9 ± 17.7, p = 0.014) and knee-related quality-of-life (QOL) in the KOOS (53.8 ± 26.4 vs 22.5 ± 30.8, p = 0.012). CONCLUSION: Magnetic targeting of MSCs was safely performed and showed complete coverage of the defects with cartilage-like tissues and significant improvement in clinical outcomes 48 weeks after treatment. The magnetic targeting of MSCs is useful as a minimally invasive treatment for cartilage repair. LEVEL OF EVIDENCE: IV.

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 Platelet-Rich Plasma on Autologous Chondrocyte Implantation for Chondral Defects: Results Using an In Vivo Rabbit Model.

Background: Articular cartilage repair remains challenging despite the availability of techniques, including autologous chondrocyte implantation (ACI) for repairing large cartilage defects. Platelet-rich plasma (PRP) therapy, a novel therapy focused on chondrocyte regeneration, needs to be investigated regarding its potential to improve the outcomes of ACI. Purpose: To examine the effect of PRP therapy on the outcomes of cartilage repair using the ACI procedure in a rabbit model of knee joint cartilage damage. Study Design: Controlled laboratory study. Methods: A total of 30 knees in 15 Japanese White rabbits (joint cartilage damage model) were divided into nontreatment (n = 7), PRP (n = 8), ACI (n = 7), and combined ACI and PRP (n = 8) groups. At 4 weeks and 12 weeks postoperatively, histological and visual examination of the surgical site was performed, and the regenerated cartilage and calcified bone areas were measured by imaging the specimens. Results: Pretransplantation evaluation in the cultured cartilage showed the histological properties of hyaline cartilage. At 4 weeks postoperatively, the regenerated cartilage area at the surgical site showed a larger safranin O-positive area in the ACI group (2.73 ± 4.46 mm2) than in the combined ACI and PRP group (1.71 ± 2.04 mm2). Calcified bone formation in the ACI group was relatively lower than that in the other groups. Cartilage repair failure occurred in all groups at 12 weeks postoperatively. Conclusion: The authors found no positive effects of PRP on the outcomes of ACI in a rabbit model. There was a smaller safranin O-positive region with the addition of PRP to ACI compared with ACI alone. In the subchondral bone, bone formation might have been promoted by PRP. Clinical Relevance: Administering PRP at the time of ACI may not have a positive effect and may have deleterious effects on cartilage engraftment and regeneration.

Augmentation of degenerated human cartilage in vitro using magnetically labeled mesenchymal stem cells and an external magnetic device.

PURPOSE: The purpose of this study was to investigate whether it is possible to regenerate degenerated human cartilage in vitro by use of magnetically labeled mesenchymal stem cells (MSCs) and an external magnetic device. METHODS: MSCs from human bone marrow were cultured and magnetically labeled. Degenerated human cartilage was obtained during total knee arthroplasty. The osteochondral fragments were attached to the sidewall of tissue culture flasks, and magnetically labeled MSCs were injected into the flasks. By use of an external magnetic device, a magnetic force was applied for 6 hours to the direction of the cartilage, and then the degenerated cartilage was cultured in chondrogenic differentiation medium for 3 weeks. In the control group a magnetic force was not applied. The specimens were evaluated histologically. RESULTS: A cell layer was formed on the degenerated cartilage as shown by H&E staining. The cell layer was also stained in toluidine blue and safranin O and with anti-collagen type II immunostaining, indicating that the cell layer contained an extracellular matrix. In the control group a cell layer was not observed on the cartilage. CONCLUSIONS: We were able to show that our system could deliver MSCs onto degenerated human cartilage and then form an extracellular matrix on the degenerated cartilage in vitro. CLINICAL RELEVANCE: Our novel cell delivery system using magnetic force may lead toward a new treatment option for osteoarthritis.

In Vitro Safety and Quality of Magnetically Labeled Human Mesenchymal Stem Cells Preparation for Cartilage Repair.

IMPACT STATEMENT: This study is very important for a preclinical assessment of the safety and quality of magnetically labeled mesenchymal stem cells (MSCs) for use in cartilage repair. The findings of this study show that magnetic labeling with an appropriate density of magnetic particles has no harmful effects on the safety and quality of MSCs.

Transplantation of tissue-engineered osteochondral plug using cultured chondrocytes and interconnected porous calcium hydroxyapatite ceramic cylindrical plugs to treat osteochondral defects in a rabbit model.

The purpose of this study was to evaluate the macroscopic and histological results of transplanting a tissue-engineered composite plug made of tissue-engineered cartilage and interconnected porous calcium hydroxyapatite ceramics (IP-CHA) with a very high porosity of 94.9% to treat osteochondral defects. Twelve 12-week-old male Japanese white rabbits were used. Fresh articular cartilage slices were taken, and isolated chondrocytes (2 x 10(6) cells) were embedded in atelocollagen gel. They were seeded on the top of IP-CHA plugs and cultured for 2 weeks. These tissue-engineered composite plugs were transplanted into the osteochondral defects in the patellar grooves (the experimental group). In the control group, the defects were treated with composite plugs without chondroytes. Twelve weeks after transplantation in the experimental group, the defects were repaired with cartilage-like tissue with good subchondral bone formation histologically. Histological scores in the experimental group were significantly better than those in the control group. This study clearly showed the defects that had been treated with tissue-engineered composite plugs.

Transplantation of meniscus regenerated by tissue engineering with a scaffold derived from a rat meniscus and mesenchymal stromal cells derived from rat bone marrow.

The purpose of this study was to assess transplantation of regenerated menisci using scaffolds from normal allogeneic menisci and bone-marrow-derived mesenchymal stromal cells (BM-MSCs) of rats. We reported that scaffolds derived from normal menisci seeded with BM-MSCs in vitro could form meniscal tissues within 4 weeks. Then, we hypothesized that our tissues could be more beneficial than allogeneic menisci regarding early maturation and chondroprotective effect. Bone marrow was aspirated from enhanced green fluorescent protein transgenic rats. BM-MSCs were isolated and seeded onto scaffolds which were prepared from Sprague-Dawley rat menisci. After 4 weeks in coculture, the tissues were transplanted to the defect of menisci. Repopulation of BM-MSCs and expression of extracellular matrices were observed in the transplanted tissues at 4 weeks after surgery. At 8 weeks, articular cartilage in the cell-free group was more damaged compared to that in the cell-seeded group or the meniscectomy group.

A novel cell delivery system using magnetically labeled mesenchymal stem cells and an external magnetic device for clinical cartilage repair.

PURPOSE: The purpose of this study was to investigate whether it is possible to successfully accumulate magnetically labeled mesenchymal stem cells (MSCs), under the direction of an external magnetic force, to the desired portion of osteochondral defects of the patellae after intra-articular injection of the MSCs. METHODS: MSCs were cultured from bone marrow and were labeled magnetically. Osteochondral defects were made in the center of rabbit and swine patellae, and magnetically labeled MSCs were injected into the knee joints either under the direction of an external magnetic force or with no magnetic force applied. In the rabbit model we evaluated the patellae macroscopically and histologically, and in the swine model we observed the patellae arthroscopically. RESULTS: Accumulation of magnetically labeled MSCs to the osteochondral defect was shown macroscopically and histologically in the rabbit model and was shown by arthroscopic observation to be attached to the chondral defect in the swine model. CONCLUSIONS: We showed the ability to deliver magnetically labeled MSCs to a desired place in the knee joint. CLINICAL RELEVANCE: Our novel approach is applicable for human cartilage defects and may open a new era of repairing cartilage defects caused by osteoarthritis or trauma by use of a less invasive technique.

Intra-articular injection of mesenchymal stromal cells in partially torn anterior cruciate ligaments in a rat model.

PURPOSE: The purpose of this study was to biomechanically and histologically evaluate whether intra-articularly injected mesenchymal stromal cells can accelerate the healing of a partially torn anterior cruciate ligament (ACL). METHODS: Ninety-eight 12-week-old male Sprague-Dawley rats were studied. The right ACL was partially transected, and a sham operation was performed on the left knee. Mesenchymal stromal cells obtained from bone marrow of green fluorescent protein (GFP) transgenic Sprague-Dawley rats were cultured for 2 weeks in medium. In the MSC(+) group, 1 x 10(6) cells were suspended in phosphate-buffered saline solution and were injected into the right knee with the partially transected ACL. In the MSC(-) group, only phosphate-buffered saline solution was injected. Six animals from each group were evaluated histologically at 1, 2, 3, and 4 weeks after surgery, and they were evaluated biomechanically immediately after surgery (time zero) (n = 9) and also at 2 weeks (n = 11) and 4 weeks (n = 12) after surgery in both groups. For biomechanical testing, the ultimate failure load of a prepared femur-ACL-tibia complex was measured. We then compared the transected side with the sham side in each group. GFP luminescence was observed with a fluorescence microscope to detect whether the injected cells mobilized into the covered tissue. RESULTS: In the MSC(-) group the transected area retracted with increasing time, and the gap remained void of any tissues at all time points after surgery. In the MSC(+) group at 2 and 4 weeks after surgery, the transected area was covered with healing tissues in which GFP-positive cells were detected. Furthermore, the histologic score of the MSC(+) group was significantly better than that of the MSC(-) group. The ultimate failure load of the femur-ACL-tibia complex in the MSC(+) group was significantly higher than that in the MSC(-) group at 4 weeks after surgery. CONCLUSIONS: We conclude from our histologic and biomechanical measurements that injected mesenchymal stromal cells can accelerate the healing of partially torn ACLs. CLINICAL RELEVANCE: The intra-articular injection of mesenchymal stromal cells can be a viable option for treating partially torn knee ACLs.

Quality Evaluation of Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair.

Quality evaluation of mesenchymal stem cells (MSCs) based on efficacy would be helpful for their clinical application. In this study, we aimed to find the factors of human bone marrow MSCs relating to cartilage repair. The expression profiles of humoral factors, messenger RNAs (mRNAs), and microRNAs (miRNAs) were analyzed in human bone marrow MSCs from five different donors. We investigated the correlations of these expression profiles with the capacity of the MSCs for proliferation, chondrogenic differentiation, and cartilage repair in vivo. The mRNA expression of MYBL1 was positively correlated with proliferation and cartilage differentiation. By contrast, the mRNA expression of RCAN2 and the protein expression of TIMP-1 and VEGF were negatively correlated with proliferation and cartilage differentiation. However, MSCs from all five donors had the capacity to promote cartilage repair in vivo regardless of their capacity for proliferation and cartilage differentiation. The mRNA expression of HLA-DRB1 was positively correlated with cartilage repair in vivo. Meanwhile, the mRNA expression of TMEM155 and expression of miR-486-3p, miR-148b, miR-93, and miR-320B were negatively correlated with cartilage repair. The expression analysis of these factors might help to predict the ability of bone marrow MSCs to promote cartilage repair.

Magnetic targeting of bone marrow stromal cells into spinal cord: through cerebrospinal fluid.

We established a new magnetic targeting system in which bone marrow stromal cells migrate through the cerebrospinal fluid to the desired site in the spinal cord in rats. Subarachnoid injection has been reported as a minimally invasive method of transplantation of bone marrow stromal cells for spinal cord injury. It may be, however, less effective than direct injection into the spinal cord in terms of cell delivery. After implantation of a magnet, subarachnoid injection of bone marrow stromal cells labeled with magnetic beads was performed. Greater numbers of bone marrow stromal cells aggregated on the surface of the spinal cord owing to the magnetic force. This targeting system may be a useful tool in minimally invasive transplantation of bone marrow stromal cells for the treatment of spinal cord injury.

Effects of CD44 antibody-- or RGDS peptide--immobilized magnetic beads on cell proliferation and chondrogenesis of mesenchymal stem cells.

We evaluated the efficacy of a novel mesenchymal stem cell (MSC) delivery system using an external magnetic field for cartilage repair in vitro. MSCs were isolated from the bone marrow of Sprague Drawley rats and expanded in a monolayer. To use the MSC delivery system, two types of MSC-magnetic bead complexes were designed and compared. Expanded MSCs were combined with small-sized (diameter: 310 nm) carboxyl group-combined (0.01-0.04 micromol/mg) magnetic beads, Ferri Sphere 100C, through either anti-rat CD44 mouse monoclonal antibodies or a synthetic cell adhesion factor, arginine (R)-glycine (G)-aspartic acid (D)-serine (S) (RGDS) peptide. Both cell complexes were successfully created, and were able to proliferate in monolayer culture up to at least day 7 after separation of magnetic beads from the cell surface, although the proliferation of the complexes was slower in the early period of culture than that of non-labeled rat MSCs (after 7 days of culture: proliferation of CD44 antibody-bead complexes, approximately 50%; RGDS peptide-bead complexes, 70% versus non-labeled rat MSCs, respectively). These complexes were seeded onto culture plates with or without an external magnetic force (magnetic flux density was 0.20 Tesla at a distance of 2 mm from plate base) generated by a neodymium magnet, and supplemented with chondrogenic differentiation medium. Both complexes could be attached and gathered effectively under the influence of the external magnet, and CD44-bead complexes could effectively generate chondrogenic matrix in monolayer culture. In a three-dimensional culture system, the production of a dense chondrogenic matrix and the expression of type II collagen and aggrecan mRNA were detected in both complexes, and the chondrogenic potential of these complexes was only a little less than that of rat MSCs alone. Thus, we conclude that due to the fact that MSC-RGDS peptide-bead complexes are composed using a biodegradable material, RGDS peptide, as a mediator, the RGDS peptide-bead complex is more useful for minimally invasive clinical applications using our design of magnetic MSC delivery system than CD44 antibody-beads.

Hemagglutinating virus of Japan-artificial viral envelope liposome-mediated cotransfer of bag-1 and bcl-2 genes protects hepatic cells against ischemic injury through BAG-1-assisted preferential enhancement of bcl-2 protein expression.

Hepatic injury subsequent to ischemia-reperfusion (I/R) was demonstrated in our previous study to be prevented by hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE) liposome-mediated gene transfer of the antiapoptotic gene, human bcl-2 (h-bcl-2). In the present study, we introduced simultaneously both mouse Bcl-2-associated athanogene 1 (m-bag-1) and the h-bcl-2 gene by the same HVJ-AVE liposome transfection method, and found that I/R-induced hepatic injuries such as release of hepatic marker enzymes into blood, cell morphological degeneration, and cellular DNA strand cleavage were suppressed more effectively than by transfection with either gene singly. In addition, the h-Bcl-2 expression level in the ischemic state, but not in the nonischemic state, was markedly higher in h-bcl-2/m-bag-1-cotransfected liver than in h-bcl-2-transfected liver. In contrast, the m-BAG-1 expression level in the ischemic state, but not in the nonischemic state, was only slightly higher in h-bcl-2/m-bag-1-cotransfected liver than in m-bag-1-transfected liver. Thus, with dual gene cotransfer, coexistent Bcl-2 protein exerts no activity to assist a marked enhancement of BAG-1 protein, whereas the function of overexpressed BAG-1 as a Bcl-2-binding protein may lead to the enhancement of efficient expression of h-Bcl-2 in I/R-treated liver as compared with nonischemic liver, which results in repression of diverse I/R-induced cell death symptoms, presumably through the formation of functional complexes of BAG-1 and Bcl-2.

Magnetically labeled human natural killer cells, accumulated in vitro by an external magnetic force, are effective against HOS osteosarcoma cells.

We evaluated the efficacy of a novel natural killer (NK) cell delivery system in vitro, and also investigated the antitumor effect of the accumulated cells on HOS osteosarcoma cells. Human peripheral blood mononuclear cells were isolated and co-cultured with inactivated K562 erythroleukaemic cells in the presence of IL-2 for 5 days. CD3- CD56+ NK cells were labeled with immunomagnetic beads and separated using a magnetic cell sorting system. Purity and cytotoxicity against K562 cells and HOS cells of the magnetically labeled NK cells were measured. To evaluate whether magnetically labeled NK cells could be accumulated in a specific area by magnetic force, the NK cells were placed in chamber slides in the presence, or not, of an external magnetic force of a neodymium magnet (diameter: 1.5 mm, height: 3 mm, total magnetic flux density: 0.282 T). Moreover, to investigate the antitumor effect on HOS cells, the magnetically labeled NK cells were added to HOS cells in chamber slides in the presence, or not, of an external magnetic force for various times. HOS cells were subsequently stained with Papanicolaou for histological examination. It was found that the magnetically labeled NK cells were highly purified and had cytotoxicity against target cells. The NK cells were accumulated effectively by the magnetic field and, when the NK cells were added to HOS cells in a chamber slide with a magnet placed beneath, a significantly larger number of HOS cells detached from the magnet zone than from other zones. Apoptosis was detected in most detached HOS cells. In conclusion, these findings indicate that magnetically accumulated NK cells efficiently induced apoptosis in HOS cells, suggesting that magnetic targeting therapy using magnetically labeled NK cells holds promise as an immunotherapy for osteosarcoma.

Neovascularization and bone regeneration by implantation of autologous bone marrow mononuclear cells.

We examined whether transplantation of autologous bone marrow mononuclear cells (BM-MNCs) can augment neovascularization and bone regeneration of bone marrow in femoral bone defects of rabbits. Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. To evaluate the in vivo effect of implanted BM-MNCs, we created bone defects in the rabbit medial femoral condyle, and implanted into them 5 x 10(6) fluorescent-labeled autologous BM-MNCs together with gelatin microspheres containing 10 microg bFGF on an atelocollagen gel scaffold. The four experimental groups, which were Atelocollagen gel (Col), Col + 5 x 10(6) BM-MNCs, Col + 10 microg bFGF, and Col + 5 x 10(6) BM-MNCs + 10 microg bFGF, were implanted into the sites of the prepared defects using Atelocollagen gel as a scaffold. The autologous BM-MNCs expressed CD31, an endothelial lineage cell marker, and induced efficient neovascularization at the implanted site 2 weeks after implantation. Capillary density in Col + BM-MNCs + bFGF was significantly large compared with other groups. This combination also enhanced regeneration of the bone defect after 8 weeks to a significantly greater extent than either BM-MNCs or bFGF on their own. In summary, these findings demonstrate that a combination of BM-MNCs and bFGF gelatin hydrogel enhance the neovascularization and the osteoinductive ability, resulting in bone regeneration.

Characterization of labeled neural progenitor cells for magnetic targeting.

For many diseases and injuries of the central nervous system, transplantation of neural progenitor cells is being evaluated as a possible treatment option. Although local, intravenous and subarachnoid injections have been reported as administration methods of neural progenitor cells, each of these methods has limitations. More effective and minimally invasive cell delivery systems are necessary for transplanting neural progenitor cells. In this study, we have developed a technique to form magnetically labeled neural progenitor cells for a magnetic targeting system. We demonstrated that neural progenitor cells can couple with magnetic beads, and that the labeled neural progenitor cells preserve the characteristics of non-labeled neural progenitor cells, and that they can be localized by magnetic force in vitro. Labeled neural progenitor cells have the potential to be used in magnetic targeting systems in-vivo models.

Meniscal regeneration using tissue engineering with a scaffold derived from a rat meniscus and mesenchymal stromal cells derived from rat bone marrow.

The purpose of this study was to regenerate a meniscus using a scaffold from a normal meniscus and mesenchymal stromal cells derived from bone marrow (BM-MSCs). Thirty Sprague-Dawley rat menisci were excised and freeze-thawed three times with liquid nitrogen to kill the original meniscal cells. Bone marrow was aspirated from enhanced green fluorescent protein transgenic Sprague-Dawley rats. BM-MSCs were isolated, cultured for 2 weeks, and 2 x 10(5) cells were then seeded onto the meniscal scaffolds. Using a fluorescent microscope and immunohistochemical staining, repopulation of enhanced green fluorescent protein positive cells was observed in the superficial zone of the scaffold after 1 week of culture, and then in the deep zone after 2 weeks. At 4 weeks, expression of extracellular matrices was detected histologically and expression of mRNA for aggrecan and type X collagen was detected. Stiffness of the cultured tissue, assessed by the indentation stiffness test, had increased significantly after 2 weeks in culture, and approximated the stiffness of a normal meniscus. From this study, we conclude that a scaffold derived from a normal meniscus seeded with BM-MSCs can form a meniscus approximating a normal meniscus.

Repair of osteochondral defect with tissue-engineered chondral plug in a rabbit model.

PURPOSE: The purpose of this study was to evaluate the macroscopic and histologic results of transplanting a tissue-engineered chondral plug made of atelocollagen sponge and PLLA mesh to treat osteochondral defects. TYPE OF STUDY: Controlled experimental study. METHODS: Twelve-week-old male Japanese white rabbits were used. Fresh articular cartilage slices were taken from the humeral head, and isolated chondrocytes were embedded in atelocollagen gel which does not have antigenic portions of collagen (2.0 x 10(6) cells/mL). They were seeded on the top of the atelocollagen sponge/PLLA mesh composite and cultured for 2 weeks. The culture medium was changed every 3 days and L-ascorbic acid (50 microg/mL) was added every 2 days. Culturing the composites for 2 weeks produced tissue-engineered chondral plugs. These tissue-engineered chondral plugs (4-mm diameter, 4-mm thick) were transplanted into the osteochondral defects (4 mm diameter, 4 mm deep) in the patellar grooves of the same rabbits from which the chondrocytes had been harvested (the experimental group). In the control group, the defects were treated with the plugs without chondrocytes. The rabbits were killed 4 and 12 weeks after transplantation. The repaired tissues were evaluated macroscopically and histologically, and analyzed immunohistochemically for expression of type II collagen. RESULTS: Four weeks after transplantation in the experimental group, the defects were partially repaired with cartilage-like tissue with good subchondral bone formation. Twelve weeks after transplantation, the defects were repaired with hyaline cartilage-like tissue densely stained by Safranin O. Well-organized subchondral bone formation was also observed. In the control group, the defects were covered with only soft fibrous tissue at 4 and 12 weeks macroscopically. Immunohistochemically, type II collagen was detected in about 90% of the repaired area. Histologic scores in the experimental group were significantly higher than those in the control group at both 4 and 12 weeks after transplantation. CONCLUSIONS: This study shows that the defects treated with tissue engineered chondral plug developed type II collagen in about 90% of the repaired area. CLINICAL RELEVANCE: The transplantation of a tissue-engineered chondral plug will be one option for treating osteochondral defects. The next step in testing our hypothesis is to evaluate the repaired tissue biomechanically and biochemically over a longer period of time.

Allogeneic bone marrow-derived mesenchymal stromal cells promote the regeneration of injured skeletal muscle without differentiation into myofibers.

Half-stratum laceration was performed on the tibialis anterior muscle of Sprague-Dawley (SD) rats as a skeletal muscle injury model. Bone marrow-derived mesenchymal stromal cells (BMMSCs), which were derived from enhanced green fluorescent protein (GFP) transgenic SD rats, were transplanted into the injured site. Tensile strength produced by nerve stimulation was measured for functional evaluation before sacrifice. Specimens of the tibialis anterior muscles were stained with hematoxylin and eosin, and immunohistochemically stained for histological evaluation. Our results showed that transplanted BMMSCs promoted maturation of myofibers histologically and made the injured muscle acquire almost normal muscle power functionally by 1 month after transplantation. However, the results of immunohistochemical staining could not prove that transplanted BMMSCs differentiated into or fused to skeletal myofibers, although it showed that transplanted BMMSCs seemed to differentiate into muscle precursor cells. Therefore, our results indicated that BMMSCs contributed to the regeneration of skeletal muscle by mechanisms other than fusion to myofibers after differentiation.

In vitro cartilage formation using TGF-beta-immobilized magnetic beads and mesenchymal stem cell-magnetic bead complexes under magnetic field conditions.

We evaluated the efficacy of transforming growth factor (TGF)-beta-immobilized magnetic beads for chondrogenesis in vitro using a mesenchymal stem cell (MSC) delivery system and an external magnetic force (EMF). MSCs isolated from the bone marrow of Sprague Dawley rats were mixed with carboxyl group-combined magnetic beads (Ferri Sphere 100C) coated with anti-rat CD44 mouse monoclonal antibodies. TGF-beta3 (10 and 1 ng/mL) was attached magnetically to such other Ferri Sphere 100C beads via an amide bond formed between a primary amino group on the TGF-beta3 and the carboxyl groups on the surface of the beads. MSC-magnetic bead complexes were centrifuged to form a pellet and cultured in chondrogenic differentiation medium (CDM) supplemented with either 10 or 1 ng/mL TGF-beta-immobilized magnetic beads (10 or 1 ng/mL TGF-beta-immobilized magnetic bead groups) or in CDM supplemented with 1 or 10 ng/mL TGF-beta (1 or 10 ng/mL TGF-beta group). TGF-beta-immobilized magnetic beads were gathered effectively under an EMF. Chondrogenesis was achieved from the MSC-magnetic bead complexes in the presence of 1 ng/mL TGF-beta-immobilized magnetic beads.