Fibrinogen supports self-renewal of mesenchymal stem cells under serum-reduced condition through autophagy activation.

Mesenchymal stem cells (MSCs) are somatic stem cells used in cell transplantation therapy for tissue injuries and inflammatory diseases because of their ability to support tissue regeneration and to suppress inflammation. While their applications are expanding, needs for automation of culture procedures with reduction of animal-derived materials to meet stable quality and suppliability are also increasing. On the other hand, the development of molecules that safely support cell adherence and expansion on a variety of interfaces under the serum-reduced culture condition remains a challenge. We report here that fibrinogen enables MSC culture on various materials with low cell adhesion property even under serum-reduced culture conditions. Fibrinogen promoted MSC adhesion and proliferation by stabilizing basic fibroblast growth factor (bFGF), which was secreted in the culture medium by autocrine, and also activated autophagy to suppress cellar senescence. Fibrinogen coating allowed MSCs expansion even on the polyether sulfone membrane that represents very low cell adhesion, and the MSCs showed therapeutic effects in a pulmonary fibrosis model. This study demonstrates that fibrinogen is currently the safest and most widely available extracellular matrix and can be used as a versatile scaffold for cell culture in regenerative medicine.

Adipose-derived exosomes block muscular stem cell proliferation in aged mouse by delivering miRNA Let-7d-3p that targets transcription factor HMGA2.

Sarcopenia is an aging-associated attenuation of muscular volume and strength and is the major cause of frailty and falls in elderly individuals. The number of individuals with sarcopenia is rapidly increasing worldwide; however, little is known about the underlying mechanisms of the disease. Sarcopenia often copresents with obesity, and some patients with sarcopenia exhibit accumulation of peri-organ or intra-organ adipose tissue as ectopic fat deposition, including atrophied skeletal muscle. In this study, we showed that transplantation of the perimuscular adipose tissue (PMAT) to the hindlimb thigh muscles of young mice decreased the number of integrin α7/CD29-double positive muscular stem/progenitor cells and that the reaction was mediated by PMAT-derived exosomes. We also found that the inhibition of cell proliferation was induced by Let-7d-3p miRNA that targets HMGA2, which is an important transcription factor for stem cell self-renewal, in muscular stem/progenitor cells and the composite molecular reaction in aged adipocytes. Reduction of Let-7 miRNA repressor Lin28 A/B and activation of nuclear factor-kappa B signaling can lead to the accumulation of Let-7d-3p in the exosomes of aged PMAT. These findings suggest a novel crosstalk between adipose tissue and skeletal muscle in the development of aging-associated muscular atrophy and indicate that adipose tissue-derived miRNAs may play a key role in sarcopenia.

Effectiveness of Rehabilitation for Cancer Patients with Bone Metastasis.

Objectives: Advances in cancer treatment have led to extended survival, and, as a result, the number of patients with bone metastases is increasing. Activities of daily living (ADL) decrease with bone metastasis and the need for rehabilitation is increasing. This study examined the effects of rehabilitation in patients with bone metastases. Methods: We retrospectively reviewed data of cancer patients with bone metastasis who received rehabilitation between 2016 and 2018. Efficacy of rehabilitation was evaluated in 92 patients as the change in the Functional Independence Measure (FIM) score divided by rehabilitation days (FIM change/day) and assessed by different metastatic sites. Results: Overall FIM scores significantly improved after rehabilitation. Moreover, FIM change/day improved in patients with pelvic metastases (n=44) more than in patients with other metastatic sites (n=48) (P=0.015). In FIM motor components, improvements in toilet, tub/shower, walk/wheelchair, and stairs were significantly greater in patients with pelvic metastasis than in those with other metastasis sites. Conclusions: Rehabilitation improved ADL status to a greater extent in patients with pelvic metastases than in those with other metastasis sites. Patients with pelvic metastases may fear fractures, limiting their ADL, but rehabilitation could eliminate this fear and improve FIM.

Induction of chondrogenesis with a RANKL-binding peptide, WP9QY, in vitro and in vivo in a rabbit model.

WP9QY (W9) is a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that inhibits osteoclastogenesis by blunting the RANKL-RANK interaction, and also increases osteoblastogenesis via RANKL reverse signaling. W9 has dual effects on osteoclasts and osteoblasts; however, it is unknown whether the peptide has an effect on chondrocytes. Here, we report that W9 induces proliferation and differentiation of chondrocytes in vitro and repairs full-thickness articular cartilage defects in vivo. W9 stimulated chondrocyte differentiation in a two-dimensional (2D) culture of human mesenchymal stem cells (hMSCs), and transforming growth factor ß3 (TGF-ß3) showed synergistic effects with W9 on chondrogenesis. W9 enlarged the size of 3D pellet cultures of hMSCs and produced chondrocyte-specific matrices, especially in combined treatment with TGF-ß3. The peptide also stimulated proliferation of hMSCs with induction of expression of chondrogenesis-related genes. Several RANKL inhibitors had no effect on chondrocytic differentiation. RANKL-knockdown experiments showed that W9 did not induce chondrogenesis through RANKL, but did induce osteoblastogenesis through RANKL. Intraarticular injection of W9 resulted in significant repair of full-thickness articular cartilage defects in rabbits. Taken together, these results suggest that W9 ameliorates the articular cartilage defects by increasing the volume of cartilaginous matrices with accompanying induction of proliferation and differentiation of chondrocytes via mechanisms independent of RANKL inhibition and RANKL reverse signaling. Since no pharmaceuticals are clinically available for treatment of cartilage damage such as osteoarthritis, our findings demonstrate the potential of W9 to address the unmet medical needs.

Depletion of NIMA-related kinase Nek2 induces aberrant self-renewal and apoptosis in stem/progenitor cells of aged muscular tissues.

Frailty of the locomotory organs has become a widespread problem in the geriatric population. The major factor leading to frailty is an age-associated decrease in muscular mass and a reduced number of muscular cells and myofibers. In aged muscular tissues, muscular satellite cells (MuSCs) are reduced due to abnormalities in their self-renewal and the induction of apoptosis. However, the molecular mechanisms connecting aging-associated physiological changes and the reduction of MuSCs are largely unknown. NIMA-related kinase 2 (Nek2), a member of the Nek family of serine/threonine kinases, was found to be downregulated in aged MuSCs/progenitors. Further, Nek2 downregulation was found to inhibit self-renewal and apoptotic cell death by activating the p53-dependent checkpoint. Attenuated NEK2 expression was also observed in the muscular tissues of elderly donors, and its function was confirmed to be conserved in humans. Overall, this study proposes a novel mechanism for inducing muscular atrophy to understand aging-associated muscular diseases.

Inflammation-associated miR-155 activates differentiation of muscular satellite cells.

Tissue renewal and muscle regeneration largely rely on the proliferation and differentiation of muscle stem cells called muscular satellite cells (MuSCs). MuSCs are normally quiescent, but they are activated in response to various stimuli, such as inflammation. Activated MuSCs proliferate, migrate, differentiate, and fuse to form multinucleate myofibers. Meanwhile, inappropriate cues for MuSC activation induce premature differentiation and bring about stem cell loss. Recent studies revealed that stem cell regulation is disrupted in various aged tissues. We found that the expression of microRNA (miR)-155, which is an inflammation-associated miR, is upregulated in MuSCs of aged muscles, and this upregulation activates the differentiation process through suppression of C/ebpß, which is an important molecule for maintaining MuSC self-renewal. We also found that Notch1 considerably repressed miR-155 expression, and loss of Notch1 induced miR-155 overexpression. Our findings suggest that miR-155 can act as an activator of muscular differentiation and might be responsible for accelerating aging-associated premature differentiation of MuSCs.

Irx3 and Bmp2 regulate mouse mesenchymal cell chondrogenic differentiation in both a Sox9-dependent and -independent manner.

Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation.

Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair: A Preclinical Study.

OBJECTIVE: The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. DESIGN: (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. RESULTS: The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. CONCLUSIONS: These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

Anatomical landmarks of the distal femoral condyles are not always parallel to the tibial bone cut surface in flexion during total knee arthroplasty.

BACKGROUND: Soft tissue balancing is crucial to the success of total knee arthroplasty (TKA). To create a rectangular flexion joint gap, the rotation of the femoral component is important. The purpose of this study is to determine whether or not anatomical landmarks of the distal femoral condyles are parallel to the tibial bone cut surface in flexion. METHODS: Forty-eight patients (three male and 45 female) with a mean age of 74years were examined. During the operation, we estimated the flexion joint gap with the following three techniques. 1) a three degree external cut to the posterior condylar line (MR1), 2) a parallel cut to the surgical transepicondylar axis (MR2), and 3) a parallel cut to the anatomical transepicondylar axis (MR3). RESULTS: The flexion joint gap was 1.1±3.0° (mean±standard deviation (SD)) in internal rotation in the case of MR1, 0.9±3.4° in internal rotation in the case of MR2, and 2.1±3.4° in external rotation in the case of MR3. An outlier (flexion joint gap >3.0°) was observed in 12 cases (25%) in MR1, 13 cases (27%) in MR2, and 15 cases (31%) in MR3. CONCLUSIONS: The anatomical landmarks of the distal femoral condyles are not always parallel to the tibial bone cut surface in flexion. To create a rectangular flexion joint gap, the rotation of the femoral component rotation is based not only on the anatomical landmarks but also on the ligament balance.

Posterior reference guides do not always maintain the size of posterior femoral condyles in TKA.

PURPOSE: Posterior reference guides are provided by many manufacturers. However, the true posterior femoral condyle bone resection thicknesses using posterior reference guides are not stated by the manufacturers. The purpose of this study was to analyse the influence of the posterior reference guide designs on the posterior femoral condyle bone resection thickness. METHODS: Thickness of posterior femoral condyle bone resection and thickness of prostheses were investigated in 8 types of total knee prostheses using the production drawings provided by the manufacturers. RESULTS: Posterior femoral condyle bone resection thickness differed between prostheses. Change in size of the medial posterior condyle ranged from -0.5 to 1.4 mm at 0° external rotation, from -2.9 to 1.4 mm at 3° external rotation, and from -5.3 to 1.4 mm at 6° external rotation. Change in size of lateral posterior condyle ranged from -0.5 to 1.4 mm at 0° external rotation, from -0.4 to 3.4 mm at 3° external rotation, and from -0.4 to 5.3 mm at 6° external rotation. CONCLUSIONS: This study showed that posterior femoral condyle bone resection thickness was different for each posterior reference guide. The rotation centre of posterior reference guides influenced the bone resection thickness in the posterior femoral condyle. The size of the posterior femoral condyles increased in some guides but decreased in other guides. The maximum differences in size changes of the posterior femoral condyles between the guides were 1.9 mm at 0° external rotation, 4.1 mm at 3° external rotation, and 6.3 mm at 6° external rotation. To control the size of posterior femoral condyles, the posterior reference guide design should be checked before use.

Mobile-bearing prosthesis and intraoperative gap balancing are not predictors of superior knee flexion: a prospective randomized study.

PURPOSE: Range of motion is a crucial measure of the outcome of total knee arthroplasty. Gap balancing technique and mobile-bearing prosthesis can improve postoperative range of motion. The purpose of this study was to determine the factors that are predictive of the postoperative range of motion. METHODS: A total of 94 knees with varus osteoarthritis were prospectively randomized to receive either a posterior-stabilized mobile-bearing or a posterior-stabilized fixed-bearing prosthesis. All preoperative and postoperative protocols and operative techniques were identical in the two groups. Extension and flexion joint gaps were measured using a tensor device during the operation. Multiple regression analysis was conducted to determine the best predictors of the knee flexion angle 2 years after the operation. The independent variables were type of prosthesis (mobile-bearing or fixed-bearing), difference between flexion and extension joint gaps (mm), age, gender, body mass index (BMI), preoperative and intraoperative knee flexion angles, change in posterior condylar offset, and posterior tilt of the tibial plateau. RESULTS: The mean difference between flexion and extension joint gaps was 0.8 ± 1.3 (mean ± SD) mm for mobile-bearing and 0.8 ± 1.9 mm for fixed-bearing prosthesis. The mean flexion angle for mobile-bearing and fixed-bearing groups was 120 ± 16° and 116 ± 20° preoperatively (n.s.), 142 ± 9° and 141 ± 12° intraoperatively (n.s.), and 129 ± 10° and 128 ± 13° at 2 years postoperatively (p = 0.773), respectively. Predictors were identified in the following three categories: (1) preoperative flexion angle, (2) intraoperative radiographic flexion angle, and (3) BMI (R = 0.603, p < 0.001). CONCLUSIONS: Mobile-bearing prosthesis and optimal gap balancing did not result in superior postoperative flexion angle. Better preoperative and intraoperative flexion angles and lower BMI were the significant predictors for better postoperative flexion angle. LEVEL OF EVIDENCE: Therapeutic study, Level I.

Systemic Administration of Granulocyte Colony-Stimulating Factor for Osteochondral Defect Repair in a Rat Experimental Model.

OBJECTIVE: The objective of this study was to assess the effect of granulocyte colony-stimulating factor (G-CSF) on osteochondral defect repair in the rat knee. DESIGN: Twenty-six 12-week-old male Lewis rats were randomly divided into 2 groups. From day 0 to day 4, the G-CSF group received glycosylated G-CSF, and the control group received phosphate-buffered saline. A 1.5-mm diameter and 1.0-mm deep osteochondral defect was introduced in the patellar groove of the bilateral femur in all rats on day 4. The peripheral blood nucleated cells were counted for 14 days from the first day of injection, the appearance of the cartilage repair was observed histologically and macroscopically for 2, 4, 8, 12, and 24 weeks after surgery. RESULTS: The number of peripheral blood leukocytes increased 3 days and returned to normal levels 7 days after the first injection. Compared with the control group, the G-CSF group had more fibrous and/or bony tissue at earlier points in time. The tissue repair rate, which is defined as the percentage of repaired osteochondral defects, was significantly higher in the G-CSF group 4 weeks after surgery. However, there were no significant differences in the cartilage repair rate and the modified Wakitani score between the 2 groups at each time point. CONCLUSIONS: The defect filling was significantly better in the G-CSF group in the early phases. Our findings suggest that G-CSF may promote the repair of osteochondral defects by mediating an increase in the number of peripheral blood nucleated cells.

Cartilage Repair With Autologous Bone Marrow Mesenchymal Stem Cell Transplantation: Review of Preclinical and Clinical Studies.

Clinical trials of various procedures, including bone marrow stimulation, mosaicplasty, and autologous chondrocyte implantation, have been explored to treat articular cartilage defects. However, all of them have some demerits. We focused on autologous culture-expanded bone marrow mesenchymal stem cells (BMSC), which can proliferate without losing their capacity for differentiation. First, we transplanted BMSC into the defective articular cartilage of rabbit and succeeded in regenerating osteochondral tissue. We then applied this transplantation in humans. Our previous reports showed that treatment with BMSC relieves the clinical symptoms of chondral defects in the knee and elbow joint. We investigated the efficacy of BMSC for osteoarthritic knee treated with high tibial osteotomy, by comparing 12 BMSC-transplanted patients with 12 cell-free patients. At 16-month follow-up, although the difference in clinical improvement between both groups was not significant, the arthroscopic and histological grading score was better in the cell-transplanted group. At the over 10-year follow-up, Hospital for Special Surgery knee scores improved to 76 and 73 in the BMSC-transplanted and cell-free groups, respectively, which were better than preoperative scores. Additionally, neither tumors nor infections were observed in all patients, and in the clinical study, we have never observed hypertrophy of repaired tissue, thereby guaranteeing the clinical safety of this therapy. Although we have never observed calcification above the tidemark in rabbit model and human histologically, the repair cartilage was not completely hyaline cartilage. To elucidate the optimum conditions for cell therapy, other stem cells, culture conditions, growth factors, and gene transfection methods should be explored.

[Cartilage repair and regenerative medicine ; past, present, and future].

Since the days of ancient Greece, it has been known that articular cartilage has poor potential to repair. In April, 2013, autologous chondrocyte implantation (ACI) was approved by the Japanese ministry of Health, Labour and Welfare, as the first orthopedic cell therapy in Japan. Half a century has passed since Chesterman's report on rabbit allogeneic chondrocyte implantation, and approximately 20 years since the first human ACI report by Brittberg. In Japan, 24 years has passed since Wakitani's allogeneic chondrocyte implantation, and over 10 years since the human ACI report by Ochi. A long-term follow-up clinical trial with the high statistical power is needed to verify the safety and efficacy of new cartilage joint therapy. Moreover, we need the infrastructure, including drug and/or device regulation and timely approved by the government, to apply new therapies in similar time frames to other developed nations.