Young-Jin Ju

Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit

We previously compared mesenchymal stem cells (MSCs) from a variety of mesenchymal tissues and demonstrated that synovium-MSCs had the best expansion and chondrogenic ability in vitro in humans and rats. In this study, we compared the in vivo chondrogenic potential of rabbit MSCs. We also examined other parameters to clarify suitable conditions for in vitro and in vivo cartilage formation. MSCs were isolated from bone marrow, synovium, adipose tissue, and muscle of adult rabbits. Proliferation potential and in vitro chondrogenic potential were compared. Toxicity of the tracer DiI for in vitro chondrogenesis was also examined. MSCs from each tissue were embedded in collagen gel and transplanted into full thickness cartilage defects of rabbits. Cartilage matrix production was compared histologically. The effects of cell density and periosteal patch on the in vivo chondrogenic potential of synovium-MSCs were also examined. Synovium- and muscle-MSCs had a higher proliferation potential than other cells. Pellets from synovium- and bone-marrow-MSCs showed abundant cartilage matrix. DiI had no significant influence on in vitro cartilage formation. After transplantation into cartilage defects, synovium- and bone-marrow-MSCs produced much more cartilage matrix than other cells. When synovium-MSCs were transplanted at a higher cell density and with a periosteal patch, more abundant cartilage matrix was observed. Thus, synovium- and bone-marrow-MSCs had greater in vivo chondrogenic potential than adipose- and muscle-MSCs, but synovium-MSCs had the advantage of a greater proliferation potential. Higher cell density and a periosteum patch were needed to obtain a high production of cartilage matrix by synovium-MSCs.

Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles.

Mesenchymal stem cells (MSCs) can be obtained from various tissues, and contain common features. However, an increasing number of reports have described variant properties dependent of cell sources. We examined (1) whether MSCs existed in several intraarticular tissues, (2) whether gene expression profiles in intraarticular tissue MSCs closely resembled each other, and (3) whether identified genes were specific to intraarticular tissue MSCs. Human synovium, meniscus, intraarticular ligament, muscle, adipose tissue, and bone marrow were harvested, and colony‐forming cells were analyzed. All these cells showed multipotentiality and surface markers typical of MSCs. Gene profiles of intraarticular tissue MSCs and chondrocytes were closer to each other than those of extraarticular tissues MSCs. Among three characteristic genes specific for intraarticular tissue MSCs, we focused on proline arginine‐rich end leucine‐rich repeat protein (PRELP). Higher expression of PRELP was confirmed in chondrocytes and intraarticular tissue MSCs among three elderly and three young donors. Synovium MSCs stably expressed PRELP, contrarily, bone marrow MSCs increased PRELP expression during in vitro chondrogenesis. In conclusion, MSCs could be isolated from various intraarticular tissues including meniscus and ligament, gene expression profiles of intraarticular tissue MSCs closely resembled each other, and the higher expression of PRELP was characteristic of intraarticular tissue MSCs.

Synovial Stem Cells Are Regionally Specified According to Local Microenvironments After Implantation for Cartilage Regeneration

We previously demonstrated that synovium‐derived MSCs had greater in vitro chondrogenic ability than other mesenchymal tissues, suggesting a superior cell source for cartilage regeneration. Here, we transplanted undifferentiated synovium‐derived MSCs into a full‐thickness articular cartilage defect of adult rabbits and defined the cellular events to elucidate the mechanisms that govern multilineage differentiation of MSCs. Full‐thickness osteochondral defects were created in the knee; the defects were filled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate‐labeled MSCs and covered with periosteum. After 4 weeks, although the cell density decreased, transplanted MSCs produced a great amount of cartilage matrix extensively. The periosteum became thinner, and chondroprogenitors in the periosteum produced a small amount of cartilage matrix. In the deeper zone, transplanted MSCs progressed to the hypertrophic chondrocyte‐like cells. In the deep zone, some transplanted cells differentiated into bone cells and were replaced with host cells thereafter. In the next phase, the border between bone and cartilage moved upwards. In addition, integrations between native cartilage and regenerated tissue were improved. Chondrocyte‐like cells derived from the transplanted MSCs still remained at least after 24 weeks. Histological scores of the MSC group improved continuously and were always better than those of two other control groups. Immunohistological analyses and transmission electron microscopy confirmed that the MSCs produced abundant cartilage matrix. We demonstrated that transplanted synovium‐derived MSCs were altered over a time course according to the microenvironments. Our results will advance MSC‐based therapeutic strategies for cartilage injury and provide the clues for the mechanisms that govern multilineage differentiation of MSCs.

Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect

IntroductionCurrent cell therapy for cartilage regeneration requires invasive procedures, periosteal coverage and scaffold use. We have developed a novel transplantation method with synovial mesenchymal stem cells (MSCs) to adhere to the cartilage defect.MethodsFor ex vivo analysis in rabbits, the cartilage defect was faced upward, filled with synovial MSC suspension, and held stationary for 2.5 to 15 minutes. The number of attached cells was examined. For in vivo analysis in rabbits, an autologous synovial MSC suspension was placed on the cartilage defect, and the position was maintained for 10 minutes to adhere the cells to the defect. For the control, either the same cell suspension was injected intra-articularly or the defects were left empty. The three groups were compared macroscopically and histologically. For ex vivo analysis in humans, in addition to the similar experiment in rabbits, the expression and effects of neutralizing antibodies for adhesion molecules were examined.ResultsEx vivo analysis in rabbits demonstrated that the number of attached cells increased in a time-dependent manner, and more than 60% of cells attached within 10 minutes. The in vivo study showed that a large number of transplanted synovial MSCs attached to the defect at 1 day, and the cartilage defect improved at 24 weeks. The histological score was consistently better than the scores of the two control groups (same cell suspension injected intra-articularly or defects left empty) at 4, 12, and 24 weeks. Ex vivo analysis in humans provided similar results to those in rabbits. Intercellular adhesion molecule 1-positive cells increased between 1 minute and 10 minutes, and neutralizing antibodies for intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and activated leukocyte-cell adhesion molecule inhibited the attachment.ConclusionPlacing MSC suspension on the cartilage defect for 10 minutes resulted in adherence of >60% of synovial MSCs to the defect, and promoted cartilage regeneration. This adherent method makes it possible to adhere MSCs with low invasion, without periosteal coverage, and without a scaffold.

Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans

OBJECTIVE The existence of mesenchymal stem cells (MSCs) in SF was previously reported. However, the behaviour and properties of MSCs derived from SF have not been fully elucidated. METHODS Human SFs were obtained from 19 knee joints with anterior cruciate ligament injury around the time of reconstruction surgery, and from three healthy volunteers. SF was plated, cultured and examined for colony-forming number, in vitro differentiation, surface epitopes and gene profiles. Also, rabbit synovium-MSCs were injected into the knee joint in a rabbit partial anterior cruciate ligament defect model, and the injected cells were traced. RESULTS SF-MSCs from IA ligament injury patients were 100 times more in number than those from healthy volunteers. Total colony number was positively correlated with post-injury period. No significant differences were observed among the cells derived from SF around the time of the surgery in relation to surface epitopes and differentiation potentials. Cluster analysis of gene profiles demonstrated that SF-MSCs were more similar to synovium MSCs than bone marrow MSCs. In rabbit experiments, the MSCs injected into the knee in which IA ligament was partially defective were observed more on the defected area than on the intact area of the ligament at 24 h. CONCLUSION We demonstrated that SF-MSCs, similar to synovium MSCs, increased in number after IA ligament injury and surgery without marked alteration of the properties.

Analysis of the chondrogenic potential of human synovial stem cells according to harvest site and culture parameters in knees with medial compartment osteoarthritis

OBJECTIVE Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage regeneration because of their high chondrogenic ability. In this study, we examined the synovium of patients with medial compartment knee osteoarthritis (OA) to determine the proportion of MSCs in relation to cellular compartmentalization, and to identify the culture parameters that could affect the chondrogenic potential of synovial MSCs. METHODS Human synovium was collected from 4 different harvest sites in the knees of patients with medial compartment OA. Each synovial tissue sample was divided into 2 parts, one for histologic assessment and the other for analysis of the cell size, surface epitopes, and chondrogenic potential of colony-forming cells in vitro. RESULTS The numbers of alpha-smooth muscle actin-positive vessels and CD31+ endothelial cells were higher in the medial outer region than in the other regions of OA synovial tissue. The numbers of these cells correlated with the number of colony-forming cells. In parallel with increasing duration of the preculture period, the size of the cells increased, while the chondrogenic potential decreased, and this was correlated with expression of CD90. CONCLUSION Medial compartment knee OA demonstrates variability in the distribution of vessels, which results in a varying distribution of MSCs. The preculture period should be utilized to assess both the potential for expansion and the chondrogenic potential of MSCs.

Effects of Local Administration of Vascular Endothelial Growth Factor on Properties of the in Situ Frozen-Thawed Anterior Cruciate Ligament in Rabbits

Background In the autogenous tendon for anterior cruciate ligament reconstruction, intrinsic fibroblasts are necrotized immediately after surgery, and repopulation and revascularization occur. Vascular endothelial growth factor is considered to be a potent mediator of angiogenesis. Hypothesis An application of vascular endothelial growth factor significantly enhances angiogenesis in the in situ frozen anterior cruciate ligament, and the application significantly affects mechanical properties of the in situ frozen anterior cruciate ligament. Study Design Controlled laboratory study. Methods Right anterior cruciate ligaments from 66 rabbits underwent the freeze-thaw treatment, and animals were then divided into 3 groups. Group I served as a freeze-thaw but otherwise untreated control. In group II, 0.2 mL phosphate-buffered saline alone was applied. In group III, 30 µ g vascular endothelial growth factor was applied. The groups were compared on the basis of histologic revascularization examinations using the Chalkley score, an indicator of the microvessel density, and mechanical evaluations, which included the anterior-posterior translation of the tibia relative to the femur during ± 10 N of anterior-posterior load and the mechanical properties of the anteromedial bundle of the anterior cruciate ligament. Results Group IIIs Chalkley score was significantly greater than that of groups I and II. The tensile strength and the tangent modulus of anterior cruciate ligaments in groups I, II, and III were significantly lower than those of a normal anterior cruciate ligament, although there were no significant differences among groups I, II, and III. Conclusion Vascular endothelial growth factor, as administered in this study, significantly promoted angiogenesis in the devitalized anterior cruciate ligament with in situ freeze-thaw treatment, but it did not affect the mechanical properties of the in situ frozen-thawed anterior cruciate ligament in the rabbit model. Clinical Relevance An application of the recombinant anterior cruciate ligament is a potential future strategy to enhance revascularization of the autograft in anterior cruciate ligament reconstruction.

Weekly intra-articular injections of bone morphogenetic protein-7 inhibits osteoarthritis progression

IntroductionWe investigated the ability of a weekly intra-articular injection of bone morphogenetic protein (BMP)-7 to prevent osteoarthritis in rabbits with anterior cruciate ligament transections.MethodsFirst, 36 knee joints were randomly divided into four groups: 50, 500, 5,000 ng BMP-7, and control. Knee cartilage was evaluated at 4, 8, and 12 weeks. Then, in order to control for individual differences, 500 ng BMP-7 was injected into one knee and phosphate-buffered saline (PBS) into the other, and the two knees were compared at 4, 8, and 12 weeks (n = 5). For pharmacokinetic analysis, cartilage was harvested at 1 hour and 1, 2, 4, and 7 days after knee injection of 500 ng BMP-7 or PBS (n = 3).ResultsHistological scores in the 500 and 5,000 ng BMP-7 groups were significantly better than those in the other groups at 12 weeks. Matched pair analysis demonstrated that both macroscopic and histological scores in the 500 ng BMP-7 group were better than those in the control group. Immunohistochemical analysis revealed higher BMP-7 expression by chondrocytes in the BMP-7 injected knees. Histology of whole knee and quantitative micro computed tomography analysis showed that weekly injections of 500 ng BMP-7 did not induce synovial fibrosis, ectopic bone, or osteophyte formation. As detected by enzyme-linked immunosorbent assay, BMP-7 concentration in the cartilage tissue was still higher in the BMP-7 treated group 7 days after the injection.ConclusionsWeekly intra-articular injections of BMP-7 inhibited progression of osteoarthritis. Obvious adverse effects were not observed. BMP-7 concentration and expression in cartilage were still higher 7 days after injection.

Intra-articular injections of bone morphogenetic protein-7 retard progression of existing cartilage degeneration

We investigated the effect of weekly intra‐articular injections of bone morphogenetic protein‐7 (BMP‐7) on prevention of progression of existing cartilage degeneration in an osteoarthritis model in rabbits. An anterior cruciate ligament transection (ACLT) model was used to create a progressive osteoarthritis model. BMP‐7 was intra‐articular injected weekly into the right knee and PBS into the left knee from 4 weeks after ACLT. Both sides of the knees were compared macroscopically, histologically, immunohistochemically, and by micro CT. Macroscopically, fibrillation in the femoral condyle was observed 4 weeks after ACLT. In the control knees, cartilage degeneration further progressed throughout the 12‐week period. In the BMP‐7 treated knee, osteoarthritis progression was milder than in the control knees. Histologically, safranin‐O staining was decreased in the surgical knees at 4 weeks. Obvious erosions in both medial and lateral condyles were revealed in the control knees at 12 weeks, while cartilage matrix was predominantly retained in the BMP‐7 treated knees. The macroscopic and microscopic OA score in the BMP‐7 treated knee was better than that in the control in each rabbit. Immunohistochemical analysis demonstrated that both type II collagen and BMP‐7 were more expressed in cartilage treated with BMP‐7. Micro CT analysis showed that osteophytes were smaller in the BMP‐7 treated knee compared to that of the control. Weekly intra‐articular injections of BMP‐7 inhibited progression of existing cartilage degeneration.

Serum keratan sulfate transiently increases in the early stage of osteoarthritis during strenuous running of rats: protective effect of intraarticular hyaluronan injection

IntroductionOsteoarthritis is influenced by genetic and environment factors, including mechanical stress; however, the relationship between running and the development of osteoarthritis remains a matter of controversy. We investigated whether osteoarthritic change could be obtained in a rat strenuous running model, whether serum keratan sulfate in rats could be detected by HPLC and was associated with onset or progression of osteoarthritis, and whether hyaluronan injection suppressed development of osteoarthritis and elevation of serum keratan sulfate.MethodsWistar rats were forced to run 30 km in 6 weeks on a treadmill machine. Articular cartilage of the knees was evaluated macroscopically and immunohistologically. Serum keratan sulfate was examined every week by HPLC. The effect of weekly knee injection of hyaluronan was also investigated.ResultsCartilage surfaces stained with India ink became irregular, metachromasia by safranin-O staining appeared to be almost lost, and Mankins score significantly worsened after 30 km of running. Serum keratan sulfate in rats was detected by HPLC and transiently increased (peaked at 3 to 4 weeks) along with depletion of keratan sulfate in cartilage tissue. Hyaluronan treatment suppressed morphological progression of osteoarthritis and elevation of serum keratan sulfate.ConclusionRat strenuous running induced osteoarthritis. Serum keratan sulfate was associated with progression of osteoarthritis. Weekly intraarticular injection of hyaluronan controlled the development of osteoarthritis, and the effect was reflected by serum keratan sulfate.