Anti-cancer effects of nitazoxanide in epithelial ovarian cancer in-vitro and in-vivo.

Epithelial ovarian cancer is one of the most lethal gynecologic malignancies and poses a considerable threat to women's health. Although the progression-free survival of patients has been prolonged with the application of anti-angiogenesis drugs and Poly (ADP-ribose) polymerases (PARP) inhibitors, overall survival has not substantially improved. Thus, new therapeutic strategies are essential for the treatment of ovarian cancer. Nitazoxanide (NTZ), an FDA-approved anti-parasitic drug, has garnered attention for its potential anti-cancer activity. However, the anti-tumor effects and possible underlying mechanisms of NTZ on ovarian cancer remain unclear. In this study, we investigated the anti-tumor effects and the mechanism of NTZ on ovarian cancer in vitro and in vivo. We found that NTZ inhibited the proliferation of A2780 and SKOV3 epithelial ovarian cancer cells in a time- and concentration-dependent manner; Furthermore, NTZ suppressed the metastasis and invasion of A2780 and SKOV3 cells in vitro, correlating with the inhibition of epithelial-mesenchymal transition; Additionally, NTZ suppressed the Hippo/YAP/TAZ signaling pathway both in vitro and in vivo and demonstrated a good binding activity with core genes of Hippo pathway, including Hippo, YAP, TAZ, LATS1, and LATS2. Oral administration of NTZ inhibited tumor growth in xenograft ovarian cancer mice models without causing considerable damage to major organs. Overall, these data suggest that NTZ has therapeutic potential for treating epithelial ovarian cancer.

GPNMB promotes peripheral nerve regeneration by activating the Erk1/2 and Akt pathways via binding Na+/K+-ATPase α1 in Schwann cells.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is ubiquitously expressed and has protective effects on the central nervous system. In particular, it is also expressed in the peripheral nervous system (PNS) and upregulated after peripheral nerve injury. However, the role and underlying mechanism of GPNMB in the PNS, especially in peripheral nerve regeneration (PNR), are still unknown and need to be further investigated. In this study, recombinant human GPNMB (rhGPNMB) was injected into a sciatic nerve injury model. It was found that rhGPNMB facilitated the regeneration and functional recovery of the injured sciatic nerve in vivo. Moreover, it was also confirmed that GPNMB activated the Erk1/2 and Akt pathways via binding with Na+/K + -ATPase α1 (NKA α1) and promoted the proliferation and migration of Schwann cells (SCs) and their expression and secretion of neurotrophic factors and neural adhesion molecules in vitro. Our findings demonstrate that GPNMB facilitates PNR through activation of the Erk1/2 and Akt pathways in SCs by binding with NKA α1 and may be a novel strategy for PNR.

Biodegradable composites of poly(propylene carbonate) mixed with silicon nitride for osteogenic activity of adipose-derived stem cells and repair of bone defects.

Absorbable polymers have attracted increasing attention in the field of bone regeneration in recent years for their degradation. Compared with other degradable polymers, polypropylene carbonate (PPC) has several advantages such as biodegradation and relatively cheap raw materials. Most importantly, PPC can degrade into water and carbon dioxide totally which does not give rise to local inflammation and bone resorption in vivo. However, pure PPC has not presented excellent osteoinductivity properties. In order to enhance the osteoinductivity of PPC, silicon nitride (SiN) was employed due to its excellent mechanical properties, biocompatibility and osteogenesis compared with the other common materials such as hydroxyapatite and calcium phosphate ceramics. In this study, composites of PPC mixed with different contents of SiN were prepared successfully (PSN10 with 10 wt% SiN content, and PSN20 with 20 wt% SiN content). The characterization of the composites suggested that PPC mixed with SiN evenly and PSN composites presented stable properties. The results in vitro revealed that the PSN20 composite possessed satisfactory biocompatibility and exerted better osteogenic differentiation effects on adipose-derived stem cells (ADSCs). In particular, the PSN20 composite accelerated the healing of bone defects better and degraded with the process of bone healing in vivo. Overall, the PSN20 composite exhibited better biocompatibility, induced osteogenic differentiation of ADSCs and promoted healing of bone defects, due to which the PSN composite is considered as a potential candidate for treating bone defects in the field of bone tissue engineering.

Retinoblastoma-Binding Protein 5 Regulates H3K4 Methylation Modification to Inhibit the Proliferation of Melanoma Cells by Inactivating the Wnt/ß-Catenin and Epithelial-Mesenchymal Transition Pathways.

Histone 3 lysine 4 methylation (H3K4me), especially histone 3 lysine 4 trimethylation (H3K4me3), is one of the most extensively studied patterns of histone modification and plays crucial roles in many biological processes. However, as a part of H3K4 methyltransferase that participates in H3K4 methylation and transcriptional regulation, retinoblastoma-binding protein 5 (RBBP5) has not been well studied in melanoma. The present study sought to explore RBBP5-mediated H3K4 histone modification and the potential mechanisms in melanoma. RBBP5 expression in melanoma and nevi specimens was detected by immunohistochemistry. Western blotting was performed for three pairs of melanoma cancer tissues and nevi tissues. In vitro and in vivo assays were used to investigate the function of RBBP5. The molecular mechanism was determined using RT-qPCR, western blotting, ChIP assays, and Co-IP assays. Our study showed that RBBP5 was significantly downregulated in melanoma tissue and cells compared with nevi tissues and normal epithelia cells (P < 0.05). Reducing RBBP5 in human melanoma cells leads to H3K4me3 downregulation and promotes cell proliferation, migration, and invasion. On the one hand, we verified that WSB2 was an upstream gene of RBBP5-mediated H3K4 modification, which could directly bind to RBBP5 and negatively regulate its expression. On the other hand, we also confirmed that p16 (a cancer suppressor gene) was a downstream target of H3K4me3, the promoter of which can directly bind to H3K4me3. Mechanistically, our data revealed that RBBP5 inactivated the Wnt/ß-catenin and epithelial-mesenchymal transition (EMT) pathways (P < 0.05), leading to melanoma suppression. Histone methylation is rising as an important factor affecting tumorigenicity and tumor progression. Our findings verified the significance of RBBP5-mediated H3K4 modification in melanoma and the potential regulatory mechanisms of melanoma proliferation and growth, suggesting that RBBP5 is a potential therapeutic target for the treatment of melanoma.

Regulation of pancreatic cancer microenvironment by an intelligent gemcitabine@nanogel system via in vitro 3D model for promoting therapeutic efficiency.

The passive targeting via nanomedicine to pancreatic tumor microenvironment (TME) is identified as an optimized therapeutic strategy for pancreatic ductal adenocarcinoma (PDAC) because lacking specific biomarkers and the intractable anatomical position. Herein, an in vitro 3D PDAC model was set up to evaluate the regulation of extracellular matrix (ECM) by an intelligent gemcitabine@nanogel system (GEM@NGH). This GEM@NGH system consisting of a reduction-sensitive core, the payloads of gemcitabine, and the coronal of hyaluronidase arrayed on the cationic surface was fabricated to improve intratumoral penetration and antitumor efficacy. The physicochemical properties, reduction sensitivity, cellular biocompatibility and cytotoxicity, intracellular distribution and therapeutic effects were all evaluated. Particularly, the GEM@NGH system showed excellent ECM eradication and in vitro/vivo solid tumor penetration ability as evaluated by home-built equipment and in vitro 3D PDAC model, which confirmed that GEM@NGH could be disintegrated in the tumoral reductive cytoplasm after internalization and release gemcitabine to exhibit promoted cytotoxicity. In the in vivo therapy, GEM@NGH displayed the highest tumor growth inhibition in PANC-1 tumor-bearing mice with the remarkably increased tumor penetration ability by TME regulation. The results obtained in this study indicate that specifically regulating TME by a well-designed intelligent gemcitabine@nanogel is promising way for the pancreatic cancer therapy.

Thermosensitive injectable decellularized nucleus pulposus hydrogel as an ideal biomaterial for nucleus pulposus regeneration.

Extracellular matrix loss is one of the early manifestations of intervertebral disc degeneration. Stem cell-based tissue engineering creates an appropriate microenvironment for long term cell survival, promising for NP regeneration. We created a decellularized nucleus pulposus hydrogel (DNPH) from fresh bovine nucleus pulposus. Decellularization removed NP cells effectively, while highly preserving their structures and major biochemical components, such as glycosaminoglycan and collagen II. DNPH could be gelled as a uniform grid structure in situ at 37°C for 30 min. Adding adipose marrow-derived mesenchymal stem cells into the hydrogel for three-dimensional culture resulted in good bioactivity and biocompatibility in vitro. Meanwhile, NP-related gene expression significantly increased without the addition of exogenous biological factors. In summary, the thermosensitive and injectable hydrogel, which has low toxicity and inducible differentiation, could serve as a bio-scaffold, bio-carrier, and three-dimensional culture system. Therefore, DNPH has an outstanding potential for intervertebral disc regeneration.

Altered expression of glycoprotein non­metastatic melanoma protein B in the distal sciatic nerve following injury.

Glycoprotein non­metastatic melanoma protein B (GPNMB) exerts neuroprotective effects on amyotrophic lateral sclerosis and cerebral ischemia reperfusion injury in the central nervous system. However, the expression and function of GPNMB in the peripheral nervous system, particularly following peripheral nerve injury, remains unknown. In the present study, the mRNAs and long non­coding RNAs of the distal sciatic nerve were profiled via microarray analysis at days 0, 1, 3, 7, 14, 21 and 28 following transection. The results revealed that the expression of GPNMB mRNA was similar to the proliferation tendency of distal acute denervated Schwann cells (SCs), the results of which were further validated by reverse transcription quantitative polymerase chain reaction, western blot analysis and immunohistochemistry. To investigate the function of GPNMB on SCs, recombinant human GPNMB (rhGPNMB) was added to cultured denervated SCs from the distal stumps of transected sciatic nerve. The proliferation, expression and secretion of neurotrophic factors (NTFs) and neural adhesion molecules (NAMs) were subsequently detected. The results demonstrated that GPNMB expression was increased in distal sciatic nerve following transection in vivo, while rhGPNMB promoted the proliferation of SCs as well as expression and secretion of NTFs and NAMs in vitro. Therefore, GPNMB could be a novel strategy for peripheral nerve regeneration.

Synergistic effects of adipose-derived stem cells combined with decellularized myocardial matrix on the treatment of myocardial infarction in rats.

AIMS: The aims of the study are to investigate whether the combination of adipose-derived stem cells (ADSCs) with decellularized extracellular matrix (dECM) of myocardium can exert synergistic therapeutic effects on acute MI and the underlying mechanism. MAIN METHODS: Myocardial dECM from fresh porcine myocardium was prepared in an injectable gel, ADSCs were seeded into the myocardial dECM gels, and then the mixture was injected into the myocardium of the infarct border zone after acute MI, which was induced by ligating the left anterior descending coronary artery in male SD rats, to assess the therapeutic potential. The degree of fibrosis was detected by Masson's trichrome. The evaluation of cardiac function was performed by Electrocardiography. KEY FINDINGS: Myocardial dECM (2.0%) had a suitable aperture and arrangement for cell growth, and also exhibited suitable biomechanical properties. Four-weeks after treatment in vivo, the combination of ADSCs and myocardial dECM could obviously increase angiogenesis, reduce the degree of fibrosis, and decrease infarct size. Furthermore, the combination treatment exerted significant functional improvement. Compared with ADSCs or dECM group alone, the left ventricular ejection fraction (LVEF) in the combination group was 13.4% and 21.8% elevated, respectively. SIGNIFICANCE: The combination of ADSCs and myocardial dECM has synergistic effects on cardiac repair in acute MI.

OCIAD1 promoted pancreatic ductal adenocarcinoma migration by regulating ATM.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplastic disease, characterized with poor outcomes and a 5-year survival rate less than 5%. Dysregulation or dysfunction of immune response factors contribute to cancer development. In this study, we found that OCIAD1 is high expressed in pancreatic cancer gene chip, and verified OCIAD1 associating with cancer malignancy in specimens from patients with PDAC. OCIAD1 down-regulation inhibited PDAC cell lines migration and vice versa. Further analysis of pancreatic cancer gene chip found OCIAD1 high expression was associating with low ATM expression. Then we proved that OCIAD1 regulated ATM to affect the migration of PDAC. Thus we concluded that high OCIAD1 levels in PDAC promoted tumor cells migration. OCIAD1 exerted its effects by regulating ATM.

UMSC-derived exosomes promote retinal ganglion cells survival in a rat model of optic nerve crush.

Traumatic optic neuropathy or glaucoma lead to retinal ganglion cells loss and cause blindness, and there is no effective therapy strategy by far. Mesenchymal cells from the Wharton's jelly of the umbilical cord (umbilical cord mesenchymal stem cells, UMSCs) and UMSC-derived exosomes (UMSC-Exos) are promising candidates for allogeneic therapy in regenerative medicine, but their effort on optic nerve injury and the underlying mechanism remains undefined. In the present study, we investigated the functions of UMSC-Exos in a rat optic nerve crush (ONC) model. After three times of treatments with an interval of one week, we found that the UMSC-Exos significantly promoted Brn3a+ retinal ganglion cells (RGCs) survival in retinal ganglion cell layer compared with PBS controls. UMSC-Exos also significantly promoted GFAP+ glia cells activation in retina and optic nerve. However, no increase of GAP43+ axon counts in the optic nerve was found after UMSC-Exos treatment. Thus, our results demonstrate that UMSC-derived exosomes may play a role in neuroprotection by promoting the RGCs survival and glia cells activation but not the axon regeneration.

Hydrogen alleviates cellular senescence via regulation of ROS/p53/p21 pathway in bone marrow-derived mesenchymal stem cells in vivo.

Senescence has become a hot point issue in recent decades and requires urgent attention. As a novel and effective antioxidant, hydrogen has been proved to alleviate cellular senescence in endothelial cells in vitro. However, the effects and mechanisms of hydrogen on senescence in vivo are still unclear. In the present study, 12-month-old Sprague Dawley (SD) rats were intraperitoneal administration of hydrogen-rich saline (HRS, 10 ml/kg). Subsequently, bone marrow-derived stem cells (BMSCs) were harvested for the detection of hydrogen antisenescence effects and mechanisms. The results showed that the number of senescence-associated ß-galactosidase (SA-ß-Gal) positive cells was reduced in BMSCs from rats treated with HRS. BMSCs in rats treated with HRS possessed a better proliferation ability, showed more effectively tri-lineage differentiation potential, and had less percentage of cells in G1 cell cycle arrest than the control cells. Additionally, HRS administration inhibited the production of intracellular reactive oxygen species (ROS) and decreased the expression of senescence-related proteins p53 and p21. Our results revealed that hydrogen could alleviate cellular senescence in vivo. And the underlying mechanism of antisenescence effects of hydrogen in BMSCs was via the ROS/p53/p21 signaling pathway. Thus, hydrogen could be a new and convenient strategy for alleviating senescence and for therapy of age-related diseases.

Bone mesenchymal stem cells attenuate radicular pain by inhibiting microglial activation in a rat noncompressive disk herniation model.

Spinal disk herniation can induce radicular pain through chemical irritation caused by proinflammatory and immune responses. Bone marrow mesenchymal stem cells (BMSCs) are a unique type of adult stem cell with the functions of suppressing inflammation and modulating immune responses. This study was undertaken to observe the effect of intrathecal BMSCs on the treatment of mechanical allodynia and the suppression of microglial activation in a rat noncompressive disk herniation model. The model was induced by the application of nucleus pulposus (NP) to the L5 dorsal root ganglion (DRG). The study found that the use of NP in the DRG can induce abnormal mechanical pain, increase the contents of the proinflammatory factors TNF-α and IL-1ß, decrease the content of the anti-inflammatory cytokine TGF-ß1 and activate microglia in the spinal dorsal horns (L5) (P < 0.05). BMSC administration could increase the mechanical withdrawal thresholds dramatically, decrease the contents of IL-1ß and TNF-α, increase the content of TGF-ß1 significantly (P < 0.05) and inhibit microglial activation in the bilateral spinal dorsal horn. Our results indicate that BMSC administration can reduce mechanical allodynia and downregulate the expression of proinflammatory cytokines by inhibiting microglial activation in the spinal dorsal horn in a rat noncompressive disk herniation model.

Reactivation of denervated Schwann cells by neurons induced from bone marrow-derived mesenchymal stem cells.

The use of neurons induced from stem cells has been introduced as an effective strategy for promoting peripheral nerve regeneration (PNR). The evolution and role of native denervated Schwann cells (SCs) were often ignored when exploring the mechanisms underlying neural transplantation therapy for PNR. The aim of this study was to understand if following injury, native denervated SCs could be reactivated by transplanting of neurons induced from bone marrow-derived mesenchymal stem cells (NI-BMSCs) to promote PNR. We co-cultured denervated SCs with NI-BMSCs in vitro, tested the proliferation of denervated SCs, and measured the expression and secretion of neurotrophic factors and neural adhesion molecules of the denervated SCs. Concurrently, 48 adult male Sprague-Dawley rats were randomly divided into 4 even groups of 12 rats each: normal group, phosphate-buffered saline (PBS) injection group, BMSCs transplantation group and NI-BMSCs transplantation group. PBS injection and cells transplantation were performed 4 weeks post-injury. After 4 weeks of NI-BMSCs transplantation, the survival of seeded NI-BMSCs was examined, proliferation and ultrastructure of native denervated SCs were detected, and myelination, axonal regeneration and the sciatic functional index measurements were also determinated. Our results demonstrated that NI-BMSCs reactivated denervated SCs both in vitro and in vivo and promoted sciatic nerve regeneration.

Comparison of mouse brown and white adipose­derived stem cell differentiation into pacemaker­like cells induced by TBX18 transduction.

The present study aimed to compare brown adipose-derived stem cell (BASC) and white adipose-derived stem cell (WASC) differentiation into pacemaker­like cells following T­box (TBX)18 transduction. Mouse BASCs and WASCs were induced to differentiate into pacemaker­like cells by adenovirus­TBX18 transduction in vitro. The transduction rate was determined by fluorescence microscopy and cell ultrastructural changes were observed by transmission electron microscopy at 48 h post­transduction. The mRNA and protein expression of pacemaker cell­associated markers, including TBX18, TBX3, sarcomeric α­actinin (Sr) and hyperpolarization­activated cyclic nucleotide­gated channel 4 (HCN4), were detected by reverse transcription­quantitative polymerase chain reaction, immunofluorescence staining and western blot analysis. The results demonstrated that no significant difference was observed in the transduction rate between BASCs and WASCs. The ultrastructure of BASCs was observed to be more complex than that of WASCs, indicating that BASCs may possess a better structural foundation to differentiate into pacemaker­like cells. TBX18, TBX3, Sr and HCN4 mRNA and protein expression in differentiated stem cells was significantly increased compared with the respective control groups. Furthermore, the expression levels were significantly higher in TBX18­BASCs compared with TBX18­WASCs. In conclusion, TBX18 gene transduction may facilitate the differentiation of BASCs and WASCs into pacemaker­like myocardial cells, and BASCs may have a higher capacity than WASCs for this differentiation. TBX18 gene may therefore act as an efficient candidate in cell transplantation therapy for diseases and for future research into the cardiovascular system.

Comparison of the neuronal differentiation abilities of bone marrow­derived and adipose tissue­derived mesenchymal stem cells.

Bone marrow­derived mesenchymal stem cells (BMSCs) and adipose tissue­derived mesenchymal stem cells (ADSCs) are able to differentiate into neuron­like cells when exposed to small molecule compounds, however the specific differences in their neuronal differentiation abilities remain to be fully elucidated. The present study aimed to compare the neuronal differentiation abilities of BMSCs and ADSCs. BMSCs and ADSCs from the same Sprague Dawley rats were isolated and cultured for use. The proliferation capacity was revealed using a cell counting method. Following BMSCs and ADSCs induction by four types of small­molecular compounds, the expression of various neuronal markers and the secretion of several neurotrophic factors were detected by immunofluorescence, western blotting, reverse transcription­quantitative polymerase chain reaction and ELISA. It was demonstrated that the ADSCs exhibited an increased proliferation capacity compared with BMSCs, according to cumulative population doubling analyses. Following a 7­day neuronal induction period, BMSCs and ADSCs exhibited a neuron­like morphology, and were termed neuronal induced (NI)­BMSCs and NI­ADSCs. They expressed neuronal markers including ß­tubulin III, microtubule associated protein 2 and choline acetyltransferase. The number of NI­BMSCs that positively expressed the neuronal markers was significantly decreased compared with NI­ADSCs, and the expression and secretion of the neurotrophic factors nerve growth factor and 3'­nucleotidase in NI­BMSCs were additionally decreased compared with NI­ADSCs. The findings of the present study indicated that the neuronal differentiation abilities and neurotrophic factor secretion abilities of ADSCs were increased compared with BMSCs. ADSCs may therefore act as efficient candidates in cell transplantation therapy for diseases and injuries of the nervous system.

Construction of vascularized pacemaker tissues by seeding cardiac progenitor cells and endothelial progenitor cells into Matrigel.

AIMS: Transplantation of a tissue engineered cardiac pacemaker (TECP) may represent a novel therapy for cardiac sinus node dysfunction. We previously reported that cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiac pacemaking cells after endothelin-1 treatment. We aimed to examine the feasibility of TECP fabricated from CPCs-derived pacemaking cells and vascularization of TECP fabricated from CPCs-derived pacemaking cells and endothelial progenitor cells (EPCs) in vitro and in vivo implantation. MAIN METHODS: TECP created using CPCs-derived pacemaking cells and vTECP created by mixing CPCs and EPCs in vitro were implanted into rat hearts. Sinus node damaged was induced by formaldehyde insult. KEY RESULTS: Spontaneous beating tissues, namely TECP, were obtained after seeding CPCs-derived pacemaking cells into Matrigel. ECG and epicardial multielectrode array (MEA) measurements confirmed implanted TECP have electrical activity. TECP implantation promoted individual survival in sinus node damage models (15/22 animals lived versus 0/17 control). vTECP fabricated by mixing the both EPCs and CPCs-derived pacemaking cells with Matrigel in equal proportions optimally formed pre-vascularization in vitro. The implantation of vTECP enhanced electrical activity in vivo, which may correlate with increased vascularization. PI3K-Akt-VEGF/VEGFR signaling was involved with vascular ingrowth in vTECP. SIGNIFICANCE: Our data supports the therapeutic potential of TECP fabricated with the CPCs-derived pacemaking cells for sinus node dysfunction. Vascularization by the addition of EPCs is an important factor to sustain viability of the TECP in vivo.

Protective Effects of Methane-Rich Saline on Rats with Lipopolysaccharide-Induced Acute Lung Injury.

Objective. The aim of this research is to evaluate the protective effects of methane-rich saline (MS) on lipopolysaccharide- (LPS-) induced acute lung injury (ALI) and investigate its potential antioxidative, anti-inflammatory, and antiapoptotic activities. Methods. LPS-induced (20 mg/kg) ALI rats were injected with MS (2 ml/kg and 20 ml/kg) before the initiation of LPS induction. Survival rate was determined until 96 h after LPS was induced. Lung injury was assayed by oxygenation index, lung permeability index (LPI), wet-to-dry weight (W/D), and histology. The cells in the bronchoalveolar lavage fluid (BALF) were counted. Oxidative stress was examined by the level of malondialdehyde (MDA) and superoxide dismutase (SOD). Inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in BALF were determined by ELISA. Lung tissue apoptosis was detected by TUNEL staining and western blotting of caspase-3. Results. It was found that methane significantly prolonged the rat survival, decreased the lung W/D ratio and the content of the inflammatory factors, and reduced the amount of caspase-3 and apoptotic index. In addition, MS increased the level of SOD and decreased the level of MDA significantly. Conclusions. MS protects the LPS-challenged ALI via antioxidative, anti-inflammatory, and antiapoptotic effect, which may prove to be a novel therapy for the clinical management of ALI.

Shaking improves the whole bone marrow adherent method of purification.

The aim of the present study was to investigate the potential effects of mechanical shaking on the purity, activity, differentiation and possible apoptosis of rat bone marrow mesenchymal stem cells (BMSCs). Based on the whole bone marrow adhesion method, different durations and frequencies of mechanical shaking were used on primary cells. The biomarkers, CD29, CD90, CD45 and CD31, in addition to the apoptosis labels, annexin V­FITC and PI, were investigated using flow­cytometric analysis. The differentiation capability following purification was evaluated. Following shaking treatment, the purity of adherent cells increased, in particular there was an increase in CD29 and CD90 positive cells, with the majority of the detached cells negative for these two markers. In addition, the apoptotic rates increased with the increasing shaking duration and frequency. Furthermore, cells following shaking were induced to differentiate into osteoblasts and adipocytes. The shaking method allows for mesenchymal stem cells at to be obtained with higher positive rates of CD29 and CD90. In addition, horizontal shaking has little influence on cell activity or differentiation, with low levels of apoptosis occurring as a result of shaking.

Biocompatibility evaluation of tissue-engineered valved venous conduit by reseeding autologous bone marrow-derived endothelial progenitor cells and multipotent adult progenitor cells into heterogeneous decellularized venous matrix.

Clinical treatment of chronic deep venous insufficiency remains difficult despite the availability of various therapies. Previous experimental efforts have demonstrated that the tissue-engineered valvedvenous conduit (TEVV) is a promising option to replace the damaged venous valve. The aim of the present study was to evaluate the TEVV by reseeding bone marrow-derived endothelial progenitor cells and multipotent adult progenitor cells into acellular matrix according to International Standard ISO10993, and to clarify their interactions with blood, the local effect after implantation both in vitro and vivo, and immunogenicity. The results showed that the 2-cm long TEVV did not cause haemolysis in vitro and remained patent without thrombosis formation in vivo. However, the luminal surface of TEVV was partially covered by multilayer cells. Compared with the native ovine femoral vein segment, the TEVV beneath the mouse skin produced significant mononuclear cell infiltration, with serum interleukin-6 and tumour necrosis factor-α similar to normal. The TEVV maintained its structural integrity, while the native ovine femoral vein segments fell apart at postoperative week nine. The TEVV implantation did not change serum immunoglobulin G. In addition, the seeds and extracts of the scaffold did not affect the proliferation of mouse lymphocytes. These findings suggest that the histocompatibility, haemocompatibility and immunogenicity of this TEVV are acceptable owing to complete removal of the cellular components of autologous seeds and residues of chemical regents, thus providing an experimental basis for further clinical translation. Copyright © 2014 John Wiley & Sons, Ltd.

Functional analysis in vivo of engineered valved venous conduit with decellularized matrix and two bone marrow-derived progenitors in sheep.

Tissue engineering has been considered a promising approach for creating grafts to replace autologous venous valves. Here, ovine bone marrow-derived endothelial progenitor cells (EPCs) and multipotent adult progenitor cells (MAPCs) were harvested and then loaded into decellularized venous matrix to create tissue-engineered (TE) valved vein. Subsequently, the ovine femoral veins containing the valve were removed and replaced by TE grafts or acellular matrix only. The morphology and function were analysed for up to 1 year by ultrasonography, angiography, H&E staining and scanning electron microscopy (SEM). The differentiation of seeded cells was traced immunofluorochemically. The results showed that decellularized venous matrix could initially and feebly attract endogenous cells, but failed afterwards and were insufficient to restore valve function. On the contrary, the seeded cells differentiated into endothelial cells (ECs) in vivo and formed a monolayer endothelium, and smooth muscle cells within the scaffold therefore produced TE grafts comparable to the native vein valve. This TE graft remained patent and sufficient after implantation into the venous circuit of the ovine lower extremity for at least 6 months. Unfortunately, cells seeded on the luminal surface and both sides of the leaflets lost their biological functions at 12 months, resulting in thrombosis formation and leading to complete occlusion of the TE grafts and impotent venous valves. These findings suggest that this TE valved venous conduit can function physiologically in vivo in the medium term. Before translating this TE venous valve into clinical practice, the durability should be improved and thrombogenicity should be suppressed. Copyright © 2016 John Wiley & Sons, Ltd.