Evaluation of the Biocompatibility of PLACL Collagen Nanostructured Matrices With Cardiomyocytes as a Model for the Regeneration of Infarcted Myocardium 2011

Shayanti Mukherjee , Jayarama Reddy Venugopal , * Rajeswari Ravichandran , Seeram Ramakrishna , and Michael Raghunath 1. Introduction Myocardial infarction (MI) is the single leading cause of deaths due to cardiovas- cular disease globally. [ 1 ] MI is caused when the supply of oxygen and nutrients to the cardiac muscle is impaired, usually due to occluded coronary arteries. A massive cell death occurs in the affected heart region, which
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   Shayanti Mukherjee , Jayarama Reddy Venugopal , * Rajeswari Ravichandran , Seeram Ramakrishna , and    Michael Raghunath 1. Introduction Myocardial infarction (MI) is the single leading cause of deaths due to cardiovas-cular disease globally. [   1   ]  MI is caused when the supply of oxygen and nutrients to the cardiac muscle is impaired, usually due to occluded coronary arteries. A massive cell death occurs in the affected heart region, which forms a rather non-contractile scar characterized by mismatch of mechan-ical and electrical properties with native myocardium. The substantial cell loss in the myocardium leads to dilation of ven-tricular wall and remodeling of the heart and, eventually, to congestive heart failure (CHF). [   2   ]  Currently, more than 10 million people suffer from CHF in the USA, UK and southeastern Asia. Drugs alone might be able slow disease progression, but they cannot prevent it. [   3   ]  Current treatments for congestive heart failure use highly invasive methods, such as open chest surgery and transplantation. [   4   ]  Given the patient mor-bidity and complications involved with cur-rent procedures, it is desirable to develop minimally invasive technologies, such as injectable therapeutics. Several modes of regenerating injured myocardium have been suggested over time, with pioneering research being Evaluation of the Biocompatibility of PLACL/Collagen Nanostructured Matrices with Cardiomyocytes as a Model for the Regeneration of Infarcted Myocardium Pioneering research suggests various modes of cellular therapeutics and biomaterial strategies for myocardial tissue engineering. Despite several advantages, such as safety and improved function, the dynamic myocardial microenvironment prevents peripherally or locally administered therapeutic cells from homing and integrating of biomaterial constructs with the inf-arcted heart. The myocardial microenvironment is highly sensitive due to the nanoscale cues that it exerts to control bioactivities, such as cell migration, proliferation, differentiation, and angiogenesis. Nanoscale control of cardiac function has not been extensively analyzed in the field of myocardial tissue engineering. Inspired by microscopic analysis of the ventricular organiza-tion in native tissue, a scalable in-vitro model of nanoscale poly( L  -lactic acid)- co  -poly( -caprolactone)/collagen biocomposite scaffold is fabricated, with nanofibers in the order of 594 56 nm to mimic the native myocar-dial environment for freshly isolated cardiomyocytes from rabbit heart, and the specifically underlying extracellular matrix architecture: this is done to address the specificity of the underlying matrix in overcoming challenges faced by cellular therapeutics. Guided by nanoscale mechanical cues pro-vided by the underlying random nanofibrous scaffold, the tissue constructs display anisotropic rearrangement of cells, characteristic of the native cardiac tissue. Surprisingly, cell morphology, growth, and expression of an interactive healthy cardiac cell population are exquisitely sensitive to differences in the composition of nanoscale scaffolds. It is shown that suitable cell–material interactions on the nanoscale can stipulate organization on the tissue level and yield novel insights into cell therapeutic science, while providing mate-rials for tissue regeneration. DOI: 10.1002/adfm.201002434 S. Mukherjee , Dr. M. Raghunath Division of Bioengineering 9 Engineering Drive 1, Block EA #03-12 National University of Singapore, Singapore S. Mukherjee , Dr. J. R. Venugopal , S. Ramakrishna HEM laboratory Nanoscience and Nanotechnology Initiative c/o Faculty of Engineering Block E3-05-29, 2 Engineering Drive 3, National University of Singapore Singapore Email: R. Ravichandran , S. Ramakrishna Department of Mechanical Engineering 9 Engineering Drive 1, Block EA, 07-08 National University of Singapore, Singapore S. Ramakrishna Institute of Materials Research and Engineering A-Star, Singapore Dr. M. Raghunath Department of Biochemistry Medical Drive Block MD7, #02-03, Yong Loo Lin School of Medicine National University of Singapore, Singapore F  UL L P AP E R ©  2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  2291  Adv. Funct. Mater.   2011 , 21 , 2291–2300  conducted in a variety of technologies including cell therapy using various cell types, injection of biomaterials, bioengineered patches, implantation of 3D construct generated in static and bioreactor culture. [   5,6   ]  Cellular therapy, involving the use of live cells to repair damaged myocardium, has been extensively studied in recent years, opening new horizons in the clinical field. Cellular therapeutics essentially involve the direct delivery of suitable cell types, such as bone marrow cells (BMCs), mesen-chymal stem cells (MSCs), endothelial progenitor cells (EPCs), cardiac stem cells (CSCs), skeletal myoblast cells (SMCs), embryonic stem cells (ESCs), or induced pluripotent stem cells (iPS), to the damaged myocardial area. [   7   ]  Currently, more than 25 new clinical trials are in progress in the United States, and a similar number in Europe, using different cell types. [   8,9   ]  How-ever, recent experimental studies fail to answer many impor-tant aspects of cell therapy. Despite several advantages, such as safety and improved function, and positive results, such as increased healing, vascular density, increased regional circula-tion and cardiac function, the efficiency of delivery and retention is lower than expected, and the retention and survival of cells at sites of delivery has been limited. [   10   ]  Once the scarred area has evolved, remodeling and collagen deposition changes the histological microenvironment of infarcted areas. This changed microenvironment may allow peripherally or locally admin-istered stem cells to home and survive. In native tissue, cell growth and structural development is supported by an extracel-lular matrix (ECM) that consistently assists in coordinating the contractility and maintenance of cardiac shape and size, as well as the function of cardiomyocytes. [   11   ]  In addition to providing growth cues, the ECM responds actively to cellular actions. It also undergoes remodeling by cells during development, homeostasis and healing processes, which includes digestion of the old matrix and deposition of a fresh one. Essentially, the ECM is made up of 80% and 10% of collagen types I and III, respectively. [   12   ]  In the healthy heart, collagen serves to maintain normal cardiac architecture by surrounding and bridging myo-cytes, which consistently assist in coordinating the contractility and maintenance of cardiac shape and size as well as the func-tion of the cardiomyocytes. ECM proteins greatly influence bio-activities, such as cell migration, differentiation, proliferation, and angiogenesis. These effects are recognizable to cells only when the ECM is suitably modified. [   13   ]  It is indeed an immense technological challenge to mimic the entire milieu of ECM arti-ficially without evoking an immune response in such sensitive surrounding. From a tissue engineering perspective, applying physiological stress on immature constructs could be a way of mimicking the natural environment. [   14   ]  Many efforts by the scientific community in the field of myocardial tissue engi-neering (MTE) are dedicated to identify materials possessing specific mechanical properties that play a pivotal role. [   15   ]  First of all, it is desirable that the mechanical performance of bioen-gineered scaffolds matches as closely as possible those of the heart extracellular matrix in terms of stiffness, since the scaf-fold should be flexible enough to promote the contraction of the growing cells. [   16   ]  The stiffness of native heart tissue ranges from 10–20 kPa at early diastole and increases to 50 kPa at the end of diastole, which may shoot up to 200 kPa or more in inf-arcted hearts. [   17,18   ]  In addition, since the myocardial tissue issubjected to cyclical and constant deformation, materials are requested to show elastomeric properties and possibly long-term elasticity. However, it is likely that the structure and function of the in vivo cardiac tissues are regulated by much smaller, nanoscale cues provided by the ECM, which is responsible for extensive control over cell and tissue function. [   19   ]  It is therefore important to investigate the effects of finer control over the cell–biomaterial interface on the nanoscale, in facilitating the creation of truly biomimetic cardiac constructs that replicate the structural and functional aspects of the in vivo ventricular organization. In addition, the ability to robustly and reproducibly generate uniformly controlled (both structurally and functionally) and precisely defined engineered cardiac tissue will likely be neces-sary for eventual therapeutic products. This makes nanofabri-cation of biomaterials for MTE an attractive strategy. Ultrafine nanofibers having an ECMlike topography can be achieved by electrospinning of biomaterials. [   20   ]  Such scaffolds of suitable biomaterials with nanoscale architecture provide larger surface areas to adsorb proteins and provide more binding sites to cell membrane receptors, unlike microscale and flat surfaces. [   21   ]  At present, there are no successful models of bioengineered car-diac implant that can satisfactorily replicate the anatomy, physi-ology, and biological stability of a healthy heart wall. [   22   ]  To address this challenge, here we report the development and analysis of a nanotopographically controlled in-vitro model using nanoscale poly( L  -lactic acid)- co  -poly( ε    -caprolactone) (PLACL) and collagen-blend biopolymer scaffolds that mimic the native myocardial environment and, specifically, the under-lying ECM architecture. The result is a nanofibrous scaffold that is scalable, biocompatible and closely imitates the myocardial ECM, allowing healthy interaction with freshly isolated cardiac cells over a span of 15 days in controlled environment. We inves-tigated the mechanical properties of the nanofibrous PLACL/collagen scaffold, demonstrating that they promote higher organizational features than unblended PLACL counterparts. Moreover, strikingly, we found the proliferation of the cells, their morphological patterns and expression of cardiac specific markers such as alpha actinin, troponin T, connexin-43, and car-diac myosin heavy chain were highly sensitive to variation in biomaterial content as well as nanoscale topographic features, revealing an unexpected level of regulation in the organization. 2. Results and Discussion 2.1. Fabrication and Characterization of Electrospun Nanofibrous Scaffolds Nanofibers of PLACL and PLACL/collagen were fabricated by electrospinning, as reported by our group previously ( Scheme 1 ), and the fiber morphology examined under a scanning electron microscope (SEM). Electrospinning enables the design and fabrication of scalable scaffolding materials, mimicking the structural and mechanical cues of the ECM. Moreover, the pres-ence of purified ECM components provides sites for integrin attachment and hence excellent binding properties to scaffolds. Therefore, we aimed at fabrication of PLACL/collagen so that nanoscale definitions could be extended to tissue dimensions.   F  U  L  L  P  A  P  E  R ©  2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2292  Adv. Funct.   Mater.   2011 , 21 , 2291–2300  We fabricated nanofibers of PLACL/collagen and PLACL with precisely defined features, extending nanoscale control of the magnitude of fiber diameter. Figure 1 shows SEM micro-graphs of electrospun PLACL and PLACL/collagen nanofibrous Scheme 1 . Myocardial tissue engineering aims at improving the structure and function of the in vivo cardiac tissue are regulated by much smaller, nanoscale cues provided by the ECM, which is responsible for extensive control over cell and tissue function. Figure 1 . Fabrication of electrospun nanofibers. Nanofiber morphology shows porous, bead-less, uniform nanofibers of: a) PLACL with diameter measuring 332 ±  31 nm, and, b) PLACL/collagen measuring 594 ±  56 nm. scaffolds as porous, beadless, uniform nanofibers with inter-connected pores with fiber diameter in the range of 332 ±  31 nm and 594 ±  56 nm, respectively. Biomaterial scaffolds are expected to provide a compliant and highly hydrated environment, similar to soft tissues having high water content, thus facilitating diffusion of nutrients and cellular waste. Thus, tailoring the surface properties of the biomaterial in terms of hydrophilicity influences its biological performance. Hydrophilicity of our fabricated nanofiber scaffolds to relative of the surfaces was ana-lyzed using water contact angle analysis. To analyze the wettability of the nanofiber scaffolds, we measured the contact angle of water on the PLACL/collagen and PLACL using the sessile drop method ( Table 1 ). The PLACL/collagen nanofibers had a contact angle of 0 °  compared to that of 120 ±  5 °  of PLACL nanofibers. This observation high-lights the hypothesis that addition of an ECM component—collagen—imparts superior F  UL L P AP E R ©  2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  2293  Adv. Funct. Mater.   2011 , 21 , 2291–2300  hydrophilic properties to the PLACL, which is otherwise hydro-phobic. Our results indicate that PLACL/collagen is completely hydrophilic, unlike PLACL, and shows good tissue-engineering potential in terms of facilitating the culture of cells. Chemical characteristics and functional groups on the nanofibers were studied by FTIR analysis ( Figure 2 ). Figure 2 indicates uniform blending of collagen with PLACL to form PLACL/collagen nanofibers. The analysis showed bands of N–H and C–H stretches at 2987.08 and 2362.08 cm −  1  , respec-tively. These form the characteristic peaks for collagen and sug-gest the presence of amide groups on the surface of PLACL/collagen nanofibers. The amide I band of collagen for C =  O stretching, the measure of secondary structure of proteins, was prominent at 1648.96 cm −  1  in PLACL/collagen. The amide II peak for N–H bending coupled with C–N stretching was obtained at 1546.53 cm −  1  and the amide III region for N–H bending was obtained between 1200 and 1250 cm −  1  . The amide region corresponds to the triple helix structure of collagen, the integral part of ECM, and greatly favours cell adhesion and proliferation. The mechanical performance of electrospun PLACL/col-lagen and PLACL mats was analyzed by tensile stress–strain measurements. It is worth noting that stress–strain data for electrospun materials depend not only on single fiber features but, most of all, on fiber arrangement in the mat. On one hand, the degree of fiber orientation affects the mechanical properties, since the load is mainly applied to those fibers oriented parallel to the direction of the deformation and, on the other hand, the presence of ‘fiber-fusion’ at the contact sites remarkably increase the elastic modulus. [   23   ]  Moreover the fiber arrangement in the mat is expected to change during the stress–strain measure-ment. In particular, Lu et al. [   24   ]  demonstrated that fibers tend to align in the direction of applied force before getting thinner and finally breaking. In this work we compared the tensile stress–strain analysis of PLACL/collagen nanofibers with electrospun PLACL nanofibers mats of similar thickness. Figure 3 reports representative stress–strain curves of both PLACL/collagen and PLACL electrospun mats obtained at RT with an elastic mod-ulus of 10 ±  2.7 MPa and 18 ±  2.3 MPa, respectively. Low stiff-ness may be a suitable property with respect to integration with native tissue, since an extremely stiff matrix inhibits the growth properties of cardiomyocytes. [   25   ]  Table 1 lists the mechanical data of the two electrospun materials measured by carrying out stress-strain analysis both at RT. With the aim of observing the surface morphologies of PLACL/collagen and PLACL nanofibrous scaffolds, atomic-force microscopy (AFM) was employed using a height mode. From AFM images, it was observed that the surface of PLACL nanofibers was much smoother than the surface of PLACL/collagen blended nanofibers. Figure 4 shows 3D nanoscope images of PLACL and PLACL/collagen. We observed that PLACL is extremely rough, exhibiting a mean surface roughness (Ra) value of 700 nm and root mean square (Rms) value of 900 nm. However, PLACL/collagen exhibit Ra value of 380 nm and Rms value of 463 nm. 2.2. Growth Profile and Characterization of Cardiac Cell Cultures Exploring the myocardial cellular structure in detail, we performed excision of ex vivo rabbit heart. The heart tissue displayed a complex organization of cell sheets of varying thick-ness. The left ventricle myocardium was the thickest, comprising aligned myocytes, which provide a natural direction for exertion of con-tractile forces. We treated the tissues with col-lagenase to breakdown the highly organized structure and successfully obtained single, highly elongated cells with identifiable aniso-tropic arrays. The freshly isolated cardiac cells Figure 3 . Tensile properties of electrospun fibrous scaffolds: PLACL/col-lagen (A), and PLACL (B) were determined to be 10 ± 2.7 and 18 ±  2.3 MPa, respectively, using stress–strain curves. Figure 2 . FTIR analysis of: PLACL/collagen showing characteristics amide I and II peaks at 1651.60 and 1547.50 cm −  1  , respectively (A); and, PLACL (B). Table 1. Characterization of fabricated PLACL/collagen and PLACL nanofibers in terms of hydrophilicity, fiber diameter and porosity. The data indicate differences in properties as a result of blending of an ECM protein, collagen, with synthetic polymer PLACL. MaterialContact Angle [º]Fiber Diameter [nm]Porosity [%]Elastic Modulus [MPa]PLACL120 ±  5332 ±  318518 ±  2.3PLACL/Collagen 0 ±  7594 ±  569410 ±  2.7   F  U  L  L  P  A  P  E  R ©  2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2294  Adv. Funct.   Mater.   2011 , 21 , 2291–2300
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