1. INTRODUCTION.

Diseases of the heart and blood vessels are the largest cause of mortality across the globe. They are the principle cause of disability and death in all industrialized nations. Currently, in USA it causes about 700000 deaths annually which is almost 40% of total mortality. Furthermore, these disease processes cause much suffering because of pain, disability and limb loss due to peripheral vascular disease (PVD).

In the brief history of modern cardiovascular medicine, it has not been uncommon for scientists, researchers and clinicians to join forces in an effort to dramatically change the development and treatment of a specific pathology. The introduction of coronary arteriography, bypass surgery, angioplasty and now, drug-eluting stents has positively influenced the care of patients suffering from lifestyle-limiting anginal symptoms due to obliterative coronary artery disease. In each of these and many other areas currently under study, the common denominator for success has been the ability to create a specific focal point where every available element of laboratory information is translated into a potential broad clinical application. About four categories of cardiac illnesses account for 85-90% of the deaths due to cardiac diseases. They are 1. Ischemic Heart diseases. (Diseases due to less blood supply to heart musculature) 2. Hypertensive and pulmonary hypertensive heart diseases. 3. Congenital heart diseases. (Birth defects like VSD, ASD, Tetrology of Fallot) 4. Valvular diseases of heart.

[1]

The etiology, pathology and different treatment modalities involved be it medical or surgical in treating these diseases is beyond the scope of this discussion since our interest lies in the knowhow of materials, technicalities and researches going on in the field of development of newer biomaterials which become a part of cardiovascular system either as prostheses or become ingenious and help in regeneration of lost cardiac or vascular tissue.

Regardless of the arena of options available to treat the cardiovascular diseases, no technique has been rated as giving 100% cure and safety. The potential risks involved in treating the cardiac diseases are many- few of which are restenosis, thrombosis, prolonged bleeding postsurgicaslly, infection and graft rejection and so on. Therefore the requirement of the day lies in designing and dispensing of newer materials which to some extent helps in overcoming these problems. During the last few decades, manmade materials and devices have been developed to the point at which they can be used to replace parts of living systems in the human body. These special materials, which are able to function in intimate contact with living tissue, with minimal adverse reaction or rejection by the body are called biomaterials. Biomaterial is any substance (other than a drug) or combination of substances, synthetic or natural in origin, which can be used for a period of time, as a whole or as a part of a system which treats, augments or replaces any tissue, organ or function in the body. Two main parameters have to be considered in choosing the biomaterial for a certain application: 1. In order to choose the right Standard design, some physical and mechanical features such as strength and deformation, fatigue and creep, friction and wear resistance, flow resistance and pressure drop, and other characteristics which may be engineered with the material, must be considered. 2. Compatibility, or biocompatibility, characterizes a set of material specifications and constraints which refer to the material tissue interactions. These
[2]

characteristics have to be specified according to the intended device application, and have to be tested and evaluated in a set of invitro and invivo experiments. Biocompatibility evaluation In order to evaluate the materials suitability for the cardiovascular application for long term implantation, the biocompatibility criteria have to include the following host reactions to the biomaterial which focus on toxicity, carcinogenicity and biostability: Foreign body reaction, Inflammatory reaction, Thrombosis, Hemolysis, Adaptation, Infection and sterilization, Carcinogenesis, Hypersensitivity and systemic effects, Long term stability, and Fatigue tests. These studies are designed according to ISO 10993 standards. Large number of materials has been tested to suit the requirements which include a variety of natural, synthetic and semi synthetic fibers, polymers, metals and also the tissue engineered materials. In the beginning let us place our interest on grafting of cardiac tissues, some techniques available and materials available.

*************

[3]

2. CARDIOVASCULAR GRAFTS WHAT IS A GRAFT? A graft is a biological material which replaces the indigenous missing tissue with the same type of tissue derived from self, other donor or synthetically prepared in a laboratory which best suits and simulates the structure and function of the tissue being replaced.

The grafts used in human body in general can be classified in following ways. 1. Autografts-Taken from ones own body. 2. Allograft-Taken from another donor living or dead. 3. Xenograft-Taken from an animal source. (commonly bovine) 4. Synthetic-Commercially prepared in laboratory.

NEED FOR CARDIOVASCULAR TISSUE GRAFTING: Diseases of heart and its valves are the major cause of death and deformity globally. The majority of surgical techniques available so far involves harvesting of the long saphenous vein from the leg or even the cephalic and basilic veins from the arm of the patient. However, in about a third of patients this proves inadequate or unsuitable. Therefore need for artificial materials which mimic the nature to maximum extent is always in demand by surgeon. In fact surgeons have already successfully tried on materials like polyethylene terephthalate (Dacron), polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE) and polyurethane.

Another important field which requires extensive research and development of new materials is the replacement of heart valves. Heart valves are nothing but gateways for the entry and exit of blood vessels into the chambers (atria and ventricles) of the heart. The specialty of the valves is that they open unidirectional, always toward the ventricles so that the regurgitation of blood to atria will never occur. The left atria-ventricles are guarded by tricuspid valve, right atria-ventricle guarded by mitral valve. The opening to pulmonary artery is guarded by pulmonary valve and aorta is guarded by aortic valve.
[4]

The current options available are mechanical and biprosthetic valve grafts both of which presents with potential difficulties of increased risk of infection, prolonged or life-long anti-coagulant therapy, increased risk of bleeding and sometime stepwise calcification leading to loss of hemodynamic performance. Therefore the currently available option to clinician is limited to treating of potential risk factors rather than alleviating it which includes plethora of drugs to be used for long duration and sometimes lifelong. The need of cardiac tissue grafting can be emphasized in following way: i. Substituting damage blood vessel of the human body- due to artificial tissue damage like in burning- here we can use dermal substitute- Integra.

ii. To replace injured blood vessel, artery, vein etc., iii. Substitute the stenosis or hardening of blood vessels, heart valves in the older patients. iv. To surgery the autogenous, saphenous veins, standard coronary and infrapopliteal bypass to many patients do not have suitable vein v. Small-caliber vascular xenograft.

[5]

REQUIREMENTS OF AN IDEAL CARDIAC GRAFT MATERIAL: The characteristics of the ideal graft have to fit the following requirements which are divided into three main parts: mechanical, biocompatibility and handling: * It must be durable, withstanding after implantation the dual threats of biodegradation and mechanical fatigue. * The ideal graft should have and maintain the same compliance as a normal artery: It should be flexible, maintaining its contour and have kinking resistance, bending without partial occlusionas it crosses joints. * The graft must not harm the host in anyway. * Its luminal surface must interact with blood elements in a minimally traumatic, nonthrombogenic fashion. * It should be resistant to infection. * It must be capable of sterilization without graft alteration. The ideal graft should have an optimal porosity, allowing for good incorporation without causing unmanageable bleeding following implantation. * Finally, from the handling point of view, it must be readily available in multiple lengths and sizes, and its handling characteristics should include an ease of suturing and maintenance of integrity.

***************

[6]

3. CARDIAC GRAFT MATERIALS

Vascular graft materials: The synthetic graft market is currently dominated by three major materials: Polyethylene Terephthalate (PET), Polytetrafluoroethylene (PTFE) and Polyurethanes. As mentioned above, vascular grafts should be chemically stable and resistant to degradation and to toxic or inflammatory by products. They have to be biocompatible and hemocompatible. Their structure has to be porotic to an extent that there wont be blood leakage. Polyester (Dacron or PET) and PTFE are chemically stable after implantation.

Dacron: Dacron: There are woven Dacron (not porous), knitted Dacron (porous), crimped grafts and newer knitted Dacron which have velour construction on their inner and outer surfaces that are thought to facilitate tissue incorporation.

In order to reduce the blood loss, knitted grafts sometimes should be pre clotted prior to insertion. The preclotting procedure is less frequently used in woven grafts because of much smaller pores produced in this technique. Dacron grafts have recently been manufactured coated with protein (collagen/albumin) to reduce the blood loss and antibiotics to prevent graft infection.

[7]

Expanded PTFE (ePTFE): Expanded PTFE (e-PTFE): ePTFE is considered a non textile technique. This is a fluorocarbon polymer, formed into sheets by a paste extrusion process, producing a porous material that has solid nodes interconnected by fine fibrils. The intranodal distance can be varied to change the graft porosity. The grafts in clinical use are impervious to blood, resistant to dilatation, and are chemically inert, highly electronegative and highly hydrophobic. ePTFE is considered to be better than Dacron for venous reconstruction if autogenous vein is not available. GORE-TEX is the newest vascular graft offering good performance by reducing kinking and compression. It is the lowest profile, radially supported graft available today. The e-PTFE are used to repair occluded arteries and veins and blood vessels in the peripheral artery. The second use of e-PTEF material is it can be used for dialysis treatment of chronic renal failure patients. These materials exhibit superior thrombo-resistence which leads to prevention of smaller blood vessels getting occluded. ePTFE grafts- the potential role of fibrin As we know that fibrin is the most important vascular endothelium growth factor (VEGF) of the blood vessels. So in this case we use a preteated heparin or fibrin seeded cells on an ePTFE or Dacron for a particular animal. Ongoing platelet activation provides high concentrations of granule products like platelet factor 3, fibrinogen and von Willebrand factor as well as dense granule contents such as calcium. At the same time, graft surfaces are continually rinsed by blood with its undiminishing concentrations of fibrinogen and other clotting factors- like TCF- (Thrombin Growth Factor). Thus, the fibrin generated on the inner surface of
[8]

vascular prostheses has a particularly high level of fibrinogen hence excellent healing properties.

Polyurethanes: Polyurethanes were first introduced to the medical device market in the earliest 50s as composites foam breast prosthesis. Since then, a lot of work and development was done to improve their characteristics and biostability. Polyurethanes advantages include a very smooth nonthrombogenic inner surface, a thin walled graft with some compliance and improved handling characteristics. Graft made by synthetic protein polymers cross-linked by -rays- new generation biopolymers: Synthetic protein polymers cross-linked by - irradiation represent a new generation of biopolymers. Preliminary reports suggest that they have similar elasticity as arteries, with a controllable rate of degradation. This recent trend illustrates the limitations of PTFE and Dacron at lower flow rates and stresses the need for alternative biomaterials. Materials such as PTFE and Dacron have little potential for micro-vessel grafts due to their poor haemodynamics caused by lack of arterial compliance. This results in thrombogenicity to the extent that in lowflow applications (as is the care for microvessels) below 4mm their usage is predelicted by their propriety for blockage. Polycarbonate-urea Urethane (CPU) Polymers:

[9]

Polyurethanes such as CPU due to their superior haemodynamics (a result of their arterial like visco-elasticity and honeycomb structure) have high applicability for microvessel development and have shown little blockage in 2mm applications. These new grafts may be constructed by using newer polymers such as CPU by coating graft lumen with anti-platelet agents or cells[most commonly endothelial cells, but also possibly fibroblasts or smooth muscle cells(SMC)] and constructing biological or bio-hybrid grafts in vitro prior to reimplantation Nylon can also be used for this type of polymer purpose-to provide good to moderate mechanical strength properties. Sol-gel systems: Gels consist of three components namely- solute, solution, and voids (air swollen macromolecules of the polymers). Example: photo-polymerisable polyvinyl alcohol (PVA), triblock co-polymers of poly (L- lactide) / PLLA and poly (ethylene oxide) (PEO) (PLLA-PEO-PLLA). These types of polymer molecules are initially functionalised with adhesion factors. These are then cross-linked via the formation of reactive termini. Cells migrating into this matrix break down the cross-linking peptide leading to local degradation and hence release of the incorporated factors such as heparin and VEGF (vascular endothelium growth factors). These synthetic gels need to be bio-degradable and cell-responsive. Using a PEG-based polymer by the shortening the block length from a mean molecular weight of 930 to 6090 kDa. The following figure shows a typical process of gel of polyethylene glycol.

[10]

Fibriller PU grafts With very rare exceptions fibrillar PU grafts are largely impenetrable for transmural tissue ingrowth. Only particularly large inter-fibrillar sp aces allow complete fibro-connective tissue penetration throughout the wall thickness.

Foamy PU grafts In microporous PU foams with less than 15 m pore size, there is relatively li ttle in growth, even over an extended period of time. Below this cut-off point, only plasma-like insulation with some erythrocytes and a few clusters of white blood cells are found in the depth of the wall. Macrophages, giant cells and a few fibroblasts may penetrate a short distance into the implant if the pore size is borderline. By introducing these well- defined, equally sized spherical pores and large, equally well defined inter connectivity.

Different approaches to achieve successful vascular graft: PROBLEMS

i. ii.

SUGGESTED SOLUTIONS

iii.

Increased risk of thrombosis and Graft made by synthetic protein polymers infection cross-linked by -rays- new generation ii. Lack of compliance or potential biopolymers. to match both of the graft and around the anastomosis Neointiminal hyperplasia Growing of EC in an unorientated fashion by transanastomotic endotheliazation rather than
[11]

excellent oriented transmural or endotheliazation

mode of blood-borne

iv. Biodegradability problem of implanted Sol-gel system graft or ECM v. Aneurismal degradation Shocking problem inside the patient body- due Fibriller PU grafts to bad implantation of arterio-venous valves Failure due to occlusion inside the blood vessel Foamy PU graft. due to abnormal obstruction

****************

[12]

4. NANOMATERIALS AS CARDIOVASCULAR GRAFTS Revolutionary advances in nanotechnology propose novel materials with superior properties for biomedical application. One of the most promising nonmaterial for biomedical application is polyhedral oligomericsilsesquioxane (POSS), an amazing nanocage consisting of an inner inorganic framework of silicon and oxygen atoms and an outer shell of organic groups. The unique properties of this nanoparticle has led to the development of a wide range of nanostructured copolymers with significantly enhanced properties including improved mechanical, chemical, and physical characteristics. Since POSS nanomaterials are highly biocompatible, biomedical application of POSS nanostructures has been intensely explored. One of the most promising areas of application of POSS nanomaterials is the development of cardiovascular implants. The incorporation of POSS into biocompatible polymers has resulted in advanced nanocomposite materials with improved hemocompatibility, antithrombogenicity, enhanced mechanical and surface properties, calcification resistance, and reduced inflammatory response, which make these materials the material of choice for cardiovascular implants.

Currently, application of POSS containing polymers in the development of new generation cardiovascular implants including heart valve prostheses, bypass grafts, and coronary stents is under intensive investigation, with encouraging outcomes. Surface modification of biomaterials used in cardiovascular grafts or implants is very much necessary since they modulate the platelet responses by directly modulating the thrombogenic proteins or by inducing antithrombogenic biomolecules. Nanotechnology is recognising a great role in such surface modification of cardiovascular implants through bio-functionalization of polymers and peptides in nanocomposites and through nanofabrication of polymers which will pave the way for finding a closer blood match through haemostasis when developing cardiovascular implants with a greater degree of patency. Silver nanoparticles or nanosilver (NS) are recognized for efficient antibacterial properties. This study aims to determine the influence of NS integrated POSS-PCU on thrombogenicity. Silver nitrate was reduced with dimethylformamide and stabilized by the inclusion of fumed silica nanoparticles to prevent aggregation of NS and were incorporated into POSS-PCU to form a range of POSS-PCU-NS
[13]

concentrations (by weight); 0.20%, 0.40%(NS32), 0.75%(NS64), and 1.50%(NS128). Surface wettability was determined with sessile-drop water contact angles. Platelets were introduced onto test samples and Alamar Blue (AB), mitochondrial-activity assay, quantified the degree of platelet adhesion whilst platelet-factor-4 (PF4) ELISA quantified the degree of platelet activation. Thromboelastography (TEG) determined the profiles of whole blood kinetics while hemolysis assay demonstrated the degree of blood compatibility. Increasing levels of NS induced greater hydrophilicity. A concentration dependant decrease in platelet adhesion and activation was observed with AB and PF4 readings, respectively.

*****************

[14]

5. TECHNICAL CONSIDERATION OF CARDIOVASCULAR GRAFTS

1. Microvascular grafts: Human micro vasculature begins with arteries dividing consecutively into smaller branches like meta-arterioles (80100m) until finally forming capillaries (10 15m). These vessels serve to redistribute blood and its nutrients whilst lowering the pressure head. This allows blood to perfuse the tissue, allowing more efficient exchange of metabolites.

* The above picture shows dual nature of vessel development; both angiogenesis (formation of blood vessels) and
arteriogenesis (formation of artery)

The vascular tree is formed during the early gestation. Angiogenic cells form clusters which coalesce to form solid tubes, which eventually canalise to form blood vessels. The outer ring consists of angioblasts which form the vessel walls. The subsequent differentiation of these precursor angioblasts into endothelial cells (EC) and the denovo formation of a vascular network ar e termed vasculogenesis. These vessels are capillary-like to begin with and eventually differentiate into either arteries or veins.

[15]

The adult vascular network remodels itself by arteriogenesis with the opening up and then the subsequent enlargement of existing collaterals (so-called collateral enlargement), as well as the formation of completely new vessels from the already existing vessels (so-called arterialisation). Micro-vascular remodelling is a mechanistic process delineated to the specific tissue type and specific stimuli. Therefore, with the exceptions of skeletal muscle responding to exercise and the female menstrual cycle itself, micro-vascular remodelling is limited solely to pathological situations, in particular inflammation, wound healing, ischemia. Microvessels divide into numerous smaller branches over a given volume of tissue, thus maximizing the available area for nutrient exchange.

Factors preventing thrombogenicity in low-flow states.

In these Microvessels the stasis (stagnation) of blood flow is prevented by repulsive charges between blood cells and vessel wall and glycocalyx film on endothelial layer. The diffusion (nutrient, oxygen exchange) through blood vessel depends on the following factors: D= - PSC. Where P=Permeability of membrane. (depends on effective pore size and pore length). C=Concentration gradient. S=Surface area. Human blood also contains plasma or aqueous liquid matrix of blood. While solvents pass unimpeded into the extracellular matrix (ECM), solutes are transported by means of convection (bulk transport) and diffusion, represented by the formula: F=Q+D. Where F is the solute flow and Q is the convective flow.

[16]

Solute efflux is dependent on the rate of solvent transfer within the medium. Solutes like glucose are transported via convection with water (solvent) Some recent studies have shown that the nutrient exchange depends on balance between the hydrostatic pressure, osmotic pressure and interstitial pressure in a microvessel. The configuration of Microvessels is something like this: The microvessels with a cross-sectional density of 1300 per mm2 and inter capillary distance of 34 m to form a rich perfusing network. Based on the above configuration the size of the microvascular grafts have internal diameter of 1mm or less and may be classified into conducting arterial and distributing capillary blood vessel. The construction of arterial conduits is based on small-calibre vascular graft technology. These new grafts may be constructed by using (a) newer polymers such as CPU (b) by coating graft lumen with anti-platelet agents or cells[most commonly endothelial cells, but also possibly fibroblasts or smooth muscle cells (SMC)] and (c) constructing biological or bio-hybrid grafts in vitro prior to reimplantation. Although the autologous vein grafts the current gold standard for micro-vascular repairs, are compliant and non-thrombogenic, they are limited by the need for additional vein-harvesting procedures. Furthermore, the construction of artificial vascularised tissue requires an inherent vascular network. Vein grafts are not suitable for this purpose as it is technically impossible to dissect out a capillary bed in its entirety. Materials used in microvascular grafts: Polyester, polyethylene terepthalate, Dacron, polytetrafluroethylene (PTFE), expanded polytetrafluroethylene (ePTFE) polyurethane etc. Poly (carbonate-urea) urethane (CPU) can also be used as the vascular graft with advanced effect of physiological shear stress. However, these prosthetic materials prove to be inferior to autologous conduits, especially when the vessel diameter is less than 5mm. The problems include increased risk of thrombosis and infection, limited durability, lack of compliance both of the graft and around the anastomosis, and failure due to restenosis, thus necessitating further interventions. Currently several groups are working towards the development of living grafts seeded grafts and hybrid grafts.

[17]

2. Macrovascular grafts: Macrovascular grafts account for the blood vessels whose diameter is more than 1 mm. The materials used in microvascular grafts have also been successfully used in grafting bigger vessels of which Dacron and PTEF are clinically more acceptable for peripheral bypass grafts where the patency is significantly lower in grafts smaller than 6 mm in diameter. The following table gives a comparative study of current status of microvascular and macrovascular grafts. Graft type. PTEF PTEF CPU Polygalactin-PGA Dimensions(mm) < 1 mm. 1 mm 5 mm 15 mm Vessels. Superficial epigastric. Femoral. Aorto-iliac Pulmonary Comments. Vein grafts have 100% patency. Vein graft has 100% patency. Human trials under way. Good tissue in growths. increase in vessel dimension. Increase in vessel dimension. Better results than plain Dacron. Better results than e-PTEF. 92% EC coverage Acute graft rejection. Graft rejection.

PHA-PGA Heparin-coated Dacron. Heparin-bonded Dacron Collagen-EC-SMFB AAM AAM

7 mm 6-7 mm 7-9 mm 4.6 mm 3-4 mm 5 mm

Aorta Infra-popletial. Femero-popletial Coronary Carotid

Coronary artery by-pass grafting: Coronary artery bypass surgery, also coronary artery bypass graft (CABG, pronounced "cabbage") surgery, and colloquially heart bypass or bypass surgery is
[18]

a surgical procedure performed to relieve angina and reduce the risk of death from coronary artery or veins from elsewhere in the patient's body are grafted to the coronary arteries to bypass atherosclerotic plaques and improve the blood supply to the coronary circulation supplying the myocardium (heart muscle). This surgery is usually performed with the heart stopped, necessitating the usage of cardiopulmonary bypass. ; techniques are available to perform CABG on a beating heart, so-called "off-pump" surgery. The first CABG was performed in the year 1960. Here the internal mammary artery was used as donor vessel and was anastamosed to right coronary artery.

The grafts are usually autografts obtained from different major arteries of the body based on requirement and surgeons choice. Typically, the left internal thoracic artery (LITA) (previously referred to as left internal mammary artery or LIMA) is grafted to the left anterior descending artery and a combination of other arteries and veins is used for other coronary arteries. The right internal thoracic artery (RITA), the great saphenous vein from the leg and the radial artery from the forearm are frequently used; in the U.S., these vessels are usually harvested endoscopically, using a technique known as endoscopic vessel harvesting (EVH).

Grafts can become diseased and may occlude in the months to years after bypass surgery is performed. Patency is the chance that a graft remains open. A graft is
[19]

considered patent if there is flow through the graft without any significant (>70% diameter) stenosis in the graft. Graft patency is dependent on a number of factors, including the type of graft used (internal thoracic artery, radial artery, or great saphenous vein), the size or the coronary artery that the graft is anastomosed with, and, of course, the skill of the surgeons performing the procedure.

However the existing materials have not been proved successful in replacing smaller diameter blood vessels. Tissue engineering offers the potential of providing vessels that can be used to replace diseased and damaged native blood vessels.

****************

[20]

6. CARDIOVASCULAR TISSUE ENGINEERING The cardiovascular diseases are becoming a major fear in the present era all over the globe. The cardiovascular tissue engineering (TE) can become an ideal substitute to replace all of these cardiac problems like: cardiovascular grafting, valves, micro vessel construction, aorta and vein formation.

Damage to heart muscle, acute or chronic, has long been considered a tipping point for individual health outlook and progression to heart failure. The problem is that adult heart muscle cells, the cardiac myocytes cannot divide to replace the injured cells. Thus despite a limited population of resident cardiac stem cells the heart cannot repair itself by any native processes. Instead, a scar tissue develops over regions of damaged myocardium. Such scar tissue keeps the organ intact but cannot contract. The ideal clinical intervention would either avoid such scar formation or simply replace formed scar tissue with functioning cardiac muscle tissue. In recent years, the emerging cardiac tissue engineering provides a new therapeutic method for heart diseases. And in the tissue engineering, the scaffold material which can mimic the structure of the extracellular matrix properly is a key factor. The rapid expansion of nano-scaffolds during the past ten years has led to new perspectives and advances in biomedical research as well as in clinical practice. The requirement of the field would include a material which will replace the damaged cardiac tissue to the fullest extent and at the same time exhibit all the properties necessary for the cardiac muscle activity namely contraction and conduction of electrical impulses. Therefore the purpose of tissue engineering in general and cardiac muscles in particular is that it has to create a viable cellular environment through the use of biologically acceptable materials. The idea is that transplantable cells can be contained and organized in so called engineering scaffolds. Such scaffolds with contained cells can then be sued to treat or replace a part of the body, say the cardiac tissue. Empty fabricated structures can also be implanted in vivo in providing a structure to condition stem cells already present. Therefore the purpose of such design is to make a livable structure. In case of cardiac tissue, healthy and functional cardiac myocytes would be the ideal inhabitants.

[21]

IMPORTANCE OF CARDIAC TISSUE ENGINEERING: The purpose of cardiac tissue engineering is to replace or repair injured heart muscle effectively. Supporting materials to create habitable spaces can provide the basic requirements of cardiac muscle cells. The design of such supporting materials influences the behavior of cells; the shape, dimensions, and chemistry of substrates affect such processes as attachment, cell signaling, and differentiation. As cardiac muscle cells flourish in artificial environments, they may become functional tissue with clinical value.

IDENTIFYING THE BASIC CELL RESPONSES Simulation of host-cell environment to its closest is of a great importance in selecting and designing any biomaterial. A tissue engineered material when taken as a scaffold for impregnation of myocytes should support the cardiac cells 3dimensionally and also effectively mimic the local microenvironment. Slight changes beyond a uniformly flat state can affect cardiac myocytes. Collagen, the most abundant constituent of the extracellular matrix, has a bundle diameter measured in the nanometer rangeV100 to 1,000 times smaller than the micrometer average cardiac myocyte diameter. Just as cells are influenced by extracellular proteins like collagen, so also are they capable of responding to artificial structures in the same size range. Meshes of electro spun nanofibers support cardiac myocyte adhesion and spontaneous contraction. Stimuli other than surface topography can also have robust cellular consequences. Mechanical forces, either generated from cell contraction or sensed from external sources, have pronounced effects on differentiation, growth, and survival of myocardial cells. For instance, experimentally exposing cardiac myocytes to repeated stretching in specific directions alters amounts of contractile proteins. when material or matrix stiffness is tuned to the range of native muscle rigidity (bearing a Youngs modulus a measure of resistance to deformation between 8 and 17 kPa), mesenchymal stem cells are induced to differentiate into a myogenic lineage. The clinical implications of controlling stiffness can be appreciated when it is recognized that collagen, a primary component of scar tissue that is overproduced in heart failure, has a relatively high stiffness. Although designing around forces and stiffnesses may seem rudimentary, the interplay of these 2 physical elements has great significance for cardiac tissue engineering.
[22]

Anatomy of cardiac musculature Before getting into the actual designing of cardiac muscle tissue it is imperative to know the anatomy of the cardiac musculature. The musculature or the muscles of the body has been divided into 3 major categories based on the functionality of the tissue. They are as follows: 1. Voluntary muscles.(Skeletal Muscles). 2. Involuntary muscles.(Visceral Muscles). 3. Cardiac muscles.(Specialized cardiac musculature). The cardiac muscle is again an involuntary muscle but classified and considered separately because of the specialized function it performs, the conduction of electrical impulses across the heart which leads to the rhythmic contraction of heart. It has structure similar to that of skeletal tissue that is it has alternate dark bands which are due to thick filaments that is made up of a contractile protein called myosin which is responsible for contraction and thin filaments made of actin, tropomyosin and troponin responsible for relaxation. The area between the dark and light is demarcated by dark Z-line.

The muscle fibres branch and interdigitate, but each is a complete unit surrounded by a cell membrane. Where the end one muscle fiber abuts on another, the membranes of both fibers parallel each other through an extensive series of folds. These areas which always occur at Z-lines are called intercalated discs. They provide a strong union between fibers maintaining cell to cell cohesion, so that the pull of one contractile unit can be transmitted along its axis to the next. Along the sides of the muscle fibres, next to the disks the cell membranes of adjacent fibers fuse for considerable distances. These
[23]

gap junctions provide low-resistance bridges for the spread of excitation from one fiber to another. Mechanical properties of cardiac muscles: Contractile response: The contractile response of cardiac muscle begins just after the start of depolarization (loss of excitation) and lasts about one and half times as long as action potential. The Calcium ion extracellular fluid as well as sarcoplasmic reticulum (network of protective fibres around muscles) contributes for contraction. Isoforms: The cardiac muscle is slow and the fibers are dependent on oxidative metabolism and hence on a continuous supply of Oxygen. Correlation between muscle fiber length and tension: The relation between initial fiber length and total tension in cardiac muscle 1is similar to skeletal muscle; there is resting length at which the tension developed upon stimuli is maximal. Metabolism: Mammalian hearts have an abundant blood supply, numerous mitochondria and a high content of myoglobin a muscle pigment that may function as an oxygen storage mechanism. Pacemaker tissue: The pacemaker tissue is a specialized bundle of heart muscle fibers which have the potentiality to initiate repetitive action potential. It makes up the conduction system that normally spreads impulses throughout the heart. The pacemaker is characterized by an unstable membrane potential.

****************

[24]

8. ENGINEERING TISSUES IN 3-DIMENSIONS

Much of cardiac tissue engineering involves the struggle to perfect cellular organization without sacrificing tissue vascularization. The in vivo myocardium has a very dense layout, with overlapping arrays of muscle cells arranged in different circumferential orientations. Individual branched cardiac myocytes in the heart are linked to others at both ends through intercalated discs (containing gap junctions and other adherent sites) that help to transfer both molecular signals (eg, electrical coupling) and forces of contraction. Replicating this cellular organization of the myocardium has proved very difficult. Therefore the complexity of engineered tissue increases from 2-D to 3-D. Here, levels of diffusion determine the degree of nutrient delivery and metabolic waste removal available to cells in the construct interior. The thickness of normal, diastolic left ventricular myocardium is a little more than 1 cm in the human heart, but there is a 200-2m tissue depth limit for the diffusion of oxygen. Therefore, cell survival in 3D cardiac tissue constructs depends on angiogenesis and functional vascular integration to serve the remaining 95% of the heart wall.

Therefore certain general factors are always considered while designing materials for cardiac conditions. They are

1. MATERIALS AND DEGRADATION-Besides non-inflammatory in composition the material should degrade steadily over the time as native supports are built around the cell. 2. ATTACHMENT: Cardiac myocytes are anchorage-dependent i.e., for proper functioning their surface attachment is very important. 3. DIFFUSION AND POROSITY: Cells contained in 3-D constructs risks poor diffusion of vital nutrients and gases while porosities can shape the capacity for the vascularization. 4. STIFNESS: The rigidity of cell substrate affects cardiac myocyte morphology and commitment of stem cells to lineage. 5. FORCES: Just as in regular heart beating the cardiac myofibrils are sensitive to mechanical forces, both in magnitude and frequency.

[25]

There are different methodologies used in making up of 3-D engineering of cardiac myocardium. Some of these are very interesting and innovative in nature. 1. One group has demonstrated that lanes of cardiac myocytes on degradable polymer films can grow up to 3 cell layers thick and form organized functional tissue. Such geometric configurations constitute the very definition of tissue -an organized collection of cells working as a unit. 2. Preparations of cardiac myocytes bound to sponge-like alginate, a natural polymer derived from seaweed have yielded vascularized cylindrical tissue in 9 weeks after implantation in rats. 3. Porous nonwoven polyglycolic patches were seeded with embryonic stem cells and implanted on the ventricular surface of infarcted hearts in mice. Within 8 weeks, the polyglycol had degraded to natural byproducts, leaving small area of active cells that contributed to an increased survival rate. 4. Gels in unpolymerized form can be mixed with cells and the resultant polymer can be shaped into desired geometric shapes. Gels and transplantable cells can also polymerize in vivo after injection, permitting the cell matrix composite to assemble and conform to specific areas of the myocardium although this relinquishes all control over final tissue shape. In total the following table gives a picture of certain clinical advantages and disadvantages of the strategies used in designing cardiac myofibrils.

METHODS Cells only-injection cells.

ADVANTAGES DISADVANTAGES of No structure to design. High amount of cell exit Less invasive to delivery. or loss; poor cell integration. Cells+Materials. Uniform support for Greater surgical risk for a) Ordered scaffolds and cells;degrade safely as implantation; limited meshes. cells incorporate. vascularization. b) ECM like gels. Similar composition to Natural components can body; cells readily attach. be inflammatory; slow
[26]

c) Cell-sheets and tissue patches. High cell density and organization; less obstructive materials. Materials only. No immunogenic a) Injectable response found. Support networks. placed where needed. b) External restraints.

vascularization. Surgery and placement difficult. Limited vascularization. May require chemical factors, inadequate repair without cells. Passive device; only prevents spread damage and stress.

II ENGINEERING ARCHITECTURE.

DESIGN

OF

HEARTS

EXTRACELLULAR

Use of extracellular architecture of the heart has been tried in cardiac cellular regeneration. It has been tried on rats wherein the heart was decellurized meaning all cell types including cardiac myocytes, fibroblasts, endothelial cells, and smooth muscle cells are removed using a detergent wash leaving only the Extracellular matrix intact. In this way, valves, chambers, and vascular channels are all preserved, allowing transplanted cells to move in and occupy familiar surroundings. A modest result was obtained in this way with a resulting weak pump function. To increase the efficiency of implantation bioengineers often treat cells in bioreactors before their use in vivo. A bioreactor is essentially a container filled with culture media that can be fine tuned to stimulate cells in a number of ways. Continuous mixing of fluid within bioreactors helps to maintain an even concentration of nutrients. External forces such as pressure, strain, and shear stress can simulate the kinetic features of the heart. By incubating for a period of around one week in bioreactor has shown cardiac myocytes seeded in polyglycolic acid assembled into a near perfect uniform electrophysiologic 3-D tissue. Limitation of 3-D tissue: The greatest disadvantages of scaffolds, constructs and gels are that they are not easily optimized for transferring contractile forces to the heart. The stacking of materials (cardiac myocytes) in animal models showed that repetitive stacking was
[27]

necessary to obtain desired properties which mean repetitive surgeries. Such a risk cannot be taken in human beings. Another biggest problem is unavailability of donors for human myocardium. Adult stem cells present the only feasible source for human treatment, but isolation, expansion, and differentiation into contractile cells remain difficult. Cell populations such as embryonic stem cells, mesenchymal stem cells from bone marrow, and umbilical cord stem cells have shown some therapeutic promise, although nonautologous cells bear an added risk of immune rejection. AN INTERESTING FACT IS HUMAN MYOCARDIAL INFARCTION CAN INJURE UPTO 50g OF CARDIAC MUSCLE TISSUE WHICH WOULD CALL FOR SUBSTITUTION OF ONE BILLION CELLS!

******************

[28]

9. TISSUE ENGINEERING OF HEART VALVES It is interesting to note that the number of patients requiring heart valve replacement will increase by 2 folds in next three decades. Currently available valves does not replace the nativity of the tissue to 100%. Heart valve tissue engineering is a promising technology to overcome these problems.

Currently available heart valve prostheses are categorized into mechanical and bioprosthetic value. Mechanical valves offer excellent structural durability but inherently prone to thromboemolic events due to high physiologic stress. Therefore the patients have to be on life-long thrombolytic drug therapy. Bioprostheses are more susceptible structural wall degradation. There are three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Advantages of an engineered tissue heart valve would likely include nonthrombogenicity, infection resistance, and cellular viability. Goal of engineering tissue heart valves: The goal to engineer functional heart valve tissue presents a unique combination of challenges. Normal heart valves are vital and dynamic tissues composed of specialized cells and extracellular matrix (ECM) that respond and remodel in response to changes in local mechanical forces. Approximately 40 million times a year, opening and closing of the leaflets induces repetitive changes in the shape, dimensions, and stress of the leaflets and supporting valvular structures. A successful tissue engineered valve and its components must not only accommodate those deformations but also have ongoing strength, flexibility, and durability, beginning at the instant of implantation and continuing indefinitely thereafter, possibly despite an evolving tissue architecture.

[29]

__HEART VALVE FUNCTION AND STRUCTURE Healthy native heart valves maintain unidirectional blood flow via an extraordinarily dynamic functional structure with several key characteristics: viability, sufficient strength to withstand repetitive and substantial mechanical stress, and ability to adapt and repair injury by connective tissue remodeling. A rational approach to heart valve tissue engineering depends on a thorough understanding of the complex normal functional elements and their coordinated interactions . The following table briefs the heart valve function and structure.

Two types of cells are present in the heart valve.

Endothelial cells covering the surface and interstitial cells with variable properties of fibroblasts, smooth muscle cells, and myofibroblasts in the interior like
[30]

endothelial cells elsewhere in the circulation, valvular endothelial cells (VEC) maintain a nonthrombogenic blood-tissue interface and regulate immune and inflammatory reactions. VEC, the most numerous valvular cell type, synthesize ECM and express matrix degrading enzymes, metalloproteinases (MMPs), and their inhibitors (TIMPs) that mediate matrix remodeling. CLINICAL STUDIES USING ENGINEERED MATRICES AS HEART VALVES One study used a decellularized pulmonary allograft seeded with autologous endothelial cells and conditioned in bioreactor to reconstruct the right ventricular outflow tract of adults undergoing the Ross procedure. According to the investigators, based on a one-year follow-up, seeded endothelial cells remained on the construct and were fully functional and the construct mechanical strength was maintained. There was no calcification and/or thrombogenesis. However, whether the seeded cells contributed to valve function is yet uncertain . Despite promising results in animal experiments using decellularized xenograft scaffolds, translation to humans has been difficult. Clinical applications of implanted decellularized xenograft tissue heart valves have been largely unsuccessful. Histological examination of decellularized porcine aortic SynerGraft valves (Cryolife Inc.) implanted for 6 months in sheep without in-vitro preseeding suggested some growth of host cells on intact leaflets and showed a lack of calcification. The decellularized valves were not seeded or conditioned in a bioreactor before implant with the hope that the unseeded scaffold could attract endogenous cells. These valves had a high rate of failure; examination of failed valves revealed incomplete initial decellularization, lack of cell repopularization, lack of endothelialization, severe inflammation, fibrous sheath formation, calcification and severe degeneration of both leaflets and wall.

Challenges for the futuristic clinical applications: Heart valve tissue engineering has exciting potential but many unanswered questions and challenges remain before human implantation can be considered. A successful tissue engineered valve must be vital, complex, dynamic, composed of
[31]

specialized cells and ECM that remodel in response to changes in local mechanical forces, and have ongoing strength, flexibility, and durability, beginning at the instant of implantation and continuing indefinitely thereafter.

*************************

[32]

10. ROLE OF NANOTECHNOLOGY IN CARDIOVASCULAR TISSUE ENGINEERING. The basic strategy of the tissue engineering is the construction of a biocompatible scaffold to replace, re-generates or repairs damaged cells or tissues. In cardiac tissue engineering, the ideal scaffold should mimic the structure of the extracellular matrix (ECM), which is very important for the proliferation and differentiation of the seeding cells. So, to seek the bionic myocardial extracellular matrix material in the myocardial tissue engineering for the cultured myocardial cells is a key factor for the translation from the tissue engineering into clinical practice. Now the common used scaffold materials include traditional scaffold material, nanometer scaffold material, and composited scaffold material. The nano-scaffolds have many unique advantages in the field of cardiac tissue engineering. Traditional scaffold materials The scaffold materials which serve as temporary 3D substrates, provide a proper microenvironment for seeding cells, and they have been shown to actively regulate cellular responses including attachment, proliferation,differentiation and matrix deposition [3-5]. The ECM-mimicking microenvironment is the place of getting nutrition, waste excretion, gas exchange and metabolism for seeding cells. Biological scaffold materials Biological scaffold materials include fibrin, collagen, hyaluronic acid and sodium alginate and so on. These natural polymers retained the normal grid structure, and they have a good biocompatibility, and they are beneficial to cell adhesion, proliferation and differentiation. The biological material is widespread, and the price is relatively cheap. Base on the advantages of good biocompatibility and cheap price, the biological material became one of the earliest applications of the scaffold materials in cardiac tissue engineering. In the laboratory, with collagen as the basis researchers have fabricated the three-dimensional myocardial model which contains a variety of extracellular matrix proteins and growth factors successfully, and by using of the model, they cultivate regeneration myocardial cells which show good differentiation like the normal myocardial cells in vivo environment. Synthetic material: Synthetic materials include polyesterselastomers and so on. The polyesters such as polylactic acid (PLA) and polyglycolic acid (PGA) are common used for myocardial tissue engineering. The reasons why they are popular are mostly
[33]

depended on the good biocompatible of their degradation products, their mechanical properties and simple to manufacture.
Nano-scaffolds:

Nanotechnology is defined by the size of a material (generally 1-100 nm) or manipulation on the molecular level, and the 3 D space of the nano-scaffold should be at least one dimensional in nanometer level. The electrospinning process produced polymer fibers in the nanometer range with an approximate diameter of 100 nm. The nanoscaffold has a better specific effect of bulk and surface advantages, which is much superior than millimeter and micrometer scaffold materials. The collagen fibers with diameters in the nanometer and submicron range are the major component of the ECM, so fabricating the nanoscaled scaffold becomes the pursuits of the researchers. While some studies have found that the smallest fibers (near 100 nm) produced by electrospining are superior. Others have concluded that slightly larger, submicron fibers (near 400 nm) offer the best performance. In contrast with traditional scaffold materials, thee 3-D nanofibrous scaffolds provide a superior microenvironment for promoting cell functions. Since nanofibrous scaffolds have nanometer pore sizes, cells are unable to penetrate by themselves, so the seeding cells must be incorporated into the scaffold during fabrication to ensure proper cell distribution. Technologies for generating nanofibrous biomaterials At present there is several major technologies for fabricating nanofibrous biomaterials, including phase separation, self-assembly, electrospinning and so on. Phase separation techniques have been used to prepare porous polymer membranes for purification and separation purposes. In laboratory, researchers have generated Nano fibrous structures by manipulating the phase separation process.

Nanocomposites in cardiac tissue engineering Recently, the function of nanocomposites in cardiac tissue engineering causes a hot discussion among the researchers. The key limitation of porous matrix used for cardiac tissue engineering is that their pore walls limit the interaction of cells, and delay electrical signal propagation. The 3D nanocomposites of gold nanowires within macroporous alginate scaffolds have been developed to bridge the nonconducting pore walls, and this can increase electrical signal propagation

[34]

throughout the cell-seeded scaffold, and enhance the organization of functioning tissue.

******************

[35]

REFERENCES: Davidsons principle and practice of medicine.-C. Davidson (21 edition). A text book of basic pathology. (4th edition)-Kumar, Cotran, Robbins. A review of medical physiology (13th edition). W.F. Ganong. Nanomaterials for Cardiac Tissue Engineering Application . Nano-micro letters (30/11/2011) Yachen Zhang1 Yong Tang1, Ying Wang2 Liying Zhang2 Tissue engineering of heart valves , advances and current chllanges.(expert review medical devices 259-275(2009). Anita Mol, Antal smiths,C.C. Bouten.F.G. Coronary artery Bypass Surgery-Wikipedia (March 2008). A text book of human histology. Tripathi and Tripathi (14th edition) Engineered heart tissue for the regeneration of diseased heart-Elselvier Biomaterials25(2004). W.H. Zimmermann1,Ivan Melnychenko2, T. Eschenhagen2. International Journal of biomedical engineering-Cardiovascular grafts existing problems and proposed solutions. (2-2-2012) Kunal singha1, Mrinal singha2. Cardiovascular application of polyhedral oligomeric silsequioxane nanomaterials-a glimpse into prospective horizons. International journal of nanomedicine(6-2011).
HosseinGhanbari, Achala de Mel, Alexander M Seifalian

Beijjeins.

US National Library of Medicine National Institutes of Health

[36]