(-)-Epigallocatechin-3-gallate (EGCG) enhances healing of femoral bone defect.

BACKGROUND: Previously, we found that (-)-epigallocatechin-3-gallate (EGCG) enhanced osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and eventually mineralization. We further found EGCG supplementation preserved bone mass and microarchitecture in female rats during estrogen deficiency in the proximal tibia and lumbar spine at least in part by increasing bone morphogenetic protein-2 (BMP2). BMP2 can enhance de novo bone formation. PURPOSE: In this study, we evaluate the effect of local EGCG application in de novo bone formation in bone defect healing. METHODS: Twenty-four rats aged 4 months were weight-matched and randomly allocated to 2 groups: defect control with vehicle treatment (control) and defect with 10 µM EGCG treatment (EGCG). Daily vehicle and EGCG were applied locally by percutaneous local injection 2 days after defect creation for 2 weeks. Four weeks after treatment, animals were sacrificed for micro-computed tomography (µ-CT) and biomechanical analysis. RESULTS: Local EGCG at femoral defect can enhance de novo bone formation by increasing bone volume and subsequently improve mechanical properties including max load, break point, stiffness, area under the max load curve, area under the break point curve and ultimate stress. CONCLUSIONS: Local EGCG may enhance bone defect healing via at least partly by the de novo bone formation of BMP-2.

Early Hyperbaric Oxygen Treatment Attenuates Burn-Induced Neuroinflammation by Inhibiting the Galectin-3-Dependent Toll-Like Receptor-4 Pathway in a Rat Model.

Hyperbaric oxygen (HBO) treatment has been proven to decrease neuroinflammation in rats. This study aimed to determine the potential mechanism underlying the anti-inflammatory effects of HBO treatment on burn-induced neuroinflammation in rats. Thirty-six adult male Sprague-Dawley (SD) rats were randomly assigned to the following six groups (n = 6 per group): (1) sham burn with sham HBO treatment; (2) sham burn with HBO treatment; (3) burn with one-week sham HBO treatment; (4) burn with two-week sham HBO treatment; (5) burn with one-week HBO treatment; and (6) burn with two-week HBO treatment. SD rats that received third-degree burn injury were used as a full-thickness burn injury model. Subsequently, we analyzed the expression of proteins involved in the galectin-3 (Gal-3)-dependent Toll-like receptor-4 (TLR-4) pathway through enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) analysis, and Western blotting. A behavior test was also conducted, which revealed that HBO treatment significantly suppressed mechanical hypersensitivity in the burn with HBO treatment group compared to the burn with sham HBO treatment group (p < 0.05). ELISA results showed that tumor necrosis factor α (TNF-α) and interleukin 1 beta (IL-1ß) levels in the dorsal horn of the spinal cord and the skin significantly decreased in the burn with HBO treatment group compared with the burn with sham HBO treatment group (p < 0.05). Western blotting results demonstrated that HBO treatment significantly reduced the expression of Gal-3 and TLR-4 in the dorsal horn of the spinal cord in the burn with HBO treatment group compared with the burn with sham HBO treatment group (p < 0.05). IHC analysis showed that the expression of Gal-3, TLR-4, CD68 and CD45 in the dorsal horn of the spinal cord was significantly lower in the burn with HBO treatment group than in the burn with sham HBO treatment group (p < 0.05), and the expression of CD68 and macrophage migration inhibitory factor (MIF) in the right hind paw skin was significantly lower. The expression of vimentin and fibroblast growth factor in the right hind paw skin was significantly higher after HBO treatment (p < 0.05). This study proved that early HBO treatment relieves neuropathic pain, inhibits the Gal-3-dependent TLR-4 pathway, and suppresses microglia and macrophage activation in a rat model.

Exercise training ameliorates glucosamine-induced insulin resistance in ovariectomized rats.

OBJECTIVE: Glucosamine (GlcN), which has been reported to induce insulin resistance (IR), is a popular nutritional supplement used to treat osteoarthritis in menopausal women. We previously demonstrated that GlcN treatment caused IR in ovariectomized rats by reducing the expression of glucose transport protein subtype 4 (GLUT-4) in skeletal muscle. In the present study, we hypothesized that endurance exercise training can reverse GlcN-induced IR. METHODS: Fifty female rats were randomly divided into five groups with 10 rats in each group: (1) sham-operated group; (2) sham-operated group with GlcN treatment for 14 days; (3) ovariectomy (OVX) group; (4) OVX with GlcN treatment; and (5) OVX with GlcN treatment followed by exercise training (running program) for 8 weeks. RESULTS: Fasting plasma glucose increased in the OVX + GlcN group, and fasting plasma insulin and the homeostasis model assessment-insulin resistance (HOMA-IR) were significantly higher only in this group. After the rats received exercise training for 8 weeks, no increase in the fasting plasma glucose, insulin, or HOMA-IR was observed. In an intraperitoneal glucose tolerance test, the plasma glucose, plasma insulin, HOMA-IR, and glucose-insulin index were significantly elevated only in the OVX with GlcN treatment group. However, the plasma glucose, plasma insulin, HOMA-IR, and glucose-insulin index decreased after exercise training for 8 weeks, implying that GlcN-induced IR in OVX rats could be reversed through exercise. A histological analysis revealed that exercise training can reduce islet hypertrophy and maintain GLUT-4 in skeletal muscle. CONCLUSIONS: Exercise training can alleviate IR in OVX rats treated with GlcN. Islet hyperplasia was subsequently prevented. Preserving GLUT-4 expression may be one of the mechanisms by which exercise prevents IR.

Synthesis and characterization of cationic polymeric nanoparticles as simvastatin carriers for enhancing the osteogenesis of bone marrow mesenchymal stem cells.

Simvastatin (SIM) can increase osteoblast activity and enhance osteogenesis. However, some limitations of SIM have been noted, such as statin-associated rhabdomyolysis and its poor solubility in water. In this study, we fabricated new cationic nanoparticles (NPs) designed for the controlled release of hydrophobic SIM and endocytosis by cells with the aim of reducing the total required amount of SIM administered and enhancing the osteogenesis of bone marrow mesenchymal stem cells (BMSCs). New copolymers of bis(poly(lactic-co-glycolic acid)-phenylalanine-polyethylene glycol)-quaternary ammonium grafted diethyltriamine (bis(PLGA-phe-PEG)-qDETA; BPPD) were created using a diethyltriamine-quaternary ammonium (qDETA) moiety, hetero-bifunctional polyethylene glycol (COOH-PEG-NH2), phenylalanine (phe) and poly(lactic-co-glycolic acid) (PLGA). SIM encapsulated in BPPD NPs (SIM/BPPD) was fabricated using a water-miscible solvent. The size distributions of BPPD NPs and SIM/BPPD NPs, the encapsulation efficacy and the in vitro release profile of SIM in SIM/BPPD NPs over 6days were investigated. Based on the results of Alizarin Red S staining, alkaline phosphatase (ALP) activity assays and quantitative polymerase chain reaction (Q-PCR) results, we propose that SIM/BPPD NPs may induce osteogenesis in BMSCs by enhancing the expression of an osteogenic gene, which subsequently elevates ALP activity and mineralization, resulting in enhanced BMSC osteogenesis. These results suggest that the SIM/BPPD NPs may be used as hydrophobic drug carriers to reduce the total required amount of SIM administered and to provide an effective SIM release mechanism for enhancing BMSC osteogenesis. Surprisingly, BPPD NPs were also shown to have the ability to promote osteogenesis in BMSCs by enhancing the expression of osteogenic genes, especially osteocalcin (OC), and subsequently elevating ALP activity and mineralization.

Low-level laser irradiation improves functional recovery and nerve regeneration in sciatic nerve crush rat injury model.

The development of noninvasive approaches to facilitate the regeneration of post-traumatic nerve injury is important for clinical rehabilitation. In this study, we investigated the effective dose of noninvasive 808-nm low-level laser therapy (LLLT) on sciatic nerve crush rat injury model. Thirty-six male Sprague Dawley rats were divided into 6 experimental groups: a normal group with or without 808-nm LLLT at 8 J/cm(2) and a sciatic nerve crush injury group with or without 808-nm LLLT at 3, 8 or 15 J/cm(2). Rats were given consecutive transcutaneous LLLT at the crush site and sacrificed 20 days after the crush injury. Functional assessments of nerve regeneration were analyzed using the sciatic functional index (SFI) and hindlimb range of motion (ROM). Nerve regeneration was investigated by measuring the myelin sheath thickness of the sciatic nerve using transmission electron microscopy (TEM) and by analyzing the expression of growth-associated protein 43 (GAP43) in sciatic nerve using western blot and immunofluorescence staining. We found that sciatic-injured rats that were irradiated with LLLT at both 3 and 8 J/cm(2) had significantly improved SFI but that a significant improvement of ROM was only found in rats with LLLT at 8 J/cm(2). Furthermore, the myelin sheath thickness and GAP43 expression levels were significantly enhanced in sciatic nerve-crushed rats receiving 808-nm LLLT at 3 and 8 J/cm(2). Taken together, these results suggest that 808-nm LLLT at a low energy density (3 J/cm(2) and 8 J/cm(2)) is capable of enhancing sciatic nerve regeneration following a crush injury.

Effects of low-level laser therapy on M1-related cytokine expression in monocytes via histone modification.

Low-level laser therapy (LLLT) has been used in the treatment of radiotherapy-induced oral mucositis and allergic rhinitis. However, the effects of LLLT on human monocyte polarization into M1 macrophages are unknown. To evaluate the effects of LLLT on M1-related cytokine and chemokine production and elucidate the mechanism, the human monocyte cell line THP-1 was treated with different doses of LLLT. The expression of M1-related cytokines and chemokines (CCL2, CXCL10, and TNF-α) was determined by ELISA and real-time PCR. LLLT-associated histone modifications were examined by chromatin immunoprecipitation (ChIP) assays. Mitochondrial involvement in the LLLT-induced M1-related cytokine expression was evaluated by quantitative real-time PCR. Flow cytometry was used to detect the cell surface markers for monocyte polarization. The results showed that LLLT (660 nm) significantly enhanced M1-related cytokine and chemokine expression in mRNA and protein levels. Mitochondrial copy number and mRNA levels of complex I-V protein were increased by LLLT (1 J/cm(2)). Activation of M1 polarization was concomitant with histone modification at TNF-α gene locus and IP-10 gene promoter area. This study indicates that LLLT (660 nm) enhanced M1-related cytokine and chemokine expression via mitochondrial biogenesis and histone modification, which may be a potent immune-enhancing agent for the treatment of allergic diseases.

Effect of low level laser therapy on chronic compression of the dorsal root ganglion.

Dorsal root ganglia (DRG) are vulnerable to physical injury of the intervertebral foramen, and chronic compression of the DRG (CCD) an result in nerve root damage with persistent morbidity. The purpose of this study was to evaluate the effects of low level laser therapy (LLLT) on the DRG in a CCD model and to determine the mechanisms underlying these effects. CCD rats had L-shaped stainless-steel rods inserted into the fourth and fifth lumbar intervertebral foramen, and the rats were then subjected to 0 or 8 J/cm2 LLLT for 8 consecutive days following CCD surgery. Pain and heat stimuli were applied to test for hyperalgesia following CCD. The levels of TNF-α, IL-1ß and growth-associated protein-43 (GAP-43) messenger RNA (mRNA) expression were measured via real-time PCR, and protein expression levels were analyzed through immunohistochemical analyses. Our data indicate that LLLT significantly decreased the tolerable sensitivity to pain and heat stimuli in the CCD groups. The expression levels of the pro-inflammatory cytokines TNF-α and IL-1ß were increased following CCD, and we found that these increases could be reduced by the application of LLLT. Furthermore, the expression of GAP-43 was enhanced by LLLT. In conclusion, LLLT was able to enhance neural regeneration in rats following CCD and improve rat ambulatory behavior. The therapeutic effects of LLLT on the DRG during CCD may be exerted through suppression of the inflammatory response and induction of neuronal repair genes. These results suggest potential clinical applications for LLLT in the treatment of compression-induced neuronal disorders.

Low-power laser irradiation suppresses inflammatory response of human adipose-derived stem cells by modulating intracellular cyclic AMP level and NF-κB activity.

Mesenchymal stem cell (MSC)-based tissue regeneration is a promising therapeutic strategy for treating damaged tissues. However, the inflammatory microenvironment that exists at a local injury site might restrict reconstruction. Low-power laser irradiation (LPLI) has been widely applied to retard the inflammatory reaction. The purpose of this study was to investigate the anti-inflammatory effect of LPLI on human adipose-derived stem cells (hADSCs) in an inflammatory environment. We showed that the hADSCs expressed Toll-like Receptors (TLR) 1, TLR2, TLR3, TLR4, and TLR6 and that lipopolysaccharide (LPS) significantly induced the production of pro-inflammatory cytokines (Cyclooxygenase-2 (Cox-2), Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), and Interleukin-8 (IL-8)). LPLI markedly inhibited LPS-induced, pro-inflammatory cytokine expression at an optimal dose of 8 J/cm². The inhibitory effect triggered by LPLI might occur through an increase in the intracellular level of cyclic AMP (cAMP), which acts to down-regulate nuclear factor kappa B (NF-κB) transcriptional activity. These data collectively provide insight for further investigations of the potential application of anti-inflammatory treatment followed by stem cell therapy.

Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro.

We tested the hypothesis that electromagnetic field (EMF) stimulation enhances chondrogenesis in human adipose-derived stem cells (ADSCs) in a chondrogenic microenvironment. A two-dimensional hyaluronan (HA)-coated well (2D-HA) and a three-dimensional pellet culture system (3D-pellet) were used as chondrogenic microenvironments. The ADSCs were cultured in 2D-HA or 3D-pellet, and then treated with clinical-use pulse electromagnetic field (PEMF) or the innovative single-pulse electromagnetic field (SPEMF) stimulation. The cytotoxicity, cell viability, and chondrogenic and osteogenic differentiations were analyzed after PEMF or SPEMF treatment. The modules of PEMF and SPEMF stimulations used in this study did not cause cytotoxicity or alter cell viability in ADSCs. Both PEMF and SPEMF enhanced the chondrogenic gene expression (SOX-9, collagen type II, and aggrecan) of ADSCs cultured in 2D-HA and 3D-pellet. The expressions of bone matrix genes (osteocalcin and collagen type I) of ADSCs were not changed after SPEMF treatment in 2D-HA and 3D-pellet; however, they were enhanced by PEMF treatment. Both PEMF and SPEMF increased the cartilaginous matrix (sulfated glycosaminoglycan) deposition of ADSCs. However, PEMF treatment also increased mineralization of ADSCs, but SPEMF treatment did not. Both PEMF and SPEMF enhanced chondrogenic differentiation of ADSCs cultured in a chondrogenic microenvironment. SPEMF treatment enhanced ADSC chondrogenesis, but not osteogenesis, when the cells were cultured in a chondrogenic microenvironment. However, PEMF enhanced both osteogenesis and chondrogenesis under the same conditions. Thus the combination of a chondrogenic microenvironment with SPEMF stimulation can promote chondrogenic differentiation of ADSCs and may be applicable to articular cartilage tissue engineering.

(-)-N-Formylanonaine from Michelia alba as a human tyrosinase inhibitor and antioxidant.

Tyrosinase is the first and rate limiting enzyme in the synthesis of melanin pigments for coloring hair, skin, and eyes. As reported in this study, a natural product, (-)-N-formylanonaine isolated from the leaves of Michelia alba D.C. (Magnolianceae), was found to inhibit mushroom tyrosinase with an IC50 of 74.3 microM and to have tyrosinase and melanin reducing activities in human epidermal melanocytes without apparent cytotoxicity to human cells, superior to the known tyrosinase inhibitors, such as kojic acid and 1-phenyl-2-thiourea (PTU). Based on homology modeling, the compound binds the active site by coordinating with two Cu2+ ions. In addition, the compound had antioxidation activities in tests for scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power, and chelating metal ions. To our knowledge, this is the first study to reveal the bioactivities of (-)-N-formylanonaine from this plant species.