Small Extracellular Vesicles Laden Oxygen-Releasing Thermosensitive Hydrogel for Enhanced Antibacterial Therapy against Anaerobe-Induced Periodontitis Alveolar Bone Defect.

Periodontitis is a bacterially induced chronic destructive inflammatory disease that leads to irreversible destruction of the tooth supporting structure, including connective tissue destruction, bone resorption, and even tooth loss. Until now, there has been no effective treatment to repair inflammatory bone loss in periodontitis. Recently, small extracellular vesicles (sEVs) emerged as the essential paracrine factors of mesenchymal stem cells (MSCs) that mediated tissue regeneration. However, limitations of antimicrobial activity associated with the use of sEVs have led to the urgency of new alternative strategies. Currently, we investigated the potential of a biocompatible oxygen-releasing thermosensitive hydrogel laded with sEVs secreted by bone marrow MSCs (BMMSCs) for the alveolar bone defect in periodontitis. The hydrogel composed of different polymers such as chitosan (CS), poloxamer 407 (P407), and cross-linked hyaluronic acid (c-HA) conglomerating is a kind of nanoporous structure material. Then, the gel matrix further encapsulated sEVs and calcium peroxide nanoparticles to realize the control of sEVs and oxygen release. Furthermore, ascorbic acid was added to achieve the REDOX equilibrium and acid-base equilibrium. The experiments in vivo and in vitro proved its good biocompatibility and effectively inhibited the growth of the periodontal main anaerobe, relieved periodontal pocket anaerobic infections, and promoted the periodontal defect regeneration. Therefore, this finding demonstrated that it was a promising approach for combating anaerobic pathogens with enhanced and selective properties in periodontal diseases, even in other bacteria-induced infections, for future clinical application.

Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling.

Increasingly natural products particularly flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. This study has reviewed previous studies on the two better known flavonoids, genistein and icariin, their structures, functions, action mechanisms, relative potency, and potential application in regulating bone remodeling and preventing bone loss. Genistein, an isoflavone abundant in soy, has dual functions on bone cells, able to inhibit bone resorption activity of osteoclasts and stimulate osteogenic differentiation and maturation of bone marrow stromal progenitor cells (BMSCs) and osteoblasts. Genistein is an estrogen receptor (ER)-selective binding phytoestrogen, with a greater affinity to ERß. Genistein inhibits tyrosine kinases and inhibits DNA topoisomerases I and II, and may act as an antioxidant. Genistein enhances osteoblastic differentiation and maturation by activation of ER, p38MAPK-Runx2, and NO/cGMP pathways, and it inhibits osteoclast formation and bone resorption through inducing osteoclastogenic inhibitor osteoprotegerin (OPG) and blocking NF-κB signaling. Icariin, a prenylated flavonol glycoside isolated from Epimedium herb, stimulates osteogenic differentiation of BMSCs and inhibits bone resorption activity of osteoclasts. Icariin, whose metabolites include icariside I, icariside II, icaritin, and desmethylicaritin, has no estrogenic activity. However, icariin is more potent than genistein in promoting osteogenic differentiation and maturation of osteoblasts. The existence of a prenyl group on C-8 of icariin molecular structure has been suggested to be the reason why icariin is more potent than genistein in osteogenic activity. Thus, the prenylflavonoids may represent a class of flavonoids with a higher osteogenic activity.

The potential therapeutic role of extracellular vesicles in critical-size bone defects: Spring of cell-free regenerative medicine is coming.

In recent years, the incidence of critical-size bone defects has significantly increased. Critical-size bone defects seriously affect patients' motor functions and quality of life and increase the need for additional clinical treatments. Bone tissue engineering (BTE) has made great progress in repairing critical-size bone defects. As one of the main components of bone tissue engineering, stem cell-based therapy is considered a potential effective strategy to regenerate bone tissues. However, there are some disadvantages including phenotypic changes, immune rejection, potential tumorigenicity, low homing efficiency and cell survival rate that restrict its wider clinical applications. Evidence has shown that the positive biological effects of stem cells on tissue repair are largely mediated through paracrine action by nanostructured extracellular vesicles (EVs), which may overcome the limitations of traditional stem cell-based treatments. In addition to stem cell-derived extracellular vesicles, the potential therapeutic roles of nonstem cell-derived extracellular vesicles in critical-size bone defect repair have also attracted attention from scholars in recent years. Currently, the development of extracellular vesicles-mediated cell-free regenerative medicine is still in the preliminary stage, and the specific mechanisms remain elusive. Herein, the authors first review the research progress and possible mechanisms of extracellular vesicles combined with bone tissue engineering scaffolds to promote bone regeneration via bioactive molecules. Engineering modified extracellular vesicles is an emerging component of bone tissue engineering and its main progression and clinical applications will be discussed. Finally, future perspectives and challenges of developing extracellular vesicle-based regenerative medicine will be given. This review may provide a theoretical basis for the future development of extracellular vesicle-based biomedicine and provide clinical references for promoting the repair of critical-size bone defects.

[Effect of osthol on apoptosis and bone resorption of osteoclasts cultured in vitro].

This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.

Erratum: Human Umbilical Cord MSCs as New Cell Sources for Promoting Periodontal Regeneration in Inflammatory Periodontal Defect: Errautm.

[This corrects the article DOI: 10.7150/thno.19888.].

Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro.

There has been a strong interest in searching for natural therapies for osteoporosis. Genistein, an isoflavone abundant in soy, and icariin, a prenylated flavonol glycoside isolated from Epimedium Herb, have both been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the relative potency in osteogenesis between the individual phytoestrogen flavonoids remains unknown. The present study compared ability of genistein and icariin in enhancing differentiation and mineralization of cultured rat calvarial osteoblasts in vitro. Dose-dependent studies in osteoblast differentiation measuring alkaline phosphatase (ALP) activity revealed optimal concentrations of genistein and icarrin for stimulating osteogenesis to be both at 10(-5) M. Time course studies comparing the two compounds both at 10(-5) M demonstrated that icariin treatment always produced higher ALP activity, more and larger areas of CFU-F(ALP) colonies and mineralized nodules, more osteocalcin secretion, and calcium deposition, and a higher level of mRNA expression of osteogenesis-related genes COL1α2, BMP-2, OSX, and RUNX-2. However, they inhibited the proliferation of osteoblasts to a similar degree. In conclusion, although future in vivo studies are required to investigate whether icariin is more efficient in improving bone mass and/or preventing bone loss, our in vitro studies have demonstrated that icariin has a stronger osteogenic activity than genistein. In addition, while the prenyl group on C-8 of icariin could be the active group that takes part in osteoblastic differentiation and explains its greater potency in osteogenesis, mechanisms of action, and reasons for the relative potency of icariin versus genistein need to be further studied.

Sinusoidal electromagnetic field stimulates rat osteoblast differentiation and maturation via activation of NO-cGMP-PKG pathway.

Nitric oxide (NO) is an important intracellular and intercellular messenger, critically affecting bone metabolism. The purpose of this research is to investigate whether the effect of sinusoidal electromagnetic field (SEMF) on the differentiation and maturation of osteoblasts is mediated by the NO-cGMP-PKG signal pathway. We examined the impact of SEMF on nitric oxide synthase (NOS) activity, and found that L-NAME, nitric oxide synthase's inhibitor, prevents SEMF-mediated increase in NOS activity and NO levels. We showed that an inhibitor of soluble guanylyl cyclase (ODQ) blocks the increase in cGMP levels triggered by exposure to SEMF. The inhibitor PDE5, which hydrolyzes 3',5'-cyclic-GMP to 5'-GMP, prevents the SEMF's stimulation of PKG activity. We also blocked the NO-cGMP-PKG pathway to determine whether the maturation and mineralization of osteoblasts, stimulated by SEMF, would be inhibited. This was evaluated by measuring alkaline phosphatase (ALP) activity, osterix gene expression and mineralized bone modulus. After treatment with SEMF, the NOS activity increases in comparison with the control group (P<0.01), reaching the highest level after 0.5h. Osterix gene expression, ALP activity and mineralized bone nodules in the SEMF experimental group also increase significantly. However, these effects are partially blocked in the L-NAME treated cultures. Surprisingly, all the osteogenic markers in the SEMF+L-NAME group were slightly higher than in the control culture, but lower than in the cells exposed to SEMF only. We conclude that the NO-cGMP-PKG signal pathway is activated by SEMF treatment, the stimulatory effect of SEMF on the differentiation and mineralization of osteoblasts is attenuated when the pathway is blocked.

Osthol, a coumarin isolated from common cnidium fruit, enhances the differentiation and maturation of osteoblasts in vitro.

The effect of osthol on osteoblasts was investigated in primary osteoblastic cells isolated from newborn Wistar rats. Osthol was supplemented into cultured medium at 10⁻7, 10⁻6, 10⁻5 and 10⁻4 mol/l, respectively. No stimulating effect was found on cell proliferation, but 10⁻5 mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity. Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol. The results showed that the ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 1.52, 2.74 and 2.0 times higher, respectively, than in the control cells. Results of ALP histochemical staining and mineralized bone nodule assays both showed that the number and area achieved in osthol-treated cells were 1.53-fold higher than in control cells. The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, bone morphogenetic protein 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with bone development, were also investigated. The increase in mRNA expression was 1.94, 1.74, 1.68, 1.83 and 2.31 times, respectively, higher compared to the control. Furthermore, osthol increased the protein expression of p38 mitogen-activated protein kinase (MAPK) and type I collagen. p38MAPK protein and collagen in osthol-treated cells were 1.42 and 1.58 times higher in osthol-treated cells compared to the control. The results of these studies support the conclusion that osthol significantly enhances the osteogenic differentiation of cultured osteoblasts. The results also indicated that osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway and that osthol may be used as an important compound in the development of new antiosteoporosis drugs.

[The changes of iNOS and NO in the osteogenic differentiation process of rat bone marrow stromal cells promoted by icariside II].

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.

[Comparative study on effect of icariin and genistein on proliferation and mineralization of osteoblasts in vitro].

OBJECTIVE: To investigated the effect of icariin and genistein on proliferation and mineralization of cultured rat osteoblasts (rat calvarial osteoblasts, ROB). And to contrast the pharmacological activity of icariin and genistein. METHOD: Bone cells were obtained by enzyme digestion from the segregated neonatal SD rat skull, and were cultured in MEM containing 10% FBS which was changed after three days later. Serial subcultivation was proceeded when cells covered with 90% culture dish. The final action concentration of icariin and genistein were both 1 x 10(-5) mol x L(-1). Proliferation was analyzed by MTT on 96-well plates, while differentiation was analyzed on 24-well plates. Under the induced condition, the alkaline phosphatase activity, calcium salt sediment yield and osteocalcin were measured at the 3, 6, 9, 12 d. At 12th day, ALP staining, alizarin red staining and calcified nodule count were preceded. Total RNA was isolated at 0, 6, 12, 24, 48, 72 h. The gene expression of bFGF, IGF-1, Osterix and Runx-2 was analyzed by Real-time RT-PCR. RESULT: With the concentration of 1 x 10(-5) mol x L(-1), icariin and genistein have no significant effect on the ROB' s proliferation. The osteogenesis, ALP activity, calcium salt sediment yield and osteocalcin, calcified tubercle amount were significantly increased. And they enhanced the mRNA level of bFGF, IGF-1, Osterix and Runx-2. On the level of osteoblasts, the activity of icariin is stronger than that of genistein. CONCLUSION: When the final concentration of icariin and genistein is 1 x 10(-5) mol x L(-1), they can significantly promoted ROB maturation. And on the level of osteoblasts, the activity of icariin is stronger than that of genistein.

[Effects of isopsoralen on bone marrow stromal stem cells differentiate and proliferate in vitro].

OBJECTIVE: To investigated the effects of isopsoralen on bone marrow stromal stem cells (BMSCs) differentiate and proliferate in vitro. METHOD: The stratum of mononuclear cells were separated and collected from the rat bone marrow sample by the all bone marrow cell culture methods. The cells were cultured in DMEM contained 10% fetal bovine serum. The culture medium was changed after three days. Nine days later, cells were treatment by isopsoralen with the concentration 1 x 10(-5), 1 x 10(-4), 1 x10(-6), 1 x 10(-7) mol x L(-1). MTT method was used for the proliferated analyzing. Under the induced condition, the alkaline phosphatase (ALP) activity, calcium salt sediment yield and osteocalcin were measured at the 4, 8, 12, 16 d. At the fifteenth day, histochemistry dyeing for calcified tubercle and ALP was proceeded. Total RNA was isolated and the gene expression of bFGF, IGF-1, Osterix and Runx-2 were investigated by Real Time PCR. RESULT: The BMSCs proliferation refrained by isopsoralen with dose dependent. But it significantly enhanced osteogenesis, which was represented by the promotion of the ALP activity, calcium salt sediment yield, osteocalcin, and calcified tubercle amount. Besides, it also enhanced the mRNA level of bFGF, IGF-1, Osterix and Runx-2. CONCLUSION: The isopsoralen with the concentration 1 x 10(-5) mol x L(-1) can promote BMSCs differentiation to osteoblasts. It demonstrated the isopsoralen can prevent antiosteoporotic, which is an active part of the traditional Chinese medicine psoralea corylifolia.

[Effect of isopsoralen on the proliferation and differentiate of osteoblasts in vitro].

OBJECTIVE: To investigate the effect of isopsoralen on proliferation, osteogenic differentiation and calcification capacity of rat calvarial osteoblasts (ROB). METHODS: Segregated neonatal SD rat skull,and digestion with enzyme to obtain bone cells and cultured in MEM containing 10% FBS. Exchange the medium after three days, proceeded serial subcultivation when cells covered with 90% culture dish. Proliferation analysis was performed in 96-well plates use MTT method, isopsoralen's final concentration were 1 x 10(-4), 1 x10(-5), 1 x 10(-6), 1 x 10(-7) mmol/L. Differentiation analysis was performed in 24-well plates, the Alkaline phosphatase activity and calcium salt sediment yield and osteocalcin measured at the 4th, 8th, 12th, 16th day. At 12th day, proceeded ALP stain, and at 14th day for alizarin red staining and calcified nodule count. RESULTS: When the Isopsoralen's final concentration was 1 x 10(-5) mmol/L, there was no significant effect on the ROB's proliferation, but it could promote osteogenesis. It also could raise the ALP activity and calcium salt sediment yield and osteocalcin, increase calcified tubercle amount. CONCLUSION: When the isopsoralen final concentration is 1 x 10(-5) mmol/L, it promoted ROB differentiation and maturation. Isopsoralen may be the active ingredients of preventing anti-osteoporosis in Psoralea corylifolia.

[Time effect of sinusoidal electromagnetic field on enhancing the maturation and mineralization of osteoblasts in vitro].

The present research was to investigate the time effect of sinusoidal electromagnetic fields (SEMFs) at different exposure time on the proliferation and differentiation of osteoblasts (OB) in vitro. The newborn rat calvarial OB were isolated by enzyme digestion and divided randomly into 7 groups after one passage. The exposure times of the SEMFs were 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h and 3.0 h, respectively, and the frequency was 50 Hz. The cells were exposed in the SEMFs of 1.8 mT. Those without SEMFs exposure were used as the control group. They were observed under the contrast phase microscope each day. After 48 h, cell proliferation was assayed by MTT method. The alkaline phosphatase (Alkaline Phosphatase, ALP) activities were measured after the exposure of SEMFs for 3 d, 6 d, 9 d and 12 d, respectively. The calcified nodules were stained by Alizarin Bordeaux after 10 d. The cells exposed in the SEMFs were arranged in Spiral appearance after 8 d. The SEMFs exposure time at 2.0 h, 2.5 h and 3.0 h significantly inhibited cell proliferation (P < 0.01) and 0.5 h, 1.0 h, 1.5 h groups more significantly than control groups (P < 0.05). When the 3 d, 6 d and 12 d the ALP activities of the 0.5 h, 1.0 h, 1.5 h and 2.0 h, times group were significantly higher than those in the control group (P < 0.05), and after 9 d the 1.0 h, 1.5 h and 2.0 h activity of ALP higher significantly than control and other groups (P < 0.01). Other groups had no effect on the ALP activity. Alizarin Bordeaux staining result showed the amounts of calcified nodules 1.0 h, 1.5 h and 2.0 h higher than control groups. The SEMFs at 50 Hz, 1.8 mT different time exposure groups inhibits the proliferation of OB, but they enhances the maturation and mineralization of the OB and SEMFs at 1.8 mT of the 1.5 h has the strongest activity.

Advancing application of mesenchymal stem cell-based bone tissue regeneration.

Reconstruction of bone defects, especially the critical-sized defects, with mechanical integrity to the skeleton is important for a patient's rehabilitation, however, it still remains challenge. Utilizing biomaterials of human origin bone tissue for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural bone tissue with regard to its properties. However, not only efficacious and safe but also cost-effective and convenient are important for regenerative biomaterials to achieve clinical translation and commercial success. Advances in our understanding of regenerative biomaterials and their roles in new bone formation potentially opened a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multicomponent construction of native extracellular matrix (ECM) for cell accommodation, the ECM-mimicking biomaterials and the naturally decellularized ECM scaffolds were used to create new tissues for bone restoration. On the other hand, with the going deep in understanding of mesenchymal stem cells (MSCs), they have shown great promise to jumpstart and facilitate bone healing even in diseased microenvironments with pharmacology-based endogenous MSCs rescue/mobilization, systemic/local infusion of MSCs for cytotherapy, biomaterials-based approaches, cell-sheets/-aggregates technology and usage of subcellular vesicles of MSCs to achieve scaffolds-free or cell-free delivery system, all of them have been shown can improve MSCs-mediated regeneration in preclinical studies and several clinical trials. Here, following an overview discussed autogenous/allogenic and ECM-based bone biomaterials for reconstructive surgery and applications of MSCs-mediated bone healing and tissue engineering to further offer principles and effective strategies to optimize MSCs-based bone regeneration.

[Icariin promotes osteogenic differentiation of rat bone marrow stromal cells in vitro].

OBJECTIVE: To investigate the effects of icariin on the osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). METHOD: rBMSCs were cultured by adherence screening method. Icariin was supplemented into the culture at 1 x 10(-5) mol x L(-1). The osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-F(ALP) and mineralized bone modulus were compared between the icariin-supplemented group and the control group. Total RNA was isolated and the gene expression of bFGF, IGF-1, Osterix(OSX) and Runx-2 was investigated by RT Real-time PCR. RESULT: Icariin significantly improved ALP activity, CFU-F(ALP) amounts and mineralized modulus. It also can enhance the mRNA level of bFGF, IGF-1, Osterix and Runx-2. CONCLUSION: Icariin enhances the osteogenic differentiation of rBMSCs significantly, which suggested that icariin has the potentiality to be a new drug of anti-osteoporosis or fracture healing.

[Comparative study on the osteogenic differentiation of rat bone marrow stromal cells effected by icariin and icariside II].

OBJECTIVE: To investigate the effects of icariin and it's main metabolites-icariside II on the osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). METHODS: rBMSCs were cultured by adherence screening method, icariin and icariside II were supplemented into the culture at 5 x 10(-5) mol/L respectively. The osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-F(ALp), osteocalcin secretion, calcium deposition and mineralized bone modulus were compared among the icariin-supplemented group, icariside II and the control. The gene expressions of bFGF, IGF-1, Osterix and Runx-2 were examined by RT-Real Time PCR. RESULTS: Both icariside II and icariin significantly improved ALP activity, CFU-F(ALP) amount, osteocalcin secretion, calcium deposition and mineralized modulus. Besides, they enhanced the gene expressions of bFGF, IGF-1, Osterix and Runx-2. Icariside II was obviously stronger than icariin at the above activities. CONCLUSION: Icariside II is stronger than icariin at enhancing the osteogenic differentiation of rBMSCs, suggesting that icariin can be administered via oral and it's metabolites are the effective constitutes for antiosteoporosis activity.

Epithelial defect repair in the auricle and auditory meatus by grafting with cultured adipose-derived mesenchymal stem cell aggregate-extracellular matrix.

BACKGROUND: Several patients experience persistent otorrhea after a flawless surgical procedure because of insufficient epithelial healing. Several efforts, such as autologous tissue allograft and xenograft, have been made to halt otorrhea. However, a stable technology to induce temporal epithelial repair is yet to be established. Therefore, this study aims to investigate whether implantation of seeding adipose-derived mesenchymal stem cell (ADMSC) aggregates on extracellular matrix (ECM; herein, ADMSC aggregate-ECM) into damaged skin wound promotes skin regeneration. METHODS: ADMSC aggregate-ECM was prepared using a previously described procedure that isolated ADMSCs from rabbits and applied to the auricle and auditory meatus wound beds of New Zealand white rabbits. Wound healing was assessed by general observation and hematoxylin and eosin (H&E) staining. Secretion of growth factor of the tissue was evaluated by western blotting. Two other groups, namely, ECM and control, were used. Comparisons of three groups were conducted by one-way analysis of variance analysis. RESULTS: ADMSCs adhered tightly to the ECM and quickly formed cell sheets. At 2 weeks, general observation and H&E staining indicated that the wound healing rates in the ADMSC aggregate-ECM (69.02 ±â€Š6.36%) and ECM (59.32 ±â€Š4.10%) groups were higher than that in the control group (43.74 ±â€Š12.15%; P = 0.005, P < 0.001, respectively) in ear auricle excisional wounds. At 7 weeks, The scar elevation index was evidently reduced in the ADMSC aggregate-ECM (2.08 ±â€Š0.87) and ECM (2.31 ±â€Š0.33) groups compared with the control group (4.06 ±â€Š0.45; P < 0.001, P < 0.001, respectively). In addition, the scar elevation index of the ADMSC aggregate-ECM group reached the lowest rate 4 weeks in advance. In auditory meatus excisional wounds, the ADMSC aggregate-ECM group had the largest range of normal skin-like structure at 4 weeks. The ADMSC aggregate-ECM and ECM groups secreted increased amounts of growth factors that contributed to skin regeneration at weeks 1 and 2, respectively. CONCLUSIONS: ADMSC aggregate-ECM and ECM are effective repair materials for wound healing, especially ADMSC aggregate-ECM. This approach will provide a meaningful experimental basis for mastoid epithelium repair in subsequent clinical trials.

Activation of the Wnt/ß-Catenin Pathway by an Inflammatory Microenvironment Affects the Myogenic Differentiation Capacity of Human Laryngeal Mucosa Mesenchymal Stromal Cells.

Various microenvironments influence the multiple differentiation potential of mesenchymal stromal cells. For example, inflammatory microenvironment can suppress the myogenic differentiation capability of laryngeal mucosa mesenchymal stromal cells (LM-MSCs). The present study therefore sought to identify the underlying molecular mechanisms regulating these processes. We isolated a novel population of MSCs, LM-MSCs, from the laryngeal mucosa tissues. The cells were cultured in osteogenic, adipogenic, and myogenic differentiation media in the presence or absence of interleukin-1ß and tumor necrosis factor α (to simulate inflammatory microenvironment). The expression of active ß-catenin, p-GSK3ß, and GSK3ß were detected by western blot and real-time polymerase chain reaction. The myogenic differentiation of LM-MSCs in inflammatory microenvironment and the regulation by Dickkopf-1 (DKK1) were tested both in vivo and in vitro. Inflammatory microenvironment could suppress the osteogenesis, adipogenesis, and myogenesis of LM-MSCs. The Wnt/ß-catenin signaling pathway was activated during myogenesis in inflammatory microenvironment. The suppressed myogenic differentiation capability of LM-MSCs in inflammatory microenvironment was reversed by DKK1. By regulating the Wnt/ß-catenin signaling pathway, DKK1 can improve the myogenic differentiation of LM-MSCs in inflammatory microenvironment. Thus, the results of this study may help improve the efficacy of LM-MSCs injection therapy for vocal fold regeneration.

Liver extracellular matrix promotes BM-MSCs hepatic differentiation and reversal of liver fibrosis through activation of integrin pathway.

In cell-based therapies for liver injuries, the clinical outcomes are closely related to the surrounding microenvironment of the transplanted bone marrow mesenchymal stem cells (BM-MSCs). However, whether liver-specific ECM (L-ECM), as one of major microenvironment signals, could regulate the therapeutic effect of BM-MSCs through changing their biological characteristics is unclear. This study aimed to investigate the hepatogenicity and underlying mechanism of L-ECM as well as its potential regulative role in the MSC-based liver recovery. L-ECM was prepared by homogenization of decellularized whole porcine liver. After three-dimensional culture with or without the presence of L-ECM, BM-MSCs expressed hepatocyte-specific genes and proteins in an L-ECM concentration-dependent manner. Further analysis showed that L-ECM could activate specific types of integrins (ITGs) as well as their downstream signalling pathways. When the cell/ECM interaction was enhanced by incorporating BM-MSCs with Mn2+ , ITGs were activated and the hepatogenic capacity of L-ECM was improved. The regeneration of rat livers from either acute or chronic fibrosis could also be accelerated after transplantation of Mn2+ -treated BM-MSCs. L-ECM therefore promotes hepatic differentiation of BM-MSCs via the ITG pathway and plays a therapeutically beneficial role for stem cell-based liver regeneration. Copyright © 2016 John Wiley & Sons, Ltd.

Human Umbilical Cord MSCs as New Cell Sources for Promoting Periodontal Regeneration in Inflammatory Periodontal Defect.

Human periodontal ligament stem cells (hPDLSCs) transplantation represents a promising approach for periodontal regeneration; however, the cell source is limited due to the invasive procedure required for cell isolation. As human umbilical cord mesenchymal stem cells (hUCMSCs) can be harvested inexpensively and inexhaustibly, here we evaluated the regenerative potentials of hUCMSCs as compared with hPDLSCs to determine whether hUCMSCs could be used as new cell sources for periodontal regeneration. Methods The characteristics of hUCMSCs, including multi-differentiation ability and anti-inflammatory capability, were determined by comparison with hPDLSCs. We constructed cell aggregates (CA) using hUCMSCs and hPDLSCs respectively. Then hPDLSCs-CA and hUCMSCs-CA were combined with ß-tricalcium phosphate bioceramic (ß-TCP) respectively and their regenerative potentials were determined in a rat inflammatory periodontal defect model. Results hPDLSCs showed higher osteogenic differentiation potentials than hUCMSCs. Meanwhile, hUCMSCs showed higher extracellular matrix secretion and anti-inflammatory abilities than hPDLSCs. Similar to hPDLSCs, hUCMSCs were able to contribute to regeneration of both soft and hard periodontal tissues under inflammatory periodontitis condition. There were more newly formed bone and periodontal ligaments in hPDLSCs and hUCMSCs groups than in non-cell treated group. Moreover, no significant differences of regenerative promoting effects between hPDLSCs and hUCMSCs were found. Conclusion: hUCMSCs generated similar promoting effects on periodontal regeneration compared with hPDLSCs, and can be used as new cell sources for periodontal regeneration.