Development of a skeletal muscle sheet with direct reprogramming-induced myoblasts on a nanogel-cross-linked porous freeze-dried gel scaffold in a mouse gastroschisis model.

PURPOSE: In this study, we attempted to create skeletal muscle sheets made of directly converted myoblasts (dMBs) with a nanogel scaffold on a biosheet using a mouse gastroschisis model. METHODS: dMBs were prepared by the co-transfection of MYOD1 and MYCL into human fibroblasts. Silicon tubes were implanted under the skin of NOG/SCID mice, and biosheets were formed. The nanogel was a nanoscale hydrogel based on cholesterol-modified pullulan, and a NanoClip-FD gel was prepared by freeze-drying the nanogel. 7 mm in length was created in the abdominal wall of NOG/SCID mice as a mouse gastroschisis model. Matrigel or NanoCliP-FD gel seeded with dMBs was placed on the biosheet and implanted on the model mice. RESULTS: Fourteen days after surgery, dMBs with Matrigel showed a small amount of coarse aggregations of muscle-like cells. In contrast, dMBs with NanoCliP-FD gel showed multinucleated muscle-like cells, which were expressed as desmin and myogenin by fluorescent immunostaining. CONCLUSION: Nanogels have a porous structure and are useful as scaffolds for tissue regeneration by supplying oxygen and nutrients supply to the cells. Combining dMBs and nanogels on the biosheets resulted in the differentiation and engraftment of skeletal muscle, suggesting the possibility of developing skeletal muscle sheets derived from autologous cells and tissues.

Enhancing Cartilage Metabolism in Rats through a Novel Thermal Stimulation Technique with Photosensitizers.

Although the moderate thermal stimulation of articular cartilage exerts chondroprotective effects, it is difficult to effectively heat deep articular cartilage with conventional methods. Photosensitizers increase the ambient temperature using near-infrared (NIR) radiation, which has high tissue permeability. We hypothesized that the intra-articular administration of photosensitizers and NIR irradiation would exert a greater heating effect on articular cartilage. We aimed to evaluate the heating effect of this method on cultured chondrocytes and rat knee cartilage. In vitro, we irradiated a photosensitizer-containing medium with NIR and measured changes in the medium temperature, cytotoxicity, and gene expression of heat shock protein (HSP) 70 and aggrecan (ACAN). In vivo, the knee joints of rats treated with photosensitizers were irradiated with NIR, and changes in intra-articular temperature and gene expression were measured, alongside histological analysis. The results showed that the medium and intra-articular temperature were raised to approximately 40 °C with no apparent disruption to articular cartilage or the immunohistochemically enhanced staining of HSP70 in chondrocytes. The gene expression of HSP70 and ACAN was increased in both cultured and articular cartilage. In summary, this method can safely heat joints and enhance cartilage metabolism by inducing HSP70 expression in articular cartilage. It presents a new hyperthermia therapy with effective cartilage protection.

TGF-ß inhibitor treatment of H2O2-induced cystitis models provides biochemical mechanism for elucidating interstitial cystitis/painful bladder syndrome patients.

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic disease for which no effective treatment is available. Transforming growth factor-ß (TGF-ß) is thought to be involved in the pathogenesis of IC/PBS, and previous studies have suggested that administrations of a TGF-ß inhibitor significantly ameliorated IC/PBS in a mouse model. However, the molecular mechanisms underlying the therapeutic effect of a TGF-b inhibitor on IC/PBS has not been comprehensively analyzed. TGF-ß has a variety of actions, such as regulation of immune cells and fibrosis. In our study, we induced IC/PBS-like disease in mice by an intravesical administration of hydrogen peroxide (H2O2) and examined the effects of three TGF-ß inhibitors, Repsox, SB431542, and SB505124, on the urinary functions as well as histological and gene expression profiles in the bladder. TGF-ß inhibitor treatment improved urinary function and histological changes in the IC/PBS mouse model, and SB431542 was most effective among the TGF-ß inhibitors. In our present study, TGF-ß inhibitor treatment improved abnormal enhancement of nociceptive mechanisms, immunity and inflammation, fibrosis, and dysfunction of bladder urothelium. These results show that multiple mechanisms are involved in the improvement of urinary function by TGF-ß inhibitor.

Development of anti-GD2 Antibody-producing Mesenchymal Stem Cells as Cellular Immunotherapy.

BACKGROUND/AIM: Using the tyrosine hydroxylase (TH)-MYCN mouse neuroblastoma (NB) model, we have previously reported the accumulation of mouse mesenchymal stem cells (mMSCs) on tumors in vivo and the antitumor effect of mMSCs transfected with a small molecule (IFN-ß) expression gene. In this study, we have developed novel MSCs secreting anti-disialoganglioside GD2 antibody (anti-GD2-MSCs) and evaluated their antitumor effects in vitro. MATERIALS AND METHODS: We generated an anti-GD2 antibody construct (14.G2a-Fcx2-GFP) incorporating FLAG-tagged single-chain fragment variable against GD2 fused to a linker sequence, a fragment of the constant portion of human IgG1, and GFP protein. The construct was lentivirally transduced into mMSCs and the transduction efficiency was assessed by GFP expression. The secretion of FLAG-tagged anti-GD2 antibody was detected by Western blotting using anti-FLAG antibody. Antibody binding capacity was confirmed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using human NB cells and human natural killer (NK) cells to assess whether the antitumor activity was enhanced in the presence of the produced antibodies. RESULTS: The transduction efficiency of anti-GD2-MSCs was more than 90%. anti-GD2-MSCs secreted antibodies extracellularly and these antibodies had high affinity to GD2-expressing human NB cells. ADCC assays showed that the addition of antibodies secreted from anti-GD2-MSCs significantly increased the cytotoxic activity of NK cells against NB cells. CONCLUSION: Newly developed anti-GD2-MSCs produced functional antibodies that have affinity to the GD2 antigen on NB cells and can induce ADCC-mediated cytotoxicity. Anti-GD2-MSCs based cellular immunotherapy has the potential to be a novel therapeutic option for intractable NB.

Prophylactic Application of Human Adipose Tissue-Derived Products to Prevent Radiation Disorders.

BACKGROUND: Radiation therapy is a mainstay treatment for malignancies, but it can induce deterministic adverse effects in surrounding healthy tissues, including atrophy, fibrosis, ischemia, and impaired wound healing. This exploratory study investigated whether prophylactic administration of products containing adipose tissue-derived stem cells immediately after radiotherapy could prevent the development of long-term functional disorders in irradiated tissues. METHODS: A total irradiation dose of 40 Gy (10 Gy, four times weekly) was delivered to the dorsal skin of nude mice. Subsequently, a prophylactic treatment with vehicle, fat tissue, stromal vascular fraction, or micronized cellular adipose matrix was injected subcutaneously into the irradiated area. Six months after these prophylactic treatments, a cutaneous punch wound was created to evaluate histologic changes and wound healing. RESULTS: Histologic assessments demonstrated dermal thickening, atrophy, and increased collagen deposits in the subcutaneous fatty layer 6 months after radiotherapy. In addition, wound healing was significantly delayed. The prophylactic treatments with three different types of human adipose tissue-derived products significantly prevented radiation-induced histologic changes and accelerated wound healing compared with the vehicle-treated irradiated group. CONCLUSIONS: This is the first study to demonstrate potential for prophylactic treatments after radiotherapy, which could prevent the progression of chronic radiation therapy disorders. The results could have a substantial impact on current anticancer radiotherapies; a next-generation radiation therapy may need to be always combined with a stem cell therapy. Such prophylactic treatments have the potential to improve wound healing of irradiated tissue and clinical outcomes of reconstructive surgery required after cancer radiotherapy. CLINICAL RELEVANCE STATEMENT: The results of this study suggest that it is clinically possible to prevent the development of radiation disorders using stem cell therapies. This study may provide a new concept of prophylactic treatment, which would be a paradigm shift in radiotherapy.

Micronized Cellular Adipose Matrix Promotes the Therapeutic Effect of an Artificial Nerve Conduit in Peripheral Nerve Gap Injury.

BACKGROUND: Stromal vascular fraction (SVF) isolated from adipose tissue has been shown to be beneficial for treating peripheral nerve injuries. Micronized cellular adipose matrix (MCAM) is an SVF-rich micronized fat tissue obtained by a series of simple mechanical processes. This study assessed the therapeutic effect of MCAM for peripheral nerve injury. METHODS: Microscopic evaluation of the cell phenotype and functions was performed to determine the adipose-derived stem cell content of the MCAM. An artificial nerve conduit (ANC) filled with MCAM was implanted into a sciatic nerve defect in immunodeficient mice. Comparisons of this treatment with an autograft, an ANC filled with SVF cells, and an ANC alone were made based on electrophysiologic characteristics, Sciatic Functional Index, and histologic analyses of regenerated nerve fiber and myelination using electron microscopy, and the preventive effect on innervated muscle atrophy. RESULTS: MCAM contained many cells with a phenotype and differentiation potency similar to those of ADSCs. The implantation experiment indicated that MCAM enhanced the efficiency of functional and structural recovery and prevented atrophy of the innervated muscle. These effects were significantly improved compared with the control group (ANC only) and comparable to those in the SVF group, whereas the improvement did not reach the same level of the autograft group. CONCLUSION: Injection of MCAM into an ANC accelerated nerve regeneration compared with use of an ANC alone, which indicates that MCAM is a promising transplant material for treatment of peripheral nerve injury and an alternative to use of SVF cells. CLINICAL RELEVANCE STATEMENT: Micronized cellular adipose matrix, which can be harvested and isolated from adipose tissue with a simple device, has been shown for the first time to be highly useful as an implantable material for new peripheral nerve regeneration.

Development of minimally invasive cancer immunotherapy using anti-disialoganglioside GD2 antibody-producing mesenchymal stem cells for a neuroblastoma mouse model.

PURPOSE: Mouse IgG anti-disialoganglioside GD2 antibody-secreting mouse mesenchymal stem cells (anti-GD2-MSCs) were developed, and their anti-tumor effects were validated in an in vivo neuroblastoma mouse model. METHODS: Anti-GD2 antibody constructs were generated, incorporating FLAG-tagged single-chain fragment variables against GD2 fused to a linker sequence, and a fragment of a stationary portion was changed from human IgG to mouse IgG and GFP protein. The construct was lentivirally introduced into mouse MSCs. A syngeneic mouse model was established through the subcutaneous transplantation of a tumor tissue fragment from a TH-MYCN transgenic mouse, and the homing effects of anti-GD2-MSCs were validated by In vivo imaging system imaging. The syngeneic model was divided into three groups according to topical injection materials: anti-GD2-MSCs with IL-2, IL-2, and PBS. The tumors were removed, and natural killer (NK) cells were counted. RESULTS: Anti-GD2-MSCs showed homing effects in syngeneic models. The growth rate of subcutaneous tumors was significantly suppressed by anti-GD2-MSCs with IL-2 (p < 0.05). Subcutaneous tumor immunostaining showed an increased NK cell infiltration in the same group (p < 0.01). CONCLUSION: Anti-GD2-MSCs using mouse IgG showed a homing effect and significant tumor growth suppression in syngeneic models. Anti-GD2-MSC-based cellular immunotherapy could be a novel therapeutic strategy for intractable neuroblastoma.

Hypoxia promotes differentiation of pure cartilage from human induced pluripotent stem cells.

While cartilage can be produced from induced pluripotent stem cells (iPSCs), challenges such as long culture periods and compromised tissue purity continue to prevail. The present study aimed to determine whether cartilaginous tissue could be produced from iPSCs under hypoxia and, if so, to evaluate its effects on cellular metabolism and purity of the produced tissue. Human iPSCs (hiPSCs) were cultured for cartilage differentiation in monolayers under normoxia or hypoxia (5% O2), and chondrocyte differentiation was evaluated using reverse transcription­quantitative PCR and fluorescence­activated cell sorting. Subsequently, cartilage differentiation of hiPSCs was conducted in 3D culture under normoxia or hypoxia (5% O2), and the formed cartilage­like tissues were evaluated on days 28 and 56 using histological analyses. Hypoxia suppressed the expression levels of the immature mesodermal markers brachyury (T) and forkhead box protein F1; however, it promoted the expression of the chondrogenic markers Acan and CD44. The number of sex­determining region Y­box 9­positive cells and the percentages of safranin O­positive and type 2 collagen­positive tissues increased under hypoxic conditions. Moreover, upon hypoxia­inducible factor (HIF)­1α staining, nuclei of tissues cultured under hypoxia stained more deeply compared with those of tissues cultured under normoxia. Overall, these findings indicated that hypoxia not only enhanced cartilage matrix production, but also improved tissue purity by promoting the expression of HIF­1α gene. Potentially, pure cartilage­like tissues could be produced rapidly and conveniently using this method.

ALK5 i II Accelerates Induction of Adipose-Derived Stem Cells toward Schwann Cells through a Non-Smad Signaling Pathway.

Schwann cells (SCs) are likely to be a vital component of cell-based therapies for nerve regeneration. There are various methods for inducing SC-like cells (SCLCs) from adipose-derived stem cells (ADSCs), but their phenotypic and functional characteristics remain unsatisfactory. Here, we report a novel efficient procedure to induce SCLCs by culturing ADSCs with ALK5 inhibitor (ALK5 i) II, a specific inhibitor of activin-like kinase 5 (ALK5) (transforming growth factor-ß receptor 1 (TGFßR1)) that is also known as Repsox. The resultant cells that we named "modified SCLCs (mSCLCs)" expressed SC-specific genes more strongly than conventional SCLCs (cSCLCs) and displayed a neurosupportive capacity in vitro, similarly to genuine SCs. Regarding the mechanism of the mSCLC induction by ALK5 i II, knockdown of Smad2 and Smad3, key proteins in the TGFß/Smad signaling pathway, did not induce SC markers. Meanwhile, expression of multipotent stem cell markers such as Sex-determining region Y- (SRY-) box 2 (Sox2) was upregulated during induction. These findings imply that ALK5 i II exerts its effect via the non-Smad pathway and following upregulation of undifferentiated cell-related genes such as Sox2. The procedure described here results in highly efficient induction of ADSCs into transgene-free and highly functional SCLCs. This approach might be applicable to regeneration therapy for peripheral nerve injury.

Treadmill running prevents atrophy differently in fast- versus slow-twitch muscles in a rat model of rheumatoid arthritis.

To investigate the effects of treadmill running on two different types of skeletal muscle, we established a rat model of collagen-induced arthritis (CIA). The skeletal muscles studied were the extensor digitorum longus (EDL), which is rich in fast-twitch muscle fibers, and the soleus, which is rich in slow-twitch muscle fibers. The histological and transcriptional changes in these muscles at 14 and 44 days after immunosensitization were compared between rats that were forced to exercise (CIA ex group) and free-reared CIA rats (CIA no group). Change in protein expression was examined on day 14 after a single bout of treadmill running. Treadmill running had different effects on the relative muscle weight and total and fiber cross-sectional areas in each muscle type. In the soleus, it prevented muscle atrophy. Transcriptional analysis revealed increased eukaryotic translation initiation factor 4E (Eif4e) expression on day 14 and increased Atrogin-1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression on day 44 in the soleus in the CIA ex group, suggesting an interaction between muscle type and exercise. A single bout of treadmill running increased the level of Eif4e and p70S6K and decreased that of Atrogin-1 in the soleus on day 14. Treadmill running prevented muscle atrophy in the soleus in a rat model of rheumatoid arthritis via activation of mitochondrial function, as evidenced by increased PGC-1α expression.

Osteogenic Response to Polysaccharide Nanogel Sheets of Human Fibroblasts After Conversion Into Functional Osteoblasts by Direct Phenotypic Cell Reprogramming.

Human dermal fibroblasts (HDFs) were converted into osteoblasts using a ALK inhibitor II (inhibitor of transforming growth factor-ß signal) on freeze-dried nanogel-cross-linked porous (FD-NanoClip) polysaccharide sheets or fibers. Then, the ability of these directly converted osteoblasts (dOBs) to produce calcified substrates and the expression of osteoblast genes were analyzed in comparison with osteoblasts converted by exactly the same procedure but seeded onto a conventional atelocollagen scaffold. dOBs exposed to FD-NanoClip in both sheet and fiber morphologies produced a significantly higher concentration of calcium deposits as compared to a control cell sample (i.e., unconverted fibroblasts), while there was no statistically significant difference in calcification level between dOBs exposed to atelocollagen sheets and the control group. The observed differences in osteogenic behaviors were interpreted according to Raman spectroscopic analyses comparing different polysaccharide scaffolds and Fourier transform infrared spectroscopy analyses of dOB cultures. This study substantiates a possible new path to repair large bone defects through a simplified transplantation procedure using FD-NanoClip sheets with better osteogenic outputs as compared to the existing atelocollagen scaffolding material.

Extracellular acidity in tumor tissue upregulates programmed cell death protein 1 expression on tumor cells via proton-sensing G protein-coupled receptors.

Acidity in the tumor microenvironment has been reported to promote cancer growth and metastasis. In our study, we examined a potential relation between extracellular acidity and expression level of the immune checkpoint molecule programmed cell death protein 1 (PD-L1) in murine squamous cell carcinoma (SCC) and melanoma cell lines. PD-L1 expression in the tumor cells was upregulated by culturing in a low pH culture medium. Tumor-bearing mice were allowed to ingest sodium bicarbonate, resulting in neutralization of acidity in the tumor tissue, a decrease in PD-L1 expression in tumor cells and suppression of tumor growth in vivo. Proton-sensing G protein-coupled receptors, T-cell death-associated gene 8 (TDAG8) and ovarian cancer G-protein-coupled receptor 1 (OGR1), were upregulated by low pH, and essentially involved in the acidity-induced elevation of PD-L1 expression in the tumor cells. Human head and neck SCC RNAseq data from the Cancer Genome Atlas also suggested a statistically significant correlation between expression levels of the proton sensors and PD-L1 mRNA expression. These findings strongly suggest that neutralization of acidity in tumor tissue may result in reduction of PD-L1 expression, potentially leading to inhibition of an immune checkpoint and augmentation of antitumor immunity.

Mechanical stimulation of chondrocytes regulates HIF-1α under hypoxic conditions.

We investigated the effects of hypoxia-inducible factor (HIF)-1α on articular cartilage under mechanical stimulation and the associated mechanisms. Chondrocytes, isolated from articular cartilage from the knee, hip, and shoulder joints of Wistar rats, were subjected to 20 % tensile stress under hypoxic (5% O2) conditions for 24 h. HIF-1α and aggrecan expression was significantly enhanced with mechanical stimulation under hypoxia but not significantly altered with mechanical stimulation under normoxia. The nuclear translocation of HIF-1α was enhanced by mechanical stress under hypoxia. Under both normoxia and hypoxia, a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS) 5 expression was significantly reduced with mechanical stimulation compared to that in the group without mechanical stimulation. However, HIF-1α knockdown mitigated changes in aggrecan and ADAM-TS5 expression mediated by mechanical stimulation under hypoxia. The effects of treadmill running on HIF-1α production in the articular cartilage of rat knee joints were also analyzed. HIF-1α production increased in the moderate running group and decreased to the same levels as those in the control group in the excessive running group. This suggests that HIF-1α regulates aggrecan and ADAM-TS5 expression in response to mechanical stimulation under hypoxia and general mechanical stimulation in articular cartilage under hypoxia, while controlling cartilage homeostasis.

Sustained Hypoxia Suppresses Joint Destruction in a Rat Model of Rheumatoid Arthritis via Negative Feedback of Hypoxia Inducible Factor-1α.

Hypoxia inducible factor (HIF)-1α has been implicated in the pathogenesis of rheumatoid arthritis (RA). HIF-1α, which is expressed in hypoxia, is reversely suppressed in sustained hypoxia. Here, we investigated the inhibitory effect of hypoxia on arthritis by controlling HIF-1α. Rheumatoid fibroblast-like synoviocyte MH7A cells were cultured in a hypoxic incubator for up to 72 h to evaluate the expression of HIF-1. Furthermore, collagen-induced arthritis (CIA) model rats were maintained under 12% hypoxia in a hypoxic chamber for 28 days to evaluate the effect on arthritis. In MH7A cells, HIF-1α protein level increased at 3 h, peaked at 6 h, and subsequently decreased in a time-dependent manner. The transcription of pro-inflammatory cytokines increased at 1 h; however, they decreased after 3 h (p < 0.05). Deferoxamine-mediated activation of HIF-1α abolished the inhibitory effect of sustained hypoxia on pro-inflammatory cytokines. In the rat CIA model, the onset of joint swelling was delayed and arthritis was suppressed in the hypoxia group compared with the normoxia group (p < 0.05). Histologically, joint destruction was suppressed primarily in the cartilage. Thus, sustained hypoxia may represent a new safe, and potent therapeutic approach for high-risk patients with RA by suppressing HIF-1α expression.

Direct Conversion of Human Fibroblasts into Adipocytes Using a Novel Small Molecular Compound: Implications for Regenerative Therapy for Adipose Tissue Defects.

There is a need in plastic surgery to prepare autologous adipocytes that can be transplanted in patients to reconstruct soft tissue defects caused by tumor resection, including breast cancer, and by trauma and other diseases. Direct conversion of somatic cells into adipocytes may allow sufficient functional adipocytes to be obtained for use in regeneration therapy. Chemical libraries of 10,800 molecules were screened for the ability to induce lipid accumulation in human dermal fibroblasts (HDFs) in culture. Chemical compound-mediated directly converted adipocytes (CCCAs) were characterized by lipid staining, immunostaining, and qRT-PCR, and were also tested for adipokine secretion and glucose uptake. CCCAs were also implanted into mice to examine their distribution in vivo. STK287794 was identified as a small molecule that induced the accumulation of lipid droplets in HDFs. CCCAs expressed adipocyte-related genes, secreted adiponectin and leptin, and abundantly incorporated glucose. After implantation in mice, CCCAs resided in granulation tissue and remained adipose-like. HDFs were successfully converted into adipocytes by adding a single chemical compound, STK287794. C/EBPα and PPARγ were upregulated in STK287794-treated cells, which strongly suggests involvement of these adipocyte-related transcription factors in the chemical direct conversion. Our method may be useful for the preparation of autogenous adipocytes for transplantation therapy for soft tissue defects and fat tissue atrophy.

Fabrication of contractile skeletal muscle tissues using directly converted myoblasts from human fibroblasts.

Transplantation of stem cell-derived myoblasts is a promising approach for the treatment of skeletal muscle function loss. Myoblasts directly converted from somatic cells that bypass any stem cell intermediary stages can avoid the problem of tumor formation after transplantation. Previously, we reported that co-transduction with the myogenic differentiation 1 (MYOD1) gene and the v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene efficiently converted human fibroblasts into myoblasts. Although the directly converted myoblasts efficiently fused into multinucleated myotubes in vitro and in vivo, it is not clear whether they have the contractile ability, which is the most significant phenotype of the muscle. In the present study, we aimed to examine the in vitro contractile ability of the myotubes differentiated from the directly converted myoblasts by the overexpression of MYOD1 and MYCL. We fabricated three-dimensional (3D) tissues on a microdevice for force measurement. The 3D culture enhanced the differentiation of the myoblasts into myotubes, which were confirmed by gene expression analysis of skeletal muscle-related genes. The tissues started to generate contractile force in response to electrical stimulation after 4 days of culture, which reached approximately 12 µN after 10 days. The addition of IGF-I decreased the contractile force of the 3D tissues, while the use of cryopreserved cells increased it. We confirmed that the tissues fabricated from the cells derived from three different donors generated forces of similar magnitude. Thus, directly converted myoblasts by the overexpression of MYOD1 and MYCL could be a promising cell source for cell therapy.

Involvement of PDGF-BB and IGF-1 in Activation of Human Schwann Cells by Platelet-Rich Plasma.

BACKGROUND: Platelet-rich plasma contains high concentrations of growth factors that stimulate proliferation and migration of various cell types. Earlier experiments demonstrated that local platelet-rich plasma administration activates Schwann cells to improve axonal regeneration at a transected peripheral nerve lesion. However, the optimal concentration of human platelet-rich plasma for activation of human Schwann cells has not been determined, and mechanisms by which platelet-rich plasma activates Schwann cells remain to be clarified. METHODS: Human Schwann cells were cultured with various concentrations of platelet-rich plasma in 5% fetal bovine serum/Dulbecco's Modified Eagle Medium. Cell viability, microchemotaxis, flow cytometry, and quantitative real-time polymerase chain reaction assays were performed to assess proliferation, migration, cell cycle, and neurotrophic factor expression of the human Schwann cells, respectively. Human Schwann cells were co-cultured with neuronal cells to assess their capacity to induce neurite extension. Neutralizing antibodies for platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor-1 (IGF-1) were added to the culture to estimate contribution of these cytokines to human Schwann cell stimulation by platelet-rich plasma. RESULTS: An addition of platelet-rich plasma at 5% strongly elevated proliferation, migration, and neurotrophic factor production of human Schwann cells. Both PDGF-BB and IGF-1 may be involved in mitogenic effect of platelet-rich plasma on human Schwann cells, and PDGF-BB may also play an important role in the migration-inducing effect of platelet-rich plasma. Neutralization of both PDGF-BB and IGF-1 cancelled the promoting effect of platelet-rich plasma on neurite-inducing activity of human Schwann cells. CONCLUSION: This study may suggest the optimal concentration of platelet-rich plasma for human Schwann cell stimulation and potential mechanisms underlying the activation of human Schwann cells by platelet-rich plasma, which may be quite useful for platelet-rich plasma therapy for peripheral nerve regeneration. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Treadmill Running in Established Phase Arthritis Inhibits Joint Destruction in Rat Rheumatoid Arthritis Models.

Exercise therapy inhibits joint destruction by suppressing pro-inflammatory cytokines. The efficacy of pharmacotherapy for rheumatoid arthritis differs depending on the phase of the disease, but that of exercise therapy for each phase is unknown. We assessed the differences in the efficacy of treadmill running on rheumatoid arthritis at various phases, using rat rheumatoid arthritis models. Rats with collagen-induced arthritis were used as rheumatoid arthritis models, and the phase after immunization was divided as pre-arthritis and established phases. Histologically, the groups with forced treadmill running in the established phase had significantly inhibited joint destruction compared with the other groups. The group with forced treadmill running in only the established phase had significantly better bone morphometry and reduced expression of connexin 43 and tumor necrosis factor α in the synovial membranes compared with the no treadmill group. Furthermore, few cells were positive for cathepsin K immunostaining in the groups with forced treadmill running in the established phase. Our results suggest that the efficacy of exercise therapy may differ depending on rheumatoid arthritis disease activity. Active exercise during phases of decreased disease activity may effectively inhibit arthritis and joint destruction.

Novel mesenchymal stem cell delivery system as targeted therapy against neuroblastoma using the TH-MYCN mouse model.

PURPOSE: Mesenchymal stem cells (MSCs) are reported to migrate toward damaged tissues or tumors. We previously reported the in vivo short-term (1 day) tumor-homing effect of xenogeneic human MSCs (hMSCs) using the TH-MYCN mouse neuroblastoma model (MYCN-TgM). In this study, we analyzed the long-term tumor-homing effect of allogeneic mouse MSCs (mMSCs) and explored the antitumor effect and drug delivery function of mMSCs. METHODS: mMSCs were administered intraperitoneally (i.p.) to MYCN-TgM and traced by an in vivo imaging system (IVIS). We administered green fluorescent protein (GFP)-transduced mMSCs into MYCN-TgM i.p. and examined the cell survival by immunohistochemistry. We also administered interferon beta-transduced mMSCs (mMSCs-IFN-ß) to MYCN-TgM i.p. and measured the concentration of IFN-ß in the tumor and organs by an enzyme-linked immunosorbent assay (ELISA). The survival curves of MYCN-TgM administered every week was analyzed. RESULTS: The IVIS revealed the accumulation of fluorescence was observed in the tumor both in vivo and after excision. Immunohistochemistry using anti-GFP antibody revealed that the mMSCs existed within the tumor until 14 days but not in the organs. The ELISA showed increased concentrations of IFN-ß only in the tumors, with the values gradually diminishing over 14 days. The mMSCs-IFN-ß group survived significantly longer than the control group (p < 0.03), while the mMSCs-alone group did not show a survival advantage. CONCLUSIONS: Allogeneic mMSCs showed a homing ability for mouse neuroblastoma and existed within the tumor for as long as two weeks. This may be a candidate drug delivery vehicle for antitumor agents against neuroblastoma.