A novel PAI-1 inhibitor prevents ageing-related muscle fiber atrophy.

Sarcopenia is among the most common medical problems of the aging population worldwide and a major social concern. Here, we explored the therapeutic potential of TM5484, a novel orally available PAI-1 inhibitor, to prevent sarcopenia. The sarcopenic phenotypes of the calf muscle of 12- and 6-month-old middle-aged mice were compared. Although significant decline of isometric gastrocnemius muscle force was detected in the older untreated mice, those administered TM5484 had significantly greater calf muscle force, as determined using isometric measurements by electrical stimulation. Histological analysis indicated that cross-sectional gastrocnemius muscle fibers in untreated older mice were thinner than those in younger mice; however, TM5484-treated group showed thicker fibers than younger mice. Treatment with TM5484 for 6 months enhanced Igf1, Atrogin-1, Mt-Co1, and Chrna1 mRNA expression in the mice gastrocnemius muscle, with increased serum IGF-1 concentration. TM5484 induced dose-dependent Igf1, Atrogin-1, and Chrna1 expression in C2C12 myoblastic cells, confirming cell autonomous effect. Further, the presence of plasmin for 72 h caused significantly increased Igf1 expression in C2C12 cells. These findings suggest that oral PAI-1 inhibitors represent a promising therapeutic candidate for preventing sarcopenia progression in humans.

Effect of transplanted mesenchymal stem cell number on the prevention of cartilage degeneration and pain reduction in a posttraumatic osteoarthritis rat model.

BACKGROUND: Mesenchymal stem cells (MSCs) transplantation therapy is considered an alternative therapy to prevent posttraumatic osteoarthritis (PTOA). However, consensus as to the sufficient number of MSCs for the prevention of PTOA is lacking. The purpose of this study was to determine the sufficient number of MSCs to achieve PTOA prevention and the reduction in pain after anterior cruciate ligament transection (ACLT). METHODS: Eight-week-old male Wistar rats were used. ACLT was conducted in the knee joint as a PTOA model. According to the species-specific knee joint volume, 104 MSCs in rats are equivalent to 3 × 107 MSCs in humans, which was clinically prepared. MSCs (104, 105, or 106 cells) or phosphate-buffered saline were injected into the knee joint at 1, 2, and 3 weeks after ACLT. Histological examinations were performed at 12 weeks after ACLT. The weight-bearing distribution improvement ratio was calculated as an assessment of pain until 12 weeks after ACLT. RESULTS: Histological evaluations showed that all the MSCs groups except for 104 MSCs group in femur were significantly improved compared to the control group at 12 weeks after ACLT. The weight-bearing distribution in the 104 and 105 MSCs groups at 12 weeks after ACLT and in the 106 MSCs group at 6, 8, 10, and 12 weeks after ACLT were significantly higher than those of the control group. CONCLUSION: A clinically feasible number of MSCs was found to reduce the articular cartilage degeneration and to decrease pain in the PTOA model. Increasing numbers of the cells further protected the articular cartilage against degeneration.

Morphological changes in synovial mesenchymal stem cells during their adhesion to the meniscus.

Synovial mesenchymal stem cells (MSCs) are an attractive cell source for transplantation because of their high chondrogenic potential, especially in areas like the meniscus of the knee. A synovial MSC suspension placed onto the meniscus for 10 min promoted healing of repaired meniscal tears that generally do not heal. Here, we quantified the proportion of human synovial MSCs that adhered to a porcine abraded meniscus, clarified their morphological changes, and revealed the mechanism by which the synovial MSCs adhered to the meniscus. The numbers of adhering cells at immediately after 10, 60 min and 6, 24 h after suspension placement were calculated. The meniscus surface was examined by scanning electron microscopy, and 50 cells were randomly selected at each time period, classified, and quantified for each of the six donors. Approximately 28% of the synovial MSCs immediately adhered to the meniscus after placement and the proportion of adhered cells increased further with time. All cells maintained a round shape for 60 min, and then transformed to a mixture of round and semi-flattened cells. By 24 h, flattened cells covered the meniscus. Microspikes were observed in 36% of the floating synovial MSCs and in 76% of the cells on the meniscus shortly after placement on the meniscus, then the proportion of cells with pseudopodia increased. The bleb-dominant cell proportion significantly decreased, and the smooth-dominant cell proportion increased within 60 min. Microspikes or the bodies of synovial MSCs were trapped by meniscal fibers immediately after placement. The proportion of adhered cells increased with time, and the cell morphology changed dynamically for 24 h as the synovial MSCs adhered to the meniscus. The MSCs in the round morphological state had a heterogeneous morphology. The microspikes, and the subsequent development of pseudopodia, may play an important role in adhesion onto the meniscus.

Synovial fluid-derived mesenchymal cells have non-inferior chondrogenic potential and can be utilized for regenerative therapy as substitute for synovium-derived cells.

Recent progress in the field of mesenchymal stem cell (MSC) biology has enabled their clinical application. In the autologous cell transplantation therapy, the source of MSCs are quite important to reduce patients' physical burden. In this study, we isolated MSCs from the synovial fluid (SF) and synovial membrane (Syn) of the same patients and compared the biological characteristics of them. In vitro and in vivo experiments indicated the non-inferior chondrocytic differentiation and articular cartilage regeneration potential of SF-MSCs compared to that of Syn-MSCs; however, SF-MSCs showed less proliferative potential than Syn-MSCs in vitro. Flow cytometry-based multiplex surface antigen expression analyses indicated that SF-MSCs exhibit fewer cells positive for CD140, which is a functional growth factor receptor for MSCs. Nevertheless, we obtained enough SF-MSCs for transplantation within several passages. Since arthrocentesis is routinely performed during outpatient care in the consultation room and is less invasive than synovial biopsy, MSC derived from synovial fluid could be considered an attractive cell source for cartilage regenerative therapy as a substitute for Syn-MSC. Developing these cells for clinical application may greatly benefit patients undergoing autologous MSC transplantation therapy.

Biomechanical analysis of the centralization procedure for extruded lateral menisci with posterior root deficiency in a porcine model.

PURPOSE: The purpose of this study was to investigate the biomechanical properties of load distribution following a centralization procedure for extruded lateral menisci with posterior root deficiency in a porcine model. METHODS: Six porcine knee joints were analyzed in a universal tester, as follows: 1) Intact; 2) Extrusion (meniscus extrusion was created by resecting the posterior root of the lateral meniscus, as well as the posterior synovial capsule); and 3) Centralization (two anchors were inserted at the lateral tibial plateau, and the meniscus was sutured to secure it close to the original position). Meniscus extrusion was evaluated using two markers put on the posterior cruciate ligament and the lateral meniscus, and the load distribution were assessed using a pressure mapping sensor system after applying a loading force of 200 N to the knee joint. RESULTS: Distance between two markers (mm, Average; 95% CI) was larger in the extrusion group (21.9; 17.8, 25.6) than in the intact (18.1; 15.1, 22.7) or the centralization (15.3; 12.9, 18.0) groups. The contact area (mm2) in the middle of the meniscus was significantly smaller in the extrusion group (45.8; 18.5, 73.2) than in the intact (85.7; 72.1, 99.2) or the centralization (98.3; 88.8, 107.8) groups. The maximum contact pressure (MPa) in the tibial plateau was significantly higher in the extrusion group (0.37; 0.35, 0.40) than in the intact (0.29; 0.21, 0.37) or the centralization (0.29; 0.22, 0.36) groups. CONCLUSIONS: The centralization procedure enabled a reduction of the meniscus extrusion in the lateral meniscus with posterior root deficiency and restored the maximum load and contact pressure to values close to those of the normal knee joint.

Cryopreservation in 95% serum with 5% DMSO maintains colony formation and chondrogenic abilities in human synovial mesenchymal stem cells.

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. The optimum cryopreservation medium has not been determined, but dimethylsulfoxide (DMSO) should be excluded, if possible, because of its toxicity. The purposes of our study were to examine the possible benefits of higher concentrations of serum and the effectiveness of 100% serum (without DMSO) for the cryopreservation of synovial MSCs. METHODS: Human synovium was harvested from the knees of four donors with osteoarthritis during total knee arthroplasty. Synovial MSCs (8 × 105 cells) were suspended in 400 µL medium and used as a Time 0 control. The same number of synovial MSCs was also suspended in 400 µL α-MEM medium containing 10% fetal bovine serum (FBS) (5% DMSO, and 1% antibiotic), 95% FBS (and 5% DMSO), or 100% FBS (no DMSO) and cryopreserved at - 80 °C for 7 days. After thawing, the cell suspensions (1.5 µL; 3 × 103 cells) were cultured in 60 cm2 dishes for 14 days for colony formation assays. Additional 62.5 µL samples of cell suspensions (1.25 × 105 cells) were added to tubes and cultured for 21 days for chondrogenesis assays. RESULTS: Colony numbers were significantly higher in the Time 0 and 95% FBS groups than in the 10% FBS group (n = 24). Colony numbers were much lower in the 100% FBS group than in the other three groups. The cell numbers per dish reflected the colony numbers. Cartilage pellet weights were significantly heavier in the 95% FBS group than in the 10% FBS group, whereas no difference was observed between the Time 0 and the 95% FBS groups (n = 24). No cartilage pellets formed at all in the 100% FBS group. CONCLUSION: Synovial MSCs cryopreserved in 95% FBS with 5% DMSO maintained their colony formation and chondrogenic abilities to the same levels as observed in the cells before cryopreservation. Synovial MSCs cryopreserved in 100% FBS lost their colony formation and chondrogenic abilities.

Time course analyses of structural changes in the infrapatellar fat pad and synovial membrane during inflammation-induced persistent pain development in rat knee joint.

BACKGROUND: Osteoarthritis (OA) is a common joint disease in aging societies, which is accompanied by chronic inflammation and degeneration of the joint structure. Inflammation of the infrapatellar fat pad (IFP) and synovial membrane (IFP surface) plays essential roles in persistent pain development in patients with OA. To identify the point during the inflammatory process critical for persistent pain development, we performed a time course histological analysis in a rat arthritis model. METHODS: Wistar rats received single intra-articular injection of monoiodoacetic acid (MIA, 0.2 or 1.0 mg/30 µL) in the right knees or phosphate-buffered saline (PBS, 30 µL) as a control in the left knees. Pain avoidance behaviors (weight-bearing asymmetry and tactile hypersensitivity of the plantar surface of the hind paw) were evaluated on days 0, 1, 3, 5, 7, and 14 after injection. Histological assessments of the knee joint were performed on days 0, 1, 3, 5, and 7 after MIA injection. RESULTS: Weight-bearing asymmetry was observed along with the onset of acute inflammation in both the low- (0.2 mg) and high-dose (1.0 mg) groups. In the low-dose group, weight-bearing asymmetry was completely reversed on day 10, indicating that joint pain seemed to alleviate between days 7 and 10. In contrast, we observed persistent joint pain after day 10 in the high-dose group. Histological assessments of the high-dose group indicated that the initial sign of inflammatory responses was observed in the perivascular region inside the IFP. Inflammatory cell infiltration from the perivascular region to the parenchymal region of the IFP was observed on day 3 and reached the IFP surface (synovial membrane) on day 7. Extensive fibrosis throughout the IFP was observed between days 5 and 7 after MIA injection. CONCLUSION: Our data indicated that acute joint pain occurs along with the onset of acute inflammatory process. Irreversible structural changes in the IFP, such as extensive fibrosis, are observed prior to persistent pain development. Thus, we consider that this process may play important roles in persistent pain development.

Persistent synovial inflammation plays important roles in persistent pain development in the rat knee before cartilage degradation reaches the subchondral bone.

BACKGROUND: The major complaint of knee osteoarthritis (OA) is persistent pain. Unlike acute inflammatory pain, persistent pain is usually difficult to manage since its pathology is not fully understood. To elucidate the underlying mechanisms of persistent pain, we established 2 different inflammation-induced arthritis models by injecting monoiodo-acetic acid (MIA) into the joint cavity and performed integrated analyses of the structural changes in the synovial tissue and articular cartilage, sensory neuron rearrangement, and pain avoidance behavior in a rat arthritis model. METHODS: Male Wistar rats received intra-articular injections of MIA (0.2 mg/30 µL, low-dose group; 1 mg/30 µL, high-dose group) in the right knee and phosphate buffered saline (PBS; 30 µL, control group) in the left knee. Fluorogold (FG), a retrograde neural tracer, was used to label the nerve fibers for the identification of sensory neurons that dominate the joints in the dorsal root ganglion (DRG). Both knees were subjected to the intra-articular injection of 2% FG in PBS (5 µL) under anesthesia 5-7 days prior to sacrifice. We performed pain avoidance behavior tests (incapacitance and von Frey tests) at 0, 1, 3, 5, 7, 14, 21, and 28 days. At 5, 14, and 28 days, the rats were sacrificed and the knee joint and DRG were excised for histological assessment. The knee joints were stained with hematoxylin and eosin, safranin O, and calcitonin gene-related peptide (CGRP). The DRG were immunostained with CGRP. RESULTS: A transient inflammatory response followed by mild articular cartilage degeneration was observed in the low-dose MIA model versus persistent inflammation with structural changes in the synovial tissue (fibrosis) in the high-dose model. In the high-dose model, full-thickness cartilage degeneration was observed within 2 weeks post-MIA injection. The pain avoidance behavior tests indicated that persistent synovial inflammation and structural changes of the infrapatellar fat pad may play important roles in persistent knee joint pain before the articular cartilage degeneration reaches the subchondral bone. CONCLUSIONS: Transient inflammation without structural changes of the synovial tissues did not induce persistent pain in the rat knee joint before degradation of the articular cartilage reached the subchondral bone plate.

[Cartilage/chondrocyte research and osteoarthritis. Emerging drugs for Osteoarthritis.]

Osteoarthritis(OA)is a group of diseases and mechanical abnormalities involving degradation of articular cartilage and subchondral bone. The major complaint of OA patients is persistent knee pain, which significantly decreases patients' activities of daily living. Therefore, most of the current treatment strategies for OA are based on symptom management by anti-inflammatory analgesics and improving joint mobility and flexibility by programed exercise and weight control. Recent progress in the field of cartilage metabolism led to develop new drug targets those play roles in structural disease modification in osteoarthritic joints.

Pretreatment with IL-1ß enhances proliferation and chondrogenic potential of synovium-derived mesenchymal stem cells.

BACKGROUND AIMS: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage regeneration because of their high proliferative ability and chondrogenic potential. We have performed clinical trials using synovial MSCs to regenerate articular cartilage. To achieve good clinical outcomes for cell transplantation therapy, it is important to control both quantity (cell number) and quality (pluripotency or chondrogenic potential) of the cells for transplantation. Interleukin (IL)-1ß is a pro-inflammatory cytokine with significant pro-proliferative potential for mesenchymal cells. However, the effects of IL-1ß on synovial MSCs remain unknown. We investigated the effects of pretreatment with IL-1ß on synovial MSCs. METHODS: Human synovial tissue was harvested during total knee arthroplasty. Nucleated cells were plated and cultured in the absence or presence of IL-1ß at 10-13, 10-12, 10-11, 10-10, 10-9 or 10-8 g/mL for 14 days. RESULTS: The number of synovial MSCs increased in a concentration-dependent manner. When cultured for 21 days in chondrogenic medium after pretreatment with 10-8 g/mL IL-1ß, pellet aggregation was observed, whereas pretreatment with 10-12, 10-11 or 10-10 g/mL IL-1ß significantly increased the weight of cartilage pellets (P <0.01). Surface markers for adhesion ability and pluripotency were reduced with high concentrations of IL-1ß. IL-6 and IL-8 expression increased, but no changes in the expression level of growth factors were indicated by cytokine array. CONCLUSIONS: We have demonstrated that pretreatment of IL-1ß increased the proliferation and chondrogenic potential of synovial MSCs, which may promote the regenerative potential of synovial MSCs.

Strenuous running exacerbates knee cartilage erosion induced by low amount of mono-iodoacetate in rats.

BACKGROUND: It is still debated whether strenuous running in the inflammatory phase produces beneficial or harmful effect in rat knees. We examined (1) the dropout rate of rats during a 30-km running protocol, (2) influences of strenuous running and/or low amounts of mono-iodoacetate injection on cartilage, and (3) the effect of strenuous running on synovitis. METHODS: Rats were forced to run 30 km over 6 weeks and the dropout rate was examined. One week after 0.1 mg mono-iodoacetate was injected into the right knee, rats were forced to run either 15 km or not run at all over 3 weeks, after which knee cartilage was evaluated. Synovium at the infrapatellar fat pad was also examined histologically. RESULTS: Even though all 12 rats run up to 15 km, only 6 rats completed 30 km of running. Macroscopically, 0.1 mg mono-iodoacetate induced erosion at the tibial cartilage irrespective of 15 km of running. Histologically, 0.1 mg mono-iodoacetate induced loss of cartilage matrix in the tibial cartilage, and an additional 15 km of strenuous running significantly exacerbated the loss. Synovitis caused by mono-iodoacetate improved after running. CONCLUSIONS: Only 50% of rats completed 30 km of running because of foot problems. Strenuous running further exacerbated tibial cartilage erosion but did not influence synovitis induced by mono-iodoacetate.

Centralization of extruded medial meniscus delays cartilage degeneration in rats.

BACKGROUND: Meniscus extrusion often observed in knee osteoarthritis has a strong correlation with the progression of cartilage degeneration and symptom in the patients. We recently reported a novel procedure "arthroscopic centralization" in which the capsule was sutured to the edge of the tibial plateau to reduce meniscus extrusion in the human knee. However, there is no animal model to study the efficacy of this procedure. The purposes of this study were [1] to establish a model of centralization for the extruded medial meniscus in a rat model; and [2] to investigate the chondroprotective effect of this procedure. METHODS: Medial meniscus extrusion was induced by the release of the anterior synovial capsule and the transection of the meniscotibial ligament. Centralization was performed by the pulled-out suture technique. Alternatively, control rats had only the medial meniscus extrusion surgery. Medial meniscus extrusion was evaluated by micro-CT and macroscopic findings. Cartilage degeneration of the medial tibial plateau was evaluated macroscopically and histologically. RESULTS: By micro-CT analysis, the medial meniscus extrusion was significantly improved in the centralization group in comparison to the extrusion group throughout the study. Both macroscopically and histologically, the cartilage lesion of the medial tibial plateau was prevented in the centralization group but was apparent in the control group. CONCLUSIONS: We developed medial meniscus extrusion in a rat model, and centralization of the extruded medial meniscus by the pull-out suture technique improved the medial meniscus extrusion and delayed cartilage degeneration, though the effect was limited. Centralization is a promising treatment to prevent the progression of osteoarthritis.

Synovial mesenchymal stem cells promote meniscus regeneration augmented by an autologous Achilles tendon graft in a rat partial meniscus defect model.

Although meniscus defects and degeneration are strongly correlated with the later development of osteoarthritis, the promise of regenerative medicine strategies is to prevent and/or delay the disease's progression. Meniscal reconstruction has been shown in animal models with tendon grafting and transplantation of mesenchymal stem cells (MSCs); however, these procedures have not shown the same efficacy in clinical studies. Here, our aim was to investigate the ability of tendon grafts pretreated with exogenous synovial-derived MSCs to prevent cartilage degeneration in a rat partial meniscus defect model. We removed the anterior half of the medial meniscus and grafted autologous Achilles tendons with or without a 10-minute pretreatment of the tendon with synovial MSCs. The meniscus and surrounding cartilage were evaluated at 2, 4, and 8 weeks (n = 5). Tendon grafts increased meniscus size irrespective of synovial MSCs. Histological scores for regenerated menisci were better in the tendon + MSC group than in the other two groups at 4 and 8 weeks. Both macroscopic and histological scores for articular cartilage were significantly better in the tendon + MSC group at 8 weeks. Implanted synovial MSCs survived around the grafted tendon and native meniscus integration site by cell tracking assays with luciferase+, LacZ+, DiI+, and/or GFP+ synovial MSCs and/or GFP+ tendons. Flow cytometric analysis showed that transplanted synovial MSCs retained their MSC properties at 7 days and host synovial tissue also contained cells with MSC characteristics. Synovial MSCs promoted meniscus regeneration augmented by autologous Achilles tendon grafts and prevented cartilage degeneration in rats.

Relationship between MRI T1 rho value and histological findings of intact and radially incised menisci in microminipigs.

PURPOSE: To examine whether the T1 rho value reflects histological changes in menisci we analyzed the relationship between T1 rho value and histological findings in intact and radially incised menisci of pigs. MATERIALS AND METHODS: Seven microminipigs were used for this experiment. A radial incision was created and repaired in the medial meniscus, which was evaluated 4 weeks after surgery. Sagittal T1 rho mapping images were taken by 3.0T magnetic resonance imaging (MRI). The region of interest was set by dividing the meniscus into six zones (from zone 1 to zone 6). For histological evaluation of intact menisci, characteristics of each zone were determined. In incised menisci, a histological score was used to evaluate pathological change. RESULTS: In intact lateral menisci, the zone where histological findings indicated fibrocartilage showed a lower T1 rho value (34.2 ± 2.3 msec) than hyaline-like cartilage (38.2 ± 2.5 msec) or fibrous tissue (37.2 ± 2.0 msec). In incised medial menisci, T1 rho values increased (about 50-90 msec) in the zone where histological findings indicated that synovial ingrowth, scar tissue formation, and degenerative changes had occurred. There were correlations between T1 rho values and histological scores in all zones (r = 0.62-0.92, P = 0.001-0.026). CONCLUSION: Zonal variations of the T1 rho value were observed in intact menisci due to varying structure in each zone. T1 rho values were correlated with histological changes such as collagen fiber organization and safranin-o stainability in incised menisci. This study supports T1 rho mapping as useful for evaluating ultrastructural composition in menisci.

Coordinate and synergistic effects of extensive treadmill exercise and ovariectomy on articular cartilage degeneration.

BACKGROUND: Although osteoarthritis (OA) is a multifactorial disease, little has been reported regarding the cooperative interaction among these factors on cartilage metabolism. Here we examined the synergistic effect of ovariectomy (OVX) and excessive mechanical stress (forced running) on articular cartilage homeostasis in a mouse model resembling a human postmenopausal condition. METHODS: Mice were randomly divided into four groups, I: Sham, II: OVX, III: Sham and forced running (60 km in 6 weeks), and IV: OVX and forced running. Histological and immunohistochemical analyses were performed to evaluate the degeneration of articular cartilage and synovitis in the knee joint. Morphological changes of subchondral bone were analyzed by micro-CT. RESULTS: Micro-CT analyses showed significant loss of metaphyseal trabecular bone volume/tissue volume (BV/TV) after OVX as described previously. Forced running increased the trabecular BV/TV in all mice. In the epiphyseal region, no visible alteration in bone morphology or osteophyte formation was observed in any of the four groups. Histological analysis revealed that OVX or forced running respectively had subtle effects on cartilage degeneration. However, the combination of OVX and forced running synergistically enhanced synovitis and articular cartilage degeneration. Although morphological changes in chondrocytes were observed during OA initiation, no signs of bone marrow edema were observed in any of the four experimental groups. CONCLUSION: We report the coordinate and synergistic effects of extensive treadmill exercise and ovariectomy on articular cartilage degeneration. Since no surgical procedure was performed on the knee joint directly in this model, this model is useful in addressing the molecular pathogenesis of naturally occurring OA.

Weekly injections of Hylan G-F 20 delay cartilage degeneration in partial meniscectomized rat knees.

BACKGROUND: Cross-linked hyaluronan--also called Hylan G-F 20--is a medical device developed to treat osteoarthritis of the knee. However, it is still controversial whether Hylan G-F 20 has a cartilage protective effect in trauma-induced osteoarthritis. We investigated whether Hylan G-F 20 delayed osteoarthritis progression in a partial meniscectomized rat model. METHODS: Lewis rats were used for the experiments. The anterior medial meniscus was resected at the level of the medial collateral ligament in both knees. From 1 week after the surgery, 50 µl of Hylan G-F 20 was injected weekly into the left knee and phosphate buffered saline was injected into the right knee. Cartilage was evaluated for macroscopic findings, histology with safranin-o, and expression of type II collagen at 2, 4, and 8 weeks. Synovitis was also evaluated, and immunohistochemical analysis was performed for ED1. RESULTS: Macroscopic findings demonstrated that India ink positive area, representing fibrillated cartilage, was significantly smaller in the Hylan G-F 20 group than in the control group at 2, 4, and 8 weeks (n = 5). There were no significant differences in osteophyte score between the Hylan G-F 20 group and the control group at 2, 4, and 8 weeks. Histologically, the cartilage in the medial tibial plateau was destroyed at 8 weeks in the control group, while type II collagen expression was still observed at 8 weeks in the Hylan G-F 20 group. OARSI score for cartilage histology was significantly lower in the Hylan G-F 20 group than in the control group at 4 and 8 weeks (n = 5). There were no significant differences in synovial cell number or modified synovitis score between the Hylan G-F 20 group and the control group at 2, 4, and 8 weeks (n = 5). In the Hylan G-F 20 group, foreign bodies surrounded by ED1 positive macrophages were observed in the synovium. CONCLUSION: Weekly injections of Hylan G-F 20 starting 1 week after surgery delayed cartilage degeneration after meniscectomy in a rat model. Synovitis induced by meniscectomy was not alleviated by Hylan G-F 20. Insoluble gels were observed in the synovium after the Hylan G-F 20 injection.

BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing.

Adult bones have a notable regenerative capacity. Over 40 years ago, an intrinsic activity capable of initiating this reparative response was found to reside within bone itself, and the term bone morphogenetic protein (BMP) was coined to describe the molecules responsible for it. A family of BMP proteins was subsequently identified, but no individual BMP has been shown to be the initiator of the endogenous bone repair response. Here we demonstrate that BMP2 is a necessary component of the signaling cascade that governs fracture repair. Mice lacking the ability to produce BMP2 in their limb bones have spontaneous fractures that do not resolve with time. In fact, in bones lacking BMP2, the earliest steps of fracture healing seem to be blocked. Although other osteogenic stimuli are still present in the limb skeleton of BMP2-deficient mice, they cannot compensate for the absence of BMP2. Collectively, our results identify BMP2 as an endogenous mediator necessary for fracture repair.

Transplantation of Achilles tendon treated with bone morphogenetic protein 7 promotes meniscus regeneration in a rat model of massive meniscal defect.

OBJECTIVE: This study was undertaken to examine whether bone morphogenetic protein 7 (BMP-7) induces ectopic cartilage formation in the rat tendon, and whether transplantation of tendon treated with BMP-7 promotes meniscal regeneration. Additionally, we analyzed the relative contributions of host and donor cells on the healing process after tendon transplantation in a rat model. METHODS: BMP-7 was injected in situ into the Achilles tendon of rats, and the histologic findings and gene profile were evaluated. Achilles tendon injected with 1 µg of BMP-7 was transplanted into a meniscal defect in rats. The regenerated meniscus and articular cartilage were evaluated at 4, 8, and 12 weeks. Achilles tendon from LacZ-transgenic rats was transplanted into the meniscal defect in wild-type rats, and vice versa. RESULTS: Injection of BMP-7 into the rat Achilles tendon induced the fibrochondrocyte differentiation of tendon cells and changed the collagen gene profile of tendon tissue to more closely approximate meniscal tissue. Transplantation of the rat Achilles tendon into a meniscal defect increased meniscal size. The rats that received the tendon treated with BMP-7 had a meniscus matrix that exhibited increased Safranin O and type II collagen staining, and showed a delay in articular cartilage degradation. Using LacZ-transgenic rats, we determined that the regeneration of the meniscus resulted from contribution from both donor and host cells. CONCLUSION: Our findings indicate that BMP-7 induces ectopic cartilage formation in rat tendons. Transplantation of Achilles tendon treated with BMP-7 promotes meniscus regeneration and prevents cartilage degeneration in a rat model of massive meniscal defect. Native cells in the rat Achilles tendon contribute to meniscal regeneration.

Mesenchymal stem cells in synovial fluid increase after meniscus injury.

BACKGROUND: Although relatively uncommon, spontaneous healing from a meniscus injury has been observed even within the avascular area. This may be the result of the existence of mesenchymal stem cells in synovial fluid. QUESTIONS/PURPOSES: The purpose of this study was to investigate whether mesenchymal stem cells existed in the synovial fluid of the knee after meniscus injury. METHODS: Synovial fluid was obtained from the knees of 22 patients with meniscus injury just before meniscus surgery and from 8 volunteers who had no history of knee injury. The cellular fraction of the synovial fluid was cultured for 14 days followed by analysis for multilineage potential and presentation of surface antigens characteristic of mesenchymal stem cells. Colony-forming efficiency and proliferation potential were also compared between the two groups. RESULTS: Cells with characteristics of mesenchymal stem cells were observed in the synovial fluid of injured knees to a much greater degree than in uninjured knees. The colony-forming cells derived from the synovial fluid of the knee with meniscus injury had multipotentiality and surface epitopes identical to mesenchymal stem cells. The average number of colony formation, obtained from 1 mL of synovial fluid, in meniscus-injured knees was 250, higher than that from healthy volunteers, which was 0.5 (p < 0.001). Total colony number per synovial fluid volume was positively correlated with the postinjury period (r = 0.77, p < 0.001). CONCLUSIONS: Mesenchymal stem cells were found to exist in synovial fluid from knees after meniscus injury. Mesenchymal stem cells were present in higher numbers in synovial fluid with meniscus injury than in normal knees. Total colony number per synovial fluid volume was positively correlated with the postinjury period. CLINICAL RELEVANCE: Our current human study and previous animal studies suggest the possibility that mesenchymal stem cells in synovial fluid increase after meniscus injury contributing to spontaneous meniscus healing.

A PAI-1 antagonist ameliorates hypophosphatemia in the Hyp vitamin D-resistant rickets model mouse.

Congenital fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets/osteomalacia is a rare bone metabolism disorder characterized by hypophosphatemia and caused by genetic abnormalities that result in excessive secretion of FGF23. Hyp mice are a model of X-linked hypophosphatemia (XLH) caused by deletion of the PHEX gene and excessive production of FGF23. The purpose of this study was to investigate the potential of TM5614 as a therapeutic agent for the treatment of congenital FGF23-related hypophosphatemic rickets and osteomalacia in humans by administering TM5614 to Hyp mice and examining its curative effect on hypophosphatemia. After a single oral administration of TM5614 10 mg·kg-1 to female Hyp mice starting at 17 weeks of age, the serum phosphate concentration increased with a peak at 6 h after administration. ELISA confirmed that TM5614 administration decreased the intact FGF23 concentration in the blood. Expression of 25-hydroxyvitamin D-1α-hydroxylase protein encoded by Cyp27b1 mRNA in the kidney was suppressed in Hyp mice, and treatment with 10 mg·kg-1 of TM5614 normalized the expression of 25-hydroxyvitamin D-1α-hydroxylase protein and Cyp27b1 mRNA in the kidneys of these mice. Our data indicate that oral administration of TM5614 ameliorates hypophosphatemia in Hyp mice, suggesting that TM5614 may be an effective treatment for congenital FGF23-related hypophosphatemic rickets and osteomalacia.