Akimoto Nimura

Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle

Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each population were CD11b (−), CD45 (−), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold higher than that for cells derived from bone marrow. With regard to expansion potential, synovium-derived cells were the highest in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues, indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages. The Oil Red-O positive colony-rate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells. Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a rat model.

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.

Increased proliferation of human synovial mesenchymal stem cells with autologous human serum: Comparisons with bone marrow mesenchymal stem cells and with fetal bovine serum

OBJECTIVE Synovial mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration due to their high chondrogenic potential. For clinical safety, autologous human serum should be used instead of fetal bovine serum (FBS). We undertook this study to compare the 2 types of serum for their enhancement of the proliferation and chondrogenic potentials of synovial MSCs and to investigate the mechanisms of the differences. Since effectiveness of the sera might depend on the origin of the MSCs, we also examined bone marrow MSCs. METHODS Synovium, bone marrow, and peripheral blood were obtained from 18 donors. Synovial and bone marrow MSCs were cultured with autologous human serum or FBS and analyzed. In addition, rabbit synovial MSCs cultured with autologous serum or FBS were transplanted into full-thickness cartilage defects of the knees of the same rabbits. RESULTS Human synovial MSCs expanded more in human serum than in FBS, and the opposite results were obtained with bone marrow MSCs. Hierarchical clustering analysis showed that the cell source, rather than the type of serum, affected the gene expression profile. Human serum contained high levels of platelet-derived growth factor (PDGF), synovial MSCs expressed higher levels of PDGF receptor alpha than did bone marrow MSCs, and neutralizing PDGF decreased the proliferation of synovial MSCs with autologous human serum. Although the in vitro chondrogenic potential of human synovial MSCs was affected by the serum source, the in vivo chondrogenic potential of rabbit synovial MSCs was similar in autologous serum and FBS groups. CONCLUSION Autologous serum predominates in increasing the proliferation of synovial MSCs with chondrogenic potential through PDGF signaling.

Subscapularis Tendon Tear: An Anatomic and Clinical Investigation

PURPOSE The purpose of this study was to clarify anatomically and clinically how the subscapularis tendon supports the superior portion of the biceps tendon to the intertubercular groove. METHODS Thirty-three embalmed shoulder girdles were examined to investigate the subscapularis tendon and the pathway of the biceps tendon. In addition, operation records of 435 consecutive arthroscopic rotator cuff repairs were retrospectively reviewed. RESULTS Anatomically, the superior-most insertion of the subscapularis tendon was located on the upper margin of the lesser tuberosity. In addition, a thin tendinous slip extended from the insertion and attached to the fovea capitis of the humerus. The insertion, the tendinous slip, and the lateral portion of the cranial part of intramuscular tendons were in direct contact with the inferior side of the biceps tendon at its corner portion. The clinical study showed that 27.4% of rotator cuff tears (119/435) had subscapularis tendon tears. In cases with an unstable biceps tendon there was no intact subscapularis tendon. The superior-most insertion of the subscapularis tendon was involved in all transverse tears. Of 29 full-thickness transverse tears, 13 (44.8%) showed intra-articular dislocation. CONCLUSIONS The trochlea-like structure was composed of the superior-most insertion, the tendinous slip, and the lateral portion of the cranial part of intramuscular tendons supporting the biceps tendon. The transverse tear of the subscapularis tendon, which included this trochlea-like structure, often leads to intra-articular dislocation of the biceps tendon. CLINICAL RELEVANCE Instability of the biceps tendon should be carefully assessed because it is associated with subscapularis tendon tears at a very high incidence. When we repair a transverse tear of the subscapularis tendon, we should widely fix sufficiently strong tissue to support the biceps tendon on the uppermost margin, not on the anteromedial portion, of the lesser tuberosity.

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.

The superior capsule of the shoulder joint complements the insertion of the rotator cuff

BACKGROUND To date, there are no studies about the attachment of the articular capsule of the superior shoulder joint. The aim of this study was to measure the width of the attachment of the articular capsule on the humerus, and to clarify the anatomy and the relationship to the footprint of the rotator cuff. METHODS The attachment of the articular capsule on the greater tuberosity was exposed. The width of the attachment of the capsule and the footprint of the rotator cuff were measured. RESULTS The maximum capsular width was located at the border between the infraspinatus and the teres minor, and measured 9.1 mm. The minimum capsular width was 3.5 mm, and it was located at 10.9 mm posterior to the anterior margin of the greater tuberosity and 1.5 mm anterior to the posterior margin of the supraspinatus. CONCLUSION Prior studies have overestimated the rotator cuff footprint width due to the lack of discrimination between the actual cuff insertion and capsule. The attachment of the articular capsule of the shoulder joint occupied a substantial area of the greater tuberosity. In particular, at the border between the infraspinatus and the teres minor, the very thick attachment of the articular capsule compensated for the lack of attachment of muscular components. The thinnest point of the articular capsule was 11 mm posterior to the anterior margin of the greater tuberosity and very close to the posterior edge of the tapered insertion of the supraspinatus, which could contribute to the etiology of degenerative rotator cuff tears.

Anatomic study of the attachment of the medial patellofemoral ligament and its characteristic relationships to the vastus intermedius

PurposeThe aim of this study was to investigate the attachment of the medial patellofemoral ligament (MPFL) using cadaver specimens and establish an anatomic basis for optimal MPFL reconstruction to achieve better patella stability.MethodsSixteen knees of eight cadavers were used in this study. The relationship of the MPFL with quadriceps muscles was investigated from outside after removal of the distal part of the vastus medialis and the rectus femoris and then evaluated from intra-articular side after release of lateral margin of the vastus lateralis muscle, patella and patella tendon.ResultsThe proximal fibres of MPFL were mainly attached to the vastus intermedius tendon, without tight adhesion to the vastus medialis. The distal fibres of MPFL were interdigitated with the deep layer of the medial retinaculum that was attached to the medial margin of the patella tendon.ConclusionThese findings imply that MPFL, which was directly attached to the vastus intermedius and patella and indirectly continued to the patella tendon, could keep pulling them medially as one unit and consequently make the patella move smoothly on the trochlea during whole movement of the knee. Clinically, dysfunction of both proximal and distal MPFL fibres should be considered in the diagnosis and treatment of patella instability after traumatic patella dislocation. MPFL reconstruction with both fibres has a possibility to lead ideal function of MPFL and better instability of the patella.