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INVITED REVIEW

3-Dimensional Structures to Enhance Cell Therapy and Engineer Contractile Tissue

Olivier Schussler, MD, PhD1,2, Juan C Chachques, MD, PhD1, Thierry G Mesana, MD2, Erik J Suuronen, PhD1, Yves Lecarpentier, MD, PhD3,4, Marc Ruel, MD1

1 Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, Canada
2 Biosurgery Laboratory, Pompidou Hospital, Paris, France
3 Cardiorespiratory Function Testing Unit, Bicêtre Hospital, Paris, France
4 Laboratory of Clinical Research, Meaux Hospital, France

Olivier Schussler, MD, PhD, Tel: +1 613 761 4893, Fax: +1 613 5367, Email: oschussler{at}ottawaheart.ca, Division of Cardiac Surgery, University of Ottawa Heart Institute, 40 Ruskin Street, Suite 3403, Ottawa, ON, K1Y 4W7, Canada.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 CURRENT LIMITATIONS OF CELLULAR...
 RATIONALE FOR ASSOCIATING CELLS...
 SCAFFOLDS UNDER DEVELOPMENT
 COLLAGEN AS A CORE...
 THE FUTURE OF COLLAGEN...
 REFERENCES
 
Experimental studies in animals and recent human clinical trials have revealed the current limitations of cellular transplantation, which include poor cell survival, lack of cell engraftment, and poor differentiation. Evidence in animals suggests that use of a 3-dimensional scaffold may enhance cell therapy and engineer myocardial tissue by improving initial cell retention, survival, differentiation, and integration. Several scaffolds of synthetic or natural origin are under development. Until now, contractility has been demonstrated in vitro only in biological scaffolds prepared from decellularized organs or tissue, or in collagenic porous scaffold obtained by crosslinking collagen fibers. While contractility of a cellularized collagen construct is poor, it can be greatly enhanced by tumor basement membrane extract. Recent advances in biochemistry have shown improved cell-matrix interactions by coupling adhesion molecules to achieve an efficient and safe bioartificial myocardium with no tumoral component. Fixation of adhesion molecules may also be a way to enhance cell homing and/or differentiation to increase local angiogenesis. Whatever the clinically successful combination ultimately proves to be, it is likely that cell therapy will require providing a supportive biochemical, physical, and spatial environment that will allow the cells to optimally differentiate and integrate within the target myocardial tissue.

Key Words: Cell Transplantation • Heart Failure • Myocardial Infarction • Tissue Engineering


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
CURRENT LIMITATIONS OF CELLULAR...
 RATIONALE FOR ASSOCIATING CELLS...
 SCAFFOLDS UNDER DEVELOPMENT
 COLLAGEN AS A CORE...
 THE FUTURE OF COLLAGEN...
 REFERENCES
 
The human heart has a limited capacity for self-repair or regeneration1; however, a very recent study confirmed that depending on the age of the patient, up to 1% of heart cells regenerate each year.2 The use of exogenous stem or progenitor cells in animals initially yielded encouraging results, but they were not followed by a successful mechanical demonstration that new contractile units are indeed derived from these cells. As the results of human cell therapy trials were reported, it became unclear whether stem cell therapy, as we currently attempt it, will ever provide significant benefit.2–7 Indeed, recent randomized controlled trials have failed to produce the same results as uncontrolled observational studies, and showed little improvement, particularly after longer follow-up times.8


   CURRENT LIMITATIONS OF CELLULAR THERAPY
TOP
 ABSTRACT
 INTRODUCTION
 CURRENT LIMITATIONS OF CELLULAR...
RATIONALE FOR ASSOCIATING CELLS...
 SCAFFOLDS UNDER DEVELOPMENT
 COLLAGEN AS A CORE...
 THE FUTURE OF COLLAGEN...
 REFERENCES
 
Several reasons may explain the minimal benefit obtained so far with cell therapy. Firstly, the capability of putative cells to differentiate into cardiomyocytes has not been demonstrated. Most studies in humans have been carried out using bone marrow cells, mesenchymal cells, circulating progenitor cells, skeletal myoblasts, or adipocyte-derived cells. While there is little concern about the angiogenic potential of these cells, many questions remain regarding their capability to become contractile cells. It is likely that most of the evidence for differentiation reported so far in animal models may in fact have been fusion events between injected cells and native cardiomyocytes. Even in animal models, there is a discrepancy between the numbers of transplanted cell and the much greater number of cells needed to replace the contractile cardiomyocytes lost.9 Therefore, the improvement in contractile function reported in many cases is not due to the contractile potential of the cells themselves, but rather to a paracrine mechanism.10 Thus contractility may be improved even if the transplanted cells are not longer present, because improving transplanted cell survival and differentiation may still increase the benefits of this paracrine effect.11 Indeed, the transplanted cells may serve as a safe and easy vehicle for local delivery of known or unknown growth factors that improve myocardial hibernation (which remains to be demonstrated), activation of cardiac-resident cells, mobilization and homing of putative regenerative cells, stabilization of extracellular matrix, endogenous repair and remodeling, as well as angiogenesis.10,12

To date, the most promising cell type, in terms of potential for proliferation, differentiation, and integration within the native myocardium, is indubitably the embryonic stem cell.7,13 In addition to ethical concerns about using embryonic stem cells in humans, there are several associated risks such as the risk of teratoma, especially because the hypothetical patient will presumably need immunosuppressant therapy as embryonic stem cells are not as non-immunogenic as initially thought. Human induced pluripotent stem cells, derived by reprogramming autologous somatic cells, are attractive because of their autologous origin, but several of the transcription factors used so far are oncogenic.14,15 Strategies to remove these transcription factors when cells are reprogrammed have been reported recently.16,17 Another interesting approach is the use of multipotent embryonic stem cells already committed toward a cardiac lineage, as it appears that the teratogenicity of this cell population is quite low.18 In-vitro culture may serve to expand and select these cells before implantation. Although initially very promising, studies in rodents mitigated previous in-vivo reports on the possibility of using embryonic stem cells alone as a free graft to treat myocardial failure. While an initial improvement in contractility at 1 month was described and confirmed by several groups, it appears that this improvement may not last long, and the initial benefits may no longer be present at 3 months.19,20 Midterm data must therefore be interpreted with caution, and long-term follow-up is essential to draw conclusions on the efficacy of cardiac cell transplantation. In addition, in many instances, the differentiation affected only a small fraction of the entire cell population. Increasing cell numbers did not change the outcome, suggesting the need to develop strategies for the long-term integration of these cells. Incomplete differentiation of these cells may be due to their surrounding environment lacking the necessary physical and biological properties to trigger their differentiation.13

The second issue concerns the low cell engraftment rate due to poor retention of cells within injection sites, poor cell survival of myogenic and angiogenic cells in the infarct zone, and their limited proliferation capabilities.21,22 Several studies have reported that cell engraftment is around 1% or even lower a couple of weeks after transplantation.23 Recent reports have also shown that this low engraftment efficiency may be partly due to suboptimal methods of cell delivery. For example, with intracoronary cell injection, only 10% of injected microspheres approximating the size of mesenchymal stem cells were retained within the site of injection after 30 min, and this rate was even lower when the heart was beating, compared to the arrested state.24 A study using a luciferase reporter gene to track bone marrow mononuclear cells confirmed very low cell retention or homing in the myocardium after intravenous, intracoronary, or intramyocardial injection.25 Low cell retention was also confirmed in peripheral muscle using injected radiolabeled cells.26 In humans, data are scarce, but studies using positron-emission tomography have shown that 50–75 min after intracoronary injection, only 1%–3% of the cells are found in the myocardium.27 For the cells that have been retained, there may still be a very high rate of death, non-differentiation, or non-integration. Potential causes of poor cell survival include ischemia due to poor vascularity at the transplant site, apoptosis due to detachment of the cells from their surrounding 3-dimensional (3D) interactive environment, inflammation with degradation products that may be toxic to the cells, and later on, immunological rejection.28 Regarding the infarct zone, the optimal time lapse for cellular transplantation is still undetermined. However, with bone marrow mononuclear cells, cell retention and survival appear to be improved if the injections are performed in the established rather than the acute infarct phase.25 Adding to the difficulties is the fact that neonatal rat cardiomyocytes transplanted into a scar area failed to become electrically coupled to native heart cardiomyocytes, while in viable myocardium, such connection was established.29 Optimizing the type of cell, possibly associating it with an angiogenic or paracrine-type cell (mesenchymal, endothelial progenitor cell, or mixed mononuclear cell populations), and selecting a cell population with enhanced resistance to hypoxia (by ischemic preconditioning in vitro or electrostimulation) are among the strategies that might enhance cell survival. The demonstration of the importance of angiogenic, anti-apoptotic and pro-survival signaling pathways to enhance angiogenic and myogenic cell engraftment was demonstrated using genetically modified cells. Notably, it was found that myogenic or angiogenic cells transplanted with a survival gene (Akt or Bcl2) or an angiogenic gene (VEGF, bFGF, or VMAP) did have improved cell survival and differentiation capability.24,30 Control of the fate of the transplanted cells and the regulation of the local delivery of concomitant growth factors remain challenging issues for future clinical purposes.

The third important limitation is the integration of the cell and its contact with adjacent cells in the native myocardium. Integration and coupling are critical to achieve contractile tissue that will beat synchronously with the rest of the myocardium. After an infarct, the myocardial extracellular matrix is altered, and through the action of local fibroblasts, a new matrix will be synthesized.31,32 The newly synthesized matrix is different from the native one not only in terms of its shape but also in its mechanical and biological properties. Post-infarct extracellular matrix is mainly composed of collagen type III, rather than type I, with lower adhesion and angiogenic properties. The survival of injected cells has been shown to be dependent of the type of collagen content in a 3D environment.33 Other important characteristics of the extracellular matrix are its orientation and stiffness. While electrical fields have been used to orient contractile cells, in-vitro experiments have demonstrated that topographical cues are the main determinant of cellular orientation.34 Furthermore, it has been shown that contractile cells such as myoblasts or cardiomyocytes differentiate and beat synchronously on a matrix with an intermediate stiffness of 8 to 11 kpa, but they do not beat on a matrix with lower or higher stiffness, such as that observed in scar (20 to 40 kpa).35,36 Cell orientation appears to be an important parameter for enhancing cell-to-cell contact to achieve an implant with increased contractility and electrical stability. Orientation has been shown in vitro to increase the conduction velocity of a neonatal rat cardiomyocyte preparation, as well as electrical integration of mesenchymal stem cells undergoing cardiomyogenic differentiation in co-culture with cardiomyocytes.37 The lack of electrical connection between myoblasts and native heart cardiomyocytes may partially explain the arrhythmogenicity of the latter cells. Intercellular contacts are directly controlled by cell-extracellular matrix interactions including the mechanical coupling of cells with extracellular matrix through integrin receptors, which are themselves mechanoreceptors sensitive to substrate stiffness, orientation, and shear stress. The importance of these interactions has been demonstrated by Valencik and colleagues38 using genetically modified mice in which the expression of an integrin receptor could be controlled in the heart. The disruption of these receptors had a drastic effect on contractility, electrical properties, and cardiomyocyte organization, including alteration of intercellular contact (including connexin 43). Electrophysiological analyses have shown that cardiomyocytes seeded in a scar environment do not connect with surrounding cardiomyocytes, whereas if the environment is viable, this connection can occur.29 All of this evidence advocates the use of scaffolds for cell transplantation, which can be modulated for the replacement of myocar-dial scar.


   RATIONALE FOR ASSOCIATING CELLS TO A 3D SCAFFOLD
TOP
 ABSTRACT
 INTRODUCTION
 CURRENT LIMITATIONS OF CELLULAR...
 RATIONALE FOR ASSOCIATING CELLS...
SCAFFOLDS UNDER DEVELOPMENT
 COLLAGEN AS A CORE...
 THE FUTURE OF COLLAGEN...
 REFERENCES
 
The use of an alternative approach to cardiac cell transplantation appears to be warranted. One promising approach is to mimic the 3D environment of the cells as if they were in native tissue, and potentially of transplanting them in a 3D scaffold.1 Although still not completely demonstrated, several mechanistic (Table 1Go) and functional (Table 2Go) advantages might be derived from the use of scaffolds or matrices. It has been shown that the presence of a 3D scaffold improves cell retention at a given site, and could be a way to improve cell delivery and limit important cell losses.12,62–65 The scaffold may thus serve as cell carrier for in-vivo cell delivery.


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  		Table 1. Possible mechanistic influences of different collagen scaffolds on behavior of contractile or endothelial cells in vitro or after implantation into muscular tissue
 

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  		Table 2. Functional characteristics of collagen scaffolds alone or with adhesion molecules, seeded with contractile or endothelial cells
 
The incorporation of immunomodulatory molecules into biomaterial designs may represent another strategy to control potential immune responses. However, there is now evidence that beyond cell retention, biomaterials are a way to expand and improve survival and differentiation of previously used cell types including neonatal cardiomyocytes and embryonic cells that do not terminally differentiate after free cell injection.41,66 Cells are very sensitive to their microenvironment in terms of mechanical (orientation/geometry, stiffness/ rigidity, porosity) as well as biological properties.67,68 In the heart, this microenvironment is the extracellular matrix (ECM) which is composed mainly of collagen type I and basement membrane proteins. The interaction of the cell is mediated by cell membrane receptors that recognize oligopeptides present in the ECM. The integrin receptors are mechanoreceptors, thus after receptor engagement and activation, there is a physical link created between the ECM and the cell cytoskeleton at the level of the Z band of the contractile apparatus in cardiomyocytes.69,70 At the same time, due to this new physical link, the cell can provoke physical cues on the ECM.71,72 The intensity and duration of this interaction translates into biological cell signals that aim to control cell proliferation, cytoskeleton and contractile apparatus reorganization, cell apoptosis and survival, and intercellular connections. The role of integrin receptors in controlling these different cellular events has been reviewed.73 Physical containment of cardiac muscle cells by the extracellular matrix maybe an important regulator of myofibrillar organization and a way to improve inter-cardiomyocyte junctions, thus potentially improving contractility and limiting the risk of arrhythmias.38,74 The same process probably occurs during embryonic development. The formation of myofibrils is a rapid and sequential process that occurs first at the level of Z-discs, then at the M-band, followed by the assembly of the thick and thin filaments.75 Myofibrils are anchored at their ends in the intercalated disc in adult cardiomyocytes, lateral contacts to the plasma membrane exist at the level of the Z-disc, called costameres. A marker of these complexes is vinculin that is known to be associated with integrin receptors. This separation between junctional and lateral membranes is not yet apparent in round-shaped embryonic cardiomyocytes. Interestingly, some cardiac pathologies are also characterized by the absence of a clear separation between distal cell-cell contacts and the lateral extracellular matrix.54

Biomaterial design has evolved from the classical first generation material that favored mechanical strength, durability, bioinertness or biocompatibility, to third generation biofunctional materials that seek to incorporate instructive signals into scaffolds to modulate cellular functions such as proliferation, differentiation, and morphogenesis. To impact the bioactivity to these biomaterials, their surface can be modified with signaling molecules such as glycoaminoglycans, proteoglycans, and glycoproteins, or loaded with soluble bioactive molecules such as chemokines, cytokines, growth factors, or hormones, which are released and act in a paracrine manner.12,66 Advances in conjugation chemistry have now widened the options for modifying natural biopolymers or synthetic biomaterials. Such biomaterials have yielded interesting results in vitro for the maintenance of large quantities of undifferentiated stem cells, by providing sufficient starting material, selecting the cell or cells of interest, as well as controlling stem cell differentiation and limiting the risk of tumor formation.


   SCAFFOLDS UNDER DEVELOPMENT
TOP
 ABSTRACT
 INTRODUCTION
 CURRENT LIMITATIONS OF CELLULAR...
 RATIONALE FOR ASSOCIATING CELLS...
 SCAFFOLDS UNDER DEVELOPMENT
COLLAGEN AS A CORE...
 THE FUTURE OF COLLAGEN...
 REFERENCES
 
Several scaffolds for cardiac tissue engineering are under development.58,76–78 Natural materials aim to mimic heart native extracellular matrix, depending on polymer composition, the density or porosity of the resulting scaffold, and the chemical production process, which must exclude the pathogen transmission risks inherent to some biological matrices, particularly when xenoproteins are used. On the other hand, all synthetic matrices lack an adequate and elaborate physiological recognition system, and they need more sophisticated modification steps to become a bioactive ECM. Among the synthetic biodegradable materials, the polyester family, including polylactic acid, polyglycolic acid, polylactones, and polyurethanes have been the most frequently explored. None of them display the ideal material properties that high elasticity, high extensibility, high robustness, and a low Young’s modulus constitute.73 Recently, due to its unique elastomeric properties, poly(glycerol sebacate) was proposed for use in soft tissue engineering, including that of cardiac tissue. However, all these polymers induce an immune response and have been hampered by limited myocyte differentiation.79 In addition, no spontaneous contractility has been demonstrated in these materials, despite the presence of a well-validated contractile cell such as neonatal rat cardiomyocytes or cardiomyocytes derived from embryonic stem cells.79,80

Several new technologies for creating smart material scaffolds exist, such as self-assembling peptides that form a nanoscale fiber network, but this was found to have a limited capacity to enhance cardiomyocyte differentiation in vitro, and no contractility was demonstrated.81 Another interesting approach is de-novo synthesis of cell-derived extracellular matrix, using reversed attachment of the cell monolayer to the culture underlay. From this, one can develop a multilayered structure.82 Immunological and biocompatibility issues are reduced, although this technique requires that cells be cultured in vitro before transplantation. While microscopic organizations along with electrophysiological and contractile performance of involved cardiomyocytes are highly preserved, the macroscopic strength and thickness of the resultant engineered tissue may turn out to be a limitation of this approach. In addition, there are some concerns about nutrient diffusion through the engineered tissue and long-term survival of cells through thick tissue.

Tissue engineering concepts that involve native ECM components appear very promising due to their strong biocompatibility, preserved bioactivity, and most prominently, their porosity which is very important for cell seeding, nutriment diffusion, and angiogenesis. Of the many biological scaffolds tested so far, including polysaccharides (dextran, hyaluronic acid, chitosan), alginate, cellulose, native decellularized tissues/organs, collagen/gelatin, and fibrin, contractility has been demonstrated only with collagen scaffolds and decellularized organs.56,83 While promising, decellularized tissues still face critical issues before they can be used as a scaffold to engineer contractile tissue.78,84 Several decellularizing processes using physical, chemical, or enzymatic treatment have been described. Progress has been made and it is now possible to remove most of the cell content from a tissue while preserving intact basal lamina, protein, and glycosaminoglycan content. The use of a scaffold for tissue engineering may similarly require treatment to achieve adequate porosity for cell seeding, but such additional treatment may alter scaffold mechanical properties and durability. In addition, the impact on tissue immunogenicity needs to be evaluated.85 The use of decellularized biological scaffolds has been proposed to reconstruct part of the myocardium by repopulation of the endocardium with cells of an endothelial phenotype. The scaffold was fully resorbed after 2 months and replaced by fibrotic tissue with cells expressing cardiomyocyte markers, but with mechanical and electrical properties largely inferior to those of native myocardium.86,87 The attachment of cells such as mesenchymal stem cells to decellularized scaffold is low, although it may be improved if the scaffold is modified with adhesion molecules or associated with growth factors.87–89 Synchronous contractility and electrical coupling with native myocardium have been demonstrated in animal models using a biological patch of collagen seeded with neonatal rat cardiomyocytes applied to the myocardium.57,61 Interestingly, both implanted and endogenous cells re-colonizing acellular biological extracellular matrices may be electrically integrated.58,86


   COLLAGEN AS A CORE FOR 3D SCAFFOLDS IN TISSUE ENGINEERING AND CELL THERAPY
TOP
 ABSTRACT
 INTRODUCTION
 CURRENT LIMITATIONS OF CELLULAR...
 RATIONALE FOR ASSOCIATING CELLS...
 SCAFFOLDS UNDER DEVELOPMENT
 COLLAGEN AS A CORE...
THE FUTURE OF COLLAGEN...
 REFERENCES
 
Collagen matrices are very attractive for many reasons. As a natural molecule, collagen presents many cell adhesion ligands, and it is the major component of the extracellular matrix of most tissues. Collagen possesses extraordinary mechanical properties in term of resistance, and it has a natural propensity to spontaneously assemble into a highly porous 3D shape. In addition, the synthesis of recombinant human-homologous collagen has been recently performed. The chemically denatured product of collagen, gelatin, does not have the same mechanical properties, and for unknown reasons, can provoke a very strong inflammatory response upon implantation in vivo.

One of the simplest biological ECM materials, porous dry collagen obtained by dehydrothermal crosslinking (DHT), has been used for many years in humans as a hemostatic agent. Recently, the possibility of using such scaffolds for regeneration of tissue, including cardiac tissue, was proposed.90 The application of an external patch of DHT collagen to the surface of the infarcted heart elicited a chronic inflammatory and angiogenic response.56 The patch was totally resorbed over a period of 2 months, accompanied by a positive effect on left ventricular remodeling.91 The use cardiomyocytes in a collagen matrix was developed by Kofidis and colleagues90 in vitro. Several cell types have been transplanted into a collagen scaffold to date. The superiority of the association of cells (cardiomyoblasts) within a collagen patch scaffold compared to free cell injection was clearly demonstrated by Kutschka and colleagues56 who found improved cell survival and cardiac function (fractional shortening and ejection fraction). The presence of an injectable collagen scaffold was also shown to improve the retention of human progenitor cells from peripheral blood.37 Similarly, the survival of embryonic cells and resultant local contractility were improved by the presence of another injectable scaffold, Matrigel, in an infarct model.92 We demonstrated the superiority of application of a collagen matrix cellularized with umbilical cord blood compared to collagen alone for controlling left ventricular remodeling and improving ejection fraction after myocardial infarction in small animals.93 The application of this approach is under investigation in humans, using whole bone marrow cells. Feasibility and initial results at one year have been reported, with improvements in ejection fraction and diastolic parameters, possibly due to the presence of the cellularized collagen-DHT matrix.94


   THE FUTURE OF COLLAGEN SCAFFOLDS
TOP
 ABSTRACT
 INTRODUCTION
 CURRENT LIMITATIONS OF CELLULAR...
 RATIONALE FOR ASSOCIATING CELLS...
 SCAFFOLDS UNDER DEVELOPMENT
 COLLAGEN AS A CORE...
 THE FUTURE OF COLLAGEN...
REFERENCES
 
In spite of these encouraging results, the survival and differentiation of angiogenic or contractile cells in collagen remain very poor.48,51–53,95–98 Contractility in the presence of neonatal rat cardiomyocytes is low, and spontaneous contraction remains rare and asynchronous even after weeks of culture.44,99 Using the animal tumor basement membrane protein extract, Matrigel, with a DHT-collagen scaffold, Eschenhagen and colleagues52,53 established one of the most convincing models of 3D cardiac cell cultures to date. To achieve terminal differentiation of neonatal rat cardiomyocytes in vitro, their model also required the use of a high dose of xenogenic horse serum, chronic physical stimuli such as shear stresses or electrical stimulation, and a bioreactor.45,48,100 The need for a bioreactor was possibly due to the presence of Matrigel that filled the pores of the scaffold and thus disturbed nutrient diffusion.48 Their in-vitro engineered tissue appeared to survive if applied to the myocardium or used to replace part of the myocardium. In a rat heart model of chronic infarction, the application of engineered constructs improved diastolic and systolic function.57 However, due to a very important immunological reaction against the associated Matrigel component, the animals needed to be immunosuppressed. Mitigated results have been reported using Matrigel for a long period with dedifferentiation of associated cells, secondary fibrosis, and poor angiogenesis.101 While the use of growth factors could compensate at some point for the requirement for Matrigel, in terms of cell survival and growth, the clear differentiation of cardiomyocytes with reorganization of the sarcomere apparatus was not demonstrated at the ultrastructural level, the spontaneous contractility of the scaffold was lost, and results were obtained by increasing the proportion of fibroblasts in the preparation, which could conversely have impaired relaxation and long-term mechanical findings.102 In addition, stimulation with growth factors and subsequent intracellular activation cascades may only partially reproduce cellular events that occur after integrin receptor engagment.103 While it has been reported that soluble growth factors can at some point mimic the effect of soluble RGD adhesion molecule (arginine-glycine-aspartic acid) which is known to be internalized in cardiomyocytes, it is most probable that cellular events triggered by the attached and presented RGD are quite different.47,104 For example, while we have shown that the presented RGD has a beneficial effect on contractility, the soluble RGD peptide has been reported to have a negative effect on contractility.54,105 Therefore, the close relationship between integrin receptors and the contractile apparatus suggests that the different signals need very fine coordination in a spatial and temporal manner to work properly. Imperfect connection between cell-cell and cell-extracellular matrix signals may lead to scaffolds that cause decreased contractility and poorer synchronization.

In recent work, we provided a way to enhance collagen functionality by covalent ligation and presentation of an integrin binding ligand that is absent on native collagen.44 By covalent coupling of RGD to collagen scaffold obtained by DHT, we were able to engineer a very efficient and safe contractile tissue that enhanced survival and differentiation of associated contractile cells in the absence of Matrigel (Figure 1Go). In addition, the engineered tissue could be electrically pulsed, and many parameters of contractility were very close to those observed in native myocardium. The low threshold observed for electrical stimulation of the engineered scaffold should facilitate electrical coupling of the construct following implantation. For the first time, we have also shown that in a cellularized scaffold, as in the native heart, both contraction and the relaxation are active processes. In addition, the fact that the efficiency of contraction and relaxation are of the same amplitude advocates the use of a cellularized scaffold instead of inert materials for the establishment of cardiac devices aimed at treating myocardial conditions. Angiogenesis and implant pre-vascularization are important to improve functionality on implantation or implant survival, as demonstrated with in-vitro engineered skeletal tissue.106 We reported the possibility of using RGD to enhance the differentiation of the progenitor for endothelial cells and differentiated endothelial cells in collagen scaffolds by interaction with transmembrane integrin receptors.107 The possibility of improving local angiogenesis in injectable collagen matrices by increasing the homing of human progenitor cells purified from peripheral blood via covalent fixation of adhesion molecules that interact with the cell surface membrane protein, L-selectin, was proposed recently by our group, and may be another way to improve local angiogenesis.50,108


Figure 1
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  		Figure 1. In-vitro contractility of neonatal rat cardiomyocytes (NC) seeded in a collagen scaffold (CS) or a collagen scaffold + RGD peptides (CS + RGD). Continuous mechanical recording of forces developed spontaneously (a and b) or under stimulation at 4Hz (c and d). Spacer bar = 1 mN. For more information, see reference no. 72.

 
In conclusion, the disappointing results of cell therapy to date should be greatly improved if the cells are associated with a 3D scaffold. For this purpose, the modification of a collagen matrix with adhesion molecules may constitute a unique platform for engineering highly efficient contractile tissues and enhancing cell therapy.

Presented at the 4th International Cardiac Bio-Assist Association Congress, Singapore, March 12–13, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 CURRENT LIMITATIONS OF CELLULAR...
 RATIONALE FOR ASSOCIATING CELLS...
 SCAFFOLDS UNDER DEVELOPMENT
 COLLAGEN AS A CORE...
 THE FUTURE OF COLLAGEN...
 REFERENCES
 

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Asian Cardiovasc Thorac Ann 2010; 18:188-198
© 2010 by SAGE Publications
DOI: 10.1177/0218492310361531



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