CCR7 expressing mesenchymal stem cells potently inhibit graft-versus-host disease by spoiling the fourth supplemental Billingham's tenet.

The clinical acute graft-versus-host disease (GvHD)-therapy of mesenchymal stem cells (MSCs) is not as satisfactory as expected. Secondary lymphoid organs (SLOs) are the major niches serve to initiate immune responses or induce tolerance. Our previous study showed that CCR7 guide murine MSC line C3H10T1/2 migrating to SLOs. In this study, CCR7 gene was engineered into murine MSCs by lentivirus transfection system (MSCs/CCR7). The immunomodulatory mechanism of MSCs/CCR7 was further investigated. Provoked by inflammatory cytokines, MSCs/CCR7 increased the secretion of nitric oxide and calmed down the T cell immune response in vitro. Immunofluorescent staining results showed that transfused MSCs/CCR7 can migrate to and relocate at the appropriate T cell-rich zones within SLOs in vivo. MSCs/CCR7 displayed enhanced effect in prolonging the survival and alleviating the clinical scores of the GvHD mice than normal MSCs. Owing to the critical relocation sites, MSCs/CCR7 co-infusion potently made the T cells in SLOs more naïve like, thus control T cells trafficking from SLOs to the target organs. Through spoiling the fourth supplemental Billingham's tenet, MSCs/CCR7 potently inhibited the development of GvHD. The study here provides a novel therapeutic strategy of MSCs/CCR7 infusion at a low dosage to give potent immunomodulatory effect for clinical immune disease therapy.

SOCS1 regulates the immune modulatory properties of mesenchymal stem cells by inhibiting nitric oxide production.

Mesenchymal stem cells (MSCs) have been shown to be highly immunosuppressive and have been employed to treat various immune disorders. However, the mechanisms underlying the immunosuppressive capacity of MSCs are not fully understood. We found the suppressor of cytokine signaling 1 (SOCS1) was induced in MSCs treated with inflammatory cytokines. Knockdown of SOCS1 did not bring much difference on the proliferation and differentiation properties of MSCs. However, MSCs with SOCS1 knockdown exhibited enhanced immunosuppressive capacity, showing as inhibiting T cell proliferation at extremely low ratio (MSC to T) in vitro, significantly promoting tumor growth and inhibiting delayed-type hypersensitivity response in vivo. We further demonstrated that SOCS1 inhibited the immunosuppressive capacity of MSCs by reducing inducible nitric oxide synthase (iNOS) expression. Additionally, we found the significantly lower SOCS1 expression and higher nitric oxide (NO) production in MSCs isolated from synovial fluid of rheumatoid arthritis patients. Collectively, our data revealed a novel role of SOCS1 in regulating the immune modulatory activities of MSCs.

[A new method for isolating and culturing mouse bone marrow mesenchymal stem cells].

This study was purposed to establish a convenient and efficient method for isolating and culturing mouse bone marrow mesenchymal stem cells (MSC). The femurs and tibias of mouse were taken under sterile condition. MSC were isolated and cultured with flushing- out bone marrow or collagenase-digested bone fragment or bone marrow plus bone fragment. MSC colony number and size were compared. Immunophenotype and differentiation ability were tested to identify MSC. The results showed that colonies from bone marrow plus bone fragment group came out earliest and the colony number was 20 ± 4 at day 4; there were 11.5 ± 2.5 colonies in collagenase-digested bone fragment group and 9.5 ± 1.5 in flushing- out bone marrow group. The total cell yields of MSC after passaging showed best in bone marrow plus bone fragment group. Flow cytometry data showed the cultured cells expressed Sca-1, CD44 and CD29, not expressed pan-leukocyte surface marker CD45 and endothelial cell marker CD31. The isolated and cultured MSC could differentiate into osteoblast at the osteogenic differentiation condition, or adipocyte at adipogenic differentiation condition. It is concluded that the method of bone marrow plus bone fragment is convenient and efficient for isolating and culturing MSC.

Temporal activation of ß-catenin signaling in the chondrogenic process of mesenchymal stem cells affects the phenotype of the cartilage generated.

Adult mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering. In vitro predifferentiation of MSCs has been explored as a means to enhance MSC-based articular cartilage repair. However, there remain challenges to control and prevent the premature progression of MSC-derived chondrocytes to the hypertrophy. This study investigated the temporal effect of transforming growth factor (TGF)-ß and ß-catenin signaling co-activation during MSC chondrogenic differentiation and evaluated the influence of these predifferentiation conditions to subsequent phenotypic development of the cartilage. MSCs were differentiated in chondrogenic medium that contained either TGFß alone, TGFß with transient ß-catenin coactivation, or TGFß with continuous ß-catenin coactivation. After in vitro differentiation, the pellets were transplanted into SCID mice. Both coactivation protocols resulted in the enhancement of chondrogenic differentiation of MSCs. Compared with TGFß activation, transient coactivation of TGFß-induction with ß-catenin activation resulted in heightened hypertrophy and formed highly ossified tissues with marrow-like hematopoietic tissue in vivo. The continuous coactivation of the 2 signaling pathways, however, resulted in inhibition of progression to hypertrophy, marked by the suppression of type X collagen, Runx2, and alkaline phosphatase expression, and did not result in ossified tissue in vivo. Chondrocytes of the continuous co-activation samples secreted significantly more parathyroid hormone-related protein (PTHrP) and expressed cyclin D1. Our results suggest that temporal co-activation of the TGFß signaling pathway with ß-catenin can yield cartilage of different phenotype, represents a potential MSC predifferentiation protocol before clinical implantation, and has potential applications for the engineering of cartilage tissue.

Zinc-finger protein 145, acting as an upstream regulator of SOX9, improves the differentiation potential of human mesenchymal stem cells for cartilage regeneration and repair.

OBJECTIVE: Human mesenchymal stem cells (hMSCs) represent one of the most promising stem cell therapies for traumatic injury and age-related degenerative diseases involving cartilage. However, few genetic factors regulating chondrogenesis of MSCs have been identified. One study showed that zinc-finger protein 145 (ZNF145), a transcription factor, was up-regulated during 3-lineage differentiation of hMSCs. The present study was undertaken to validate whether this novel transcription factor is useful for the repair and regeneration of cartilage. METHODS: Human MSCs were transfected with lentiviral short hairpin RNA (for small interfering RNA knockdown of ZNF145) and a lentiviral vector for overexpression of ZNF145, and the effects of ZNF145 on chondrogenesis were studied using quantitative polymerase chain reaction and immunostaining. Microarray and transient expression analyses were used to determine whether ZNF145 is a factor operating upstream of SOX9. Allogeneic transplantation of hMSCs into osteochondral defects in rats was performed to determine the effects of ZNF145 on repair of cartilage in vivo. RESULTS: Small interfering RNA-mediated gene silencing of ZNF145 slowed down chondrogenesis, whereas overexpression of ZNF145 enhanced chondrogenesis. Global gene expression profiling showed up-regulated gene expression in ZNF145-overexpressing MSCs, and transient overexpression of ZNF145 enhanced the expression of SOX9, suggesting that ZNF145 acts as a factor upstream of SOX9, the master regulator of chondrogenesis. Moreover, allogeneic transplantation of hMSCs into osteochondral defects of rat knees showed that ZNF145-overexpressing MSCs repaired cartilage defects better and earlier than empty control MSCs. CONCLUSION: These findings suggest that ZNF145 gene therapy may be a very useful strategy for improving the quality of cartilage regeneration and repair.

Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells.

Human embryonic stem cells (hESCs) have the potential to offer a virtually unlimited source of chondrogenic cells for use in cartilage repair and regeneration. We have recently shown that expandable chondrogenic cells can be derived from hESCs under selective growth factor-responsive conditions. In this study, we explore the potential of these hESC-derived chondrogenic cells to produce an extracellular matrix (ECM)-enriched cartilaginous tissue construct when cultured in hyaluronic acid (HA)-based hydrogel, and further investigated the long-term reparative ability of the resulting hESC-derived chondrogenic cell-engineered cartilage (HCCEC) in an osteochondral defect model. We hypothesized that HCCEC can provide a functional template capable of undergoing orderly remodeling during the repair of critical-sized osteochondral defects (1.5 mm in diameter, 1 mm depth into the subchondral bone) in a rat model. In the process of repair, we observed an orderly spatial-temporal remodeling of HCCEC over 12 weeks into osteochondral tissue, with characteristic architectural features including a hyaline-like neocartilage layer with good surface regularity and complete integration with the adjacent host cartilage and a regenerated subchondral bone. By 12 weeks, the HCCEC-regenerated osteochondral tissue resembled closely that of age-matched unoperated native control, while only fibrous tissue filled in the control defects which left empty or treated with hydrogel alone. Here we demonstrate that transplanted hESC-derived chondrogenic cells maintain long-term viability with no evidence of tumorigenicity, providing a safe, highly-efficient and practical strategy of applying hESCs for cartilage tissue engineering.

Differentiation and enrichment of expandable chondrogenic cells from human embryonic stem cells in vitro.

Human embryonic stem cells (hESCs) are considered as useful tools for pre-clinical studies in regenerative medicine. Although previous reports have shown direct chondrogenic differentiation of mouse and hESCs, low yield and cellular heterogenicity of the resulting cell population impairs the generation of sufficient numbers of differentiated cells for further testing and applications. Based on our previously established high-density micromass model system to study hESC chondrogenesis, we evaluated the effects of transforming growth factor (TGF)-beta(1) and bone morphogenetic protein-2 on early stages of chondrogenic differentiation and commitment by hESCs. Significant chondrogenic induction of hESCs, as determined by quantitative measurements of cartilage-related gene expression and matrix protein synthesis, was achieved in the presence of TGF-beta(1). By means of selective growth factor combination (TGF-beta(1), FGF-2 and platelet-derived growth factor-bb) and plating on extracellular matrix substratum, we report here the reproducible isolation of a highly expandable, homogenous and unipotent chondrogenic cell population, TC1, from chondrogenically committed hESCs. Like primary chondrocytes, TC1 rapidly dedifferentiates upon isolation and monolayer expansion but retains the chondrogenic differentiation potential and responds to TGF-beta(1) for cartilaginous tissue formation both in vitro and in vivo. In addition, TC1 displays a somatic cell cycle kinetics, a normal karyotype and does not produce teratoma in vivo. Thus, TC1 may provide a potential source of chondrogenic cells for drug testing, gene therapy and cell-based therapy.

Effects of ectopic Nanog and Oct4 overexpression on mesenchymal stem cells.

Mesenchymal stem cells (MSCs) represent a source of pluripotent cells that are already in various phases of clinical application. However, the use of MSCs in tissue engineering has been hampered largely due to their limitations, including low proliferation, finite life span, and gradual loss of their stem cell properties during ex vivo expansion. Nanog and Oct4 are key transcription factors essential to the pluripotent and self-renewing phenotypes of undifferentiated embryonic stem cells (ESCs). To determine whether Nanog and Oct4 improve human bone marrow-MSC quality, we therefore established stable Nanog and Oct4 overexpressing MSCs using a lentiviral system and showed that this promoted cell proliferation and enhanced colony formation of MSCs. In differentiating MSCs, Nanog, and Oct4, overexpression had converse effects on adipogenesis of MSCs and Nanog overexpression slowed down adipogenesis, whereas Oct4 overexpression improved adipogenesis. Nanog and Oct4 overexpression both improved chondrogenesis. Microarray data showed many differences in transcriptional targets in undifferentiated MSCs overexpressing Nanog and Oct4. These results provide insight into the improvement of the stemness of MSCs by genetic modification with stemness-related genes.

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

OBJECTIVE: To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). METHODS: HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. RESULTS: Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. CONCLUSION: The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

Engineering cardiac tissue from embryonic stem cells.

Restoration of cardiac function by replacement of diseased myocardium with functional cardiac myocytes may offer a potential cure for cardiac disease and will likely revolutionize treatment methods. During the past 20 years, we have seen the development of tissue engineering; among these types of tissue engineering is cardiac tissue engineering. This type of cardiac tissue engineering includes growing neonatal cardiomyocytes on preformed polymers, liquid collagen, and temperature-responsive surfaces. It also includes the application of neonatal rat or chick cardiomyocytes to skeletal myoblasts, mesenchymal stem cells and embryonic stem cells, static culture, and bioreactor and stretching cultivation. Progress has come step-by-step, but, in recent years, with great technological advances, the progress has been accelerating, moving this area of research from dream to reality. The engineered cardiac tissue not only reproduces in vitro, but it can also be shaped so that it will, at some time, be able to form valves or endothelial lining. This chapter describes the currently used protocols for cardiac tissue engineering: liquid collagen-based cardiac tissue engineering and cell sheet-based cardiac tissue engineering, especially cardiac tissue engineering using cardiomyocytes derived from embryonic stem cells.

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.

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.

Comparison of two types of alginate microcapsules on stability and biocompatibility in vitro and in vivo.

Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases, especially diabetes. Range-scale application of the technique, however, is hampered by insufficient stability of the capsules. It is difficult to find the optimal membrane to meet all the properties required for cell transplantation. To overcome these difficulties, it is necessary to compare characteristics such as mechanical strength, cell proliferation and biocompatibility of different membranes. We prepared Ca-alginate-poly-L-lysine-alginate (APA) and Ba-alginate-poly-L-lysine-alginate (BPA) microcapsules using the electrostatic droplet method. The integrity of the microcapsules was measured by suspending them in a saline buffer and shaking at 150 rpm for 48 h. The microcapsules were cultured in simulated body fluid to analyze the osmotic pressure stability and implanted in the leg muscle pouch of SD rats to test in vivo transplantation stability. The microcapsules were implanted in the intraperitoneal cavity; then the biocompatibility of microcapsules was identified through analyzing fibrosis formation of microcapsules. The proliferation of cells (Cos-7 and HL-60) cultured in the microcapsules was measured by MTT assay. After 48 h shaking at 150 rpm, the percentages of intact microcapsules of BPA and APA microcapsules were 98.5 +/- 0.248% and 95.7 +/- 0.221% (p < 0.05), respectively. The intact percentages of APA and BPA microcapsules were 96.9% and 97.7%, respectively, after being soaked in SBF at 37 degrees C for 15 days. The empty APA and BPA microcapsules were not adhered to the muscle and there was light cellular overgrowth. There is no difference on biocompatibility in implantation into peritoneal cavities. After the cells were cultured in microcapsules, A(490 nm) of the 8th week was significantly higher than that of 1 day, and the 4th week was at the peak of the cell proliferation curve. After culture for 2 to 6 weeks, spheroids started to develop gradually within the beads. The mechanical strength of BPA microcapsules was higher than that of APA microcapsules. However, there was no difference between the two kinds of capsules in biocompatibility. Microencapsulation did not affect cell proliferation or increase the quantity of cells. In conclusion, BPA microcapsules were more suitable for transplantation in vivo.

[Reconstruction of dentin-pulp complex structure by tissue engineering technology].

OBJECTIVE: To investigate the possibility of reconstruction of dentin-pulp complex by tissue engineering technology. METHODS: Rat dental pulp stem cells were seeded into HA-TCP scaffold and incubated for 20 hours in vitro. Then the cell-scaffold complex was implanted subcutaneously into the dorsal side of nude mice. 8 weeks postimplantation, the samples were extracted for histological and immunohistochemical examinations. RESULTS: Three strata of tissue were observed in the hole of HA-TCP scaffold. They were dentin-like tissue, predentin-like tissue and pulp-like tissue respectively from the inner surface of the pore to the center. Dentin tubules were obvious in predentin-like and dentin-like tissue lining from the pulp-like tissue through predentin-like tissue and dentin-like tissue. Cells localized along the edge of pulp-like tissue were dense and polarized, resembling odontoblasts. Immunohistochemical study demonstrated DSP and DMP1 expression in these odontoblast-like cells and in the area of predentin-like tissue. CONCLUSIONS: Tissue-engineered rat dentin-pulp complex was reconstructed by seeding HA-TCP scaffold with rat dental pulp stem cells.

[Scalable production of embryoid bodies with the rotay cell culture system.].

Embryonic stem (ES) cells are pluripotent cells capable of extensive proliferation while maintaining their potential to differentiate into any cell type in the body. ES cells can therefore be considered a renewable source of therapeutically useful cells. While ES-derived cells have tremendous potential in many experimental and therapeutic applications, the scope of their utility is dependent on the availability of relevant cell quantities. Therefore, most of the researches are being focused on the differentiation of ES cells. ES cell aggregation is important for embryoid body (EB) formation and the subsequent generation of ES cell derivatives. EB has been shown to recapitulate aspect of early embryogenesis, including the formation of a complex three-dimensional architecture wherein cell-cell and cell-matrix interactions are thought to support the development of the three embryonic germ layers and their derivatives. Standard methods of EB formation include hanging drop and liquid suspension culture. Both culture systems maintain a balance between allowing ES cell aggregation necessary for EB formation and preventing EB agglomeration for efficient cell growth and differentiation. However, they are limited in their production capacity. In this paper, we established a new approach for the mass production of EBs in a scalable culture system. The rotary cell culture system (RCCS, STLV type) was adopted to produce EBs. The vessel was placed on its rotary base and the experiment started with a beginning rotation rate of approximately 8 r/min which has been previously determined empirically as the optimal initial speed to yield randomized gravitational vectors while minimizing fluid shear stress. To keep the aggregations pfloating in simulated microgravityq, the rotation rate was increased as the EBs visibly grew. The EB production efficiency was calculated when different cell densities were inoculated. The kinetic change of EBs was measured during the time course of EB formation. Compared with the traditional method of producing EBs with hanging drop, the multi-potential of the resulting EBs in RCCS was analyzed by the capability of cardiomyocyte genesis. The results showed that EBs could be produced by RCCS with high efficiency. The optimal cell density inoculated in RCCS was 10000 cells/ml, in which EB production was about twice higher than that in the suspending culture. Day 4-5 was the optimal time point for harvesting EBs. To clarify whether the differentiated potential of EBs might be affected by the microgravity produced by the rotary cell culture system, cardiogenic induction during ES cell differentiation was evaluated in our study. It was manifested by appearance of spontaneously and rhythmically contracting myocytes. In addition, immuno-histological and RT-PCR detection showed that the harvested EBs in RCCS exhibited the expected cardiac genesis and morphology. So, scalable production of EBs is obtained by RCCS. It will provide a useful approach to generate a large quantity of ES-derived cells for further research or application.

Construction of a unidirectionally beating 3-dimensional cardiac muscle construct.

BACKGROUND: Cardiac tissue engineering aims to construct cardiac tissue with characteristics similar to those of the native tissue. Engineered cardiac tissues (ECTs) can be constructed using synthetic scaffold or liquid collagen. We report an initial study using our own newly designed cardiac muscle device to construct heart tissue. We investigated the effects of cell seeding density and collagen quantity on the formation of liquid collagen-based cardiac muscle. METHODS: We obtained cardiac myocytes from neonatal rats mixed with collagen type I and matrix factors cast in circular molds to form circular strands. Cell densities (0.1 x 10(7) to 6 x 10(7)) and collagen quantity (0.3 to 1.0 ml/ECT) were tested. Cell gross morphology, cell orientation, spatial distribution and ultrastructure were evaluated using histologic analyses, confocal laser scanning microscopy and transmission electron microscopy. RESULTS: Histologic analyses of ECTs revealed that cardiac cells reconstituted longitudinally oriented, cardiac bundles with morphologic features characteristic of the native tissue. Confocal and electron microscopy demonstrated that, using optimized cell density and collagen quantity, we made ECTs with characteristic features similar to those of native differentiated myocardium. CONCLUSIONS: ECTs comparable to native cardiac tissue can be engineered under optimized conditions. This construct is a first step in the development of cardiac tissue engineered in vitro, and may be used as a basis for studies of cardiac development, drug testing and tissue replacement therapy.

A study on biomineralization behavior of N-methylene phosphochitosan scaffolds.

Biomimetic growth of calcium phosphate over natural polymer may be an effective approach to constituting an organic/inorganic composite scaffold for bone tissue engineering. In this work, N-methylene phosphochitosan (NMPCS) was prepared via formaldehyde addition and condensation with phosphoric acid in a step that allowed homogeneous modification without obvious deterioration in chitosan (CS) properties. The NMPCS obtained was characterized by using FT-IR and elemental analysis. The macroporous scaffolds were fabricated through a freeze-drying technique. A comparative study on NMPCS and CS scaffold biomimetic mineralization was carried out in different media, i.e, a simulated body fluid (SBF) or alternative CaCl(2) and Na(2)HPO(4) solutions respectively. Apatite formation within NMPCS and CS scaffolds was identified with FT-IR, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) and X-ray diffractometery (XRD). The results revealed alternate soaking of the scaffolds in CaCl(2) and Na(2)HPO(4) solutions was better than soaking in SBF solution alone in relation to apatite deposition on the scaffold pore walls. Biomineralization provides an approach to improve nature derived materials, e.g., chitosan derivative NMPCS properties e.g., compressive modulus, etc. SEM image of a NMPCS/apatite composite scaffold.

[Experimental study of tissue-engineered heart tissue using type I collagen as scaffold].

OBJECTIVE: To construct tissue-engineered heart tissue (EHT) using liquid collagen as scaffold. METHODS: Neonatal rat cardiac myocytes were isolated, cultured, and mixed with liquid collagen type I and matrix factors and then cast in circular molds to construct circular cardiac myocytes/collagen strand. After a 7-day culture in circular molds, the strands were removed, and subjected to 10% static stretch for another 7 days. Microscopy and transmission electron microscopy, routine HE staining and immunohistochemical staining were used to analyze the engineered heart tissue. RESULTS: Beating areas could be seen on the surface of the EHTs at the second day after stretching; more beating areas could be seen thereafter. These areas beat stronger and stronger, and finally came to synchronzation. Histological and immunohistochemical analyses showed that the cardiac myocytes in the EHTs distributed evenly in the whole strand and the majority of the cells, with elongated nuclei, stretched along the stretching direction. The morphology of EHTs resembled that of the native adult cardiac tissue. Transmission electron microscopy revealed that the cardiac myocytes in EHTs contained arranged myofibrils oriented parallel to the longitudinal cell axis. Clearly defined sarcomeres and Z lines were observed. CONCLUSION: Liquid type I collagen is a good scaffold for generation of EHTs similar to the native heart tissue.

[Experimental study of cardiac muscle tissue engineering in bioreactor].

OBJECTIVE: This study investigates construction of cardiac muscle cell-porous collagen scaffold complex in a bioreactor so as to unveil the possibility of generating 3-dimensional cardiac muscle tissue under the environment that mimics microgravity in vitro. METHODS: 1-2-day old neonatal rat cardiac muscle cells were isolated by sequential digestion and pre-plating methods, then seeded onto porous collagen scaffold. The cell-collagen complex was transferred into rotary cell culture system (RCCS) and incubated for 7 days. Cells cultured in 75 ml flasks and constructs cultures on plates served as control. Morphological changes of the cells were observed by light microscope and metabolic rate was recorded. Ultrastructure of the cells growing in porous collagen was observed by transmission electron microscopy. Content of total DNA and protein in the newly-formed tissue were analyzed. H-E and anti-sarcomeric alpha-actin stains were performed in comparison with native cardiac muscle. RESULTS: The isolated cardiac muscle cells adhered to the bottom of the flasks 24 hours after plating and began to beat spontaneously. When incubated for 7 days in RCCS, cell-collagen constructs of form a continuous outer tissue layer containing cells aligned with each other. The cell population in the interior of the construct was less in density than the outer part. Transmission electron microscopy demonstrated that subcellular elements characteristic of cardiac myocytes were in the outermost layer of constructs. A strongly positive stains of anti-sarcomeric alpha-actin suggested presence of cell population of differentiated cardiac myocytes in these constructs. Construct biomass was not significantly different from that in neonatal rat ventricle and approximately 40% of that in adult rat ventricles. Construsts in plates contained a few of cells which were less than those in RCCS. Metabolic activity of cells cultured in RCCS was higher than that in flasks and plates. CONCLUSIONS: Dissociated cardiac muscle cells cultured on 3-dimensional scaffolds in RCCS under favorable conditions can form engineered constructs with structural and functional features resembling those of native cardiac tissue.