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.

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.

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.].

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.

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.

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.

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.

Apocynin suppression of NADPH oxidase reverses the aging process in mesenchymal stem cells to promote osteogenesis and increase bone mass.

Because of the reduced potential for osteogenesis in aging bone marrow stromal cells, the balance of bone metabolism becomes disrupted, leading to various bone diseases. An increase in reactive oxygen species has been determined to be one of the key factors that accelerates the aging process in BMSCs. In these cells, increased expression of NADPH oxidases is the major source of ROS. In the current study, we suppressed the expression of NOX using apocynin, an effective antioxidant and free radical scavenger, and the results showed that aging BMSCs exhibited an enhanced potential for osteogenesis. The expression of potential key targets influencing this reversal was evaluated using qRT-PCR, and the expression of p53 was shown to be reduced with the suppression of NOX. We speculate that this may be one of the major reasons for the reversal of the aging process. We also examined the effect of apocynin in vivo, and the results showed that in SAMP6 mice, bone mineral density and total bone volume were increased after 3 months of apocynin treatment. In conclusion, our results demonstrate that in aging BMSCs, suppression of NADPH oxidase by apocynin partially reverses the aging process and enhances osteogenic potential.

Bone marrow mesenchymal stem cell aggregate: an optimal cell therapy for full-layer cutaneous wound vascularization and regeneration.

Cutaneous wounds are among the most common soft tissue injuries. Wounds involving dermis suffer more from outside influence and higher risk of chronic inflammation. Therefore the appearance and function restoration has become an imperative in tissue engineering research. In this study, cell-aggregates constructed with green fluorescent protein-expressing (GFP(+)) rat bone marrow mesenchymal stem cells (BMMSCs) were applied to rat acute full-layer cutaneous wound model to confirm its pro-regeneration ability and compare its regenerative efficacy with the currently thriving subcutaneous and intravenous stem cell administration strategy, with a view to sensing the advantages, disadvantages and the mechanism behind. According to results, cell-aggregates cultured in vitro enjoyed higher expression of several pro-healing genes than adherent cultured cells. Animal experiments showed better vascularization along with more regular dermal collagen deposition for cell-aggregate transplanted models. Immunofluorescence staining on inflammatory cells indicated a shorter inflammatory phase for cell-aggregate group, which was backed up by further RT-PCR. The in situ immunofluorescence staining manifested a higher GFP(+)-cell engraftment for cell-aggregate transplanted models versus cell administered ones. Thus it is safe to say the BMMSCs aggregate could bring superior cutaneous regeneration for full layer cutaneous wound to BMMSCs administration, both intravenous and subcutaneous.

Licochalcone A up-regulates of FasL in mesenchymal stem cells to strengthen bone formation and increase bone mass.

The role of bone marrow-derived mesenchymal stem cells(BMSCs)in the pathogenesis and therapy of osteoporosis has drawn increasing attention in recent years. In the development of osteoporosis, it has been demonstrated that many changes occurred in the behavior of BMSCs. For example, the biological system of FasL pathways mediated differentiation of ERK and GSK-3ß-catenin pathway was damaged. Here we found that 0.35 mg/L Licochalcone A (L-A) had a strong effect in increasing the osteogenic differentiation and mineralization of BMSCs both in vivo and in vitro by up-regulating FasL and further playing a role in regulating the ERK and GSK-3ß-catenin systems. It has also demonstrated that the administration of L-A could restore the biological function of the damaged BMSCs differentiation by recovering or protecting bone mass in a disease state through activating the endosteal bone formation and partially inhibiting bone resorption in acute estrogen deficiency model. Results of our study suggested that careful titration of MSC was response to L-A and up-regulated FasL pathways mediating differentiation of ERK and GSK-3ß-catenin biological systems under disease state in vivo, restore the impaired function, is one of the ways of L-A relieve or treatment osteoporosis.

[Myogenic differentiation of laryngeal mucosal mesenchymal stem cells].

OBJECTIVE: To investigate the myogenic differentiation of laryngeal mucosal mesenchymal stem cells (LM-MSCs) and the possibility of LM-MSCs as new alternative seed cells for laryngeal tissue engineering. METHODS: LM-MSCs were separated from normal epiglottis mucosa and the cell surface markers including CD44, CD105, CD90, CD29, CD34 and CD45 were analyzed through flow cytometry. The osteogenesis and adipogenesis differentiation of LM-MSCs were investigated by oil red staining and alizarin red S staining. Immunofluorescence staining and RT-PCR were used to detect the expressions of myogenic differentiation markers including Myod1, Myogenin and myosin heavy chain (MyHc). RESULTS: The separated LM-MSCs were in a fibrocyte-like form with long fusiform shape and grew adherent. The expression rates of cell surface markers LM-MSCs were CD44 (100.0%), CD105 (90.4%), CD90(99.9%), CD29 (93.0%), CD34 (0.4%) and CD45(1.3%) respectively. A number of beaded lipid drops and mineral deposition were observed after 14 days of adipogenesis differentiation and 21 days of osteogenesis differentiation. Myod1, Myogenin and MyHc genes appeared after 1 week and 3 weeks of myogenesis differentiation respectively. CONCLUSIONS: The LM-MSCs have the properties of mesenchymal stem cells and could be differentiated into myoblasts, providing with the possibility to repair the damaged vocal cords with LM-MSCs through tissue engineering techniques.

The effect of licochalcone A on cell-aggregates ECM secretion and osteogenic differentiation during bone formation in metaphyseal defects in ovariectomized rats.

Treatment of weight-bearing bones fractures with defects is critical for patients with osteoporosis's rehabilitation. Although various tissue engineering methods were reported, the best treating strategy for tissue engineering remains to be identified as the limitation of enhancing the ability of the osteogenetic differentiation potential of seed cell is one of the cardinal issues to be solved. The objective of this study is to investigate the feasibility of applying licochalcone-A (L-A) and bone marrow mesenchymal stem cells (BMSC)-aggregate in bone repairing tissue engineering and further study the biological effects of L-A on the cell aggregate formation and osteogenic properties. 80 female Sprague Dawley rats underwent bilateral ovariectomy were made with a 3.5 mm femurs bone defects without any fixation. These rats were then randomly assigned to five different treatment groups: (1) empty defect (n = 16), (2) CA-LA (n = 16), (3) CA-N (n = 16), (4) CA-L (n = 16), (5) CA-S (n = 16) and 16 female SD rats were treated as a control. Data showed that L-A administrated cell aggregate had a stronger osteogenic differentiation and mineralized formation potential than non-administrated group both in vitro and in vivo. For in vitro study, L-A administrated group had a more significant expression of ECM, osteogenic associated maker in addition with more mineralized area and higher ALP activity compared with the control group. For in vivo study, 3D reconstruction of micro-CT, HE staining and bone strength results showed that newly formed bone in groups administrated by L-A was significant higher than that in Sham group at 2, 4, 8 and 12 weeks after transplantation, especially for groups which was systematically injected with L-A at 8 weeks. Results of our study demonstrated that LA could positively affect cell behavior in cell-aggregate engineering and could be a promising strategy in treating osteoporotic weight-bearing bones fractures with defects.

Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects.

Reconstruction of large area bone defect with mechanical integrity to the skeleton is important for patient's rehabilitation. However with the limitation of scaffold material and suitable seed cell sources, the best treating strategy remains to be identified though various tissue engineering methods were reported. In this study, we investigated the feasibility of applying calcined bovine bone (CBB) which was coated by allograft bone marrow mesenchymal stem cells (BMSC)-sheet as a 3D scaffold material in bone repairing tissue engineering. The new scaffold material was implanted into osteoporosis rat cranial bone defects and repairing critical size bone defects (8 mm diameter). Data showed that CBB-BMSC-sheet combination had a stronger potential in osteogenic differentiation and mineralized formation both in vitro and in vivo than CBB-BMSC combination. In in vitro study BMSC-sheet had a more feasible characteristic upon bone repairing including richer ECM, larger mineralized area and stronger ALP activity in addition with a significant higher mRNA expression of osteogenic maker such as BMP-2, b-FGF, Col 1a1, OSX and Runx-2 than the control group. In in vivo study 3D reconstruction of micro CT, HE staining and bone strength results showed that newly formed bone in CBB-BMSC-sheet group was significant higher than that in CBB-BMSC group at 4, 8 and 12 weeks after transplantation in the aspect of area and volume. What was more, results indicated that allograft BMSC-sheet had survivaled in the scaffold material and participated in the newly formed bone which had the same thickness with surrounding autologous bone tissues after transplantation. Results of our study demonstrated that CBB-BMSC-sheet combination was a promising strategy in healing of large area bone defect in osteoporosis.

The use of laryngeal mucosa mesenchymal stem cells for the repair the vocal fold injury.

Stem cell transplantation is a kind of attractive and new approach that complements traditional restorative or surgical techniques for the regeneration of injured or pathologically damaged laryngeal tissues. However, the best cell delivery strategy remains to be identified. The objective of this study was to establish a new strategy to the healing of injured vocal fold, using laryngeal mucosa mesenchymal stem cells differentiating into myofibroblasts or fibroblasts and improving the reconstruction microenvironment in the vocal fold injury as a new alternative as seed cells for laryngeal tissue engineering. After isolation and expansion, cells were identified as adherent mesenchymal cells with substantial proliferation potential in vitro, and were also characterized by flow cytometry. The differentiation potential of mesenchymal cells was maintained during proliferation as confirmed by culturing for adipogenesis, osteogenesis and chondrocyte. When LM-MSC was transplanted into the injured vocal fold, it has the potent differentiated into myofibroblasts and fibroblasts, which could regulate extracellular matrix, block collagen and the fibronectin rapid increased, inhibit the rapidly decrease of elastic fiber and HA, decrease the microenvironment inflammatory reaction, and prevent the formation of vocal fold scar.

The prenyl group contributes to activities of phytoestrogen 8-prenynaringenin in enhancing bone formation and inhibiting bone resorption in vitro.

Previous studies have found that 8-prenylflavonoids have a higher osteogenic activity than do flavonoids, which suggested that the 8-prenyl group may play an active role in bone-protective properties. To address this hypothesis, activities of 8-prenylnaringenin (PNG) and naringenin (NG) in osteoblast and osteoclast differentiation and function were compared in vitro. PNG was found to have a stronger ability than NG to improve osteoblast differentiation and osteogenic function in cultured rat calvarial osteoblasts, as demonstrated by levels of alkaline phosphatase activity, osteocalcin, calcium deposition, and the number and area of mineralized bone nodules, as well as mRNA expression of osteogenesis-related genes Bmp-2, OSX, and Runx-2. In addition, although expression of osteoclastogenic inducer receptor activator of nuclear factor kappa-B ligand (RANKL) was not affected, that of osteoclastogenesis inhibitor osteoprotegerin (OPG) and consequently the OPG/RANKL ratio were increased, more potently by PNG than NG. PNG was also found to have a higher potency than NG in inhibiting the osteoclast formation in rabbit bone marrow cells and their resorptive activity, as revealed by lower numbers of osteoclasts formed, lower numbers and areas of bone resorption pits, and lower mRNA expression levels of tartrate-resistant acid phosphatase and cathepsin K. Furthermore, PNG induced apoptosis of mature osteoclasts at a higher degree and at an earlier time than did NG. These results indicate that the 8-prenyl group plays an important role and contributes to the higher bone-protective activity of PNG in comparison with NG.

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.

[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.

[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.