An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult-they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

The effect of low-level laser irradiation on hyperglycemia-induced inflammation in human gingival fibroblasts.

Hyperglycemia-induced inflammation can greatly increase the risk of periodontal disease in people with diabetes. Low-level laser irradiation (LLLI) has been used for wound healing and anti-inflammation in many cases, and LLLI is known to inhibit the lipopolysaccharide (LPS)-stimulated inflammatory response. However, the therapeutic effect of LLLI in diabetes patients with periodontitis remains unknown. In this study, we cultured human gingival fibroblasts (HGFs) in high-glucose medium (35 mM) to mimic a hyperglycemic environment, and then measured the anti-inflammatory effect of LLLI by assessing the expression of pro-inflammatory genes including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-8 by quantitative real-time polymerase chain reaction. The results demonstrated no significant inflammatory response in HGFs cultured in mannitol medium and in those treated only with LLLI. However, HGFs cultured only in high-glucose medium showed significantly higher expression of pro-inflammatory cytokine than in those treated together with LLLI. We then observed that LLLI reduced the expression of pro-inflammatory cytokines in HGFs cultured in high-glucose medium by modulating cAMP signaling. We also investigated whether antioxidant (vitamin C) treatment reduced the inflammatory effect of oxidative stress in HGFs cultured in high-glucose medium but found no additive effect upon co-treatment with LLLI, suggesting that LLLI may activate cAMP signaling, but not reactive oxygen species (ROS) signaling, to reduce the high glucose-induced inflammation. In conclusion, LLLI may have an anti-inflammatory effect on HGFs in a high glucose environment and may benefit the treatment of periodontal disease in diabetes patients.

Low power laser irradiation and human adipose-derived stem cell treatments promote bone regeneration in critical-sized calvarial defects in rats.

Both stem cell therapy and physical treatments have been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli for large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low-power laser irradiation (LPLI) and human adipose-derived stem cell (ADSC) treatments during fracture repair using a comparative rat calvarial defect model. We evaluated the viability of human ADSCs, which were cultured on a porous PLGA scaffold using an MTS assay. The critical-sized calvarial bone defect rats were divided into 4 groups: control group, LPLI group, ADSC group, and ADSC+LPLI group. Bone formation was evaluated using micro-CT. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. Our data showed that PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses revealed that both the LPLI and ADSC groups showed improved calvarial bone defect healing compared to the control group. In addition, the ADSC+LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC+LPLI group. There were significant differences between the groups. In addition, both ADSC and ADSC+LPLI groups showed strong signals of vWF expression. ADSC and LPLI treatments improved fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and LPLI further enhances the bone healing process.

Anti-inflammatory effects of low-level laser therapy on human periodontal ligament cells: in vitro study.

Periodontal disease is a chronic inflammatory disease that is commonly treated with surgical and nonsurgical techniques. However, both approaches have limitations. Low-level laser therapy (LLLT) has been widely applied in reducing inflammatory reactions, and research indicates that LLLT induces an anti-inflammatory effect that may enhance periodontal disease therapy. The purpose of this study was to investigate the anti-inflammatory effect of LLLT on human periodontal ligament cells (hPDLCs) in an inflammatory environment and aimed to determine the possible mechanism of action. Cells were cultured and treated with or without lipopolysaccharide (LPS) from Porphryromonas gingivalis or Escherichia coli, followed by irradiation with a gallium-aluminum-arsenide (GaAlAs) laser (660 nm) at an energy density of 8 J/cm2. Quantitative real-time polymerase chain reactions were used to assess the expression of pro-inflammatory genes, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-8. The dual-luciferase reporter assay was used to examine nuclear factor-κB (NF-κB) transcriptional activity. An enzyme-linked immunosorbent assay was used to monitor the concentration of intracellular cyclic adenosine monophosphate (cAMP). Both LPS treatments significantly induced the mRNA expression of pro-inflammatory cytokines. However, LLLT inhibited the LPS-induced pro-inflammatory cytokine expression and elevated intracellular levels of cAMP. The LLLT inhibitory effect may function by downregulating NF-κB transcriptional activity and by increasing the intracellular levels of cAMP. LLLT might inhibit LPS-induced inflammation in hPDLCs through cAMP/NF-κB regulation. These results should be further studied to improve periodontal therapy.

Liposomal Encapsulation for Systemic Delivery of Propranolol via Transdermal Iontophoresis Improves Bone Microarchitecture in Ovariectomized Rats.

The stimulatory effects of liposomal propranolol (PRP) on proliferation and differentiation of human osteoblastic cells suggested that the prepared liposomes-encapsulated PRP exerts anabolic effects on bone in vivo. Iontophoresis provides merits such as sustained release of drugs and circumvention of first pass metabolism. This study further investigated and evaluated the anti-osteoporotic effects of liposomal PRP in ovariectomized (OVX) rats via iontophoresis. Rats subjected to OVX were administered with pure or liposomal PRP via iontophoresis or subcutaneous injection twice a week for 12 weeks. Changes in the microarchitecture at the proximal tibia and the fourth lumbar spine were assessed between pure or liposomal PRP treated and non-treated groups using micro-computed tomography. Administration of liposomal PRP at low dose (0.05 mg/kg) via iontophoresis over 2-fold elevated ratio between bone volume and total tissue volume (BV/TV) in proximal tibia to 9.0% whereas treatment with liposomal PRP at low and high (0.5 mg/kg) doses via subcutaneous injection resulted in smaller increases in BV/TV. Significant improvement of BV/TV and bone mineral density (BMD) was also found in the fourth lumbar spine when low-dose liposomal PRP was iontophoretically administered. Iontophoretic low-dose liposomal PRP also elevated trabecular numbers in tibia and trabecular thickness in spine. Enhancement of bone microarchitecture volumes has highlighted that liposomal formulation with transdermal iontophoresis is promising for PRP treatment at the lower dose and with longer duration than its clinical therapeutic range and duration to exhibit optimal effects against bone loss in vivo.

The Fractionated Toona sinensis Leaf Extract Induces Apoptosis of Human Osteosarcoma Cells and Inhibits Tumor Growth in a Murine Xenograft Model.

BACKGROUND: Osteosarcoma is a malignant bone tumor prevalent in adolescents with poor prognosis. Toona sinensis showed potent antiproliferation effect on lung, melatonin, ovary, colon, and liver cancers. However, the effects of the species on osteosarcoma cells are rarely investigated. RESULTS: In this study, we found fraction 1 of Toona sinensis leaf (TSL-1) resulted in inhibition of cell viability in MG-63, Saos-2, and U2OS osteosarcoma cell lines, while it only caused a moderate suppressive effect on normal osteoblasts. In addition, TSL-1 significantly elevated lactate dehydrogenase leakage and induced apoptosis and necrosis in Saos-2 cells. TSL-1 increased mRNA expression of pro-apoptotic factor Bad. Most important, TSL-1 significantly suppressed Saos-2 xenograft tumor growth in nude mice by increasing caspase-3. The IC-50 of TSL-1 for the 3 tested osteosarcoma cells is around 1/9 of that for lung cancer cells. CONCLUSION: We demonstrated that TSL-1, a fractionated extract from TSL, caused significant cytotoxicity to osteosarcoma cells due to apoptosis. In vivo xenograft study showed that TSL-1 suppressed the growth of osteosarcoma cells at least in part by inducing apoptosis. Our results indicate that TSL-1 has potential to be a promising anti-osteosarcoma adjuvant functional plant extract.

Characterization of a Novel Dermal Fibrosis Model Induced by Areca Nut Extract that Mimics Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is an oral potentially malignant disorder and areca quid chewing is the main etiological factor. However, the molecular mechanism underlying OSF remains unclear, partly due to the lack of an appropriate animal model. The present study aimed to establish and characterize an animal model of areca nut extract (ANE)-induced skin fibrosis that mimics OSF. Mice were divided into 4 groups: the control group; the bleomycin group; and the ANE10 and ANE20 groups, which received 10mg/ml and 20mg/ml subcutaneous (SC) injection of ANE, respectively. Skin fibrosis was evaluated by histological analyses. Additionally, the expression levels of the fibrotic marker genes were determined by immunohistochemical staining and immunoblotting. ANE administration significantly increased dermal thickness and collagen deposition compared with the control group. Moreover, ANE induced the expression of the fibrotic marker genes alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in the skin lesions. The SC injection of ANE successfully induced skin fibrosis, exhibiting characteristics similar to those of OSF. This model may facilitate future studies of the mechanism underlying OSF.

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.

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.

Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line.

Green tea has been reported to possess antioxidant, antitumorigenic, and antibacterial qualities that regulate the endocrine system. Previous epidemiological studies found that the bone mineral density (BMD) of postmenopausal women with a habit of tea drinking was higher than that of women without habitual tea consumption. However, the effects of green tea catechins on osteogenic function have rarely been investigated. In this study, we tested (-)-epigallocatechin-3-gallate (EGCG), one of the green tea catechins, on cell proliferation, the mRNA expressions of relevant osteogenic markers, alkaline phosphatase (ALP) activity and mineralization. In a murine bone marrow mesenchymal stem cell line, D1, the mRNA expressions of core binding factors a1 (Cbfa1/Runx2), osterix, osteocalcin, ALP increased after 48 h of EGCG treatment. ALP activity was also significantly augmented upon EGCG treatment for 4 days, 7 days and 14 days. Furthermore, mineralizations assayed by Alizarin Red S and von Kossa stain were enhanced after EGCG treatment for 2-4 weeks in D1 cell cultures. However, a 24-h treatment of EGCG inhibited thymidine incorporation of D1 cells. These results demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation. This finding suggests that the stimulatory effects of EGCG on osteogenesis of mesenchymal stem cells may be one of the mechanisms that allow tea drinkers to possess higher BMD.