Qiuxia Lin

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.

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.

Tumourigenesis in the infarcted rat heart is eliminated through differentiation and enrichment of the transplanted embryonic stem cells.

The therapeutic potential of embryonic stem cells (ESCs) in ischaemic heart disease has been widely explored. However, tumourigenesis upon implantation interferes with the clinical application of ESC transplantation. This study aims to evaluate the influence of differentiation and enrichment of transplanted ESCs on tumourigenesis in infarcted rat hearts.

Self-assembly of renal cells into engineered renal tissues in collagen/Matrigel scaffold in vitro.

To tissue engineer a kidney is a formidable task because of the complex cell composition and structures in the kidney. This study reconstructed renal tissues using mixed renal cells in collagen/Matrigel® scaffolds in vitro. Neonatal rat renal cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During in vitro culture, the renal constructs were observed under microscope and analyzed by histological and immunofluorescent examinations. Results showed that the mixed renal cells reconstituted renal tubular and glomeruli‐like structures with different appearances at varying developmental stages. Tubular structures were formed by CK18‐positive cells with similar appearances lining the surrounding hollow centres. The glomeruli‐like structures were tufts of cell aggregates containing Flk‐1‐positive cells. These results show that neonatal rat renal cells self‐assembled into engineered renal tissues containing both tubules and glomeruli‐like structures when cultured in 3D collagen/Matrigel scaffold in vitro. Copyright

Carbon nanotubes enhance intercalated disc assembly in cardiac myocytes via the β1-integrin-mediated signaling pathway

Carbon nanotubes (CNTs) offer a new paradigm for constructing functional cardiac patches and repairing myocardial infarction (MI). However, little is known about how CNTs enhance the mechanical integrity and electrophysiological function of cardiac myocytes. To address this issue, we investigated the regularity and precise mechanism of the influence of CNTs on the assembly of intercalated disc (IDs). Here, single walled CNTs incorporated into collagen substrates were utilized as growth supports for neonatal cardiomyocytes, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, western blotting, transmission electron microscopy, and intracellular calcium transient measurement, we discovered that the addition of CNTs remarkably increased ID-related protein expression and enhanced ID assembly and functionality. On that basis, we further explored the underlying mechanism for how CNTs enhanced ID assembly through the use of immunohistochemical staining and western blotting. We found that the β1-integrin-mediated signaling pathway mediated CNT-induced upregulation of electrical and mechanical junction proteins. Notably, CNTs remarkably accelerated gap junction formation via activation of the β1-integrin-mediated FAK/ERK/GATA4 pathway. These findings provide valuable insight into the mechanistic effects that CNTs have on neonatal cardiomyocyte performance and will have a significant impact on the future of nanomedical research.

Molecular Imaging of Induced Pluripotent Stem Cell Immunogenicity with In Vivo Development in Ischemic Myocardium

Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.

The reconstruction of lung alveolus‐like structure in collagen‐matrigel/microcapsules scaffolds in vitro

This study attempted to use collagen–Matrigel as extracellular matrix (ECM) to supply cells with three‐dimensional (3D) culture condition and employ alginate‐poly‐l‐lysine‐alginate (APA) microcapsules to control the formation of alveolus‐like structure in vitro. We tested mice foetal pulmonary cells (FPCs) by immunohistochemistry after 2D culture. The alveolus‐like structure was reconstructed by seeding FPCs in collagen–Matrigel mixed with APA microcapsules 1.5 ml. A self‐made mould was used to keep the structure from contraction. Meanwhile, it provided static stretch to the structure. After 7, 14 and 21 days of culture, the alveolus‐like structure was analysed histologically and immunohistochemically, or by scanning transmission electron microscopy (TEM). We also observed these structures under inverted phase contrast microscope. The expression of pro‐surfactant protein C (SpC) was detected by reverse transcription‐polymerase chain reaction (RT‐PCR). We obtained fibroblasts, epithelial cells and alveolar type II (AE2) cells in FPCs. In the reconstructed structure, seeding cells surrounding the APA microcapsules constructed alveolus‐like structures, the size of them ranges from 200 to 300 μm. In each reconstructed lung tissue sheet, microcapsules had integrity. Pan‐cytokeratin, vimentin and SpC positive cells were observed in 7‐ and 14‐day cultured structures. TEM showed lamellar bodies of AE2 cells in the reconstructed tissues whereas RT‐PCR expressed SpC gene. Primary mice FPCs could form alveolus‐like structures in collagen–Matrigel/APA microcapsules engineered scaffolds, which could maintain a differentiated state of AE2 cells.