Shear Flow-Assembled Janus Membrane with Bifunctional Osteogenic and Antibacterial Effects for Guided Bone Regeneration.

Guided bone regeneration (GBR) membranes that reside at the interface between the bone and soft tissues for bone repair attract increasing attention, but currently developed GBR membranes suffer from relatively poor osteogenic and antibacterial effects as well as limited mechanical property and biodegradability. We present here the design and fabrication of a bifunctional Janus GBR membrane based on a shear flow-driven layer by a layer self-assembly approach. The Janus GBR membrane comprises a calcium phosphate-collagen/polyethylene glycol (CaP@COL/PEG) layer and a chitosan/poly(acrylic acid) (CHI/PAA) layer on different sides of a collagen membrane to form a sandwich structure. The membrane exhibits good mechanical stability and tailored biodegradability. It is found that the CaP@COL/PEG layer and CHI/PAA layer contribute to the osteogenic differentiation and antibacterial function, respectively. In comparison with the control group, the Janus GBR membrane displays a 2.52-time and 1.84-time enhancement in respective volume and density of newly generated bone. The greatly improved bone repair ability of the Janus GBR membrane is further confirmed through histological analysis, and it has great potential for practical applications in bone tissue engineering.

Surface demineralized freeze-dried bone allograft followed by reimplantation in a failed mandibular dental implant.

The removal of a failed implant with high torque causes significant damage to the surrounding tissue, compromising bone regeneration and subsequent osseointegration in the defect area. Here, we report a case of carrier screw fracture followed by immediate implant removal, bone grafting and delayed reimplantation. A dental implant with a fractured central carrier screw was removed using the bur-forceps technique. The resulting three-wall bone defect was filled with granular surface demineralized freeze-dried bone allograft (SD-FDBA). Cone-beam computerized tomography was performed at 1 week, 6 months and 15 months postoperatively and standardized for quantitative evaluation. The alveolar bone width and height at 15 months post-surgery were about 91% of the original values, with a slightly lower bone density, calculated using the gray value ratio. The graft site was reopened and was found to be completely healed with dense and vascularized bone along with some residual bone graft. Reimplantation followed by restoration was performed 8 months later. The quality of regenerated bone following SD-FDBA grafting was adequate for osseointegration and long-term implant success. The excellent osteogenic properties of SD-FDBA are attributed to its human origin, cortical bone-like structure, partly demineralized surfaces and bone morphogenetic protein-2-containing nature. Further investigation with more cases and longer follow-up was required to confirm the final clinical effect.

Effective Scald Wound Functional Recovery Patch Achieved by Molecularly Intertwined Electrical and Chemical Message in Self-Adhesive Assemblies.

Bioactive materials that communicate with bio-tissues via simultaneous chemical and electrical information promise an advanced medical treatment strategy. Rational design of simultaneous chemically and electrically active materials is still challenging. In this study, we develop a bioactive wound healing patch that enables functional recovery of scald skin wounds by integrating electrically and chemically active units at the molecular level. The patch should be used with massages for 10 min daily during the recovery process. This healing patch consists of a closely intertwined piezoelectric poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF) film and a self-adhesive poly(N,N-dimethylacrylamide) (PDMAA) hydrogel layer, which permits itself to adhere on skin wounds reversibly. Frequency-dependent electrical and chemical dose delivery is achieved in response to mechanical stimuli via the electrical-chemical crosstalk within the healing patch. Animal scald experiments show that the patch can effectively guide the functional recovery of grade I and shallow grade II scald wounds, promoting proper collagen deposition and hair follicle, blood vessel, and gland regeneration. Integrating electrically and chemically active units at the molecular level in treatment devices provides a new design concept for tissue engineering and medical treatment materials.

Effect of local application of bone morphogenetic protein -2 on experimental tooth movement and biological remodeling in rats.

Background: This study attempts to detect the potential effects of local bone morphogenetic protein -2 (BMP-2) on orthodontic tooth movement and periodontal tissue remodeling. Methods: Forty adult SD rats were randomly divided into four groups: blank control group, unilateral injection of BMP-2 on the pressure side or tension side of orthodontic teeth and bilateral injection of BMP-2. Their maxillary first molar was moved by a 30 g constant force closed coil spring. 60 µL of BMP-2 with a concentration of 0.5 µg/mL was injected into each part at a time. In addition, three rats were selected as healthy control rats without any intervention. Fluorescent labeled BMP-2 was used to observe the distribution of exogenous BMP-2 in tissues. Micro-CT was used to measure the microscopic parameters of tooth displacement, trabecular bone and root absorption volume. Three different histological methods were used to observe the changes of tissue remodeling, and then the number of osteoclasts and the content of collagen fibers were calculated. Results: Compared with the blank control group, BMP-2 injection reduced the movement distance and increased the collagen fiber content and bone mass (p < 0.01). There was no significant difference in tooth movement distance, BV/TV ratio and BMD between injection sites in unilateral injection group (p > 0.05). In the case of bilateral injection of BMP-2, the osteogenesis is enhanced. Unilateral injection of BMP-2 did not promote root resorption, but double injection showed root resorption (p < 0.01). Conclusion: Our study does show that the osteogenesis of BMP-2 is dose-dependent rather than site-dependent when a certain amount of BMP-2 is applied around orthodontic teeth. Local application of BMP-2 around orthodontic teeth in an appropriate way can enhance bone mass and tooth anchorage without increasing the risk of root absorption volume. However, high levels of BMP-2 may cause aggressive root resorption. These findings are of great significance, that is, BMP-2 is an effective target for regulating orthodontic tooth movement.

Construction of artificial periosteum with methacrylamide gelatin hydrogel-wharton's jelly based on stem cell recruitment and its application in bone tissue engineering.

The presence of periosteum can greatly affect the repair of a bone fracture. An artificial periosteum imitates the biological function of natural periosteum, which is capable of protecting and maintaining bone tissue structure and promoting bone repair. In our artificial periosteum, biocompatible methacrylate gelatin was used to provide the mechanical support of the membrane, E7 peptide added bioactivity, and Wharton's jelly provided the biological activity support of the membrane, resulting in a hydrogel membrane (G-E-W) for the chemotactic recruitment of bone marrow mesenchymal stem cells (BMSCs) and promoting cell proliferation and osteogenic differentiation. In an in vitro experiment, the G-E-W membrane recruited BMSCs and promoted cell proliferation and osteogenic differentiation. After 4 weeks and 8 weeks of implantation in a rat skull defect, the group implanted with a G-E-W membrane was superior to the blank control group and single-component membrane group in both quantity and quality of new bone. The artificial G-E-W membrane recruits BMSC chemotaxis and promotes cell proliferation and osteogenic differentiation, thereby effectively improving the repair efficiency of fractures and bone defects.

Biomineralized Piezoelectrically Active Scaffolds for Inducing Osteogenic Differentiation.

There is an endogenous electric field in living organisms, which plays a vital role in the development and regeneration of bone tissue. Therefore, self-powered piezoelectric material for bone repair has become hot research in recent years. However, the current piezoelectric materials for tissue regeneration still have the shortcomings of lack of biological activity and three-dimensional structure. Here, we proposed a three-dimensional polyurethane foam (PUF) scaffold coated with piezoelectric poly (vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) and modified by a calcium phosphate (CaP) mineralized coating. The preferred scaffold has an open circuit voltage and short circuit current output of 5 V and 200 nA. Combining the physical and chemical properties of the CaP coating, the piezoelectric signal of PVDF-HFP and the three-dimensional structure of PUF, the scaffold exhibits superior promotion of cell osteogenic differentiation and ectopic bone formation in vivo. The mechanism is attributed to an increase in intracellular Ca2+ levels in response to chemical and piezoelectric stimulation with the material. This research not only paves the way for the application of piezoelectric scaffolds to stimulate osteoblasts differentiation in situ, but also lays the foundation for the clinical treatment of long-term osteoporosis.

Comparison of osteogenesis of bovine bone xenografts between true bone ceramics and decalcified bone matrix.

Xenograft bone scaffolds have certain advantages such as mechanical strength, osteoinductive properties, sufficient source and safety. This study aimed to compare osteogenesis of the two main bovine bone xenografts namely true bone ceramics (TBC) and decalcified bone matrix (DBM), and TBC or DBM combined with bone morphogenetic protein (BMP)-2 (TBC&BMP-2 and DBM&BMP-2). The characteristics of TBC and DBM were investigated by observing the appearance and scanning electron microscopic images, examining mechanical strength, evaluating cytotoxicity and detecting BMP-2 release after being combined with BMP-2 in vitro. The femoral condyle defect and radial defect models were successively established to evaluate the performance of the proposed scaffolds in repairing cortical and cancellous bone defects. General observation, hematoxylin and eosin (HE) staining, mirco-CT scanning, calcein double labeling, X-ray film observation, three-point bending test in vivo were then performed. It indicated that the repair with xenograft bone scaffolds of 8 weeks were needed and the repair results were better than those of 4 weeks whatever the type of defects. To femoral condyle defect, TBC and TBC&BMP-2 were better than DBM and DBM&BMP-2, and TBC&BMP-2 was better than TBC alone; to radial defect, DBM and DBM&BMP-2 were better than TBC and TBC&BMP-2, and DBM&BMP-2 was better than DBM alone. This study has shown that TBC and DBM xenograft scaffolds can be more suitable for the repair of cancellous bone and cortical bone defects for 8 weeks in rats, respectively. We also have exhibited the use of BMP-2 in combination with DBM or TBC provides the possibility to treat bone defects more effectively. We thus believe that we probably need to select the more suitable scaffold according to bone defect types, and both TBC and DBM are promising xenograft materials for bone tissue engineering and regenerative medicine. Graphical abstract.

A Shapable Alginate Hydrogel Resolving the Conflicts between Multifunctionality and Fabrication Simplicity.

Alginate is a naturally derived biocompatible polymer widely used as a drug or food adjuvant. However, its usage as a biofunctional material has been confounded by the lack of shapable strategies. In this study, we report an easily applied ionic cross-linking strategy for fabricating shapable multifunctional SA-Ca(II) hydrogels employing the process of regulated diffusion. The fabrication proceeds in neutral solutions under ambient conditions. The obtained SA-Ca(II) hydrogel presents tunable moduli ranging from 4 to 30 kPa, resembling a series of human tissues. The tunable mechanical strength provides differentiation signals for stem cell polarization. The hydrogel film can lift a weight of 10 kg. The hydrogel can be prepared into various shapes and remains stable over one year upon rinsing in deionized water, but rapidly degrades in alginate lyase solutions. Subcutaneously embedded SA-Ca(II) hydrogels in mice show high biocompatibility and degrade over 4 weeks accompanied by hair follicle regeneration. Wearable protections as well as stimuli-responsive electronic circuits are then achieved, which not only protect the model body against high-temperature environments but also show warning signals when the protection loses effectiveness because of high temperatures. Overall, these results demonstrate that our SA-Ca(II) hydrogel offers appealing comprehensive functionalities from multifaceted perspectives, including mechanical strength, economic and environmental considerations, transparency, forming capability, biocompatibility, and conductivity.

Biological evaluation of acellular bovine bone matrix treated with NaOH.

We mainly proceed from the view of biological effect to study the acellular bovine bone matrix (ABBM) by the low concentration of hydrogen oxidation. After cleaning the bovine bone routinely, it was cleaned with different concentrations of NaOH and stained with hematoxylin-eosin (HE) to observe the effect of decellulization. The effect of bovine bone matrix treated with NaOH were observed by optical microscopy and scanning electron microscopy (SEM), and compared by DNA residue detection. Cell toxicity was also evaluated in MC3T3-E1 cells by CCK-8. For the in vitro osteogenesis detection, alkaline phosphatase (ALP) staining and alizarin red (AR) staining were performed in MC3T3-E1 cells. And the in vivo experiment, Micro CT, HE and Masson staining were used to observe whether the osteogenic effect of the materials treated with 1% NaOH solution was affected at 6 and 12 weeks. After the bovine bone was decellularized with different concentrations of NaOH solution, HE staining showed that ultrasonic cleaning with 1% NaOH solution for 30 min had the best effect of decellularization. The SEM showed that ABBM treated with 1% NaOH solution had few residual cells on the surface of the three-dimensional porous compared to ABBM treated with conventional chemical reagents. DNA residues and cytotoxicity of ABBM treated with 1% NaOH were both reduced. The results of ALP staining and AR staining showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis effect. The results of micro-CT, HE staining and Masson staining in animal experiments also showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis ability. The decellularization treatment of ABBM with the low concentration of NaOH can be more cost-effective, effectively remove the residual cellular components, without affecting the osteogenic ability. Our work may provide a novelty thought and a modified method to applicate the acellular bovine bone matrix clinically better. Graphical abstract.

A healing promoting wound dressing with tailor-made antibacterial potency employing piezocatalytic processes in multi-functional nanocomposites.

Developing a novel antibiotics-free antibacterial strategy is essential for minimizing bacterial resistance. Materials that not only kill bacteria but also promote tissue healing are especially challenging to achieve. Inspired by chemical conversion processes in living organisms, we develop a piezoelectrically active antibacterial device that converts ambient O2 and H2O to ROS by piezocatalytic processes. The device is achieved by mounting nanoscopic polypyrrole/carbon nanotube catalyst multilayers onto piezoelectric-dielectric films. Under stimuli by a hand-held massage device, the sterilizing rates for S. aureus and E. coli reach 84.11% and 94.85% after 10 minutes of operation, respectively. The antibacterial substrate at the same time preserves and releases drugs and presents negligible cytotoxicity. Animal experiments demonstrate that daily treatment for 10 minutes using the device effectively accelerates the healing of infected wounds on the backs of mice, promoting hair follicle generation and collagen deposition. We believe that this report provides a novel design approach for antibacterial strategies in medical treatment.

Bone induction and defect repair by true bone ceramics incorporated with rhBMP-2 and Sr.

OBJECTIVE: To study the bone induction and defect repair of true bone ceramics (TBC) combined with rhBMP-2 and Sr. METHODS: MC3T3-E1 cells were used to evaluate the bioactivity of the composite. Cell proliferation activity was detected by CCK-8, ALP activity was detected by p-nitrophenyl phosphate (PNPP), and the differences of material surface topography were observed by scanning electron microscopy (SEM). Bone induction was verified by the implantation in nude mice. The rabbit femoral condyle defect model was achieved to verify the bone defect repair ability of the material. RESULTS: SEM results showed nearly the same surface morphology and cell proliferation quantified by CCK-8 showed that compared with TBC, both TBC&Sr and TBC&BMP-2&Sr had a significant promoting effect (P < 0.05). ALP activity result showed that the ALP activity of TBC&BMP-2&Sr was significantly higher than that of TBC alone (P < 0.05). The bone induction result showed that TBC&Sr had a small amount of new bone formation, and the new bone area was only 2.5 ± 0.11%. The bone induction activity of TBC&BMP-2&Sr was the highest, the new bone area was up to 75.36 ± 4.21%. Histological result of bone defect repair showed that TBC&BMP-2&Sr was also the highest, the new bone area was up to 72.42 ± 3.14%. The repair effect of TBC& BMP-2 was second, and better than that of TBC&Sr. CONCLUSION: TBC combined with rhBMP-2 and Sr had the good bioactivity, obvious bone conduction and bone defect repair performance, laying the foundation of clinical application potentially.

The construction of a novel xenograft bovine bone scaffold, (DSS)6-liposome/CKIP-1 siRNA/calcine bone and its osteogenesis evaluation on skull defect in rats.

BACKGROUND: Xenograft bone scaffolds have advantages such as mechanical strength, sufficient source and safety. Combined with siRNA properly targeting CKIP-1, a negative regulator of osteogenesis, may contribute to the repair result of calcine bone alone. METHODS: Herein, we constructed a novel xenograft bovine bone scaffold namely (DSS)6-liposome/CKIP-1 siRNA/calcine bone, the characteristics of which were investigated by confirming the effect of (DSS)6-liposome, observing the appearance and testing mechanical strength of calcine bone, and observing the combined result of CKIP-1 siRNA by FAM immunofluorescence. In addition, cytotoxicity by CCK-8 and LDH activity of L929 â€‹cells and MC3T3-E1 osteoblasts cultured with the scaffold were tested in vitro, primary osteoblasts proliferation, the mRNA expressions of CKIP-1, ALP, COL1-α and OCN, the protein expressions of CKIP-1, BMP-2, COL-1 and Runx2 and calcium nodules were also determined by CCK-8, RT-qPCR, western-blot and Alizarin Red staining in vitro. Then, we successively established the skull defect model for evaluating the repair result of the novel scaffold by HE staining of 2, 4, 8 and 12 weeks, immumohistochemical stainings of 2, 4, 8 and 12 weeks such as ALP, COL-1α and OCN, Mirco-CT scanning of 4 and 12 weeks and the relative parameters and so on in vivo. RESULTS: It indicated that (DSS)6-liposome/CKIP-1 siRNA/calcine bone could successfully knock down the CKIP-1 mRNA and protein expressions, promote osteoblasts proliferation with the little cytotoxicity in vitro, increase the protein expressions of BMP-2, COL-1 and Runx2 in vitro, increase mRNA expressions of ALP, COL-1α and OCN in vitro and in vivo, and have a better bone defect repair effect with few side effects in rats after 12 weeks. CONCLUSION: Our research indicates (DSS)6-liposome/CKIP-1 siRNA/calcine bone could repair skull defects well in rats, and it may lay the foundation of applicating the novel xenograft bone scaffold in the clinical. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: These findings provide evidence that (DSS)6- liposome/CKIP-1 siRNA/calcine bone could be used as a novel xenograft bone scaffold for osteogenesis with the good safety.

Synergy effect of Sr and rhBMP-2: A potential solution to osteolysis caused by rhBMP-2.

As an approach to enhance autogenous bone grafting, the fusion rate of recombinant human bone morphogenetic protein-2 (rhBMP-2) is close to 100%, which is significantly higher than other bone graft methods. However, there are some obvious problems in applying rhBMP-2 clinically. Among them, early endplate osteolysis frequently occurs in the lumbar interbody fusion, which readily leads to cage subsidence or shift, thus influencing clinical effects. Moreover, robust bone formation activity and serious osteolysis coexist. What is the internal mechanism? How do we solve this problem? Strontium (Sr) is now widely used for the treatment of osteoporosis. It elicits a double effect in that it simultaneously enhances bone formation and inhibits bone resorption. We propose that Sr might be a solution for osteolysis induced by rhBMP-2 in spinal interbody fusion. Whether this synergistic effect leads to new metabolic pathway activation remains to be explored. Clarifying the synergistic effect and mechanism will be of great importance in improving both the osteogenic effect and reducing the dose amount of rhBMP-2, as well as corresponding costs.

GRM4 inhibits the proliferation, migration, and invasion of human osteosarcoma cells through interaction with CBX4.

In recent years, the survey of metabolic glutamate receptor 4 (GRM4) in tumor biology has been gradually concerned. There are currently few studies on GRM4 in osteosarcoma, and the biological function is not clear. Analysis of TCGA database showed that there was no substantial deviation in the expression of GRM4 between osteosarcoma and normal tissues. In the subsequent experiments, there is no significant difference in either mRNA or protein levels among immortalized human osteoblasts and various osteosarcoma cells. With the overexpression of GRM4, cell proliferation, migration and invasion were inhibited obviously. It was further revealed that GRM4 can interact with CBX4 to restrict the nuclear localization of CBX4 and affect the transcriptional activity of HIF-1α. This is the evidence supporting the interaction between GRM4 and CBX4, which could inhibit the malignant behavior of osteosarcoma cells through the GRM4/CBX4/HIF-1α signaling pathway.

Iron oxide nanoparticles in liquid or powder form enhanced osteogenesis via stem cells on injectable calcium phosphate scaffold.

The objectives of this study were to incorporate iron oxide nanoparticles (IONPs) into calcium phosphate cement (CPC) to enhance bone engineering, and to investigate the effects of IONPs as a liquid or powder on stem cells using IONP-CPC scaffold for the first time. IONP-CPCs were prepared by adding 1% IONPs as liquid or powder. Human dental pulp stem cells (hDPSCs) were seeded. Subcutaneous implantation in mice was investigated. IONP-CPCs had better cell spreading, and greater ALP activity and bone mineral synthesis, than CPC control. Subcutaneous implantation for 6 weeks showed good biocompatibility for all groups. In conclusion, incorporating IONPs in liquid or powder form both substantially enhanced hDPSCs on IONP-CPC scaffold and exhibited excellent biocompatibility. IONP incorporation as a liquid was better than IONP powder in promoting osteogenic differentiation of hDPSCs. Incorporating IONPs and chitosan lactate together in CPC enhanced osteogenesis of hDPSCs more than using either alone.

Use of polyvinylpyrrolidone-iodine solution for sterilisation and preservation improves mechanical properties and osteogenesis of allografts.

Allografts eliminate the disadvantages associated with autografts and synthetic scaffolds but are associated with a disease-transmission risk. Therefore, allograft sterilisation is crucial. We aimed to determine whether polyvinylpyrrolidone-iodine (PVP-I) can be used for sterilisation and as a new wet-preservation method. PVP-I-sterilised and preserved allografts demonstrated improved mechanical property, osteogenesis, and excellent microbial inhibition. A thigh muscle pouch model of nude mice showed that PVP-I-preserved allografts demonstrated better ectopic formation than Co60-sterilised allografts (control) in vivo (P < 0.05). Furthermore, the PVP-I-preserved group showed no difference between 24 h and 12 weeks of allograft preservation (P > 0.05). PVP-I-preserved allografts showed more hydrophilic surfaces and PVP-I-sterilised tendons showed higher mechanical strength than Co60-sterilised tendons (P < 0.05). The level of residual PVP-I was higher without washing and with prolonged preservation (P < 0.05). In vitro cellular tests showed that appropriate PVP-I concentration was nontoxic to preosteoblast cells, and cellular differentiation measured by alkaline phosphatase activity and osteogenic gene markers was enhanced (P < 0.05). Therefore, the improved biological performance of implanted allografts may be attributable to better surface properties and residual PVP-I, and PVP-I immersion can be a simple, easy method for allograft sterilisation and preservation.

The protective mechanism of Ginkgolides and Ginkgo flavonoids on the TNF-α induced apoptosis of rat hippocampal neurons and its mechanisms in vitro.

OBJECTIVE: To explore the neuroprotective mechanism of Ginkgolides or Ginkgo flavonoids on the TNF-α induced apoptosis of cultured rat hippocampal neurons. MATERIALS AND METHODS: Primary hippocampal neurons were isolated from rat brains and cultured with (model group) or without (control group) addition of tumor necrosis factor-α (TNF-α, final concentration of 40 ng/ml) to induce apoptosis. TNF-α induced cultures were divided into model group, Ginkgolides pre-treatment group (20 µg/ml) and Ginkgo flavonoids pre-treatment group (12 µg/ml). CCK8 was used to assess cell viability while Hoechst 33258 staining, Flow cytometry and TUNEL kits were all employed to determine apoptotic neurons. Expression levels of Bcl-2, Bax, Caspase-3, Caspase-7, Caspase-8, Caspase-9 and Cytc were estimated by qRT-PCR. RESULTS: Cell viability was significantly improved in Ginkgolides pre-treatment group or Ginkgo flavonoids pre-treatment group compared with that in model group. Apoptotic neurons were significantly less in Ginkgolides pre-treatment group or Ginkgo flavonoids pre-treatment group than those in model group. Transcription levels of caspase-7, caspase-8, caspase-3, caspase-9, Bax and Cytc were down-regulated, while transcription levels of Bcl-2 was up-regulated in Ginkgolides pre-treatment or Ginkgo flavonoids pre-treatment group than those in model group. CONCLUSIONS: Ginkgolides and Ginkgo flavonoids might protect against apoptosis of hippocampal neurons through inhibiting death receptor pathway or mitochondrial pathway under TNF-α background.

A cocktail method for promoting cardiomyocyte differentiation from bone marrow-derived mesenchymal stem cells.

A growing body of evidence supports the argument that bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into cardiomyocyte-like cells in an appropriate cellular environment, but the differentiation rate is low. A cocktail method was designed: we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (SalB), and cardiomyocyte lysis medium (CLM) in inducing MSCs to acquire the phenotypical characteristics of cardiomyocytes. The fourth-passage MSCs were treated with 5-aza, SalB, CLM, 5-aza+salB, 5-aza+CLM, SalB+CLM, and 5-aza+SalB+CLM for 2 weeks. Immunofluorescence results showed that cTnT expression in the 5-aza+salB+CLM group was stronger than other groups. Real-time qPCR and Western blotting analyses showed that cTnT, alpha-cardiac actin, mef-2c, Cx43, and GSK-3beta expression increased while beta-catenin expression decreased. The salB+5-aza+CLM group had the most evident effects. SalB combined with 5-aza and CLM improved cardiomyocyte differentiation from MSCs. In the MSCs differentiation process, the Wnt/beta-catenin signaling pathway had been inhibited.

Cardiomyocyte-like cells differentiation from non ß-catenin expression mesenchymal stem cells.

Recent studies have shown that block wnt/ß-catenin signaling pathway is integrant for cardiomyocytes differentiation from bone marrow mesenchymal stem cells (MSCs). By transducing the MSCs with lentivirus which contain ß-catenin interference RNA, we screened out the non ß-catenin expression clone. In the establishment of knockdown ß-catenin in MSCs, we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (salB), and cardiomyocytes lysis medium (CLM) in inducing MSCs to differentiate into cardiomyocyte-like cells. A method for culturing MSCs and cardiomyocytes was established. Purified MSCs were investigated by flow cytometry. The MSCs were positive for CD90 and CD29, but negative for CD34 and CD45. Meanwhile, the cardiomyocytes contracted spontaneously after 24 h of seeding into the plates. The fourth-passage non-ß-catenin expression MSCs were divided into eight groups: control group, 5-aza, salB, CLM, 5-aza + salB, 5-aza + CLM, salB + CLM, and 5-aza + salB + CLM. The gene and protein expression of cTnT, α-actin, ß-myosin, ß-catenin, and GSK-3ß were detected by quantitative real-time PCR and Western blotting. Our results showed that cTnT expression in 5-aza + salB + CLM group was ninefold higher than in the control group in the non-ß-catenin MSCs model, implying that cardiomyocytes differentiation from MSCs is an extremely complicated process and it is necessary to consider the internal and external environmental conditions, such as suitable pharmaceutical inducers, cardiomyocytes microenvironments, inhibition of the negative signaling pathway and so on.