DNMT1-mediated epigenetic suppression of FBXO32 expression promoting cyclin dependent kinase 9 (CDK9) survival and esophageal cancer cell growth.

Esophageal cancer (EC) is a common and serious form of cancer, and while DNA methyltransferase-1 (DNMT1) promotes DNA methylation and carcinogenesis, the role of F-box protein 32 (FBXO32) in EC and its regulation by DNMT1-mediated methylation is still unclear. FBXO32 expression was examined in EC cells with high DNMT1 expression using GSE163735 dataset. RT-qPCR assessed FBXO32 expression in normal and EC cells, and impact of higher FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with EMT-related proteins. The xenograft model established by injecting EC cells transfected with FBXO32 was used to evaluate tumor growth, apoptosis, and tumor cells proliferation and metastasis. Chromatin immunoprecipitation (ChIP) assay was employed to study the interaction between DNMT1 and FBXO32. HitPredict, co-immunoprecipitation (Co-IP), and Glutathione-S-transferase (GST) pulldown assay analyzed the interaction between FBXO32 and cyclin dependent kinase 9 (CDK9). Finally, the ubiquitination assay identified CDK9 ubiquitination, and its half-life was measured using cycloheximide and confirmed through western blotting. DNMT1 negatively correlated with FBXO32 expression in esophageal cells. High FBXO32 expression was associated with better overall survival in patients. Knockdown of DNMT1 in EC cells increased FBXO32 expression and suppressed malignant phenotypes. FBXO32 repressed EC tumor growth and metastasis in mice. Enrichment of DNMT1 in FBXO32 promoter region led to increased DNA methylation and reduced transcription. Mechanistically, FBXO32 degraded CDK9 through promoting its ubiquitination.

Esophageal metastases from primary lung cancer: a case report.

BACKGROUND: Primary lung cancer is one of the most frequently diagnosed cancers. The common metastatic sites are the liver, bones, brain, adrenal glands and central nervous system. However, gastrointestinal metastases, particularly esophageal metastases, from lung cancer are rare. There are no cases of esophageal metastases from lung cancer which refer to its particular treatment. CASE PRESENTATION: We report a case of esophageal metastases from lung cancer. The patient was a 55-year-old Han Chinese man who first attended our hospital due to dry cough and was diagnosed with late-stage lung cancer. Three months later, the patient complained of dysphagia. Endoscopic ultrasonography (EUS) and pathological examination of the biopsy specimen was performed to confirm the lesion was metastases from lung cancer. Thyroid transcription factor 1 (TTF-1), cytokeratin 7 (CK-7) and napsin A were positive by immunohistochemistry examination. These results reconfirmed the diagnosis of esophageal metastases from lung cancer. CONCLUSIONS: Esophageal metastasis from lung cancer is very rare. It may be alleviated with personalized chemotherapy. In addition, molecular targeted therapy for patients with epidermal growth factor receptor (EGFR) mutations may be reasonable.

Mechanism of Cxc Chemokine Ligand 5 (CXCL5)/Cxc Chemokine Receptor 2 (CXCR2) Bio-Axis in Mice with Acute Respiratory Distress Syndrome.

BACKGROUND Acute respiratory distress syndrome (ARDS) is a common acute and severe disease in clinic. Recent studies indicated that Cxc chemokine ligand 5 (CXCL5), an inflammatory chemokine, was associated with tumorigenesis. The present study investigated the role of the CXCL5/Cxc chemokine receptor 2 (CXCR2) bio-axis in ARDS, and explored the underlying molecular mechanism. MATERIAL AND METHODS The pathological morphology of lung tissue and degree of pulmonary edema were assessed by hematoxylin-eosin staining and pulmonary edema score, respectively. Real-time PCR and Western blot analysis were performed to detect the expression levels of CXCL5, CXCR2, Matrix metalloproteinases 2 (MMP2), and Matrix metalloproteinases 9 (MMP9) in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the expression levels of CXCL5 and inflammatory factors (IL-1ß, IL-6, TNF-alpha, and IL-10) in serum. RESULTS The results demonstrated that diffuse alveolar damage and pulmonary edema appeared in lipopolysaccharide (LPS)-induced ARDS and were positively correlated with the severity of ARDS. In addition, CXCL5 and its receptor CXCR2 were overexpressed by upregulation of MMP2 and MMP9 in lung tissues of ARDS. In addition, CXCL5 neutralizing antibody effectively alleviated inflammatory response, diffuse alveolar damage, and pulmonary edema, and decreased the expression levels of MMP2 and MMP9 compared to LPS-induced ARDS. CONCLUSIONS We found that CXCL5/CXCR2 accelerated the progression of ARDS, partly by upregulation of MMP2 and MMP9 in lung tissues with the release of inflammatory factors.

Membrane-Binding Adhesive Particulates Enhance the Viability and Paracrine Function of Mesenchymal Cells for Cell-Based Therapy.

Understanding the fundamental cell-material interactions is essential to designing functional materials for biomedical applications. Although mesenchymal stromal cells (MSCs) are known to secrete cytokines and exosomes that are effective to treat degenerative diseases, the inherent property of biomaterials to modulate the therapeutic function of MSCs remains to be investigated. Here, a multivalent cell-membrane adhesive conjugate was generated through polyamindoamine (PAMAM) and an oligopeptide, IKVAV, and the conjugate was further complexed with hyaluronic acid (HA). The adhesive particulates were used to coat the surface of adipose-derived mesenchymal stromal cells (Ad-MSCs) and studied in the MSC spheroid culture. The analysis showed that the adhesive complexes formed via PAMAM conjugates and HA significantly promoted the proliferation and the gene expression of pro-angiogenesis cytokines in MSCs; the production of anti-inflammatory miRNAs in exosomes could also be elevated. The transplantation of the Ad-MSCs primed with PAMAM-IKVAV/HA composite particulates in a rat myocardial infarction model further demonstrated the beneficial effects of membrane-binding materials on improving the cell retention and tissue angiogenesis. The new function of membrane-binding adhesive materials potentially provides useful ways to improve cell-based therapy.

Covalent bonding of YIGSR and RGD to PEDOT/PSS/MWCNT-COOH composite material to improve the neural interface.

The development of coating materials for neural interfaces has been a pursued to improve the electrical, mechanical and biological performances. For these goals, a bioactive coating was developed in this work featuring a poly(3,4-ethylenedioxythiophene) (PEDOT)/carbon nanotube (CNT) composite and covalently bonded YIGSR and RGD. Its biological effect and electrical characteristics were assessed in vivo on microwire arrays (MWA). The coated electrodes exhibited a significantly higher charge storage capacity (CSC) and lower electrochemical impedance at 1 kHz which are desired to improve the stimulating and recording performances, respectively. Acute neural recording experiments revealed that coated MWA possess a higher signal/noise ratio capturing spikes undetected by uncoated electrodes. Moreover, coated MWA possessed more active sites and single units, and the noise floor of coated electrodes was lower than that of uncoated electrodes. There is little information in the literature concerning the chronic performance of bioactively modified neural interfaces in vivo. Therefore in this work, chronic in vivo tests were conducted and the PEDOT/PSS/MWCNT-polypeptide coated arrays exhibited excellent performances with the highest mean maximal amplitude from day 4 to day 12 during which the acute response severely compromised the performance of the electrodes. In brief, we developed a simple method of covalently bonding YIGSR and RGD to a PEDOT/PSS/MWCNT-COOH composite improving both the biocompatibility and electrical performance of the neural interface. Our findings suggest that YIGSR and RGD modified PEDOT/PSS/MWCNT is a promising bioactivated composite coating for neural recording and stimulating.

The paracrine effects of adipose-derived stem cells on neovascularization and biocompatibility of a macroencapsulation device.

The foreign-body response to biomaterials compromises the performance of many biomedical devices by severe fibrosis and limited neovascularization. Mesenchymal stem cells are known to secrete cytokines for treating inflammatory conditions. In this study, we aim to investigate whether the paracrine products of adipose-derived mesenchymal stem cells (ADSCs) can affect the microenvironment of biomaterials and improve tissue responses to biomaterial implants. A model system was built by loading ADSC spheroids into a macroencapsulation device composed of polytetrafluoroethylene (PTFE) filtration membranes. Soluble ADSC factors that diffused out of the device in vitro promoted the angiogenetic activity of endothelial cells and affected the secretion pattern of macrophages. In vivo study was carried out by subcutaneously embedding blank or ADSC-laden devices in rats. Following a 4 week implantation, the ADSC-laden devices were better vascularized and induced significantly less fibrotic tissue formation in comparison to the non-cellular controls. This study may facilitate our understanding of foreign-body responses and suggest new ways to improve the tissue reaction of biomedical devices for cell-based therapy.

The spatiotemporal development of intercalated disk in three-dimensional engineered heart tissues based on collagen/matrigel matrix.

Intercalated disk (ID), which electromechanically couples cardiomyocytes into a functional syncitium, is closely related to normal morphology and function of engineered heart tissues (EHTs), but the development mode of ID in the three-dimensional (3D) EHTs is still unclear. In this study, we focused on the spatiotemporal development of the ID in the EHTs constructed by mixing neonatal rat cardiomyocytes with collagen/Matrigel, and investigated the effect of 3D microenvironment provided by collagen/Matrigel matrix on the formation of ID. By histological and immmunofluorescent staining, the spatiotemporal distribution of ID-related junctions was detected. Furthermore, the ultra-structures of the ID in different developmental stages were observed under transmission electron microscope. In addition, the expression of the related proteins was quantitatively analyzed. The results indicate that accompanying the re-organization of cardiomyocytes in collagen/Matrigel matrix, the proteins of adherens junctions, desmosomes and gap junctions redistributed from diffused distribution to intercellular regions to form an integrated ID. The adherens junction and desmosome which are related with mechanical connection appeared earlier than gap junction which is essential for electrochemical coupling. These findings suggest that the 3D microenvironment based on collagen/Matrigel matrix could support the ordered assembly of the ID in EHTs and have implications for comprehending the ordered and coordinated development of ID during the functional organization of EHTs.

Real-time tracking of adipose tissue-derived stem cells with injectable scaffolds in the infarcted heart.

Adipose tissue-derived stem cells (ADSCs) has shown promise in the emerging field of regenerative medicine. Many studies have highlighted the importance of coadministering a "scaffold" for increasing intramyocardial retention of stem cells. In this work, an optimized method was developed for efficient transduction of ADSCs with a lentiviral vector carrying a triple-fusion reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). The transduced ADSCs were assessed on biological performance and transplanted into infarcted heart with fibrin scaffolds. In vivo cell retention was tracked by bioluminescence imaging (BLI) and micro positron emission tomography/computed tomography (PET/CT) imaging. Histological assessment was performed for regeneration potentials. The results showed that lentiviral transduction did not influence cell functions. In vitro imaging analysis showed a robust linear correlation between cell numbers and BLI signals (R (2) = 0.99) as well as between cell numbers and radiotracer uptakes (R (2) = 0.98). Transduced ADSCs were visualized in the heart under both BLI and PET/CT imaging, contributing to cardiomyocyte regeneration and angiogenesis in the implanted areas. Compared with BLI monitoring, PET/CT data provided precise localization for cell retention. Thus, a combination of imaging modalities can assist in reliable and efficient monitoring of transplanted cells, holding great potential for the transplantation of injectable scaffolds encapsulating stem cells in treating heart disease.

RCCS enhances EOE cell proliferation and their differentiation into ameloblasts.

In this article we report on the culturing of dental enamel organ epithelia (EOE) using a rotary cell culture system (RCCS) bioreactor associated with a cytodex-3 microcarrier. This culture system enhanced the proliferation and differentiation of the EOE into ameloblasts. Primary dental EOE trypsinized from 4-day old post-natal rat pups were cultured in the RCCS associated with Cytodex-3. The results were analyzed in comparison to a conventional plate system (control). Cells grown in RCCS have shown higher viabilities (above 90%) and final cell densities in terms of cells/ml than in the control system. In the case of RCCS, 46±2 manifold increases were obtained, while significantly lower yields of 10.8±2.5 manifod were obtained for control plates. Throughout the experiments, glucose levels were maintained within the accepted physiological range. In this case, LDH levels are kept low (below 150 mmol/ml), which is in accordance with the low cell death observed in the RCCS. Scanning electron microscopy revealed cells that were spread and forming three dimensional aggregates on the surface of cytodex-3. Cells cultured in the RCCS exhibited a stronger positive immunofluorescence staining for ameloblastin than those in control plates. RT-PCR results revealed that cells cultured in RCCS have higher amelogenin mRNA levels compared to controls. We have done an exploratory study on biological characteristics and self-assembling of epithelium cellula intersitialis, which demonstrated that the special 3D environment enhanced the rat dental EOE cell proliferation and differentiation into ameloblasts. The study has revealed that RCCS could be used to study the reaction of the EOE cells, tooth enamel organ cells and mesenchymal cells under the spacial 3D culture system, which will also provide a novel hypothesis for dental regeneration.

Posterolateral spinal fusion with nano-hydroxyapatite-collagen/PLA composite and autologous adipose-derived mesenchymal stem cells in a rabbit model.

Spinal fusion is routinely performed to treat low back pain caused by degeneration of intervertebral discs. An autologous bone graft derived from the iliac crest is the standard procedure used for spinal fusion. However, several shortcomings, including pseudarthrosis, pain and the need for blood transfusion are known to be associated with the procedure. Our study analysed the effectiveness of a new mineralized collagen matrix, nano-hydroxyapatite-collagen-polylactic acid (nHAC-PLA), combined with autologous adipose-derived mesenchymal stem cells (ADMSCs) as a graft material for posterolateral spinal fusion in a rabbit model. Forty rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC-PLA composite group (nHAC-PLA), autologous iliac crest bone mixed with nHAC-PLA composite group (ACB + nHAC-PLA), and nHAC-PLA composite combined with ADMSCs (ADMSCs + nHAC-PLA). The viability and the proliferation of the ADMSCs seeded on the scaffolds were evaluated by live/dead kit and MTT assay in vitro, respectively. Lumbar posterolateral fusions were assessed by manual palpation, radiographical and histological procedures, mechanical strength and scanning electronic microscopy (SEM) in 10 weeks of observation. The results showed that the rate of fusion was significantly higher in the ACB and ADMSCs + nHAC-PLA groups than that in the nHAC-PLA and ACB + nHAC-PLA groups. It was not significantly higher in the ACB group than in the ADMSCs + nHAC-PLA group. From microstructural analysis of the samples using histological staining methods, there was more new bone-like tissue formation in the ACB and ADMSCs + nHAC-PLA groups than that in the other two groups at the 10th postoperative week. Our study demonstrated the effective impact of nHAC-PLA combined with ADMSCs in rabbit posterolateral spinal fusion.

Reconstruction of endometrium in vitro via rabbit uterine endometrial cells expanded by sex steroid.

OBJECTIVE: To culture rabbit endometrial cells by using sex steroids to provide adequate seeding cells for endometrium reconstruction and uterine tissue engineering. DESIGN: Prospective experimental study. SETTING: Beijing Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences. ANIMAL(S): New Zealand rabbit and Kunming white strain mice. INTERVENTION(S): Rabbits were primed with pregnant mare serum gonadotropin and hCG. Endometrial cells were cultured with E(2) and P(4) of different concentrations. The endometrium was reconstructed by using endometrial cells as seeding cells and collagen-basement membrane matrix as scaffolds. MAIN OUTCOME MEASURE(S): Assay with 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, immunofluorescence staining, flow cytometric analysis, hematoxylin and eosin and immunohistochemical staining, and developmental rate of embryos. RESULT(S): The expression patterns of estrogen receptor and P receptor of rabbit endometrium were different before and after treatment with pregnant mare serum gonadotropin-hCG. One hundred nanomolar E(2) with 10 nmol/L P(4) facilitated the proliferation of epithelial cells whereas 100 nmol/L P(4) facilitated that of stromal cells. The epithelial cells could be stable if cultured for seven or eight passages. Cells in the epithelial layer of the reconstructed endometrium were cytokeratin positive. Some showed columnar morphology akin to the luminal epithelium in vivo. Reconstructed endometrium could improve the developmental rate and quality of one-cell mice embryos. CONCLUSION(S): Rabbit endometrial cells could be cultured with a long-standing proliferation capability by sex steroids and applied in uterine tissue engineering. Reconstructed endometrium with proliferated endometrial cells was akin to native endometrium in structure and function.

Functional improvement of infarcted heart by co-injection of embryonic stem cells with temperature-responsive chitosan hydrogel.

Transplantation of embryonic stem cells (ESCs) can improve cardiac function in treatment of myocardial infarction. The low rate of cell retention and survival within the ischemic tissues makes the application of cell transplantation techniques difficult. In this study, we used a temperature-responsive chitosan hydrogel (as scaffold) combined with ESCs to maintain viable cells in the infarcted tissue. Temperature-responsive chitosan hydrogel was prepared and injected into the infarcted heart wall of rat infarction models alone or together with mouse ESCs. The result showed that the 24-h cell retention and 4 week graft size of both groups was significantly greater than with a phosphate buffered saline control. After 4 weeks of implantation, heart function, wall thickness, and microvessel densities within the infarct area improved in the chitosan + ESC, chitosan, and ESC group more than the PBS control. Of the three groups, the chitosan + ESC performed best. Results of this study indicate that temperature-responsive chitosan hydrogel is an injectable scaffold that can be used to deliver stem cells to infarcted myocardium. It can also increase cell retention and graft size. Cardiac function is well preserved, too.

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice.

AIM: To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. METHODS: Mice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured. RESULTS: Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia. CONCLUSION: E2 has a dose-related mitogenic effect on spermatogonia.

[Differential analysis of proteomic profiles between cryptorchid and normal mouse testes].

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.

Cardiomyocytes.

Derivation of cardiomyocytes from embryonic stem cells would be a boon for treatment of the many millions of people worldwide who suffer significant cardiac tissue damage in a myocardial infarction. Such cells could be used for transplantation, either as loose cells, as organized pieces of cardiac tissue, or even as pieces of organs. Eventual derivation of human embryonic stem cells via somatic cell nuclear cloning would provide cells that not only may replace damaged cardiac tissue, but also would replace tissue without fear that the patient's immune system will reject the implant. Embryonic stem cells can differentiate spontaneously into cardiomyocytes. In vitro differentiation of embryonic stem cells normally requires an initial aggregation step to form structures called embryoid bodies that differentiate into a wide variety of specialized cell types, including cardiomyocytes. This chapter discusses methods of encouraging embryoid body formation, causing pluripotent stem cells to develop into cardiomyocytes, and expanding the numbers of cardiomyocytes so that the cells may achieve functionality in transplantation, all in the mouse model system. Such methods may be adaptable and/or modifiable to produce cardiomyocytes from human embryonic stem cells.

[Cloning of Bmi1 cDNA from mouse testis and its expression in E. coli BL21].

OBJECTIVE: To produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21. METHODS: Bmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET-28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot. RESULTS: Mouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody. CONCLUSION: There was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly.

Creation of engineered cardiac tissue in vitro from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cells can terminally differentiate into all types of somatic cells and are considered a promising source of seed cells for tissue engineering. However, despite recent progress in in vitro differentiation and in vivo transplantation methodologies of ES cells, to date, no one has succeeded in using ES cells in tissue engineering for generation of somatic tissues in vitro for potential transplantation therapy. METHODS AND RESULTS: ES-D3 cells were cultured in a slow-turning lateral vessel for mass production of embryoid bodies. The embryoid bodies were then induced to differentiate into cardiomyocytes in a medium supplemented with 1% ascorbic acid. The ES cell-derived cardiomyocytes were then enriched by Percoll gradient centrifugation. The enriched cardiomyocytes were mixed with liquid type I collagen supplemented with Matrigel to construct engineered cardiac tissue (ECT). After in vitro stretching for 7 days, the ECT can beat synchronously and respond to physical and pharmaceutical stimulation. Histological, immunohistochemical, and transmission electron microscopic studies further indicate that the ECTs both structurally and functionally resemble neonatal native cardiac muscle. Markers related to undifferentiated ES cell contamination were not found in reverse transcriptase-polymerase chain reaction analysis of the Percoll-enriched cardiomyocytes. No teratoma formation was observed in the ECTs implanted subcutaneously in nude mice for 4 weeks. CONCLUSIONS: ES cells can be used as a source of seed cells for cardiac tissue engineering. Additional work remains to demonstrate engraftment of the engineered heart tissue in the case of cardiac defects and its functional integrity within the host's remaining healthy cardiac tissue.

Enrichment of cardiomyocytes derived from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cell-derived cardiomyocytes transplantation and tissue engineering together represent a promising approach for the treatment of myocardial infarction, despite the limited supply of cardiac myocytes. This study examines whether functional cardiomyocytes can be efficiently enriched from mouse embryonic stem (mES) cells. METHODS: mES cells were induced by ascorbic acid to differentiate into cardiomyocytes. Beating cells were observed after 1 week and increased in number with time while under differentiation conditions. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll gradient density centrifugation. RESULTS: The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac troponin T (cTnT). The enriched population contained 88.7% cardiomyocytes and showed expression of cardiomyocyte markers of troponin T and cardiac genes, including alpha-MHC, beta-MHC, ANF and Nkx2.5. However, Oct-4, a marker of early-stage ES cells, was not expressed in the mES cell-derived cardiac cell clusters. Moreover, the mES cell-derived and Percoll-enriched cardiomyocytes responded appropriately to cardioactive drugs, as did normal neonatal rat cardiomyocytes. CONCLUSIONS: mES cell-derived functional cardiomyocytes can be enriched by the method of discontinuous Percoll gradient centrifugation. The ability to differentiate and enrich for functional mouse cardiomyocytes makes it possible for further development of these cells as a model of myocardial repair through cell transplantation or tissue engineering.

[The study of tissue-engineering bone for repair of segmental bone defects].

OBJECTIVE: To investigate the effect of tissue-engineering bone on repair of segmental bone defects. METHODS: Segmental bone defect of 21mm was created at sheep left metatarsus, which was then implanted with tissue-engineering bone (the experimental group) and pure porous beta-TCP (the control group) respectively. The bone defect in the blank group was left without treatment. After the sheep were sacrificed at the 1st, 3rd, or 6th month postoperatively, the samples were taken and examined by radiological, histological and biomechanical methods as well as scanning electron microscopy. The sheep in the blank group were sacrificed at the 6th month postoperatively. RESULTS: The osteoid tissue, woven bone and lamellar bone in the defect of the experimental group occurred earlier than in the control group. The new bone formed directly without through a cartilaginous intermediate in the experimental group, while the defect was repaired in a "creep substitution" way in the control group. At the 6th month, radiological and biomechanical tests revealed nearly complete repair of the bone defect of the experimental group, partial repair in the control group and non-healing in the blank group. CONCLUSIONS: Tissue-engineering bone can repair bone defect, accelerating healing and without "creep substitution", which is a good option in repair of critical segmental bone defects. This study set up a basis for clinical applications in the future.

[Repair of cranial defects with bone marrow derived mesenchymal stem cells and beta-TCP scaffold in rabbits].

OBJECTIVE: To determine whether culture expanded bone marrow derived mesenchymal stem cells (MSCs) in combination with beta-tricalcium phosphate(beta-TCP) can repair critical cranial defects in New Zealand rabbits. METHODS: In group A(n = 20), MSCs from homogeneous rabbits were isolated and expanded in vitro and then implanted onto the pre-molded porous beta-TCP. The MSCs-beta-TCP complexes were implanted into rabbit critical cranial defects. In group B (n = 10), The defects were repaired with beta-TCP only. In group C(n = 4), the defects were left un-repaired. Samples were extracted 6 and 12 weeks after operation for histological, histochemical and immunohistochemical analysis. RESULTS: In group A, bone-like tissue formation could be seen on the surface of the implants. Microscopic analysis demonstrated certain degradation of beta-TCP and extensive new bone filling in rich extracellular matrix after 6 weeks. The cells were stained positively for type I collagen. After 12 weeks, the bioceramics had almost completely degraded and abundant bone formation could be seen in the whole defects. In group B, marginal bone ingrowth was observed after 6 weeks and the number of osteoblasts increased significantly after 12 weeks. However, no new bone formation could be detected in the middle of the material. In group C, only a small quantity of new bone formation was found along the margin of defects. CONCLUSION: Transplantation of MSCs with beta-TCP can serve as an example of a cell-based treatment for bone regeneration in skeletal defects.