Haibin Wang

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.

The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment

One challenge of cellular cardiomyoplasty for myocardial infarction (MI) is how to improve MI microenvironment to facilitate stem cell engraftment, survival and homing for myocardial repair. The application of injectable hydrogels is an effective strategy. However, it has not been thoroughly investigated on the role of the injectable scaffolds, in improving MI microenvironment, providing space and guidance for cell survival, engraftment and homing. We explored an injectable chitosan hydrogel for stem cell delivery into ischemic heart and investigated the beneficial effects and mechanisms of the hydrogel. In vitro, H(2)O(2)-treatment was used to mimic reactive oxygen species (ROS) microenvironment. The influence of ROS and protection of chitosan components on adipose-derived mesenchymal stem cells (ADSCs) was analyzed too. In vivo, MI was induced by the left anterior descending artery ligation in SD rats. PBS, chitosan hydrogel, ADSC/PBS and ADSC/chitosan hydrogel were injected into the border of infracted hearts respectively. Multi-techniques were used to assess the beneficial effects of chitosan hydrogel after transplantation. We observed that ROS generated by ischemia would impair ADSC adhesion molecules, including integrin-related adhesion molecules integrin αV and β1, focal adhesion-related molecules p-FAK and p-Src, and corresponding ligands of host myocardium ICAM1 and VCAM1. Chitosan hydrogel could rescue these molecules through ROS scavenging and recruit key chemokine for stem cell homing, such as SDF-1. The results suggest that chitosan hydrogel could improve MI microenvironment, enhance stem cell engraftment, survival and homing in ischemic heart through ROS scavenging and chemokine recruitment, contributing to myocardial repair.

The support of matrix accumulation and the promotion of sheep articular cartilage defects repair in vivo by chitosan hydrogels

OBJECTIVE Chitosan has been widely used as an injectable scaffold in cartilage tissue engineering due to its characteristic biocompatibility and biodegradability. In this study, chitosan was used in its hydrogel form as a scaffold for chondrocytes that act to reconstruct tissue-engineered cartilage and repair articular cartilage defects in the sheep model. This study aims to find a novel way to apply chitosan in cartilage tissue engineering. METHODS Temperature-responsive chitosan hydrogels were prepared by combining chitosan, beta-sodium glycerophosphate (GP) and hydroxyethyl cellulose (HEC). Tissue-engineered cartilage reconstructions were made in vitro by mixing sheep chondrocytes with a chitosan hydrogel. Cell survival and matrix accumulation were analyzed after 3 weeks in culture. To collect data for in vivo repair, reconstructions cultured for 1 day were transplanted to the freshly prepared defects of the articular cartilage of sheep. Then at both 12 and 24 weeks after transplantation, the grafts were extracted and analyzed histologically and immunohistochemically. RESULTS The results showed that the chondrocytes in the reconstructed cartilage survived and retained their ability to secrete matrix when cultured in vitro. Transplanted in vivo, the reconstructions repaired cartilage defects completely within 24 weeks. The implantation of chitosan hydrogels without chondrocytes also helps to repair cartilage defects. CONCLUSIONS The chitosan-based hydrogel could support matrix accumulation of chondrocytes and could repair sheep cartilage defects in 24 weeks. This study showcased the success of a new technique in its ability to repair articular cartilage defects.

Improved myocardial performance in infarcted rat heart by co-injection of basic fibroblast growth factor with temperature-responsive Chitosan hydrogel

BACKGROUND Basic fibroblast growth factor (bFGF) stimulates neoangiogenesis. The sustained release of bFGF by using biomaterials helped to enhance its angiogenic activity in vivo. In this study we investigated the effects of co-injection of bFGF with temperature-responsive chitosan hydrogel on myocardial performance in a rat model of infarction. METHODS Myocardial infarction was induced in rats using coronary artery ligation. Temperature-responsive chitosan hydrogel was prepared and injected intramyocardially into the left ventricular wall of rat infarction models alone or together with bFGF. Detailed histologic analysis and echocardiography were used to determine the structural and functional consequences 4 weeks after injection. RESULTS Heart function improved significantly in the chitosan+bFGF group compared with the phosphate-buffered saline (PBS)+bFGF group with regard to left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS) 4 weeks after transplantation (p < 0.05, n = 8 per group). In addition, arteriole densities within the infarcted area improved significantly (p < 0.01) in the chitosan+bFGF group (259 +/- 22/mm(2)) compared with the PBS+bFGF group (95 +/- 18/mm(2); n = 8 per group) at 4 weeks after transplantation. Infarct size and fibrotic area decreased significantly (p < 0.05) in the chitosan+bFGF group (39.64 +/- 1.75% and 25.09 +/- 3.31%, respectively) compared with the PBS+bFGF group (48.91 +/- 1.39% and 48.0 +/- 3.83%, respectively; n = 8 per group). No significant difference (p > 0.05) was noted between the PBS and PBS+bFGF groups. CONCLUSIONS Co-injection of bFGF with temperature-responsive chitosan hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling and cardiac function. Our findings suggest a new approach to improve infarcted repairs in the prevention of adverse remodeling after myocardial infarction.

Injectable cardiac tissue engineering for the treatment of myocardial infarction.

•  Introduction •  Seeding cell sources •  Embryonic stem cells •  Induced pluripotent stem cells •  Adult stem cells ‐  Skeletal myoblasts (SkM) ‐  Bone marrow‐derived stem cells ‐  Cardiac stem cells ‐  Other adult stem cells •  Biomaterials in the injectable cardiac tissue engineering •  Fibrin •  Alginate •  Matrigel •  Collagen •  Chitosan •  Other biomaterials •  Clinical applications •  Future perspectives

Preservation of the cardiac function in infarcted rat hearts by the transplantation of adipose-derived stem cells with injectable fibrin scaffolds

Cell-based therapy can improve cardiac function but is limited by the low cell retention and survival within ischemic tissues. Injectable cardiac tissue engineering aims to support cell-based therapies and enhance their efficacy for cardiac diseases. So far, no research has been devoted to studying the usefulness of the combination of fibrin glue (as scaffold) and adipose-derived stem cells (ADSCs) to treat myocardial infarction. In our study, the rat ADSCs were isolated from subcutaneous adipose tissues. The surface phenotype of these cells was analyzed by flow cytometry. The fibrin glue was then co-injected with ADSCs into the left ventricular wall of rat infarction models. The structure and functional consequences of transplantation were determined by detailed histological analysis and echocardiography. Most cultured ADSCs expressed CD105 and CD90, and were negative for CD34 and CD45. After injection, both the 24-h cell retention and four-week graft size were significantly higher and larger in the Fibrin + ADSCs group than those of the ADSCs group alone (P < 0.01). The heart function improved significantly in the Fibrin + ADSCs group compared with that of the ADSCs group four weeks after transplantation (P < 0.01). In addition, the arteriole densities within the infarcted area improved significantly in the Fibrin + ADSCs group compared with those in the ADSCs group four weeks after transplantation (P < 0.01). In conclusion, the ADSCs with the fibrin glue has the therapeutic potential to improve the function of infarcted hearts. The method of in situ injectable tissue engineering combining fibrin glue with ADSCs is promising clinically.

Promotion of cardiac differentiation of brown adipose derived stem cells by chitosan hydrogel for repair after myocardial infarction

The ability to restore heart function by replacement of diseased myocardium is one of the great challenges in biomaterials and regenerative medicine. Brown adipose derived stem cells (BADSCs) present a new source of cardiomyocytes to regenerate the myocardium after infarction. In this study, we explored an injectable tissue engineering strategy to repair damaged myocardium, in which chitosan hydrogels were investigated as a carrier for BADSCs. In vitro, the effect and mechanism of chitosan components on the cardiac differentiation of BADSCs were investigated. In vivo, BADSCs carrying double-fusion reporter gene (firefly luciferase and monomeric red fluorescent protein (fluc-mRFP)) were transplanted into infarcted rat hearts with or without chitosan hydrogel. Multi-techniques were used to assess the effects of treatments. We observed that chitosan components significantly enhanced cardiac differentiation of BADSCs, which was assessed by percentages of cTnT(+) cells and expression of cardiac-specific markers, including GATA-4, Nkx2.5, Myl7, Myh6, cTnI, and Cacna1a. Treatment with collagen synthesis inhibitors, cis-4-hydroxy-D-proline (CIS), significantly inhibited the chitosan-enhanced cardiac differentiation, indicating that the enhanced collagen synthesis by chitosan accounts for its promotive role in cardiac differentiation of BADSCs. Longitudinal in vivo bioluminescence imaging and histological staining revealed that chitosan enhanced the survival of engrafted BADSCs and significantly increased the differentiation rate of BADSCs into cardiomyocytes in vivo. Furthermore, BADSCs delivered by chitosan hydrogel prevented adverse matrix remodeling, increased angiogenesis, and preserved heart function. These results suggested that the injectable cardiac tissue engineering based on chitosan hydrogel and BADSCs is a useful strategy for myocardium regeneration.

Injectable biodegradable hydrogels for embryonic stem cell transplantation: improved cardiac remodelling and function of myocardial infarction

In this study, an injectable, biodegradable hydrogel composite of oligo[poly(ethylene glycol) fumarate] (OPF) was investigated as a carrier of mouse embryonic stem cells (mESCs) for the treatment of myocardial infarction (MI). The OPF hydrogels were used to encapsulate mESCs. The cell differentiation in vitro over 14 days was determined via immunohistochemical examination. Then, mESCs encapsulated in OPF hydrogels were injected into the LV wall of a rat MI model. Detailed histological analysis and echocardiography were used to determine the structural and functional consequences after 4 weeks of transplantation. With ascorbic acid induction, mESCs could differentiate into cardiomyocytes and other cell types in all three lineages in the OPF hydrogel. After transplantation, both the 24‐hr cell retention and 4‐week graft size were significantly greater in the OPF + ESC group than that of the PBS + ESC group (P < 0.01). Four weeks after transplantation, OPF hydrogel alone significantly reduced the infarct size and collagen deposition and improved the cardiac function. The heart function and revascularization improved significantly, while the infarct size and fibrotic area decreased significantly in the OPF + ESC group compared with that of the PBS + ESC, OPF and PBS groups (P < 0.01). All treatments had significantly reduced MMP2 and MMP9 protein levels compared to the PBS control group, and the OPF + ESC group decreased most by Western blotting. Transplanted mESCs expressed cardiovascular markers. This study suggests the potential of a method for heart regeneration involving OPF hydrogels for stem cell encapsulation and transplantation.

Telocytes accompanying cardiomyocyte in primary culture: two- and three-dimensional culture environment

Recently, the presence of telocytes was demonstrated in human and mammalian tissues and organs (digestive and extra‐digestive organs, genitourinary organs, heart, placenta, lungs, pleura, striated muscle). Noteworthy, telocytes seem to play a significant role in the normal function and regeneration of myocardium. By cultures of telocytes in two‐ and three‐dimensional environment we aimed to study the typical morphological features as well as functionality of telocytes, which will provide important support to understand their in vivo roles. Neonatal rat cardiomyocytes were isolated and cultured as seeding cells in vitro in two‐dimensional environment. Furthermore, engineered myocardium tissue was constructed from isolated cells in three‐dimensional collagen/Matrigel scaffolds. The identification of telocytes was performed by using histological and immunohistochemical methods. The results showed that typical telocytes are distributed among cardiomyocytes, connecting them by long telopodes. Telocytes have a typical fusiform cell body with two or three long moniliform telopodes, as main characteristics. The vital methylene blue staining showed the existence of telocytes in primary culture. Immunohistochemistry demonstrated that some c‐kit or CD34 immuno‐positive cells in engineered heart tissue had the morphology of telocytes, with a typical fusiform cell body and long moniliform telopodes. Also, a significant number of vimentin+ telocytes were present within engineered heart tissue. We suggest that the model of three‐dimensional engineered heart tissue could be useful for the ongoing research on the functional relationships of telocytes with cardiomyocytes. Because the heart has the necessary potential of changing the muscle and non‐muscle cells during the lifetime, telocytes might play an active role in the heart regeneration process. Moreover, telocytes might be a useful tool for cardiac tissue engineering.

Reconstruction of Engineered Uterine Tissues Containing Smooth Muscle Layer in Collagen/Matrigel Scaffold In Vitro

OBJECTIVE This study attempted to reconstruct engineered uterine tissues (EUTs) containing smooth muscle layer, akin to the normal uterine wall. METHODS EUTs were reconstructed by seeding epithelial cells on top of the constructed stromal layer over smooth muscle layer. A self-made mold was used to keep the EUTs from contraction. At the same time, it provided static stretch to the EUTs. After 14 days of culture, the structure of the EUTs was analyzed histologically and immunohistochemically, or by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of integrin beta3 subunit, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and HOXA-10 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The ability of the EUTs supporting the development of embryos was estimated by coculturing embryos on the EUTs. We also tried a new method to reconstruct EUTs by mixing epithelial cell and stromal cells (1:2) in collagen/Matrigel to form an endometrial layer and putting it on top of the smooth muscle layer. The self-assembling ability of the endometrial epithelial cells and stromal cells in the reconstructed EUTs was analyzed histologically and immunohistochemically. RESULTS The results found that the constructed EUTs with the first and the second method showed three-layered structures. The epithelial layer, stromal layer, and smooth muscle layer were stained by cytokeratin 18, vimentin, and alpha-actin, respectively. TEM showed that the cells in the EUTs reconstructed by the first method were attached to each other by apical tight junctions and rivet-like desmosomes. SEM showed protruded pinopodes, microvilli, and cilium of epithelial cells. The RT-PCR analysis showed that integrin beta3 subunit, HB-EGF, and HOXA-10 were expressed in EUTs. The coculture system of EUTs improved the development rate and quality of murine embryo significantly in comparison with those of control Chatot Ziomek Bavister culture. In the EUTs reconstructed by the second method, the epithelial cells demonstrated self-assembling ability and formed epithelial cell layer on top of the stromal layer and glandular tube-like structures in the stromal layer. Columnar epithelial cells existed in some parts of the epithelial layer. CONCLUSION We engineered EUTs containing smooth muscle layer by two methods. The reconstructed EUTs could support the development of embryos. The epithelial cells showed self-assembling ability in the EUTs.