Neocardiogenesis through myocardial tissue engineering ©2017, Keesee WW et al

Neocardiogenesis through myocardial tissue engineering

©2017, Keesee WW et al

Classical tissue engineering is based on seeding cells into biodegradable polymer scaffolds or gels, culturing and expanding them in bioreactors, and finally implanting the resulting tissue into the recipient organism, where the maturation of the new organ takes place. Capitalizing on this basic concept, tissue engineering has rapidly evolved in the past decade into an integrating discipline in which every organ forms a science of tissue engineering: Each of these sciences are interfacing with different scientific communities, including biotechnology, biopharmaceutical manufacturing , chemical engineering, cell biology, developmental biology, gene therapy, medical sciences, and organic chemistry. With so many “tissue engineers” at work on this globe…managing and covering all specialized methods implemented in current tissue engineering activities is a mission impossible.
Hansjorg Hauser & Martin Fussenegger Methods in Molecular Medicine: Tissue Engineering 2nd Edition 2007.

Congestive heart failure is a major medical challenge in developed countries and heart disease is still the number one killer and biggest health crisis of all, with millions of deaths each year globally. No longer does it afflict only the old. More than 64 million Americans suffer from it, but only 25 million are over 65 years or older. The total cost of treating cardiovascular diseases and stroke in the U.S. in 2004 was estimated to reach $368 billion 86. Current therapeutic strategies to treat CHF are limited to surgical transplantation, pharmaceutical therapies, and artificial mechanical support. These treatment options have substantially improved the quality of the care, but there are notable limitations to each approach.22

Heart transplantation has shown to be the most successful treatment option to treat severe CHF. As of March 2000, over 55,000 heart transplants had been performed. A rather small number considering the magnitude of the pathology. Short term survival rates remain high at 81%; however the widespread usage has been limited by the shortage of donor organs. Each day, it has been reported, 100 patients waiting for transplants actually die. We have seen the factual evidence and know, that new studies (+175 clinical studies and published papers) in regenerative cardiology have demonstrated that direct intramyocardial reinfusion of autologous cell sources, to be safe and have, relatively high percentages 50-80% efficacy for patients, adding years to their life and life to their years, that would not of been possible without it.

Advances in regenerative biology have spurred the development of cellular based therapies at a rate that could not have been imagined even five years ago. Cells from many sources are being purified, cultured, genetically modified, frozen, thawed, and infused to treat a host of diseases.

New pharmaceuticals such as vasodilators, ACE inhibitors, statins, B-blockers, diuretics, inotropes, and antiarrhythmic drugs have reduced mortality of CHF
patients, but there has been little improvement in the functional performance of compromised myocardiums. Substantial percentages of patients receiving pharmacological treatments develop more severe congestive heart failure, leading to more invasive treatments. A tiny population of patients, estimated at 300-400 per year, use mechanical circulatory support devices as a bridge after cardiac surgery. Current limitations include infections, device size and weight, thromobogenicity, and electrical power and product wiring defects.
A recent search of the national database for HD, identified more than 250,000 patients had been rejected for a third coronary bypass operation. The very fact that they were rejected for bypass would most likely also mean, that they would of already tried or been excluded from qualifying for balloon angioplasty. These end-stage patients have exhausted all medical options. These are questions that articulate the concerns of those particular patients whose disease was, or is now, becoming far advanced, and have found themselves among the 5 M congestive heart failure patients in our country. These patients pass each day with the conscious and subconscious realization that their magnificent heart, that has served them well, 100,000 times per day, 36 million times per year and 3 billion times in their long lifetime, is now losing its ability to function.

Cardiac aging, myocardial infarction and ischemic cardiomyopathy are characterized by various forms of structural damage that are common to all cardiac diseases. The identification of a pluripotent cardiac stem cell (CSC) requires a new evaluation of possible interventions that can repair the myocardium damaged by age, stress, infarct, or combinations. Advanced age is typically associated with multiple sites of injury, which appear as areas of myocardial loss in various phases of healing, scattered throughout the ventricular wall. Neo-cardio-matrix-engineering (NMTE) may provide viable solutions to the ever escalating pandemic amounts’ of heart patients that have no further options.

Bioengineered scaffolds and cardiac repair

A substantial effort in cardiac regeneration research has focused on identifying suitable cell types for replacing lost cardiomyocytes and the proliferation and
differentiation capabilities of those cells. The rational behind this research is that the lack of spontaneous cardiac regeneration after myocardial infraction is due to cardiomyocyte terminal differentiation and inability to proliferate, coupled with inadequate availability of progenitor cells capable of forming new cardiomyocyte. Supplementing the damaged heart with progenitor cells or another source of cardiomyocytes is a logical option following this rationale.

However, the vast majority of cells implanted in the myocardium do not survive, and those that do survive do not fully integrate into the myocardium as functional cardiomyocytes or endothelial cells, failing to couple electrically or mechanically with remaining myocardium. 58
Vincent F.M. Segers, MD & Richard T. Lee, MD

Engineered Heart Tissue

The field of tissue engineered heart tissue is relatively new with the first published journal article appearing in 1997 1. Within the last decade, there have been approximately 60 journal articles and some reviews published in the field. The various strategies in current literature are described as follows.
Utilize polymeric scaffolding material as a support matrix
Incorporating the various types of stem cells within the biodegradable gels
Incorporating various polymeric temperature sensitive surfaces
Eschenhagen, a patriarch of neo-cardio-matrix engineering (NCME), developed a 3-D model of a tissue engineered heart muscle in a collagen matrix. The initial model was refined to generate engineered heart tissue (EHT) by casting a mixture of neonatal cardio myocytes and collagen into plastic molds.
According to Eschenhagen and Zimmermann:
In 1994, our idea to generate 3-dimensional cardiac tissue constructs came from the observation that it is fairly easy to genetically manipulate cultured immature cardiac myocytes in the classic 2-dimesional culture format (and to observe
biochemical and molecular consequences) but difficult to obtain reliable information from these cells in terms of contractile function. The experiments quickly yielded success, and cardiac myocytes spontaneously generated a coherently and regularly beating cardiac tissue-like structure (cardiac myocyte-populated matrix CMPM) that developed measurable forces when suspended in organ baths and connected to a force transducer (Eschenhagen et al 1997)1-23-24-34.

The Dynamic Heart

As opposed to static structures of the body, like bones, cartilage, and various relatively uncomplicated organs, the heart is a dynamic organ with a complex mechanical function. The restoration via tissue engineering of the heart poses unique challenges and solutions. The heart is a heterogenesis, complex helical structure with asymmetric and anisotropic features, with spiraling muscle bands, valves, vessels and electrical conduction system. The key mechanism underlying the hearts durable and strong performance is the vortex function of the myocardial muscle spiral during systole (Buckberg et al 2002) 35. Furthermore, the myocardium is a highly anisotropic structure, that is, the orientation of muscle fibers may be varying from site to site, as do the vascular density and the contractile pattern. With high metabolic output demands the heart is angiotropic and densely vascularized.

Recent studies have reported on the use of various types of scaffolds and the combinational use of divergent stem/progenitor cells in seeding and expansion in 3-D bioreactors.
In addition, two fundamental types of bioengineered heart valves are being produced: decellularized xenogenic valves and recellularized valves with the recipients own cells. Also, bioartificial blood vessels are being produced with collagens and cells from previously harvested endothelial cells from the patients own autologous fibroblasts.
Also quite notably, the recent discoveries that demonstrate the use/function of designer self-assembling peptide nanofiber scaffolds for 3-D dimensional cultures
will be used in many promising new medical applications, such as spinal cord injuries, as well as the rapidly expanding field of neo-cardio-matrix-engineering (NCME).
It is certainly not hard to imagine, a direct transendocardial re-infusion of an expanded autologous stem cell cocktail in a slurry of self assembling nano-peptides. WW Keesee Trust

Healthy tissue can be considered comprising three dynamic components.
(i) cells (ii) the extra-cellular matrix or natural scaffold and (iii) other elements such as growth factors that signal to the cells. Although non conclusive, the evidence strongly suggests that successful cardiac regeneration may, not be, enabled by injection of one cell type alone. Newly formed cells require an extracellular environment providing growth factors, nutrients, mechanical support, and physiological integration.58

Creation of Cardiac Tissue

The most elemental approach in the creation of cardiac tissue has been to control the microenvironment of the proposed cells designated for later infusion in vitro, growing in 3-dimensional scaffolds. These scaffolds can be constructed in different sizes and shapes and display functional and morphological properties of differentiated cardiac muscle in vitro. (Eschenhagen) 1 Spontaneous contractions start a few days after construction, and applying cyclical stretch in order to mechanically condition the matrix can improve functional and mechanical properties. These three-dimensional blocks of tissue can be used for clinical studies and for in vitro drug testing. Many attempts at engineering myocardium have used collagen or matrigel as a scaffold and neonatal cardiomyocytes as a cell source.

Methodology for Cardioid Formation

The method for cardioids formation has been described in detail (Baar K, Birla, R, et al) 19-26. Cardioids are formed from the spontaneous delamination of a
confluent monolayer of neonatal cardiac myocytes. The cardioid model has several unique characteristics that make it an attractive model for cardiac muscle. Cardioid formation is dependent on the self-organization of individual cardiac cells into a 3-dimensional tissue construct. This process eliminates the need for synthetic scaffolding material in the contractile region of the tissue construct, and allows cardioids to exhibit uninhibited repetitive contractions.
Secondly, during cardioid formation, extracellular matrix (ECM) components are generated by cardiac fibroblasts that are plated with the cardiac myocytes.
Thirdly, the entire process is spontaneous and allows for the generation of new cells to dictate the new 3-D environment. The 3-D organization of cardioids is not dictated by the geometry of pre-fabricated polymeric scaffolds.
According to R.K. Birla, Section of Cardiac Surgery/University of Michigan.
The cell monolayer initiates spontaneous contractions and the entire cell monolayer beats as a continuum during the entire culture period. Here we have described an in vitro method for the formation of contractile 3-D cardiac muscle, which we have termed cardioids. One of the most attractive features is that isolated cardiac cells self organize to form 3-D cardiac muscle. Cardioids exhibit several physiologically relevant metrics of function. Cardioids can be electrically stimulated to generate active force and can be electrically paced at frequencies of 1-7Hz. In addition, they are responsive to calcium and various cardio-active drugs, and may provide viable cardiac tissue for clinical or research applications.
Dr. Birla’s work has been funded by DARPA.19-26

Tissue Engineering With Adult Stem Cells
Dr. Anthony Atala MD

Investigators around the world, including our institution, have been working towards the development of several cell types, tissues and organs for clinical application. The predominant cell type for tissue engineering applications today is
the predetermined progenitor cells, which are present in almost every tissue. Our institute has successfully engineered multiple tissues for organ reconstruction using specific progenitor cells, including urinary bladder (Atala, 2001), uterus (Duel et al, 1996), vagina (De Filippo et al, 2003), and penile tissue (Falke et al, 2003).
The first tissue engineered hollow organ successfully transplanted into patients was the urinary bladder, a composite tissue, engineered to form smooth muscle cells and urothelial cells (Atala et al., 2006). 59-60-61-62-63
Former Harvard University surgeon and medical researcher, currently at Wake Forest University, Anthony Atala expresses it as follows:
“I have one goal…to cure the patient. They said 10 years ago, that human organs could not be built.” One of the new generations of physician-scientists, Atala created the FIRST HUMAN ORGAN, a bladder, using tissue engineering. Other organs followed and he was able to form uteruses, vaginas, and large blood vessels.
Believe it or not, Atalas’ group has constructed a fully functioning RABBIT PENIS. Apparently, randy rabbits with newly engineered members chase their female counterparts around the cage, barely four weeks after surgery.
Atala continues, “Why is it that surgeon’s think that if a piece of your heart gives out, you have to change the whole heart? You don’t! Our organs have tremendous reserves. When someone comes to a doctor with heart pain or kidney trouble, you don’t need much repair to get back to a normal lifestyle. And a stem cell patch may be the best approach.”
When asked about whether making customized organs and tissues patient-by-patient will be cost effective, Atala replies:
“You can’t argue with autologous, and it’s the best way to go, regardless of the cost. Immunosuppressant drugs are nasty things. I think that people who suggest that we can control rejection with better HLA matches haven’t spent much time
at the bedside of someone on prednisone. There are too many genes that make us different, and more are discovered every year.”
“I don’t believe that we’ll solve immune rejection in my lifetime or perhaps ever…” 81.
When compared to embryonic stem cells, adult stem cells have many similarities. They can differentiate into all three germ layers, that express common markers, and they preserve telomere length. However, the adult stem cells demonstrate considerable advantages. They easily differentiate into specific cell lineages; they do not form teratomas if injected in vivo; they do not need feeder layers to grow; and they do not require the sacrifice of human embryos for their isolation. Adult stem cells are negative for tissue-specific markers and can be positive for embryonic cell antigens (Peterson et al., 1999). These cells are able to differentiate into committed cells of other tissues, a feature defined as plasticity. This would allow for engineering of composite tissues composed of multiple cells types using one single source of adult stem cells. The preferred cells to use are autologous cells, in which a biopsy of tissue is obtained from the host, the cells are dissociated and expand in culture, and the expanded cells are implanted into the same host. (Atala, 2001, 2005). Therefore, use of adult stem cells opens a new avenue for cellular therapy and for the engineering of tissues and organs.

Great clinical and scientific interest in the use of adult stem cells for cardiac tissue engineering has emerged over the last decade. In addition, as we reported infra, more than 175 clinical studies and papers have been published for various cellular therapies for heart disease, mainly on the quest for the holy grail of stem cell cardio-biology. A German group (Strauer et al., 2001)65 reported the first human application of adult stem cells for the treatment of myocardial infraction. Mononuclear bone marrow cells of the patient were prepared, and 6 days after infraction, 12 million cells were injected in the infract-related artery. At 10 weeks after the stem cell transplantation, the transmural infract area had been reduced from 24.6-to-15.7% of left ventricle circumference, whereas ejection fraction,
cardiac index, and stroke volume had increased 20-30%. More research in a randomized setting and more clinical studies are critically needed now.
Atala claims to be able to build a hollow organ in just five weeks: four weeks to expand the cells and one week to seed and build the “construct”, the three dimensional structure of the organ.
Major advances have been achieved in engineering of tissues within the past decade. However, like every new evolving field, regenerative medicine and tissue engineering are expensive. Several of the clinical trials involving bioengineered products have been placed on hold because of the costs involved with the specific technology. As with any therapy, the cost that the medical health care system can allow for a specific technology is limited, therefore, the costs of bioengineered products have to be reduced for them to have an impact clinically.
Tissue engineering and Regenerative Medicine: Methods in Enzymology
Vol. 420 Stem Cell Tools and Other Experimental Protocols 2006

The Role of Endothelial Cells in Cardiac Regeneration and
Tissue Engineering
The structural complexity of the myocardium goes far beyond the mere viability of cardiomyocytes. Other cell types like endothelial cells have important roles in both the embryonic and adult heart. In the normal myocardium, every cardiomyocyte is surrounded by a network of capillaries and cardiomyocytes interact directly with adjacent endothelial cells. Cardiomyocytes depend on endothelial cells not only for blood supply but also for paracrine signals and growth factors that control contractility and promote cardiomyocyte organization and survival. 7
The most obvious role of endothelial cells in cardiac regeneration and tissue engineering in general is oxygen and nutrient supplied by vascularization of the newly formed tissue. Therefore the ability to support vascularization is one of the most important roles and requirements of tissue engineering scaffolds. 8
This concept applies also to injectable scaffolds. Cell survival will depend upon recruitment of endothelial cells and newly growing functional vessels, within these matrix scaffolds. Some materials support vascularization better than others.

Self-assembling peptides are short peptides that can form a solid scaffold within the myocardium after injection. In many materials including collagen gels and alginate gels, cultured endothelial cells undergo apoptosis, while endothelial apoptosis in self assembling peptides in the heart is less prominent. Segers 58 showed that, after injection of self assembling peptides in the heart, endothelial cells are recruited and form functional vessels within 4 weeks, as opposed to few endothelial cells that are recruited in basement membrane material called matrigel. (Graham et al 2002)
The endocardial endothelium plays an important role in cardiac development. Endothelial cells can also regulate myocardial performance (Hoshijima et al 2005). Cardiomyocytes also depend upon endothelial cells not only for blood supply but also for paracrine signals and growth factors that control contractility and promote cardiomyocyte organization and survival. Physiological observations of secreting endolthelin I and angiotensin conversions suggest that vascularization is essential for physiological performance.
According to Segers et al, in fact, engineered heart tissue (EHT) develops a two-fold higher contractile force if generated from an unpurified cell population than that generated from a pure cardiomyocyte fraction (Wang et al 2006). 58
It has been shown that co-culturing endothelial cells with cardiomyocytes promotes cell survival (Narmoneva et al) 40. And Hsieh et al showed that PDGF-BB is a CRUTIAL FACTOR in the signaling of endothelial and cardiomyocytes that are beneficial in injectable scaffolds because of decreased apoptosis and increased cardiac function after MI.
Endothelial cells form typical networks of vascular connections with capillary-like tubes cultured in 3-D dimensional scaffolds, whereas cardiomyocytes cultured alone form small islands of dying cells. Most interesting, when both cells are cultured together, the endothelial cells form tubular structures and the cardiomyocytes attach to these vascular tubes. (Narmoneva et al) 40 This data shows that endothelial cells promote the assembly of cardiomyocytes on the capillary endothelial tubes by their direct physical interaction with the cardiomyocytes.
This suggests that endothelial cells as well as various cells included in the cellular spectrum of homogeneous bone marrow, and other noncardiomyocytes must be considered in strategies for cardiac regeneration. For instance, cardiac fibroblasts, another important cell population, both quantitatively and qualitatively, but are regarded suspiciously because of the consequences of excessive fibrous; contribute and have functional roles in the myocardium, such as passive mechanical properties, and organization of the collageneous matrix, and may be essential in the future considerations of neo-cardio-matrix engineering (NCME).

Myocardial Restoration and Tissue Engineering

According to Theo Kofidis et al, the following caveats and requirements arise for the myocardial restoration using bioartificial, cell-enriched tissues: 9-10
1. Is the production of natural and efficient heart muscle possible with the currently available tools? If so, what type of cell and what kind of bioartificial matrix is better suited for the production of bioartificial heart tissue?
2. The engineered tissue graft should exhibit strong viability and stability for a sustained myocardial replacement.
3. The inoculated cells should preferably bear differentiation potential to cardiomyocytes or other contractile elements.
4. The engineered tissue should be conservable in a viable and functional state to be implanted into the diseased heart muscle at a latter time point and in the frame of an elective and well-controlled procedure.
5. The myocardial tissue construct should encompass vessel and neuronal networks to allow for perfusion and signal conduction.
6. The bioartificial matrix , which forms the framework for cellular seeding, has to exhibit adequate porosity and biodegradability to facilitate cell engraftment and infiltration, without losing integrity in a given time span following implantation in vivo.

Aspects of Cardiac Electrical Stimulation

Recently Cannizzaro, 12 have shown that electrical stimulation directs neonatal cardiomyocytes to assemble into native-like tissue in vitro, and by employing this ‘biomimetic’ approach, discusses how electrical field stimulation can influence cell behavior. The utility of engineered cardiac grafts depends on cell survival, integration, functionality, and electrical coupling.
Native heart tissue has low-resistance pathways for electrical signal propagation due to the presence of gap junctions and high cell density. Individual cells are packed tightly together and held in place by tight junctions, such that the myocardium acts like a synctium. To induce synchronous contractions of the cultured cardiac constructs, electrical signals are applied and designed to mimic the orchestrating and synchronous native electrical contractions of the heart.

Bioreactor electrical characteristics

Stimulation efficiency is determined by the ability to attain the desired physiological responses with minimal damage to the surrounding tissue. For each application, electrical stimulation conditions should be optimized by not only choosing appropriate electrode geometry but also studying electrode material properties and the charge-transfer characteristics at the electrode-electrolyte interface. Electrodes must be biocompatible to avoid toxic shock or immune responses in the adjacent tissue or medium, and they should efficiently transfer
charge from the electrode material where it is carried by free electrons to the medium where it is carried by free ions.13
In a microelectrode array (MEA) study on cardiomyocyte activity, Giovangrandi et al 14 clearly showed how cardiomyocyte beat closely follows imposed temperature profiles; as beat rate increases the temperature increases and also conversely as the temperature increases the hearts beat rate increases.
Electrical field stimulation apparatus is available commercially or custom-designed hardware with computer controller may be custom made, which will require additional software and circuit design expertise. 44-45
In addition, more advanced bioreactors that integrate electrical stimulation with medium perfusion are needed to engineer tissue of sufficient thickness for clinical applications.39

Tissue engineering using human embryonic stem cells

The possibility of using stem cells for tissue engineering has greatly encouraged scientists to design new platforms in the fields of regenerative and reconstructive medicine. Among cell types suggested as a cell source for tissue engineering (TE), human embryonic stem cells (hESC’s) are one of the most promising. Isolated from the inner mass of preimplantation stage blastocysts, they possess the ability to differentiate into practically all adult cell types. In addition, their unlimited self renewal capacity enables the generation of sufficient amounts of cells for cell based TE applications. Several important challenges need to be addressed, such as isolation of the desired cell type and gaining control over its differentiation and proliferation. As for having the ultimate cell source to accomplish these goals, human embryonic stem cells (hESCs) hold great promise. Ever sense they were identified by Thomson et al, they have shown /exhibited capacity to differentiate into cells representing all three embryonic germ layers (Itskovitz-Eldor et al 2000), including neurons (Reubinoff et al., 2001), and endothelial cells (Levenberg et al.,2002), and have an almost unlimited self-renewal capacity. Never the less, they
also possess critical limitations because undifferentiated hESC’s have the inherent capacity to form tumors in vitro.
HESCs are the most potent stem cells available for TE, and controlling their differentiation is a notable challenge. In general, hESCs can be induced to differentiate once removed from the MEF feeders and introduced with bioactive signals. This is done either directly or through the formation of embryonic bodies (EB’s). EB’s are small clumps of hESC colonies grown in suspension, which form three-dimensional (3-D) spheroid bodies representing a differentiation model with the widest possible spectrum of cell types. Differentiating cells within the EB requires the interaction of paracrine effects of the 3 embryonic germ layers, in developing 3-D micro- environments that reproduce in vivo native structures as faithfully as possible. Although direct differentiation is possible, most differentiation systems rely on EB formations.
The next challenge is the isolation of the desired cell type. Methods for isolating specific cell types include either positively selecting the desired cells or negatively removing the undesired cells. Defining the target cell population is crucial when using these strategies and planning the next stages and appropriate time points for seeding cells into scaffolds and transplanting them into animal models.
Lavik and Langer, 2004, used TE scaffolds designed to provide cells with a solid 3-D matrix.

Scaling up a bioprocess

Large-scale production of functional tissues requires bioprocesses that are scalable, tightly controlled, and easily managed. Each step of the hESC-based systems should be scaled up in a regulated and systematic fashion. Perhaps the most challenging process is to control the formation and cultivation of EBs. Eb’s remain the preferable approach in many differentiation protocols. Made of a small aggregate of hESC’s grown in suspension, EB’s are independently growing and differentiating units. In addition, cells within the EBs at times, respond differently and at times unpredictably to bioactive cues, stimuli, and growth factors. The desired target cell population has been either somatic cell types or
progenitor cells, although some laboratories followed protocols for mesenchymal linage. Shahar Cohen, Lucy Leshanski, and Joseph Itskovitz-Eldor
Engineering cardiac tissue from embryonic stem cells
Cardiac tissue engineering is an emerging field that holds great promise for developing revolutionary treatments for heart disease. A large number of patients who are not candidates for transplants and other interventions may benefit from smaller structures of muscles, valves, or vessels. Cardiac tissue engineering may not only provide advanced surgical implants in these patients, but the development of these cells in vitro, provides a substantially improved method for testing new drugs and therapeutic agents, and will dramatically enhance our understanding of cardiac cell biology. Li et al., 1999, 2000, and Sakai et al, have grown small 3-dimensional cardiac grafts and implanted them in host myocardia.
Two important critical observations from this work were. The survival of the grafted material, in the right ventricular outflow tract, and the apparent vascularization of the implants. In conclusion, engineered heart tissue prevented further dialation, induced systolic wall thickening of infarcted myocardial segments, and improved fractional area shortening of infarcted hearts compared with controls. (Xi-Min Guo et al 2006)70
Based on these methods, Guo et al, created cardiac tissue using cardiomyocytes derived from mouse embryonic stem cells (mESCs). In this study, a step-by-step strategy was used to derive cardiomyocytes as described infra, to produce embryoid bodies in large scale ramp up, in their bioreactors, inducing cardiac differentiation of ESCs, and enriching the cardiomyocytes in Percoll density gradients. The cells were then mixed with liquid collagen to construct heart tissue and subsequently stretched mechanically, in vitro, which ultimately proved to be a reasonably fair copy, of structurally and functional native heart constructs.
In their innovative study, neonatal rat cardiomyocyte sheets detached from PIPAPm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell
sheets began to pulse simultaneously, and morphological communications by connexin43 were established between the layered sheets. In vivo, layered cardiomyocyte sheets were then transplanted into subcutaneous tissues of the animals. Three weeks after transplantation, electromyograms detected spontaneous beating in the transplanted tissues. Histological studies, in addition, showed, characteristic structures of heart tissue and of multiple neo -vascularizations within the contractile tissue.
Long term survival of pulsitile cardiac grafts was confirmed for up to 12 weeks. Miyahara et al., 2006, 71 cultured adipose tissue-derived mesenchymal stem cells using temperature-responsive culture dishes. Four weeks after coronary ligation, the monolayered mesenchymal were transplanted into scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells, and a few cardiomyocytes, and triggered angiogenesis. Unlike fibroblast cell sheeting, the monolayer mesenchymal cells reversed wall thinning, in the infract areas, of the rats. In brief, cell sheet engineering, using temperature responsive cell cultures has been very effective in fabricating electrically bioactively pulsating, cardiac grafts in both in vitro and in vivo models. This technology should have an enormous potential for constructing functional cardiac engineered tissue for clinical tissue repair. Liquid collagen and cell sheeting has proven so far to be the best strategy for engineering cardiac tissue (Guo et al 2006).70

Tissue engineering with mesenchymal stem cells (MSCs)

One of the most researched stem cells for therapies involving diseased and damaged tissue, the mesenchymal stem cell, is certainly most promising. Mesenchymal stem cells are named for the mesenchyme during embryonic development or the embryonic mesodermal, and can differentiate into all types of connective tissue phenotypes , such as bone marrow stromal, bone, cartilage , skeletal muscle, dense fiberous tissues such as tendons, and ligaments and
interstitial tissue and adipose tissues. MSC’s are capable of self replication, and able to differentiate successfully into multiple cell lineages that resemble myoblasts, osteoblasts, fibroblasts, adipocytes, chrondrocytes, and have key markers of endothelial, cardiomyocytes, and neural like cells. MSC’s have been used successfully alone in stem cell therapies and may be seeded into matrix for tissue engineering applications. Mesenchymal cells reside in various stem cell niches in the body and are believed to be an excellent choice for tissue engineering and regeneration of diminished organs like the heart.
MSCs -how do I love thee-let me count the ways…
MSC’s demonstrated therapeutic capacity has captured the imagination of the cellular biologist/medical regenerative communities, and many new biomedical companies have been created recently based on the potential efficacy of MSC related products, and therapies.
Recently bone marrow-derived cells have been shown to differentiate into nonmesenchymal cell lineages, such as cardiac, neural, renal,(Alhadlaq and Mao, 2004) 72-73
Why are MSC’s perceived to be superior to autologous tissue grafts in the generation of human tissue and organs? Autologous tissue grafts represent the the current “gold standard” for the reconstruction of defects resulting from trauma, chronic diseases, congenital anomalies, and tumor resection.
However, autologous tissue grafting is based on the concept that a diseased or damaged tissue must be replaced by like tissue that is healthy. Thus, the key drawback of autologous tissue grafting is donor site trauma and morbidity. Also, spare healthy tissue is scarce because of biological design during evolution. In contrast,
MSC-based therapeutic approaches may circumvent the key deficiencies associated with autologous grafting procedures. Second, MSC’s can differentiate into multiple cell lineages thus providing the possibility that a common cell source can heal many tissues. Finally, MSC’s or MSC-derived cells can be seeded in biocompatible scaffolds, which can be shaped into anatomical structure that is to
be replaced by MSCs. The construct is then surgically implanted to heal the defect. 72-73

Nicholas Marion and Jeremy Mao
Mesenchymal Stem Cells and Tissue Engineering
As we know, MSC’s can be autologous and eliminate the issues of immunorejection and alien pathogens or they may prove to be very immuno- friendly as an allogenic; generically available as a jar of spaghetti sauce on your refrigerator shelf.
This aside, the ability of MSC’s to revolutionize bone and joint replacement is not only possible but a reality, as the FDA has approved the first autologous cellular-based product which has been used on thousands of patients. In addition, the EMEA, and CAT, Committee for Advanced Therapies, as of late 2009-early 2010, has established a special regulatory pipeline and a possible epediated approval process for the entire EU, with a call for companies with similar and related osteo-cellular products to get a pre-submission review and eventually certified by CAT.
For example , the general life span of a surgically successful total joint replacement is 8-10 years, which is as far to short of a duration, and creates further questions about what are the options for the patients, after the replaced joints wear out? Current hip replacement therapy involves the sawing off the patient’s upper and lower leg bone, cutting off the native ball joint and attaching a steel ball joint. Many patients never fully recover and most live with constant pain. Compare this to a tiny sponge-like-matrix, seeded with the patients own cells; a tiny incision at the hip, and the little sponge is placed in the deteriorating hip bone, and the incision is closed. The hip repairs itself and there are no adverse side-effects.
Recently Mao et al, as well as others, reported an entirely new articular condyle with both cartilage and bone layers from a single population of MSCs. Clusters of MSCs initiate joint osteogenesis, which leads to apparently superior outcomes,
and may prove to be the long-term solution. (Archer et al., 2003; Dowthwaite et al., 2003).

Isolation and expansion

Friedenstein et al, 75 back in the 70’s observed, that when bone-marrow was isolated and cultured, there were subsets of various cells, and fibroblast-like cells capable of multiple doublings and differentiations; and they developed protocols for the isolation and expansion of these newly discovered cells. From that time our understanding of MSCs has advanced tremendously. Numerous studies on the fibroblast-like-cells have reported upon the potential of differentiations and expansion. The protocol by centrifugation in a density gradient to separate bone marrow-derived mononucleated cells from plasma and red blood cells is still widely used today. Enrichment techniques using positive selection of cell surface markers have been used and flow cytometry has been an additional enrichment tool (Marion et al., 2005; Lee et al., 2004)74. However, others point out that there still is no reliable phenotype to allow prospective isolation of purified MSCs by FACS analysis. As a consequence, the isolation of more specific antibodies must be a priority in ongoing and future research. Having isolated cells from the aspirate of human bone marrow, how does an investigator verify the ‘stem nature’ of the cells?
According to Lennon and Caplan, 80-87 the in vivo ceramic cube assay, is the standard for identifying MSCs. The in vivo ceramic cube assay system is complemented by the use of a number of in vitro assays, each of which is specific for different differentiated cells. Although no cell surface marker unique to MSCs have been identified to date, an extensive extension profile of cytokines and their receptors , adhesion and extracellular matrix , shared by these cells has been described.

The CD34, which has been extensively researched, and cited in 7000 published studies, can be used as specific markers for hematopoietic stem cells. Once the enriched bone marrow sample is placed atop the Percoll or Ficoll gradient and centrifuged, the dense cells and the antibody units are drawn to the bottom,

leaving the desired cells on the top of the gradient (Marion et al., 2005) 74. The enriched layer will contain a high concentration of MSCs, which can be plated and then expanded. Although the hematopoietic stem cell marker CD34 is not expressed by ex vivo culture expanded mesenchymal stem cells, it is possible to directly isolate early MSCs from fresh bone marrow on the basis of CD34 expression, 124 but oddly enough, not after culture, as the CD34 antigen is rapidly lost.
According to Lennon et al, perhaps the most important parameter in MSC technology, is the selection of the proper lot of fetal bovine serum (FBS), and in their experience, is not a good idea to merely purchase serum ‘off the shelf’, without testing it, and additionally discovered that, a batch of serum from one species will probably not support proliferation of MSCs from another.
Human MSCs (hMSCs) demonstrate initial lag during the beginnings of expansion, but is subsequently followed by rapid expansion doublings of 12-24 hours. Further the estimated number of hMSCs, in a 2 ml of bone marrow aspirate is between 12.5 and 35.5 billion (Spees et al., 2004)76. In addition, the multipotency of hMSCs, is further retained up to 23 population doublings (Banfi et al., 2000)77. No changes in the morphology of the MSCs are observed until after 38 doublings (Bruder et al., 1997)78. Immortalized MSC cell lines have been developed (Osyczka et al., 2002), and although not usable for clinical studies, are valuable as experimental tools.

Isolation protocol
Human bone marrow-derived MSCs
1. Ficoll-Paque-room temperature (e.g. Stem Cells Inc.,Vancover,BC)
2. Bone marrow sample-room temperature (e.G. AllCells, LLC, Berkeley, Ca.)
3. 10 ml marrow +5ml DPBS +125 U/ml heparin (total vol., 15ml).
4. Basal culture media (89%) DMEM-low glucose, 10% fetal bovine serum (FBS),1% antibiotics.
5. RosetteSep MSC enrichment cocktail (Stem Cells Inc.)
6. 100 ml PBS with 2% FBS and 1mM EDTA.
7. Transfer the bone marrow sample to a 50-ml conical tube. Add 750u1 RosetteSep(50 u1/1 ml of bone marrow, 50u1 x 15 ml=750 u1)
8. Incubate for 20 minutes at room temperature.
9. Add 15 ml of PBS 2% FBS 1 mM EDTA solution to bone marrow. Total volume is 30 ml.
10. Add 15 ml Ficoll-Paque to two new 50-ml conical tubes.
11. Layer bone marrow solution gently on top of the Ficoll-Paque in each tube. Do not allow marrow to mix with the Ficoll-Paque.
12. Centrifuge for 25 min at 300g with brake off at room temperature.
13. Remove enriched cells from the Ficoll-Paque interface.
14. Wash enriched cells with PBS-FBS-EDTA solution in a 50ml tube and centrifuge at 1000rpm for 10 min, with brake off.
15. Plate cells approximately 0.5-1 million total per petri dish with basal culture media.(now referred to as primary cultures or passage 0 (P0),)
16. Change medium every 2 days. Remove nonadherent cells during medium changes. Some of the adherent colonies are of mesenchymal linage
Additionally, it was discovered that MSCs could be induced to overcome their mesenchymal-ness, and differentiate after a relatively simple treatment of fully defined media, into cells with neuronal characteristics within 5 hours of induction 89 109; surprisingly, this differentiation was reversible. After incubation of the cells in neuronal induction medium for up to 24 hours and reculturing of the cells in standard growth medium, cells readapted to an MSC-like phenotype. Additionally, several groups have shown that BM-derived MSCs are able to differentiate into functional cardiomyocytes, both in vitro and in vivo 9-93- 94-117-118-119
Expanding human MSCs
Plating the MSCs are allowed to adhere to the flasks overnight. The next morning, non-adherent cells can be gently flushed from the flasks and replated in a second flask, in the same medium. The initial flask is refed with fresh medium for MSC expansion without differentiation. DOM is the richest medium for MSC expansion.
Genetic Engineering of Mesenchymal Stem Cells 2006
Osteogenic differentiation
As bone marrow is a rich source of many subsets of cells including MSCs, it is certainly not surprising that osteogenic differentiation was the first identied lineage of MSC s. According to Marion et al, several well-explored cocktails have been shown to induce MSCs to differentiate into osteoblasts. Among others, ascorbic acid, the bioactivecomponent of AsAP as an osteogenic supplement. The osteogenic differentiation of MSCs is verified by several osteogenic matrix molecules, and ultimately by the regeneration of bone in vivo. Tissue engineered bone must have the appropriate structural characteristics that approximate natural bone. Total joint replacement is one of the many examples of MSC therapies whose proof of concept has been verified (Rahaman and Mao 2005)72-73.
Recent studies by Caplan and Dennis (2006)80, suggest that new aspects of the MSCs , mainly tropic effects, that secret a variety of cytokines that function by both paracrine and autocrine pathways, create interactions between the synthesized MSCs , enhancing our understanding of these fundamental pathways and advancing practical approaches in MSC-based tissue engineering.
New clinical studies in the area of cardiac infract, stroke regeneration, and joint restoration will provide preliminary data, testing the tropic effects of MSCs. Although bone marrow is the most customarily available source of MSCs, they may also be isolated from adipose tissue, fresh or banked human umbilical cord blood, placental tissue, and human teeth (Mao, 2005)72. MSCs will certainly play an important role in future tissue engineering advances.
Genetic engineering of MSCs
MSCs are relatively easy to expand in culture and can be transduced with exogenous genes without the need for additional cytokines and conversely appear to have several advantages over the use of HSCs in gene therapy. Additionally, transduction of MSCs does not seem to impair their ability to home to several organs, 146-147, nor does it appear to affect their ability for self renewal. The in vivo use of genetically engineered MSCs in animal models is the first steps in this development. In a number of studies of gene transfer into MSCs, it was demonstrated that human stromal cells could be efficiently transduced with exogenous genes employing a variety of vectors, without a marked effect on their inherent stem cell characteristics. 149-150-151-152 In addition, it was shown that the transfer of genes into human MSCs by the use of retroviral vectors resulted in long term in vitro and in vivo expression of GFP 153-154-155. Subsequently, transduction protocols have been developed to reach transduction percentages of 80-90%. 151
Several strategies under investigation using MSCs therapeutically are ongoing. MSCs can be transplanted into poorly healing bone fractures, and also into damaged heart muscle, where they can actively participate or support reparation processes. 162-163-164
Gene therapy can be employed for the correction of osteogenesis imperfta, or brittle bone disease; by transplantation of autologous MSCs containing a wild-type gene for type I collagen 83-165-166. Long term cultured stromal bone marrow cells, with more than 20 passages, retrovirally transduced with LacZ and neoR genes were shown to form bone and to express exogenous genes after
transplantation into femurs.114 In addition to many notable examples, it also seems feasible to use engineered MSCs as a delivery vehicle of genes for the treatment of both hemophilia A and B, as well as other genetic diseases.
Adenoviral-mediated infections have the advantage over the use of retroviruses sense they do not require cell divisions for gene insertion and have a low toxicity. Multiple copies of the intended gene can be inserted into the genome of the target cell. However the down side appears to be that these copies transferred to the MSCs cannot be controlled and are very unpredictable.
Cryopreservation of hMSCs
Bruder et al, 78 reportedly cryopreserved in liquid nitrogen, a portion of cells at the end of primary culture, and then thawed the batch. These cells were then taken through the same protocol of extensive subcultivation as the unfrozen, same-batch of cells. Cryopreservation was not found to have an adverse effect on further cell expansion. Thawed hMScs are similar in morphology to those that have never been frozen and then thawed. They may have elongated , slender processes or may be somewhat more compact one day after seeding, however after an adjustment to the medium, become identical to the fresh-unfrozen cells.
Tissue Engineering of Heart Valves
Heart valve replacement represents the most common surgical therapy for end-stage valvular heart disease. The major drawback of all contemporary heart valve replacements, which they all have in common, is their lack of growth, repair and remodeling capabilities. To overcome these limitations, the emerging field of tissue engineering is currently focused on the in vitro generation of functional, living heart valve replacements.
Currently available heart valve prostheses for the treatment of advanced heart disease represent non-living foreign material and therefore are inherently
different from the tissue they replace. Thus, they are associated with substantial morbidity and mortality with regard to increased risks of thromboembolism, increased rates of infections, immunological reactions, and assorted mechanical failures. In the future of in vitro heart valve engineering and replacement, we will see living autologous replacements with the capacity of regeneration and reparation (R&R). 46-47-48-50
State-of-the-art heart valves replacements exhibits disadvantages such as mechanical failure, structural deterioration, and the need for permanent anticoagulation. Allogenic homo grafts are reported to be more efficient in their long term performance but are also not free of morphological changes, such as dilations, regurgitation, or structural failure (Staab et al 1999)54.
Therefore, scientists and surgeons have shifted their interest in generating a valve, which can be engineered and costumed to the patient’s needs in vitro and which can be implanted into the host and grow with him. It should require little or no anticoagulant and exhibit enough mechanical stability to withstand the hemodynamic load at the outflow tract. There are currently two main approaches to tissue engineered heart valves.
Regeneration and repopulation
Regeneration is based upon implantation of a biologic or otherwise absorbable matrix with advantageous biodegradation characteristics. Repopulation involves implantation of a decellularized animal valve. After a cleaning process from the native animal cells, leaving only native connective tissue matrix left for implantation, which is implanted with the patients cells and align along the valve surface to form an endothelial layer. Both methods have advantages and also disadvantages, both in the manufacturing and in vivo level. Other recent approaches involve seeding scaffolds with endothelial cells, endothelial progenitors, and mesenchymal stem cells.
Rieder et al, have demonstrated that the lowest level of immune system reaction was with decellurized human tissues. The cellularized porcine valves triggered a much stronger macrophage response. Thus they favor the human allograft, not
xenografts, as a basis of their decellularized technology for tissue engineering heart valves.
Another alternate approach, involves polylactic acid which is reabsorbable in synthetic scaffolds. The dominant material is polyglycolic (PLA/PGA), initially used for skin substitutes, which is FDA approved. Vacanti et al, of Harvard’s Children’s Hospital utilized a more compliant polyhydroxyalkanoate scaffold. Another material that allows for cell seeding and cell matrix is HA, which have been modified into hydrogels (Masters et al).53
Several groups have demonstrated the feasibility of creating living cardiovascular structures by cell seeding on synthetic polymers, collagen, as well as xenogenic, previously mentioned scaffolds.
Advancement milestones have been achieved, which comprise the successful replacement of a single pulmonary valve leaflet by an autologous tissue engineered leaflet. Hoerstrup et al 48, and Sutherland et al, followed this work with an in vitro completely autologous trileaflet heart valves based on ovine and human stromal cells.
The process of in vitro engineering of heart valves requires the harvest of autologous cells. After isolation, cells are expanded in vitro, 49 until a sufficient number of cells for tissue engineering are obtained. Cells are seeded onto the biodegradable heart valve scaffolds. Bioreactors are used to guide tissue maturation and formation, applying mechanical stimulation to the growing tissue, which have shown to result in an improved and implantable heart valve.3
Alternative human cell sources
Several alternative human cell sources have been investigated for their use in heart valve replacement engineering. Among the most promising are the vascular-derived cells, bone marrow-derived cells, and umbilical cord-derived cells. The obtained mixed-cell population consisting of myofibroblasts and endothelial cells is sorted by fluorescence-activated cell sorting (FACS), and the pure cell populations are cultivated and used for seeding and fabrication.
Additional thoughts
Heart valve scaffolds can be produced on several matrices including synthetic and biological. Several synthetic biodegradable polymers, such as polyglactin 1, polyglycolic acid (PGA) 5, polylactid acid (PLA), as well as PHA, have been investigated. Another approach for fabricating complete, trileaflet heart valves is the use of PGA coated with poly-4-hydroxybutyrate (P4HB). This biologically derived and rapidly absorbable copolymer is strong and pliable. Owing to its thermoplasticity, in can be molded into almost any shape, and complete degradation has been observed within 8 weeks after implantation (Hoerstrup et al).
Different types of bioreactors have been developed for this process such as pulsatile flow reactors, and diastolic pulse duplicators (Mol et al 2005), and as we noted infra, new and better bioreactors need to be designed and engineered50-39.
Tissue engineering of vascular grafts
Atherosclerotic vascular disease is the primary cause of morbidity and mortality in the U.S. Treatment of cardiovascular disease leads to more than 1.8 million surgical procedures annually that require arterial prostheses, including both coronary and peripheral vascular grafts (American Heart Lung Association, 2002)22-85 .Typically autologous vein or, less frequently, artery is employed in surgical revascularization procedures. In the coronary system, the saphenous vein and internal mammary artery are the most commonly selected. The internal mammary offers the highest long term graft patency rates, with saphenous vein grafts being more prone to progressive intimal hyperplasia and accelerated atherosclerotic change. Individual patients often require multiple coronary artery bypasses, and the more preferable saphenous vein or internal mammary grafts
are insufficient or unsuitable because of intrinsic vessel disease or prior use in revascularization (Eagle et al) 55
Alternatives for graft material include autologous venous or arterial sources and cryopreserved nonautologous saphenous vein or umbilical vein; however both options have inferior patency rates. Although synthetic grafts have been relatively successful in the replacement of larger-diameter vessels, i.e. 6-10mm, they are rarely used for coronary bypass surgery, because they routinely thrombose early after implantation (Nerem and Seliktar).57 These limitations in quality and quantity of ideal graftable materials have lead to a situation, in which approximately 100,000 patients per year, which require imminent revascularization, are rejected each year.22
These patients face a slow tedious death of palliative medical therapy and often suffer myocardial infractions (MI) and /or endure limb amputations as blood flow becomes progressively restricted.
Thus the critical need to develop small-diameter , less than 6 mm vascular grafts has prompted many investigators to develop tissue based vascular replacements that more closely mimic native vascular tissue.
L’Heureaux et al 1998 28-29 described the first entirely autologous vascular grafts. Their approach was to layer continuous sheets of fibroblasts and human umbilical smooth muscle cells around a central mandrel to form tubular vessels. After a 6-13 week culture period, the inner lumen of the tubular construct was seeded with endothelial cells. Although these artificial vessels were able to withstand high pressures, displaying rupture strengths greater than 2000 mmHg, their strength is derived primarily from the adventitial layer rather than the medial layer, which carries the majority of the load in a native artery (Nerem and Selikar)57. These vessels suffered a 50% thrombosis rate after one week, in transplantation procedures into canines.
Niklason et al, 1999, 2001,27-31-32 developed a technique for engineering arteries from explanted autologous vascular cells that are cultured on highly porous , degradable polyglycolic acid (PGA) scaffolds in specifically designed bioreactors
that subject the tubular scaffolds to physiological pulsatile radial distension that mimics the human cardiovascular system. After 8 weeks of culture time, the polymer scaffold has largely degraded and has been replaced by a dense medial layer consisting of smooth muscle cells and collagenous extracellular matrix. Once the lumen has been seeded with endothelial cells, the resulting vascular structure histologically resembles artery and remains patent for up to 4 weeks after implantation in miniature swine. Burst strengths of over 2000 mmHg and good contractile response have been reported, in these engineered vessels.
Fundamental requirements
Specific mechanical requirements for artificial vascular prostheses have been outlined by the American National standards Institute and the Association for the Advancement of Medical Instrumentation (ANSI/AAMI VP20-19994). According to these guidelines, any implantable arterial construct must be able to withstand normal physiological pressures of 80-120 mmHG, have burst strength of at least 1680 mmHg, and suture retention strength of over 273g (Barron et al 2003).37
Niklason’s et al, 27-31-32, overall goal is to develop an arterial graft that closely resembles the structure and properties found in native vessels. Arteries are composed of three distinct layers, each of which confers unique functional properties. The inner most layer, the endothelial cell layer functions to prevent spontaneous thrombosis in the vessel, as well as regulate the cell tone of vascular smooth muscle. The medial layer is composed of smooth muscle cells and their secreted extracellular matrix components including elastin, proteoglycans, and collagen. It has been observed that it is the media that contributes to the mechanical strength of the vessel as well as inherent contractibility and relaxation in response to external stimuli. The adventitial layer is composed mostly of fibroblasts as well as extracellular matrix. Within this outer layer also lie the microscopic blood vessels that supply blood to the artery via vasa vasorum. The ideal tissue designed blood vessel will certainly mimic this time tested model. The fabrication of tissue-engineered vessels that appear to reproduce the native arterial structures is not empirical evidence that a clinically equivalent
replacement has been achieved. Only in vivo implantation clinical studies provide a definitive measurement of biocompatibility, and we await results of these trials.
Much further work is necessary, however Nikason et al, have moved the field several important steps forward in the process of achieving a tissue engineered graft, with the necessary mechanical and physiologically properties. In time, this course of discovery will have enormous impact in the lives of thousands of patients.
Clinical studies are ongoing. Such grafts could be used as bypass grafts or dialysis shunts. Common arguments about the lengthy process of weeks to isolate and expand the endothelial cells of the patient in sufficient numbers required for seeding are rendered moot by the culture of stem cells in new generations of 3-D bioreactor systems, which will expedite vessel preconditioning, mechanical and electrical coupling improvements.
Injectable biomaterials
Cell therapy does not generally allow control of the local cellular environment. However injectable scaffolds can combine advances in materials sciences and progenitor cell biology to modify the cellular microenvironment in vivo. Ideally an injectable myocardial scaffold promotes repair after infarction by providing a matrix in support of neocardiogenesis.
Christman et al 2 showed that, fibrin glue improved myoblast graft retention and survival, reduced infract expansion, and induced neovascularization in the infarcted myocardium. These findings, supported by Ryu et al 2005, 3 showed that the implantation of bone marrow mononuclear cells using injectable fibrin matrix enhanced revascularization in the infarcted myocardium compared to cell implantation without matrix.
Synthetic biomaterials can be designed with a variety of biological features. Some short peptides have the unique properties of self-organization and self assembly (Zhang et al 2005).11 Once injected these materials respond to the physiological
environment within the tissue and self-associates in particular patterns to form organized structures. These self-assembling peptides, because of their regular repeats of alternating hydrophilic and hydrophobic amino acids, form stable structures based on protein B-sheets. Scaffold formation occurs within minutes after exposure to physiological salt conditions, a property that greatly enhances the viability of the materials in cardio-matrix assembly.
Cardiac myocytes cultured in these scaffolds exhibit a high degree of special organization and differentiation similar to myocardium, with spontaneous contractions. Upon injection in vivo, these nanofiber microenvironments are spontaneously vascularized (Davis et al 2005).5
Key factors in material selection
The bioscaffold should be able to provide not only physical support for the cells but also the chemical and biological cues needed for functional tissue generation.
Optimal features of materials for cardiac tissue engineering
Innate properties
1. Noncytotoxic
2. Nonimmunogenic
3. Noninfectious
4. Biodegradable with nontoxic products
5. Porosity
6. Mechanical strength/elasticity
7. Injectable
Biological properties
1. Support cell attachment
2. Support cell survival and proliferation
3. Drive migration of progenitors
4. Support development of functional vasculature
5. Support mechanical and electrical integration
Smart properties
1. Shape memory
2. Trigger release of growth factors
(Segers & Lee) 58
Management of the microenvironment by tissue engineering consists of attracting the progenitor cells, enhancing their survival; driving their differentiation and promoting their mechanical and electrical coupling. Biomaterials such as self-assembling peptides form stable hydrogels. Upon injection to a physiological environment, they form a scaffold of interwoven nano-fibers because of the changes in ionic strength and ph.
Materials with shape memory transform to a present form at a given temperature. Bioactive factors can be incorporated by noncovalent binding to the scaffold. Depending upon the type of reactions between the delivered cells and the scaffold, binding can be weak or strong, with respectively, fast and slow release. Factors can be linked to the scaffold by substrate peptide sequences, and may be released after myocardial infarction (MI).
Natural materials
Drawbacks of natural materials isolated by extraction, e.g., collagen can be overcome by selecting fragments with specific functions instead of using crude materials (Ito et al). Hence, although biological materials can be difficult and expensive to engineer and purify, known properties of natural biomaterials can be used as a template for synthetic materials to circumvent drawbacks associated with natural scaffolds and to select and amplify the desired properties.
Synthetic materials
Synthetic materials can be manufactured on a large scale, and their structure and mechanical properties can be more easily controlled and manipulated than natural materials. To improve cellular adhesion, cell-adhesion motifs may be engineered into biomaterials. A potential drawback of most synthetic materials is their lack of specific cell-recognition signals.
Surface properties of biomaterials affect cell spreading and proliferation and differentiation. For instance, differentiation of mesenchymal stem cells is dependent upon specific coatings of the biomaterials.
Mesenchymal stem cells are sensitive to surface chemistry, and the N-enriched surfaces by means of glow discharge plasma techniques can influence human mesenchymal responses, and potentially affecting differentiation towards osteoblasts. 16
Another important element in the development of bioactive materials is the control of porosity. This property of porosity has not been studied extensively of date, for cardiac tissue engineering17. Pores smaller than 100nn result in decreased diffusion of oxygen, nutrients, and growth factors, leading to poor survival of implanted cells. Larger pores eg100um, are beneficial for cell migration and proliferation in the scaffold, but pores larger than the size of an endothelial cell are difficult to bridge , and may lead to decreased angiogenesis. 17
A balance of porosity size is an important element which can affect critical aspects such as angiogenesis, migration, remodeling, and scaffold material degradation.
Smart biomaterials
Smart biomaterials have been defined as materials that are responsive to environmental cues, such as ionic strength, temperature, enzymes, pH; that cause or react to responses, including protein release. Besides protein delivery, engineered scaffolds have potential for controlling or improving gene transfer. Synthetic materials, including peptide-based delivery systems, are being developed at several laboratories that include alternatives to using viruses as
vectors. Peptide vectors are, because of their exceptional adaptiality, particularly applicable to this approach. (Zhang et al) 11
Combination products and final thoughts
With a combination of specific cells, such as full spectrum, mononuclear bone marrow stem cells, mesenchymal, endothelial, fibroblasts, neural, autologous, allogenic, embryonic and/or others selected for requisite properties; combined with designer self-assembling nano-cardio-matrix scaffolds, and/or other suitable construction modalities, both natural and synthetic in the future will include all of these elements for bionatural engineering efficacy.
Transendocardial (Perin et al) catheter-based combination injectables, forming living-cardio-matrix will eventually circumvent the need for routine invasive surgery, making draconian open-heart surgery as it has been practiced today and through our lifetimes, a historical passing reference and footnote in old medical journals.
Statistical realities:
By 2011, over 2 million Americans are projected to contract end-stage renal disease, with a cost of $1 trillion. In 2009, nearly 80,000 people needed organ transplants, fewer than 24, 000 were performed; and 6000 died waiting. Of those receiving organs, 40% died within the first three years after surgery.2 One in five of our elders 65 and over will require an organ transplant during their remaining years.83- Heart disease and stroke figures top 380 billion dollars per year.
In 2013, over 19 million people or 7.3% of the population, suffered with diabetes, at a cost of $140 billion. Add to this list cancer, Alzheimer’s, spinal cord injuries, Parkinson’s, MS, etc, Behind these sobering facts, patients and their families ask, “Will there be a cure? And will it be in time for us?”

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