Cardiac Tissue Engineering Based On Carbon Nanotubes

Acute Myocardial Infarction (AMI) is myocardial ischemic necrosis whichresults from a sharp shortage or stop of blood supply of coronary artery. Theregeneration of the myocardial tissue is rather limited, whose necrosis will force theventricle to pathologically rebuild itself, while the necrotic myocardium will be takenplace by fibrotic scar. All these will lead to a loss of the myocardial contractility orheart failure and even death till the worst. At present, the therapeutic approachestoward AMI conclude mainly medicine treatment, interventional treatment, coronaryartery bypass surgery and heart transplantation. However, all the treatments haveobvious limitations, for example, medicine treatment, interventional treatment,coronary artery bypass surgery can ease the symptom to some extent while hearttransplantation may result in immunological rejection and complication and someother problems. Therefore, it is one of the urgent tasks to explore new treatingstrategy and method in the field of ischemic heart diseases research.With the development and blending of the research of stem cells, biomaterialsand tissue engineering, the cardiac tissue engineering, which aims to repair andrebuild cardiac tissue, has undergone a rapid development, and it brings a new hope tothe treatment of ischemic heart diseases. Seed cells、scaffolds and the construction andtransplantation of engineered cardiac tissue are the main content of cardiac tissueengineering.As for the seed cells, stem cells is an important cell resource of the cardiac tissueengineering. In the present research, some kinds of stem cells are used in the research,which include ESCs, iPSCs and dozens of tissue stem cells. Though these seedingcells show the potentiality of treating MI, it has been found that there still exists somelimitations in the treatment and application of the cells, for example, there are someproblems, such as immunological rejection and ethics matters about ESCs, some riskof oncogenicity about iPSCs, the low capacity to cardiac differentiation of MSCs, andthe limited source of CSCs and so on. By far, consensus hasn’t been reached as to theideal cell; therefore, to try to find a new stem cell source still remains the focus of thepresent cardiac tissue engineering research. Recent research shows that brown adiposetissue derived stem cells (BASCs) contains plenty of heart stem cells and has highcardiac differentiation potential. Meanwhile, in the field of transplantation, it is also found out that BASCs can be diverted into cardiac cells, thus can reduce the infarctionsurface and improve the function of the left ventricle. It follows that BASCs can beregarded as one of the source of the important seeding cells in the cardiac tissueengineering research. However, for it is new, the application of BASCs to the MItreatment need deep research.As for the scaffolds, it is a key point to develop the suitable ones. Recently, therapid development of biomaterials and nanotechnology has been highly concerned,and their application in cardiac tissue engineering has also become a focus. Thesuccessful integration of nanomaterials and traditional scaffolds has become animportant method of optimizing traditional materials. The heart is an organ which hasconductivity and lots of conducting Purkinje fibers, in shapes of mesh, are distributedin myocardium. Single myocardial cell generated excitement and contraction couplingwhen receiving electrical impulses from heart conduction system. Ventricular musclesfunction as to pump blood by synchronous contraction. However, traditional scaffoldmaterials failed to conduct electricity, but the conductivity of scaffold materials can besignificantly increased when integrated with conductive nanomaterials. Therefore, thestrategy which integrates conductive nanomaterials with traditional scaffolds has beenpaid more and more attention.Carbon nanotubes(CNTs) is a kind of cylinder lamellar structure which hassuperior mechanical properties, conductivity and thermostability. It can increasesalvation, improve biocompatibility, decrease cytotoxicity for CNTs and improvemechanical properties and conductivity for compound materials to a large extentwhen made into compound materials using CNTs and other materials throughdifferent methods. Compared with those of multi-wall carbon nanotubes (MWCNTs),the range of diameter is narrower, the defects in tubal wall are fewer and theuniformity is better in single-wall carbon nanotubes (SWCNTs). SWCNTs possess alarge specific surface area for the diameter of it is about0.6-2nm while the length of itis within micrometer scale. At present, the compound conducting nanomaterialsscaffolds based on CNTs have been applied widely and are appealing in tissueengineering area.As for the construction and transplantation of cardiac tissue, there are two mainlyapproaches, which are injectable cardiac tissue engineering and engineered cardiactissue patch.injectable cardiac tissue engineering：It is a kind of modalities by using an injectable biomaterials to deliver cells with or without growth factor directly into theinfarcted wall to improve the damaged myocardial function. Injectable biomaterialscan also be utilized in acellular approaches to support the LV wall and avoid thenegative remodeling after an MI, or for the controlled delivery of therapeutic genesand proteins to ischemic myocardium. Injectable biomaterials can improve themicroenvironment around MI and engrafted cells can promote myocardialregeneration after injectable biomaterials are transplanted with stem cells. Moreover,the approach of injectable cardiac tissue engineering is another research directionbecause it needs no special scaffolds and is minimally invasive and is benefit forclinically appealing.Many biomaterials, including fibrin, collagen, matrigel and chitosan hydrogel,have been successfully applied in injectable cardiac tissue engineering in the pastyears. However, these materials all have some drawbacks. Among these reportedscaffolds, poly(N-isopropylacrylamide)(PNIPAAm) hydrogel is an attractive one thathas been extensively studied. It is fluid below32℃and can be gelated rapidly in situwhen injected in vivo, favorable for cell engraftment. PNIPAAm hydrogel also enjoysgood biocompatibility for harvesting cell sheet. However, no report has been madeabout combining PNIPAAm hydrogel modification with CNTs as a carrier for thetreatment of MI.engineered cardiac tissue patch：It can offer proper microenvironment andstructure for cells, promote differentiation of engrafted cells and prevent their anoikis.Thus, cardiac patch has been paid widespread attention and has become an importantresearch trend in the therapy of MI. However, numerous studies are concerned aboutthe construction and transplanted application of engineered cardiac tissue, while thestudy of intercalated disc (ID) affected by cardiac tissue patch is rarely reported,which is special features architecture between the myocardial cells.ID was an important structure which connects and communicates amongmyocardial cells. ID is closely related to the conductivity and rhythmic contraction incardiac and is mainly made up by adhering junction, desmosomes and gap junction.Adhering junction and desmosomes are mainly responsible for the mechanicalcoupling among myocardial cells, while gap junctions are related to electricalcoupling and metabolic coupling among myocardial cells. These three kinds ofconnected modes are indispensable and complementary for ensuring the normalfunctioning of the ID. We tried to construct engineered cardiac tissue sheet using collagen/matrigel hydrogel material in vitro in the previous study, and made in-depthstudy of the spatial and temporal distribution of ID self-assembly during the formationof three connections. However, no relevant report is available as for the influence ofthe construction of the electrical conductivity of CNTs engineered cardiac tissue sheetupon myocardial cells ID formation, assembly and polarization.Based on the above-mentioned analysis, this study starts from the twoapproaches in myocardial tissue engineering research, and aims to make the researchdeeper in the two aspects. On the one hand, it presents how to constructPNIPAAm/SWCNTs injectable scaffolds and how to repair the damaged myocardialfunction with BASCs in vivo. On the other hand, it tries to explore the construction ofengineered myocardial tissue by using collagen functional CNTs film, and analyze theinfluence of constructing CNTs upon ID forming, assembling and functionalmaturing.Therefore, this study can be divided into two parts:Part1: Injected cardiac tissue engineering based on thePNIPAAm/SWCNTs hydrogelIn this study, first, PNIPAAm/SWCNTshydrogel is made and then is evaluated interms of physical properties, chemical properties and biocompatibility. With BASCsas the seed cells, using PNIPAAm/SWCNTs hydrogel as the injected scaffolds, weevaluate the effect of PNIPAAm/SWCNTs hydrogel over the adhesion, spread andproliferation of BASCs in vitro. Moreover, after transplantation of thePNIPAAm/SWCNTs hydrogel with BASCs, the improvement of the myocardialfunction after MI is evaluated by the methods of echocardiogram and histology; theretention and differentiation of BASCs around the edge of MI are observed byimmunofluoresence stain.The results are: the surface roughness increases obviously and has betterconductivity in the PNIPAAm/SWCNTs group, compared with PNIPAAm group.What’s more, PNIPAAm/SWCNTs has higher biocompatibility, and adhesion andproliferation of BASCs can be improved in vitro, and this can also be achieved underthe circumstance of H2O2(as exogenous reactive oxygen species, ROS). The retentionof BASCs and thickness of LV wall can be increased, and infarct size can bedecreased after the injection of BASCs using the PNIPAAm/SWCNTs scaffold, thuscan improve the myocardial function. Part2: Fabrication of SWCNTs/collagen compound scaffold-basedengineered cardiac tissue patch and influence of SWCNTs for ID development ofreconstracated cardiac tissueIn this study, we combine collagen I with SWCNTs to make compound scaffolds,and evaluate for their physical properties, chemical properties and biocompatibility.Then, we construct the cardiac patch by SWCNTs/collagen compound scaffold withneonate rat ventricular myocytes(NRVM). In different time point, we analyze theexpression, distribution, assembly and development of relevant protein to the ID ofreconstructional cardiac patch using immunofluorescence staining, western blotting,TEM and intracellular Ca2+transient.The results are as follows: SWCNTs in collagen solution are clustered indifferent levels. When the concentration is0.1mg/mL, SWCNTs are distributedevenly in collagen solution, with no obvious cluster, which is proved by SEM. Whilewith the increase of the level, the surface roughness of the compound and theconductivity are also increased by AFM and electrochemistry workstation. Comparewith those of other4groups, the results of Live/Dead staining are best when the levelof SWCNTs is0.1mg/mL. It can be indicate that the effect upon the cells is the bestwhen the level is0.1mg/mL.In the reconstructed cardiac patch group based on the SWCNTs/collagen, theexpression of α-actinin were reinforced obviously by immunofluorescence stainingand TEM detection and the polarization of Cx43were promoted significantly, whichshows that it possessed better structure and constriction, compared with those of incollagen group.In summary, this study successfully prepared SWCNTs modified PNIPAAmhydrogel. The results show that PNIPAAm/SWCNTs hydrogel promotes the adhesionand proliferation of BASCs in vitro, and improves the contractive function ofdamaged myocardium after transplantation with BASCs. Moreover, we also preparedengineered cardiac tissue patch by using collagen functionalized SWCNTs composites.The results from this study indicate that the assembly and development ofreconstructed cardiac tissue can be promoted by SWCNTs/collagen compoundscaffolds. Thus, the injectable cardiac tissue engineering and engineered cardiac tissuepatch based on SWCNTs in conjunction with current treatment modalities may helpreduce mortality and improve the quality of life in MI patients.