Nonthermal atmospheric plasma enhances myoblast differentiation by eliciting STAT3 phosphorylation.

The use of nonthermal atmospheric plasma (NTP) in the biomedical field has recently expanded into cell death induction in cancer, infection prevention, inflammation treatment, and wound-healing enhancement. NTP has been demonstrated to enhance skin and muscle regeneration, but its effects on tissue regeneration, following deep tissue or muscle damage, remains underinvestigated. In this study, we determined the effects of NTP on muscle differentiation and the mechanisms of NTP's contribution to differentiation and regeneration. NTP treatment enhanced cell differentiation in primary normal human skeletal muscle myoblast cells and increased the relative expression of mRNA levels of MyoD which is one of the earliest markers of myogenic commitment, and myogenin, which are important transcription factors required for myogenic differentiation. Furthermore, NTP treatment induced increases in the levels of myosin heavy chain, a differentiated muscle-specific protein, and in myotube formation of myoblasts. We observed that signal transducer and activator of transcription 3 (STAT3) activation induced by NTP treatment affects the myogenic differentiation. In addition, STAT3 phosphorylation was also enhanced by NTP treatment in injured animal muscle. These findings indicate that NTP could enhance musculoskeletal differentiation by acting as an external stimulus for myoblast differentiation, suggesting its treatment potential in promoting regeneration of damaged muscle.-Park, J. K., Kim, Y. S., Kang, S. U., Lee, Y. S., Won, H.-R., Kim, C.-H. Nonthermal atmospheric plasma enhances myoblast differentiation by eliciting STAT3 phosphorylation.

N2 non-thermal atmospheric pressure plasma promotes wound healing in vitro and in vivo: Potential modulation of adhesion molecules and matrix metalloproteinase-9.

Advances in physics and biology have made it possible to apply non-thermal atmospheric pressure plasma (NTP) in the biomedical field. Although accumulating evidence suggests that NTP has various medicinal effects, such as facilitating skin wound healing on exposed tissue while minimizing undesirable tissue damage, the underlying molecular mechanisms are not fully understood. In this study, NTP generated from N2 optimized wound healing in the scratch wound healing assay. In addition, matrix metalloproteinase (MMP)-9 expression and enzyme activity increased and the urokinase-type plasminogen activator (uPA) system was activated after NTP treatment. We also showed that NTP treatment increased Slug and TCF8/ZEB1 expression and decreased that of E-cadherin, suggesting induction of the epithelial-to-mesenchymal transition (EMT). The effect of N2 NTP was verified on rat wound model. Taken together, these results suggest that N2 NTP promotes wound healing by inducing the EMT and activating the MMP-9/uPA system. These findings show the therapeutic potential of NTP for skin wound healing.

Protective effects of Korean red ginseng on radiation-induced oral mucositis in a preclinical rat model.

Numerous studies' attempts to improve radiation-induced oral mucositis have not produced a qualified treatment yet. Our aim was to investigate the effectiveness of Korean red ginseng (KRG) on radiation-induced damage in an in vivo rat model. After 20 Gy of irradiation, rats were divided randomly into the following 4 groups: control, KRG only, radiotherapy (RT) only, and RT + KRG group. The rats were monitored in terms of survival rate, activity, mucositis grade, oral intake, and body weight. The tongue, buccal mucosa, and submandibular gland (SMG) were harvested, and the weight of the SMG was analyzed. The samples then underwent hematoxylin and eosin, TUNEL, and immunohistochemical staining. Radiation-induced severe oral mucositis and SMG injury led to poor oral intake and delayed healing, resulting in the death of some rats. We found that survival rate, oral intake, and body weight increased. Moreover, rats treated with KRG showed less severe mucositis and decreased histologic changes of the oral mucosa and SMG. Furthermore, we showed that the protective effects of KRG were caused by inhibition of the apoptotic signal transduction pathway linked to caspase-3. In conclusion, KRG protects the oral mucosa and SMG from radiation-induced damage by inhibiting caspase-mediated apoptosis in rats.

Tissue-engineered tracheal reconstruction using three-dimensionally printed artificial tracheal graft: preliminary report.

Three-dimensional printing has come into the spotlight in the realm of tissue engineering. We intended to evaluate the plausibility of 3D-printed (3DP) scaffold coated with mesenchymal stem cells (MSCs) seeded in fibrin for the repair of partial tracheal defects. MSCs from rabbit bone marrow were expanded and cultured. A half-pipe-shaped 3DP polycaprolactone scaffold was coated with the MSCs seeded in fibrin. The half-pipe tracheal graft was implanted on a 10 × 10-mm artificial tracheal defect in four rabbits. Four and eight weeks after the operation, the reconstructed sites were evaluated bronchoscopically, radiologically, histologically, and functionally. None of the four rabbits showed any sign of respiratory distress. Endoscopic examination and computed tomography showed successful reconstruction of trachea without any collapse or blockage. The replaced tracheas were completely covered with regenerated respiratory mucosa. Histologic analysis showed that the implanted 3DP tracheal grafts were successfully integrated with the adjacent trachea without disruption or granulation tissue formation. Neo-cartilage formation inside the implanted graft was sufficient to maintain the patency of the reconstructed trachea. Scanning electron microscope examination confirmed the regeneration of the cilia, and beating frequency of regenerated cilia was not different from those of the normal adjacent mucosa. The shape and function of reconstructed trachea using 3DP scaffold coated with MSCs seeded in fibrin were restored successfully without any graft rejection.

Controlled Release of Hepatocyte Growth Factor from MPEG-b-(PCL-ran-PLLA) Diblock Copolymer for Improved Vocal Fold Regeneration.

An in situ-forming gel system comprised of diblock copolymer formed from polyethylene glycol (PEG) and polycaprolactone (PCL) {MPEG-b-(PCL-ran-PLLA)} could be used in controlled drug delivery for tissue remodeling. The purpose of this study is to demonstrate favorable vocal folds (VF) regeneration by using MPEG-b-(PCL-ran-PLLA) diblock copolymers (C97L3; CL/LA ratio 97:3) incorporating hepatocyte growth factor (HGF). Gradual release of HGF from C97L3 is detected and biochemical properties of released HGF are maintained. A scar is made with microscissors on both VFs in 32 rabbits, followed by injection of HGF-only, C97L3-only, or HGF-C97L3 composite gel in the left side VF, while the right side VF is left untreated. In vivo fluorescence live imaging system demonstrates that C97L3 enables the sustained release of injected HGF in the scarred VF for 12 weeks. The histological analysis shows increased glycosaminoglycan including hyaluronic acid accumulation and decreased collagen deposition. Videokymographic analysis shows more favorable vibrations of HGF-C97L3 treated VF mucosa, compared to other treatment groups. In conclusion, the controlled HGF release helps to regulate extracellular matrix synthesis, and leads to the eventual functional improvement of the scarred VF.

Opposite effects of non-thermal plasma on cell migration and collagen production in keloid and normal fibroblasts.

Recent progress in the understanding non-thermal plasma (NTP) properties prompted its application in the treatment of various diseases. However, therapeutic effect of NTP on keloid cells has not been reported previously. We sought to investigate the effect of NTP treatment on keloid by comparing cell migration and collagen production of keloid (KFs) and normal fibroblasts (NFs) and determined the regulatory pathways involved. We assessed NTP effects on cell migration in KFs and NFs by the wound healing assay and measured the expression of the epidermal growth factor receptor (EGFR), signal transducer and activator of transcription-3 (STAT3), and collagen by western blot. Expression of the transforming growth factor-ß and Type I collagen following NTP treatment was determined by reverse transcription-polymerase chain reaction, immunofluorescence staining, and the Sircol collagen assay. NTP treatment increased cell migration and collagen production of NFs. However, it reduced these parameters in KFs. NTP reduced the expression of EGFR, STAT3, and Type I collagen in KFs but increased their levels in NFs. We revealed that NTP suppressed KF cell migration via down-regulation of EGFR and STAT3 and reduced collagen production via supressing transforming growth factor-ß. Our data suggest that NTP may be a new therapeutic strategy for keloids.

Anti-cancer effect of (-)-epigallocatechin-3-gallate (EGCG) in head and neck cancer through repression of transactivation and enhanced degradation of ß-catenin.

BACKGROUND AND PURPOSE: Aberrant expression of ß-catenin is highly associated with progression of various cancers including head and neck cancer (HNC). Green tea is most commonly used beverage in the world and one of the more bioactive compounds is the antioxidant epigallocatechin gallate (EGCG). This study was performed to investigate the mechanism by which EGCG inhibits the growth of HNC, focusing on the modulation of the expression and activity of ß-catenin. METHODS: In vitro effects of EGCG on the transcription, translation, or degradation of ß-catenin were investigated. Antitumor effects of EGCG in vivo were evaluated in a syngeneic mouse model and ß-catenin expression was checked in HNC patients' samples. RESULTS: ß-catenin expression was elevated in tumor samples of HNC patients. EGCG induced apoptosis in KB and FaDu cells through the suppression of ß-catenin signaling. Knockdown of ß-catenin using siRNA enhanced the proapoptotic activities of EGCG. EGCG decreased mRNA and transcriptional activity of ß-catenin in p53 wild-type KB cells. EGCG also enhanced the ubiquitination and proteasomal degradation of ß-catenin. The suppression of ß-catenin and consequent apoptosis were observed in response to EGCG treatment in a syngeneic mouse model. In conclusion, we report that EGCG inhibits ß-catenin expression through multiple mechanisms including decreased transcription and increased ubiquitin-mediated 26S proteasomal degradation. CONCLUSION: This study proposes a novel molecular rationale for antitumor activities of green tea in HNCs.

Tissue-engineered artificial oesophagus patch using three-dimensionally printed polycaprolactone with mesenchymal stem cells: a preliminary report.

OBJECTIVES: There has been a recent focus on 3D printing with regard to tissue engineering. We evaluated the efficacy of a 3D-printed (3DP) scaffold coated with mesenchymal stem cells (MSCs) seeded in fibrin for the repair of partial oesophageal defects. METHODS: MSCs from rabbit bone marrow were cultured, and a 3DP polycaprolactone (PCL) scaffold was coated with the MSCs seeded in fibrin. The fibrin/MSC-coated 3DP PCL scaffold was implanted on a 5 × 10 mm artificial oesophageal defect in three rabbits (3DP/MSC group) and 3DP PCL-only scaffolds were implanted in three rabbits (3DP-only group). Three weeks post-procedure, the implanted sites were evaluated radiologically and histologically. RESULTS: None of the rabbits showed any infection, stenosis or granulation on computed tomography. In the 3DP/MSC group, the replaced scaffolds were completely covered with regenerating mucosal epithelium and smooth muscle cells as determined by haematoxylin and eosin and Desmin staining. However, mucosal epithelium and smooth muscle cell regeneration was not evident in the 3DP-only group. CONCLUSIONS: Use of the 3DP scaffold coated with MSCs seeded in fibrin resulted in successful restoration of the shape and histology of the cervical oesophagus without any graft rejection; thus, this is a promising material for use as an artificial oesophagus.

Novel Therapeutic Effects of Non-thermal atmospheric pressure plasma for Muscle Regeneration and Differentiation.

Skeletal muscle can repair muscle tissue damage, but significant loss of muscle tissue or its long-lasting chronic degeneration makes injured skeletal muscle tissue difficult to restore. It has been demonstrated that non-thermal atmospheric pressure plasma (NTP) can be used in many biological areas including regenerative medicine. Therefore, we determined whether NTP, as a non-contact biological external stimulator that generates biological catalyzers, can induce regeneration of injured muscle without biomaterials. Treatment with NTP in the defected muscle of a Sprague Dawley (SD) rat increased the number of proliferating muscle cells 7 days after plasma treatment (dapt) and rapidly induced formation of muscle tissue and muscle cell differentiation at 14 dapt. In addition, in vitro experiments also showed that NTP could induce muscle cell proliferation and differentiation of human muscle cells. Taken together, our results demonstrated that NTP promotes restoration of muscle defects through control of cell proliferation and differentiation without biological or structural supporters, suggesting that NTP has the potential for use in muscle tissue engineering and regenerative therapies.

Downregulation of Nrf2 by the combination of TRAIL and Valproic acid induces apoptotic cell death of TRAIL-resistant papillary thyroid cancer cells via suppression of Bcl-xL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents an effective agent for the treatment of many cancers, though the majority of thyroid cancers are found to be resistant. Therefore it would be necessary to identify agents capable of increasing the sensitivity of these cancers to TRAIL-mediated cell death. Here, we examined the therapeutic effect and its underlying mechanism of combination treatment of TRAIL and histone deacetylase inhibitor, Valproic acid (VPA) in vitro using human papillary thyroid cancer (PTC) cells and in vivo using an orthotopic mouse model of PTC. TRAIL-VPA combination therapy synergistically induced apoptotic cell death in TRAIL-resistant PTC through caspase activation. In addition, downregulation of antioxidant transcription factor, Nrf2 by co-treatment of TRAIL-VPA induces cell death via suppression of Bcl-xL in vitro and in vivo; these effects were further enhanced following siRNA inhibition of these proteins in combination with TRAIL or TRAIL-VPA. Taken together, VPA sensitized TRAIL-resistant PTC cells to apoptotic cell death through involvement of Nrf2 and Bcl-xL. Thus, the combination of VPA and TRAIL may be a promising therapy for TRAIL-resistant PTC.

Non-thermal plasma induces AKT degradation through turn-on the MUL1 E3 ligase in head and neck cancer.

Recent research on non-thermal plasma (NTP, an ionized gas) has identified it as a novel cancer therapeutic tool. However, the molecular mechanism remains unclear. In this study, we demonstrated NTP induced cell death of head and neck cancer (HNC) through the AKT ubiquitin-proteasome system. NTP increased the gene expression of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), an E3 ligase for AKT, and NTP-induced HNC cell death was prevented by MUL1 siRNA. We also showed that MUL1 inhibited the level of AKT and p-AKT and MUL1 expression was increased by NTP-induced ROS. Furthermore, we optimized and manufactured a new type of NTP, a liquid type of NTP (LTP). In syngeneic and xenograft in vivo tumor models, LTP inhibited tumor progression by increasing the MUL1 level and reducing p-AKT levels, indicating that LTP also has an anti-cancer effect through the same mechanism as that of NTP. Taken together, our results suggest that NTP and LTP have great potential for HNC therapy.

Efficiency and durability of hyaluronic acid of different particle sizes as an injectable material for VF augmentation.

CONCLUSION: The results of the present investigation suggest that modification of HA could improve efficiency and durability in augmentation laryngoplasty. OBJECTIVES: Injection laryngoplasty (IL) is one of the most suitable options for treatment of glottic insufficiency, which is caused by vocal fold (VF) paralysis, atrophy, or scarring. Hyaluronic acid (HA) is a widely used material for VF injection. This study was intended to evaluate the durability and efficiency of HA of different particle sizes for VF augmentation. METHODS: Three types of HA, Restylane®, monophasic low-viscosity, and unequal particle-sized middle-viscosity HA were injected into the left VF of three groups with eight rabbits each. RESULTS: After 6 and 10 weeks, the injected site was evaluated endoscopically, histologically, radiologically, and functionally. None of the 24 rabbits showed any signs of respiratory distress. Computed tomography (CT) images and endoscopic evaluation revealed sufficient augmented volume of the injected VF in all treated groups 6 weeks after the injection. Histological data at week 10 showed that unequal particle-sized HA did not migrate from its original injection site, while other HAs migrated to the periphery of the arytenoid cartilage. Videokymographic analysis showed more favorable vibrations of unequal particle-sized HA injected VF mucosa 10 weeks post-injection, compared to the other treatment groups.

Tissue-engineered tracheal reconstruction using mesenchymal stem cells seeded on a porcine cartilage powder scaffold.

Tissue engineering using a biocompatible scaffold with various cells might be a solution for tracheal reconstruction. We investigated the plausibility of using mesenchymal stem cells (MSCs) seeded on a porcine cartilage powder (PCP) scaffold for tracheal defect repair. PCP made with minced and decellularized porcine articular cartilage was molded into a 5 × 12 mm (height × diameter) scaffold. MSCs from young rabbit bone marrow were expanded and cultured with the PCP scaffold. After 7 weeks culture, the tracheal implants were transplanted on a 5 × 10 mm tracheal defect in six rabbits. 6 and 10 weeks postoperatively, the implanted area was evaluated. None of the six rabbits showed any sign of respiratory distress. Endoscopic examination revealed that respiratory epithelium completely covered the regenerated trachea and there were no signs of collapse or blockage. A patent luminal contour of the trachea was observed on the computed tomography scan in all six rabbits and the reconstructed areas were not narrow compared to normal adjacent trachea. Histologic examination showed that neo-cartilage was successfully produced with minimal inflammation or granulation tissue. Ciliary beating frequency of the regenerated epithelium was not significantly different from the normal adjacent mucosa. MSCs cultured with a PCP scaffold successfully restored not only the shape but also the function of the trachea without any graft rejection.

Tracheal reconstruction using chondrocytes seeded on a poly(L-lactic-co-glycolic acid)-fibrin/hyaluronan.

Reconstruction of trachea is still a clinical dilemma. Tissue engineering is a recent and promising concept to resolve this problem. This study evaluated the feasibility of allogeneic chondrocytes cultured with fibrin/hyaluronic acid (HA) hydrogel and degradable porous poly(L-lactic-co-glycolic acid) (PLGA) scaffold for partial tracheal reconstruction. Chondrocytes from rabbit articular cartilage were expanded and cultured with fibrin/HA hydrogel and injected into a 5 × 10 mm-sized, curved patch-shape PLGA scaffold. After 4 weeks in vitro culture, the scaffold was implanted on a tracheal defect in eight rabbits. Six and 10 weeks postoperatively, the implanted sites were evaluated by bronchoscope and radiologic and histologic analyses. Ciliary beat frequency (CBF) of regenerated epithelium was also evaluated. None of the eight rabbits showed any sign of respiratory distress. Bronchoscopic examination did not reveal stenosis of the reconstructed trachea and the defects were completely recovered with respiratory epithelium. Computed tomography scan showed good luminal contour of trachea. Histologic data showed that the implanted chondrocytes successfully formed neocartilage with minimal granulation tissue. CBF of regenerated epithelium was similar to that of normal epithelium. Partial tracheal defect was successfully reconstructed anatomically and functionally using allogeneic chondrocytes cultured with PLGA-fibrin/HA composite scaffold.

Small intestine submucosa and mesenchymal stem cells composite gel for scarless vocal fold regeneration.

The purpose of this study is to demonstrate scarless vocal fold (VF) regeneration by using a composite gel composed of small intestine submucosa (SIS) and mesenchymal stem cells (MSCs). A scar was made with an electrocoagulator on both VFs in 24 rabbits, followed by injection of either MSCs, SIS, or MSCs-SIS composite gel in the right side VF, while the left side VF was left untreated. VF scars were evaluated with in vivo fluorescence live imaging system (IFLIS), endoscopy, histology, and videokymography (VKG) after eight weeks. IFLIS demonstrated that SIS enabled the MSCs to survive and be engrafted in the VF. The histological analysis showed increased hyaluronic acid accumulation and controlled collagen deposition by MSCs-SIS composite gel. VKG analysis showed more favorable vibrations of MSCs-SIS injected VF, compared to other treatment group. In conclusion, the injectable SIS supplied a niche for the MSCs to stably settle down in scarred VFs and helped to regulate ECM synthesis. The ECM remodeling underwent by the surviving MSCs eventually led to the functional improvement of the VF. The results of the present investigation suggest that SIS-MSCs composite gel is a plausible biomaterial for prolonged survival of MSCs in VFs and promotes scarless VF healing.