A New Science: Scaffolding and Tissue Engineering Heart Valves

           More than 5 million of the adult U.S. population suffers from heart disease and heart valve dysfunction plays a large part which leads to 20,000 deaths annually. The third most common cardiovascular (CV) disease is aortic valve dysfunction. Heart valves maintain one-way flow of blood to our body. Newborns can be born with heart valve malfunction. Heart valve dysfunction can also be caused by mineralized calcium deposits that lead to calcification and genetic defects in matrix protein structure. The medical treatment for a valve malfunction is to replace the original valve with an artificial one. Cardiac surgeries to replace heart valves are common and improve life expectancy. Clinically, only mechanical and biological artificial valves are used with some drawbacks. A new technique is tissue engineering (TE) that is being researched and not yet performed clinically. This new method is a 3-D scaffold that is fabricated as the template for neo-tissue development; appropriate cells are then seeded to the matrix in vitro. This new technique can eliminate the need for lifelong anti-coagulation (blood thinners), durability, and reoperation problems.

Heart valve analysis: valve opening and closing

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           I chose this complex topic of heart valves and scaffolding because I believe that this technology and science is amazing that we can create heart valves out of existing human cells as well as polymers and biological materials. I find this topic intriguing because of how important it is to have functioning heart valves to make sure we have circulation throughout our body, to and from our heart. I have always found the heart to be very interesting and the cardiac muscles and heart valves are a major part of development to keep the body perfusing. Working as a medical scribe in the ED I see people with cardiovascular issues on a daily basis and sometimes have valve issues such as mitral valve prolapse and mitral valve regurgitation and different valve malfunctions that require a valve replacement. These valve replacements are usually referred to as "pig valves" which are tissue valves that typically last 10-15 years. These valve replacements have extended people's life expectancy and they were able to live a full life with this procedure.

            
          There are 4 valves in a mammalian heart that determine the direction of blood flow: aortic, pulmonary, mitral (bicuspid), and tricuspid valve. The aortic and pulmonary valves are in arteries that leave the heart (semilunar valves). The mitral and tricuspid valves are between the atria and ventricles referred to as atrioventricular valves. The valve leaflets (cusps) are composed of the extracellular matrix (ECM) of 3 layers: fibrosa, spongiosa, and ventricularis. The fibrosa is composed of parallel, dense collagen which gives the mechanical properties of stiffness and strength of the cusps. Spongiosa is the middle surface composed of proteoglycans and lower abundance of collagen which allows movement. The ventricularis is composed of aligned fiber of elastin and short collagen fiber to give elasticity of the leaflets.  

           A major drawback of mechanical heart valves is that prostheses are foreign materials which may cause inflammation, infection, and thromboembolic (blood clot) complication because of the high sheer stresses of blood flow. A blood thinner such as Warfarin is used to combat thromboembolism that is required for the rest of the mechanical valve recipient’s life. Although blood thinners are an answer to anti-coagulation there can also be negative aspects such as risk of hemorrhage and embryo toxicity in fertile women. However, there is no need for anti-coagulation for biological valves or tissue engineering heart valves.  A downfall is artificial prostheses offer no capacity to grow, remodel, or repair especially in infant patients. 50-60% of patients will experience the problem with artificial valves which requires reoperation. Because of these drawbacks, the research of tissue engineering heart valves looks promising. 

           The goal of tissue engineering is to fabricate a neotissue from cellular combination that has most characteristics of original tissues such as non-inflammation and non-immunogenic reaction, mechanical properties, and durability. The tissue can be broken down into three parts: 3-D biomaterial scaffold fabrication, cell source and cell cultivation, and development conditions of the TE before implantation. The scaffold is very important because it determines the shape of a native particular tissue and space for meiosis. The structure is important because it needs to provide nutrients and oxygen to the cells and materials need to be biocompatible and biodegradable. One obstacle of TE is possibility of leakage of these valves.

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            TE is classified into 2 parts: polymer based scaffold constructed from synthetic polymeric materials (PLA, PGA, PCL, PF, and PGS) and the natural based scaffold constructed from natural substances (collagen, intestine submucosa, fibrin gel, and hydrogel). Material selection for tissue engineering heart valves (TEHV) is based on biocompatibility, biodegradability, thermoplasticity, and elasticity characteristics. Along with these characteristics the pore size of the scaffold is also important. All of these polymeric materials mentioned above are accepted by the FDA as biocompatible and reported to be useful in TE. The main advantage of synthetic scaffold is that biomechanics and degradation properties can be chemically controlled. Drawbacks of synthetic polymeric scaffold are biocompatibility, low degradation rate, and inflammation.

            Collagen is the main ECM protein of the original heart valves and is found in intestine submucosa, fibronectin, and glycosaminoglycans. All of these biological materials are used for scaffold fabrication. Intestine submucosa was successful in porcine (pig) pulmonary heart valves; it showed longevity without need for anti-coagulation or immunosuppression. Fibrin gel is another natural scaffold that is easily prepared using the patient’s blood without any inflammation, but the gel shrinkage (mechanical property is the drawback). A hydrogel natural scaffold was constructed using hyaluronic acid that increased ECM production and cell proliferation.

            There are four main techniques of scaffold fabrication: solvent casting, freeze drying, electrospinning & fiber knitting, and solid free form. Solvent casting is useful for tissue with thin membrane: A polymer solution is cast with water soluble particulates into a proper mold; once the solvent is evaporated the scaffold is leached into the water to form the pores. Freeze drying is used for collagen based scaffold; freezing it at -30ºC forming ice crystal pores immersed in ethanol and once crystals evaporate a porous scaffold is formed. Electrospinning: A high electric field ejects the conductive polymer jet from a needle connected to polymer solution supplier. The fibrous scaffold matrix prepares a layout for cell seeding and meiosis. High surface area to volume ratio and high porosity are the advantages. Solid free form translates computer data such as computer aided design, computed tomography, and MRI data that is converted through layered manufacturing SFF machines to the 3-D scaffold. Major drawbacks of this are cost and accuracy. The best choice of fabrication is the electrospinning technique because of the controllable setting and ultrafine quality of produced fibers with porous and interconnecting structure.  

Micro- and nanofiber composite PCL scaffolds fabricated by electrospinning

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            Different cells that are used for TEHV are adipose-derived cells, valve interstitial cells(VICs), and bone marrow stem cells.  Adipose tissues are harvested from abdominal liposuction, isolated and washed, digested, centrifuged, and filtered through a nylon mesh. The VICs are both smooth muscle and the fibroblast, called myofibroblast harvested from semilunar valves of a recipient during the cardiac transplantation with no previous background of heart valve disease. Human bone marrow cells express smooth muscle actin and produce collagen. 


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Diagram of tissue engineering process.

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A primary article: Metre-long cell-laden microfibres exhibit tissue morphologies and functions

            This study involved electrospinning, artificial reconstruction of fibre-shaped cellular constructs that could improve tissue assembly in vitro. A microfluidic device with double-coaxial laminar flow was used and metre-long core-shell hydrogel microfibres encapsulated ECM proteins and differentiated cells or somatic cells were fabricated. These microfibres have different morphologies and functions of living tissues and can be assembled by weaving and reeling, into macroscopic cellular structures with various spatial patterns. A study was done using diabetic mice; fibres encapsulating primary pancreatic islet cells were transplanted through a microcatheter into the subrenal capsular space of diabetic mice and this normalized there glucose level for 2 weeks. These microfibres may be useful as a template for reconstruction of fibre-shaped functional tissues that are similar to muscle fibers, blood vessels, or nerve networks in vivo.

            In conclusion, this study successfully fabricated microfibres using ECM proteins which provided a desired microenvironment for cells to function as they normally do in vivo. This study developed cellular level morphology and functions such as contractile motion of primary myocytes, the tubular shape of primary endothelial cells, and the synaptic connections of highly oriented primary neural cells. With this research it may be possible to reconstruct functional complex 3-D macroscopic tissues involving vascular and neuronal networks. This strategy also improved the safety of medical transplantation using regenerated cells with minimally invasive procedures: endoscopic surgery and catheter intervention. This study was eye-opening to tissue engineering and a break through to continue this research so it can be used clinically one day. 

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Works Cited:

Fallahiarezoudar, Ehsan, Mohaddeseh Ahmadipourroudposht, Ani Idris, and Noordin Mohd Yusof. "A Review Of: Application of Synthetic Scaffold in Tissue   Engineering Heart Valves." Materials Science and Engineering: C: 556-65. Print.

Onoe, Hiroaki, Teru Okitsu, Akane Itou, Midori Kato-Negishi, Riho Gojo, Daisuke Kiriya, Koji Sato, Shigenori Miura, Shintaroh Iwanaga, Kaori Kuribayashi-Shigetomi, Yukiko T. Matsunaga, Yuto Shimoyama, and Shoji Takeuchi. "Metre-long Cell-laden Microfibres Exhibit Tissue Morphologies and Functions." Nature Materials Nat Mater 12 (2013): 584-90. Print.