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Cyclic strain generated at the cell-material interface is critical for the engraftment of biomaterials. Mechanosensitive immune cells, macrophages regulate the host-material interaction immediately after implantation by priming the environment and remodeling ongoing regenerative processes. This study investigated the ability of mechanically active scaffolds to modulate macrophage function

Cancer is driven by both genetic aberrations in the tumor cells and fundamental changes in the tumor microenvironment (TME). These changes offer potential targets for novel therapeutics, yet lack of in vitro 3D models recapitulating this complex microenvironment impedes such progress. Here, we generated several tumor-stroma scaffolds reflecting the dynamic in vivo breast TME, using a high throughput microfluidic system. Alginate (Alg) or alginate-alginate sulfate (Alg/Alg-S) hydrogels were used as ECM-mimics, enabling the encapsulation and culture of tumor cells, fibroblasts and immune cells (macrophages and T cells, of the innate and adaptive immune systems, respectively). Specifically, Alg/Alg-S was shown capable of capturing and presenting growth factors and cytokines with binding affinity that is comparable to heparin. Viability and cytotoxicity were shown to strongly correlate with the dynamics of cellular milieu, as well as hydrogel type. Using on-chip immunofluorescence, production of reactive oxygen species and apoptosis were imaged and quantitatively analyzed. We then show how macrophages in our microfluidic system were shifted from a proinflammatory to an immunosuppressive phenotype when encapsulated in Alg/Alg-S, reflecting in vivo TME dynamics. LC-MS proteomic profiling of tumor cells sorted from the TME scaffolds revealed upregulation of proteins involved in cell-cell interactions and immunomodulation in Alg/Alg-S scaffolds, correlating with in vivo findings and demonstrating the appropriateness of Alg/Alg-S as an ECM biomimetic. Finally, we show the formation of large tumor-derived vesicles, formed exclusively in Alg/Alg-S scaffolds. Altogether, our system offers a robust platform for quantitative description of the breast TME that successfully recapitulates in vivo patterns. Statement of significance Cancer progression is driven by profound changes in both tumor cells and surrounding stroma. Here, we present a high throughput microfluidic system for the generation and analysis of dynamic tumor-stroma scaffolds, that mimic the complex in vivo TME cell proportions and compositions, constructing robust in vitro models for the study of the TME. Utilizing Alg/Alg-S as a bioinspired ECM, mimicking heparin's in vivo capabilities of capturing and presenting signaling molecules, we show how Alg/Alg-S induces complex in vivo-like responses in our models. Alg/Alg-S is shown here to promote dynamic protein expression patterns, that can serve as potential therapeutic targets for breast cancer treatment. Formation of large tumor-derived vesicles, observed exclusively in the Alg/Alg-S scaffolds suggests a mechanism for tumor survival.

Self-assembled nanocomplexes composed of individual molecules that spontaneously connect via noncovalent interactions have recently emerged as versatile alternatives to conventional controlled drug delivery systems because of their unique bioinspired properties (responsiveness, dynamics, etc.). Characterization of such nanocomplexes typically includes their size distribution, surface charge, morphology, drug entrapment efficiency, and verification of the coexistence of labeled components within the nanocomplexes using a colocalization study. Less common is the direct examination of the molecular interactions between the different components in the coassembled nanocomplex, especially in nanocomplexes composed of hygroscopic components, because convenient methods are still lacking. Here, we present a detailed experimental protocol for determining the surface composition and the chemical bonds by X-ray photoelectron spectroscopy (XPS) after drying the deposit hygroscopic sample overnight under UHV. We applied this method to investigate the surface chemistry of binary Ca

Replication of physiological oxygen levels is fundamental for modeling human physiology and pathology in in vitro models. Environmental oxygen levels, applied in most in vitro models, poorly imitate the oxygen conditions cells experience in vivo, where oxygen levels average ∼5%. Most solid tumors exhibit regions of hypoxic levels, promoting tumor progression and resistance to therapy. Though this phenomenon offers a specific target for cancer therapy, appropriate in vitro platforms are still lacking. Microfluidic models offer advanced spatio-temporal control of physico-chemical parameters. However, most of the systems described to date control a single oxygen level per chip, thus offering limited experimental throughput. Here, we developed a multi-layer microfluidic device coupling the high throughput generation of 3D tumor spheroids with a linear gradient of five oxygen levels, thus enabling multiple conditions and hundreds of replicates on a single chip. We showed how the applied oxygen gradient affects the generation of reactive oxygen species (ROS) and the cytotoxicity of Doxorubicin and Tirapazamine in breast tumor spheroids. Our results aligned with previous reports of increased ROS production under hypoxia and provide new insights on drug cytotoxicity levels that are closer to previously reported in vivo findings, demonstrating the predictive potential of our system.

Background: Despite intensive research, regeneration of articular cartilage largely remains an unresolved medical concern as the clinically available modalities still suffer from long-term inconsistent data, relatively high failure rates and high prices of more promising approaches, such as cell therapy. In the present study, we aimed to evaluate the feasibility and long-term efficacy of a bilayered injectable acellular affinity-binding alginate hydrogel in a large animal model of osteochondral defects. Methods: The affinity-binding alginate hydrogel is designed for presentation and slow release of chondrogenic and osteogenic inducers (transforming growth factor-β1 and bone morphogenic protein 4, respectively) in two distinct and separate hydrogel layers. The hydrogel was injected into the osteochondral defects created in the femoral medial condyle in mini-pigs, and various outcomes were evaluated after 6 months. Results: Macroscopical and histological assessment of the defects treated with growth factor affinity-bound hydrogel showed effective reconstruction of articular cartilage layer, with major features of hyaline tissue, such as a glossy surface and cellular organisation, associated with marked deposition of proteoglycans and type II collagen. Microcomputed tomography showed incomplete bone formation in both treatment groups, which was nevertheless augmented by the presence of affinity-bound growth factors. Importantly, the physical nature of the applied hydrogel ensured its shear resistance, seamless integration and topographical matching to the surroundings and opposing articulating surface. Conclusions: The treatment with acellular injectable growth factor–loaded affinity-binding alginate hydrogel resulted in effective tissue restoration with major hallmarks of hyaline cartilage, shown in large animal model after 6-month follow-up. The translational potential of this article: This proof-of-concept study in a clinically relevant large animal model showed promising potential of an injectable acellular growth factor–loaded affinity-binding alginate hydrogel for effective repair and regeneration of articular hyaline cartilage, representing a strong candidate for future clinical development. Keywords: Affinity-binding alginate, Bone morphogenic protein 4, Hyaline cartilage, Osteochondral defect, Transforming growth factor-β1

Osteochondral defects (OCDs) are conditions affecting both cartilage and the underlying bone. Since cartilage is not spontaneously regenerated, our group has recently developed a strategy of injecting bioactive alginate hydrogel into the defect for promoting endogenous regeneration of cartilage via presentation of affinity-bound transforming growth factor β1 (TGF-β1). As in vivo model systems often provide only limited insights as for the mechanism behind regeneration processes, here we describe a novel flow bioreactor for the in vitro modeling of the OCD microenvironment, designed to promote cell recruitment from the simulated bone marrow compartment into the hydrogel, under physiological flow conditions. Computational fluid dynamics modeling confirmed that the bioreactor operates in a relevant slow-flowing regime. Using a chemotaxis assay, it was shown that TGF-β1 does not affect human mesenchymal stem cell (hMSC) chemotaxis in 2D culture. Accessible through live imaging, the bioreactor enabled monitoring and discrimination between erosion rates and profiles of different alginate hydrogel compositions, using green fluorescent protein-expressing cells. Mathematical modeling of the erosion front progress kinetics predicted the erosion rate in the bioreactor up to 7 days postoperation. Using quantitative real-time polymerase chain reaction of early chondrogenic markers, the onset of chondrogenic differentiation in hMSCs was detected after 7 days in the bioreactor. In conclusion, the designed bioreactor presents multiple attributes, making it an optimal device for mechanistical studies, serving as an investigational tool for the screening of other biomaterial-based, tissue engineering strategies.

Heart failure (HF) after myocardial infarction (MI) due to blockage of coronary arteries is a major public health issue. MI results in massive loss of cardiac muscle due to ischemia. Unfortunately, the adult mammalian myocardium presents a low regenerative potential, leading to two main responses to injury: fibrotic scar formation and hypertrophic remodeling. To date, complete heart transplantation remains the only clinical option to restore heart function. In the last two decades, tissue engineering has emerged as a promising approach to promote cardiac regeneration. Tissue engineering aims to target processes associated with MI, including cardiomyogenesis, modulation of extracellular matrix (ECM) remodeling, and fibrosis. Tissue engineering dogmas suggest the utilization and combination of two key components: bioactive molecules and biomaterials. This chapter will present current therapeutic applications of biomaterials in cardiac regeneration and the challenges still faced ahead. The following biomaterial-based approaches will be discussed: Nano-carriers for cardiac regeneration-inducing biomolecules; corresponding matrices for their controlled release; injectable hydrogels for cell delivery and cardiac patches. The concept of combining cardiac patches with controlled release matrices will be introduced, presenting a promising strategy to promote endogenous cardiac regeneration.

Alginate biomaterial is widely utilized for tissue engineering and regeneration due to its biocompatibility, non-thrombogenic nature, mild and physical gelation process, and the resemblance of its hydrogel matrix texture and stiffness to that of the extracellular matrix. In this review, we describe the versatile biomedical applications of alginate, from its use as a supporting cardiac implant in patients after acute myocardial infarction (MI) to its employment as a vehicle for stem cell delivery and for the controlled delivery and presentation of multiple combinations of bioactive molecules and regenerative factors into the heart. Preclinical and first-in-man clinical trials are described in details, showing the therapeutic potential of injectable acellular alginate implants to inhibit the damaging processes after MI, leading to myocardial repair and tissue reconstruction.

Cardiovascular tissue engineering (CTE) aims to provide regenerative solutions in the creation of contractile and vascularized tissues capable of replacing damaged or dysfunctional myocardium. Additionally, CTE aims to offer cell-based strategies to regenerate injured arteries and heal vascular lesions, mitigating the complications of an obstructive coronary vascular disease, the key factor leading to an acute myocardial infarction and heart failure. Magnetic-responsive biomaterials in the form of scaffolds or nanoparticles offer unique opportunities to create stimulatory conditions or means for efficient cell delivery, as a part of multi-component microenvironment promoting tissue development and regeneration in vitro and in vivo. This chapter will discuss strategies and principles that leverage the unique properties of magnetic materials, enabling the creation of functional and vascularized tissue constructs, generating directionally-guided tissue structures, and forming physical means for efficient delivery, homing and engraftment of cell-based therapies in cardiovascular applications.

The therapeutic application of autologous cardiac-derived progenitor cells (CPCs) requires a large cell quantity generated under defined conditions. Herein, we investigated the applicability of a three-dimensional (3D) perfusion cultivation system to facilitate the expansion of CPCs harvested from human heart biopsies and characterized by a relatively high percentage of c-kit(+) cells. The cells were seeded in macroporous alginate scaffolds and after cultivation for 7 days under static conditions, some of the constructs were transferred into a perfusion bioreactor, which was operated for an additional 14 days. A robust and highly reproducible human CPC (hCPC) expansion of more than seven-fold was achieved under the 3D perfusion culture conditions, while under static conditions, the expansion of CPCs was limited only to the first 7 days, after which it leveled-off. On day 21 of perfusion cultivation, the expanded cells exhibited a higher expression level of the progenitor marker c-kit, suggesting that the c-kit-positive CPCs are the main cell population undergoing proliferation. The profile of the spontaneous differentiation in the perfused construct was different from that in the static cultivated constructs; genes typical for cardiac and endothelial cell lineages were more widely expressed in the perfused constructs. By contrast, the differentiation to osteogenic (Von Kossa staining and alkaline phosphatase activity) and adipogenic (Oil Red staining) lineages was reduced in the perfused constructs compared with static cultivated constructs. Collectively, our results indicate that 3D perfusion cultivation mode is an appropriate system for robust expansion of human CPCs while maintaining their progenitor state and differentiation potential into the cardiovascular cell lineages.

Extended partial hepatectomy may be needed in cases of large hepatic mass, and can lead to fulminant hepatic failure. Macroporous alginate scaffold is a biocompatible matrix which promotes the growth, differentiation and long-term hepatocellular function of primary hepatocytes in vitro. Our aim was to explore the ability of implanted macroporous alginate scaffolds to protect liver remnants from acute hepatic failure after extended partial hepatectomy. An 87% partial hepatectomy (PH) was performed on C57BL/6 mice to compare non-treated mice to mice in which alginate or collagen scaffolds were implanted after PH. Mice were scarified 3, 6, 24 and 48 h and 6 days following scaffold implantation and the extent of liver injury and repair was examined. Alginate scaffolds significantly increased animal survival to 60% vs. 10% in non-treated and collagen-treated mice (log rank = 0.001). Mice with implanted alginate scaffolds manifested normal and prolonged aspartate aminotransferases and alanine aminotransferases serum levels as compared with the 2- to 20-fold increase in control groups (P < 0.0001) accompanied with improved liver histology. Sustained normal serum albumin levels were observed in alginate-scaffold-treated mice 48 h after hepatectomy. Incorporation of BrdU-positive cells was 30% higher in the alginate-scaffold-treated group, compared with non-treated mice. Serum IL-6 levels were significantly decreased 3 h post PH. Biotin-alginate scaffolds were quickly well integrated within the liver tissue. Collectively, implanted alginate scaffolds support liver remnants after extended partial hepatectomy, thus eliminating liver injury and leading to enhanced animal survival after extended partial hepatectomy.

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate–sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate–sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10–14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ. Copyright © 2013 John Wiley & Sons, Ltd.

Enhancing vascularization of cell-transplantation devices is necessary for maintaining cell viability and integration within the host, but it also increases the risk of allograft rejection. Here, we investigated the feasibility of generating an immunoregulatory environment in a highly vascularized macroporous alginate scaffold by affinity-binding of the transforming growth factor-β (TGF-β) in a manner mimicking its binding to heparan sulfate. Using this device to transplant allofibroblasts under the kidney capsule resulted in the induction of local and peripheral TGF-β-dependent immunotolerance, characterized by higher frequency of immature dendritic cells and regulatory T cells within the device and by markedly reduced allofibroblast-specific T-cell response in the spleen, thereby increasing the viability of the transplanted cells. Culturing whole splenocytes in the TGF-β-bound scaffold indicated that the regulatory function of TGF-β is IL-10-dependent. We thus demonstrate a novel platform for transplantation devices, designed to promote an immunoregulatory microenvironment suitable for cell transplantation and autoimmune regulation. Statement of Significance Allogeneic cell graft transplantation is a potentially optimal treatment for many clinical deficiencies. It is yet challenging to overcome chronic rejection without compromising host immunity to pathogens. We present the features and function of a cell transplantation device designed based on the principle of affinity binding of angiogenic and immunoregulatory factors to extracellular matrix in aim to achieve sustained release of these factors. We show that presentation of these factors in such manner generates the infrastructure for device vascularization and induces profound local allocell-specific tolerance, which then evokes peripheral T-cell tolerance. The tolerance is antigen specific, does not cause immune deficits and may thus serve to improve allocell survival as well as a platform to mitigate pathogenic autoimmunity.

A noninvasive, effective approach for immediate and painless heart pacing would have invaluable implications in several clinical scenarios. Here we present a novel strategy that utilizes the well-known mechano-electric feedback of the heart to evoke cardiac pacing, while relying on magnetic microparticles as leadless mechanical stimulators. We demonstrate that after localizing intravenously-injected magnetic microparticles in the right ventricular cavity using an external electromagnet, the application of magnetic pulses generates mechanical stimulation that provokes ventricular overdrive pacing in the rat heart. This temporary pacing consistently managed to revert drug-induced bradycardia, but could only last up to several seconds in the rat model, most likely due to escape of the particles between the applied pulses using our current experimental setting. In a pig model with open chest, MEF-based pacing was induced by banging magnetic particles and has lasted for a longer time. Due to overheating of the electromagnet, we intentionally terminated the experiments after 2 min. Our results demonstrate for the first time the feasibility of external leadless temporary pacing, using injectable magnetic microparticles that are manipulated by an external electromagnet. This new approach can have important utilities in clinical settings in which immediate and painless control of cardiac rhythm is required.

Cardiac tissue engineering presents a challenge due to the complexity of the muscle tissue and the need for multiple signals to induce tissue regeneration in vitro. We investigated the effects of compression (1 Hz, 15% strain) combined with fluid shear stress (10(-2) -10(-1) dynes/cm(2) ) provided by medium perfusion on the outcome of cardiac tissue engineering. Neonatal rat cardiac cells were seeded in Arginine-Glycine-Aspartate (RGD)-attached alginate scaffolds, and the constructs were cultivated in a compression bioreactor. A daily, short-term (30 min) compression (i.e., "intermittent compression") for 4 days induced the formation of cardiac tissue with typical striation, while in the continuously compressed constructs (i.e., "continuous compression"), the cells remained spherical. By Western blot, on day 4 the expression of the gap junction protein connexin 43 was significantly greater in the "intermittent compression" constructs and the cardiomyocyte markers (α-actinin and N-cadherin) showed a trend of better preservation compared to the noncompressed constructs. This regime of compression had no effect on the proliferation of nonmyocyte cells, which maintained low expression level of proliferating cell nuclear antigen. Elevated secretion levels of basic fibroblast growth factor and transforming growth factor-β in the daily, intermittently compressed constructs likely attributed to tissue formation. Our study thus establishes the formation of an improved cardiac tissue in vitro, when induced by combined mechanical signals of compression and fluid shear stress provided by perfusion.

Tissue engineering holds the promise of providing new solutions for heart transplant shortages and pediatric heart transplantation. The aim of this study was to evaluate the ability of a peritoneal-generated, tissue-engineered cardiac patch to replace damaged myocardium in a heterotopic heart transplant model. Fetal cardiac cells (1 × 106/scaffold) from syngeneic Lewis rats were seeded into highly porous alginate scaffolds. The cell constructs were cultured in vitro for 4 days and then they were implanted into the rat peritoneal cavity for 1 week. During this time the peritoneal-implanted patches were vascularized and populated with myofibroblasts. They were harvested and their performance in an infrarenal heterotopic abdominal heart transplantation model was examined ( n = 15). After transplantation and before reperfusion of the donor heart, a 5-mm left ( n = 6) or right ( n = 9) ventriculotomy was performed and the patch was sutured onto the donor heart to repair the defect. Echocardiographical studies carried out 1–2 weeks after transplantation showed normal LV function in seven of the eight hearts studied. After 1 month, visual examination of the grafted patch revealed no aneurysmal dilatation. Microscopic examination revealed, in most of the cardiac patches, a complete disappearance of the scaffold and its replacement by a consistent tissue composed of myofibroblasts embedded in collagen bundles. The cardiac patch was enriched with a relatively large number of infiltrating blood vessels. In conclusion, cardiac patches generated in the peritoneum were developed into consistent tissue patches with properties to seal and correct myocardial defects. Our study also offers a viable rat model for screening and evaluating new concepts in cardiac reconstruction and engineering.

We explored the possibility of entrapping retroviral vector producing cells (VPC) within porous 3D matrix to induce a local and sustained release of viral particles to the malignant milieu. PA317/STK, which constantly shed reroviral vectors, was used to transduce cancer cells with the herpes simplex virus thymidine kinase (HSV-tk) gene. Once HSV-tk is expressed, it preferentially phosphorylates nucleoside analog prodrugs, such as ganciclovir (GCV) and N-methanocarbathymidine (N-MCT), to their active triphosphate metabolites, which when incorporated into cellular DNA cause cell death. PA317/STK cells were seeded within 3D alginate scaffold at two different cell densities via static seeding procedure. In vitro assays determined that PA317/STK seeded at high-cell density in scaffolds maintained constant cell number, low cell leakage, and spheroid morphology with viral vector transfection activity. Postcell-seeding viral vector activity was confirmed by transfection of murine colon cancer cells (MC38) with conditioned media originated from VPC-containing scaffolds and the subsequent ability to generate N-MCT triphosphate. Preliminary in vivo transplantation of VPC-containing scaffolds into the peritoneal cavity of mice bearing intraperitoneal MC38 tumors with 2 weeks subsequent GCV administration resulted in a significantly higher survival rate relative to control groups. Our results demonstrate the feasibility of employing alginate scaffolds to efficiently entrap and support PA317/STK cells for cancer gene therapy. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007

Tissue engineering entails the in vitro or in vivo generation of replace-ment tissues from cells with the aid of supporting scaffolds and stimulatingbiomolecules, in order to provide biological substitutes for restoration and mainte-nance of human tissue functions. In this review, we summarize the main classesof degradable polymeric scaffolds, natural and synthetic ones, and the evolutionmade in this field from adaptation of materials in clinical use to the fabrication of“designer” scaffolds. TISSUE ENGINEERING—BASIC PRINCIPLES Tissue engineering is an interdisciplinary field thatapplies the principles of engineering, materials, and lifesciences to the development of biological substitutes forrestoration and maintenance of human tissue functions. 1 This new and emerging technology entails the in vitro orin vivo generation of biological tissue from individualcells with the aid of supporting scaffolds and stimulat-ing biomolecules. The supporting scaffold temporarilyreplaces the function of the natural extracellular matrix(ECM), thus enabling the seeded individual cells toregenerate cell–cell and cell–matrix interactions, lead-ing to the formation of a functioning tissue. As potentialreplacements of ECM, implanted scaffolds can alsofunction as in vivo guided-regenerative matrices by in-ducing remaining healthy patient’s cells to populatethem.A large portion of the research in this field has beendevoted to designing the ideal scaffold that would bestmimic the native extracellular matrix. In this review, wedescribe the main classes of degradable polymeric scaf-folds that have been developed since the “birth” oftissue engineering until today. Our goal is to describethe evolution of this field, from adaptation of materialsin clinical use to the fabrication of “designer” scaffolds.

Hepatocyte transplantation within porous scaffolds (HT) is being explored as a treatment strategy for end-stage liver diseases and enzyme deficiencies. One of the main issues in this approach is the limited viability of transplanted cells because vascularization of the scaffold site is either too slow or insufficient. We now address this by enhancing scaffold vascularization before cell transplantation via sustained delivery of vascular endothelial growth factor (VEGF), and by examining the liver lobes as a platform for transplanting donor hepatocytes in close proximity to the host liver. The vascularization kinetics of unseeded VEGF-releasing scaffolds on rat liver lobes were evaluated by analyzing the microvascular density and tissue ingrowth in implants harvested on days 3, 7, and 14 postimplantation. Capillary density was greater at all times in VEGF-releasing scaffolds than in the control scaffold without VEGF supplementation; on day 14, it was 220 +/- 33 versus 139 +/- 23 capillaries/mm2 (p < 0.05). Furthermore, 35% of the newly formed capillaries in VEGF-releasing scaffolds were larger than 16 microm in diameter, whereas in control scaffolds only 10% exceeded this size. VEGF had no effect on tissue ingrowth into the scaffolds. HT onto the implanted VEGF-releasing or control scaffolds was performed after 1 week of prevascularization on the liver lobe in Lewis rats. Fifty implants were harvested on days 1, 3, 7, and 12 and the area of viable hepatocytes was evaluated. The enhanced vascularization improved hepatocyte engraftment; 12 days after HT, the intact hepatocyte area (136,910 microm2/cross-section) in VEGF-releasing scaffolds was 4.6 higher than in the control group. This study shows that sustained local delivery of VEGF induced vascularization of porous scaffolds implanted on liver lobes and improved hepatocyte engraftment.

Tissue engineering with three-dimensional biomaterials represents a promising approach for developing hepatic tissue to replace the function of a failing liver. Herein, we address cell seeding and distribution within porous alginate scaffolds, which represent a new type of porous biomaterial for tissue engineering. The hydrophilic nature of the alginate scaffold as well as its pore structure and interconnectivity enabled the efficient seeding of hepatocytes into the scaffolds, that is, 70-90% of the initial cells depending on the seeding method. Utilization of centrifugal force during seeding enhanced cell distribution in the porous scaffolds, consequently enabling the seeding of concentrated cell suspensions (>1 x 10(7) cells/mL). Cell density in scaffolds affected hepatocyte viability as judged by MTT assay. At a cell density of 0.28 x 10(6) cells/cm3 scaffold, the number of viable hepatocytes decreased to 33% of its initial value within 7 days, whereas at the denser cultures, 5.7 x 10(6) cells/cm3 scaffold and higher, the cells maintained higher viability while forming a network of connecting spheroids. In the high-density cellular constructs, hepatocellular functions such as albumin and urea secretion, and detoxification (cytochrome P-450 and phase II conjugating enzyme activities), remained high during the 7-day culture. Collectively, the results of the present study highlight the importance of cell density on the hepatocellular functions of three-dimensional hepatocyte constructs as well as the advantages of alginate matrices as scaffoldings.

Site-specific delivery of angiogenic growth factors from tissue-engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would ensure cell survival and function. In the present article, we describe the construction of a novel porous alginate scaffold that incorporates tiny poly (lactic-co-glycolic acid) microspheres capable of controlling the release of angiogenic factors, such as basic fibroblast growth factor (bFGF). The microspheres are an integral part of the solid alginate matrix, and their incorporation does not affect the scaffold porosity or pore size. In vitro, bFGF was released from the porous composite scaffolds in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of cardiac fibroblasts. The controlled delivery of bFGF from the three-dimensional scaffolds accelerated the matrix vascularization after implantation on the mesenteric membrane in rat peritoneum. The number of penetrating capillaries into the bFGF-releasing scaffolds was nearly fourfold higher than into the control scaffolds (those incorporating microspheric BSA and heparin but not bFGF). At day 10 posttransplantation, capillary density in the composite scaffolds was 45 +/- 3/mm(2) and it increased to 70 +/- 7/mm(2) by day 21. The released bFGF induced the formation of large and matured blood vessels, as judged by the massive layer of mural cells surrounding the endothelial cells. The control over bFGF delivery and localizing its effects to areas of need, may aid in the wider application of bFGF in therapeutic angiogenesis as well as in tissue engineering.

Cardiac tissue engineering has emerged as a promising approach to replace or support an infarcted cardiac tissue and thus may hold a great potential to treat and save the lives of patients with heart diseases. By its broad definition, tissue engineering involves the construction of tissue equivalents from donor cells seeded within 3-D biomaterials, then culturing and implanting the cell-seeded scaffolds to induce and direct the growth of new, healthy tissue. In this review, we present an up-to-date summary of the research in cardiac tissue engineering, with an emphasis on the design principles and selection criteria that have been used in two key technologies employed in tissue engineering, (1) biomaterials technology, for the creation of 3-D porous scaffolds which are used to support and guide the tissue formation from dissociated cells, and (2) bioreactor cultivation of the 3-D cell constructs during ex-vivo tissue engineering, which aims to duplicate the normal stresses and flows experienced by the tissues.

Cardiac tissue engineering has evolved as a potential therapeutic approach to assist in cardiac regeneration. We have recently shown that tissue-engineered cardiac graft, constructed from cardiomyocytes seeded within an alginate scaffold, is capable of preventing the deterioration in cardiac function after myocardial infarction in rats. The present article addresses cell seeding within porous alginate scaffolds in an attempt to achieve 3D high-density cardiac constructs with a uniform cell distribution. Due to the hydrophilic nature of the alginate scaffold, its >90% porosity and interconnected pore structure, cell seeding onto the scaffold was efficient and short, up to 30 min. Application of a moderate centrifugal force during cell seeding resulted in a uniform cell distribution throughout the alginate scaffolds, consequently enabling the loading of a large number of cells onto the 3D scaffolds. The percent cell yield in the alginate scaffolds ranged between 60-90%, depending on cell density at seeding; it was 90% at seeding densities of up to 1 x 10(8) cells/cm(3) scaffold and decreased to 60% at higher densities. The highly dense cardiac constructs maintained high metabolic activity in culture. Scanning electron microscopy revealed that the cells aggregated within the scaffold pores. Some of the aggregates were contracting spontaneously within the matrix pores. Throughout the culture there was no indication of cardiomyocyte proliferation within the scaffolds, nor was it found in 3D cultures of cardiofibroblasts. This may enable the development of cardiac cocultures, without domination of cardiofibroblasts with time.

Among the greatest hurdles hindering the successful implementation of tissue-engineered cardiac patch as a therapeutic strategy for myocardial repair is the know-how to promote its rapid integration into the host. We previously demonstrated that prevascularization of the engineered cardiac patch improves cardiac repair after myocardial infarction (MI); the mature vessel networks were generated by including affinity-bound angiogenic factors in the patch and its transplantation on the blood vessel-enriched omentum. Here, we describe a novel in vitro strategy to promote the formation of capillary-like networks in cell constructs without supplementing with angiogenic factors. Endothelial cells (ECs) were seeded into macroporous alginate scaffolds impregnated with magnetically responsive nanoparticles (MNPs), and after pre-culture for 24 h under standard conditions the constructs were subjected to an alternating magnetic field of 40 Hz for 7 days. The magnetic stimulation per se promoted EC organization into capillary-like structures with no supplementation of angiogenic factors; in the non-stimulated constructs, the cells formed sheets or aggregates. This chapter describes in detail the preparation method of the MNP-impregnated alginate scaffold, the cultivation setup for the cell construct under magnetic field conditions, and the set of analyses performed to characterize the resultant cell constructs.

Tissue regeneration after myocardial infarction (MI) represents a major challenge in cardiovascular therapy, as current clinical approaches are limited in their ability to regenerate damaged myocardium. This chapter presents an overview of two emerging strategies based on the use of biomaterials, as stand-alone therapy or in combination with regeneration signals and cells, for regenerating the infarcted heart. One strategy is cardiac tissue engineering, which creates cardiac patches from functional cells seeded in a biomaterial scaffold, bio-inspired to provide the appropriate interface for cellular interactions. Implementation of perfusion bioreactors and pre-vascularization strategies can nowadays produce thicker cardiac patches that are better integrated into the host, thus advancing the realization of this strategy in the clinics. The second strategy, injection of biomaterials, has shown great promise as a stand-alone therapy. The intracoronary delivery of alginate solution that undergoes gelation only at the infarct in the presence of elevated calcium ions, was shown to increase scar thickness and LV dimensions in acute MI models in rats and pigs and was proven safe in phase I/II clinical studies. To enable the spatio-temporal presentation of regeneration factors, the alginate was modified with sulfate groups to mimic the binding of heparin-binding proteins to heparin/heparan-sulfate. When combining alginate-sulfate with the in-situ formed alginate hydrogel, multiple factor delivery was prolonged in ischemic tissues and enabled regeneration and cardiac repair after MI. The chapter emphasizes the increasing important role of biomaterials in various therapeutic strategies aimed at cardiac regeneration.

This paper describes the preparation and characterization of a three-dimensional, porous sponge made from the marine polysaccharide alginate for creating a cell-matrix transplant to replace damaged organs or tissues. The sponge is prepared by a three-step procedure: first gelation of the alginate with bivalent cations, followed by freezing of the hydrogel and finally lyophilization to produce a porous sponge. The pattern and the extent of sponge porosity, as well as its mechanical properties, were influenced by the concentration and the type of alginate (guluronic to mannuronic ratio and viscosity), the type and concentration of the cross-linkers and the freezing regime. By controlling these variables, macroporous sponges (pore size of 70-300 microns) that are suitable for cell culture and neovascularization were achieved. Fibroblasts seeded within the sponges preferred the pores, where they maintained a spherical shape. The alginate sponges conserved their initial volume for at least 3 months. It appears that alginate sponges may provide an excellent support for cell transplantation.

The design of focal spatiotemporal delivery systems for multiple regeneration-inducing factors represents a crucial step in the development of effective therapies for osteochondral injuries, osteoarthritis, and other pathologies of cartilage and bone. While endogenous bone regeneration is an established process, cartilage has very limited intrinsic regeneration ability. Thus, cartilage defects progressively affect subchondral bone and alter osteochondral interface homeostasis, leading to pain and disability. Spatiotemporal delivery of osteo- and chondroinductive factors by biomaterial-based systems represents an attractive therapeutic strategy for the currently available clinical therapies that still cannot provide a superior functional replacement for the damaged or lost tissue. This chapter offers an up-to-date review of the acellular biomaterial-based strategies, aimed at simultaneous regeneration of bone and cartilage by the controlled focal delivery of the appropriate factors. It describes the various factors and delivery systems (microspheres, hydrogels, and macroporous scaffolds) developed and tested in animals and presents a novel biomaterial approach, developed by our group, for the affinity binding of TGF-β1 and BMP-4 in two separate layers, which promoted the regeneration of osteochondral interface in rabbits with osteochondral defects.

Cardiac tissue engineering (CTE) aims to create contractile tissues to replace damaged or missing myocardial tissues. Biomaterials/scaffolds constitute a major component in various strategies of CTE, as stand-alone treatments or in combination with cells and/or bioactive molecules. This chapter describes the recent applications of nanomaterials and nanofabrication methods in CTE scaffolds to mimic the structural and architectural properties of the cardiac extracellular matrix (ECM) and to enable controllable and efficient single cell stimulation. Emphasis will be given to the implementation of magnetic field stimulation in CTE, attained by impregnating nano-magnetites into the CTE scaffold. Nanotechnology has already led to engineered cardiac tissues better suited for implantation.

Magnetic-based systems utilizing superparamagnetic nanoparticles and a magnetic field gradient to exert a force on these particles have been used in a wide range of biomedical applications. This review is focused on drug targeting applications that require penetration of a cellular barrier as well as strategies to improve the efficacy of targeting in these biomedical applications. Another focus of this review is regenerative applications utilizing tissue engineered scaffolds prepared with the aid of magnetic particles, the use of remote actuation for release of bioactive molecules and magneto–mechanical cell stimulation, cell seeding and cell patterning.

Tissue engineering research is increasingly relying on the use of advanced cultivation technologies that provide rigorously-controlled cell microenvironments. Herein, we describe the features of a micro-fabricated Multi-Shear Perfusion Bioreactor (MSPB) designed to deliver up to six different levels of physiologically-relevant shear stresses (1-13 dyne cm(-2)) to six cell constructs simultaneously, during a single run. To attain a homogeneous fluid flow within each construct, flow-distributing nets photo-etched with a set of openings for fluid flow were placed up- and down-stream from each construct. Human umbilical vein endothelial cells (HUVECs) seeded in alginate scaffolds within the MSPB and subjected to three different levels of shear stress for 24 h, responded accordingly by expressing three different levels of the membranal marker Intercellular Adhesion Molecule 1 (ICAM-1) and the phosphorylated endothelial nitric oxide synthetase (eNOS). A longer period of cultivation, 17 d, under two different levels of shear stress resulted in different lengths of cell sprouts within the constructs. Collectively, the HUVEC behaviour within the different constructs confirms the feasibility of using the MSPB system for simultaneously imposing different shear stress levels, and for validating the flow regime in the bioreactor vessel as assessed by the computational fluid dynamic (CFD) model.

Human hepatocyte transplantation has not been routinely established as an alternative to liver transplantation in liver disease due to low cell engraftment rates. Preimplantation in vitro engineering of liver tissue using primary human hepatocytes on three-dimensional scaffolds could be an alternative model. Alginate bioscaffolds were seeded with 1×10(6) hepatocytes freshly isolated from the livers of three children suffering from different metabolic disorders. During a culture period of 14 days only a marginal loss of hepatocytes was observed via measurement of DNA content per scaffold. Formation of hepatocyte spheroids was detected from day 3 onward using transmission light microscopy. Biochemical assays for albumin, α1-antitrypsin, and urea revealed excellent metabolic function with its maximum at day 7. Low lactate dehydrogenase enzyme release demonstrated minor cellular membrane damage. Hematoxylin and eosin and periodic acid Schiff staining displayed high cell viability and well-preserved glycogen storage until day 7. Immunofluorescent staining of hepatocyte nuclear factor 4, zonula occludens protein 1, and cytokeratin 18 revealed highly differentiated hepatocytes in spheroids with a tissue-like structure on scaffolds. Fluorescent labeling of cytochrome P450 and bile canaliculi demonstrated detoxification ability as well as a well-shaped bile canaliculi network. Almost constant expression levels in most target genes were detected by quantitative real-time polymerase chain reaction. The results of TUNEL reaction implicated a safe scaffold-dissolving procedure. Our results indicate that alginate scaffolds provide a favorable microenvironment for liver neo-tissue recreation and regeneration. Further, we demonstrate that livers from children with inherited metabolic disorders could serve as an alternative cell source for in vitro experiments.

One of the major challenges in engineering thick, complex tissues such as cardiac muscle, is the need to pre-vascularize the engineered tissue in vitro to enable its efficient integration with host tissue upon implantation. Herein, we explored new magnetic alginate composite scaffolds to provide means of physical stimulation to cells. Magnetite-impregnated alginate scaffolds seeded with aortic endothelial cells stimulated during the first 7 days out of a total 14 day experimental course showed significantly elevated metabolic activity during the stimulation period. Expression of proliferating cell nuclear antigen (PCNA) indicated that magnetically stimulated cells had a lower proliferation index as compared to the non-stimulated cells. This suggests that the elevated metabolic activity could instead be related to cell migration and re-organization. Immunostaining and confocal microscopy analyses supported this observation showing that on day 14 in magnetically stimulated scaffolds without supplementation of any growth factors, cellular vessel-like (loop) structures, known as indicators of vasculogenesis and angiogenesis were formed as compared to cell sheets or aggregates observed in the non-stimulated (control) scaffolds. This work is the first step in our understanding of how to accurately control cellular organization to form tissue engineered constructs, which together with additional molecular signals could lead to a creation of an efficient pre-vascularized tissue construct with potential applicability for transplantation.

Herein we describe a bio-inspired, affinity binding alginate-sulfate scaffold, designed for the presentation and sustained release of transforming growth factor beta 1 (TGF-β1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-β1 loading was significantly greater and its initial release from the scaffold was attenuated compared to its burst release (>90%) from scaffolds lacking alginate-sulfate. The sustained TGF-β1 release was further supported by the prolonged activation (14 d) of Smad-dependent (Smad2) and Smad-independent (ERK1/2) signaling pathways in the seeded hMSCs. Such presentation of TGF-β1 led to hMSC chondrogenic differentiation; differentiated chondrocytes with deposited collagen type II were seen within three weeks of in vitro hMSC seeding. By contrast, in scaffolds lacking alginate-sulfate, the effect of TGF-β1 was short-term and hMSCs could not reach a similar differentiation degree. When hMSC constructs were subcutaneously implanted in nude mice, chondrocytes with deposited type II collagen and aggrecan typical of the articular cartilage were found in the TGF-β1 affinity-bound constructs. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation.

Tissue regeneration following myocardial infarction (MI) represents a major challenge in cardiovascular therapy, as current clinical approaches are limited in their ability to regenerate or replace damaged myocardium. The lack of clinically-relevant cell sources, and the growing importance of paracrine effects of cell therapy, mediated by soluble growth factors and cytokines, favors the use of acellular biomaterials for myocardial tissue engineering. While the efficacy of acellular scaffold-based approaches have already been shown, applying the biomaterial in an injectable form represents a more clinically-appealing strategy, where only minimally invasive interventions are required to deliver the biopolymer solution. However, in order to enhance the passive effects mediated by the injected biomaterial on infarct stabilization and mechanical support, and achieve long-term functional improvement and regeneration of the cardiac muscle, the combination with controlled spatio-temporal delivery of bioactive molecules is required. Biomaterial-based growth factor delivery has already been shown to improve therapeutic outcome after MI. Affinity-binding alginate represents an example of such a system. This strategy has promising potential for myocardial repair and regeneration, as it provides mechanical support conferred by in situ hydrogel formation, and can affect multiple processes of myocardial regeneration by controlled delivery of multiple proteins. In conclusion, as the development of novel polymer schemes and approaches continues, the application of biomaterials that can instruct a favorable tissue reconstruction, facilitate self-repair, tissue salvage and regeneration, represents a platform for future modifications and combinations (for instance, with cell therapy). Hopefully, such efforts will have major clinical consequences on the treatment of MI and improve long-term outcome in heart failure patients.

We describe herein the features of a novel cultivation system, combining electrical stimulation with medium perfusion for producing thick, functional cardiac patches. A custom-made electrical stimulator was integrated via inserting two carbon rod electrodes into a perfusion bioreactor, housing multiple neonatal Sprague-Dawley rat cardiac cell constructs between two 96% open-pore-area fixing nets. The stimulator produced adjustable stimulation waveform (i.e., duty cycle, number of stimulating channels, maximum stimulation amplitude, etc.), specially designed for cardiac cell stimulation. The cell constructs were subjected to a homogenous fluid flow regime and electrical stimulation under conditions optimal for cell excitation. The stimulation threshold in the bioreactor was set by first determining its value in a Petri dish under a microscope, and then matching the current density in the two cultivation systems by constructing electric field models. The models were built by Comsol Multiphysics software using the exact three-dimensional geometry of the two cultivation systems. These models illustrate, for the first time, the local electric conditions required for cardiomyocyte field excitation and they confirmed the uniformity of the electrical field around the cell constructs. Bioreactor cultivation for only 4 days under perfusion and continuous electrical stimulus (74.4 mA/cm², 2 ms, bipolar, 1 Hz) promoted cell elongation and striation in the cell constructs and enhanced the expression level of Connexin-43, the gap junction protein responsible for cell-cell coupling. These results thus confirm the validity of the electrical field model in predicting the optimal electrical stimulation in a rather complex cultivation system, a perfusion bioreactor.

Cardiac tissue engineering aims to regenerate damaged myocardial tissues by applying heart patches created in vitro. The present study was undertaken to explore the possible role of matrix-attached RGD peptide in the engineering of cardiac tissue within macroporous scaffolds. Neonatal rat cardiac cells were seeded into RGD-immobilized or unmodified alginate scaffolds. The immobilized RGD peptide promoted cell adherence to the matrix, prevented cell apoptosis and accelerated cardiac tissue regeneration. Within 6 days, the cardiomyocytes reorganized their myofibrils and reconstructed myofibers composed of multiple cardiomyocytes in a typical myofiber bundle. The nonmyocyte cell population, mainly cardiofibroblasts, benefited greatly from adhering to the RGD–alginate matrix and consequently supported the cardiomyocytes. They often surrounded bundles of cardiac myofibers in a manner similar to that of native cardiac tissue. The benefits of culturing the cardiac cells in RGD-immobilized alginate scaffolds were further substantiated by Western blotting, revealing that the relative expression levels of α-actinin, N-cadherin and connexin-43 were better maintained in cells cultured within these scaffolds. Collectively, the immobilization of RGD peptide into macroporous alginate scaffolds proved to be a key parameter in cardiac tissue engineering, contributing to the formation of functional cardiac muscle tissue and to a better preservation of the regenerated tissue in culture.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are promising cell candidates for cartilage regeneration. Building the appropriate microenvironment for cell differentiation in response to exogenous stimuli is a critical step towards the clinical utilization of hMSCs. In this study, the effects of RGD peptide immobilization onto macro-porous alginate scaffolds on TGF-beta1-induced hMSC chondrogenesis were evaluated. The results revealed different cell morphology, viability and proliferation extent in the RGD-immobilized vs. un-modified scaffolds. The TGF-beta1-induced activation of both Smad-dependent (SMAD2) and Smad-independent (ERK1/2) signaling pathways was stronger and persisted for over 3 weeks in the RGD-immobilized scaffolds, indicating greater accessibility of the cells to the inducer. By contrast, in the un-modified alginate scaffolds, the cells aggregated into compacted clusters resulting in lesser effects of TGF-beta1. The efficient and prolonged exposure to the chondrogenic inducer in the RGD-modified scaffolds ensured the appropriate progression of MSC differentiation from the initial phase of cell condensation until the appearance of committed chondrocytes, at 3 weeks of cultivation. Taken together, our results highlight the fundamental importance of the microenvironment design of the scaffold as well as the presentation of the inductive cue for inducing efficient stem-cell controlled differentiation.

Enhancing tissue self-repair through the use of active acellular biomaterials is one of the main goals of regenerative medicine. We now describe the features of an injectable alginate biomaterial designed to affinity-bind heparin-binding proteins and release them at a rate reflected by their association constant to alginate-sulfate. The interactions of hepatocyte growth factor (HGF) with alginate-sulfate resulted in factor protection from proteolysis, as shown by mass spectroscopy analysis after trypsin digestion. When the HGF/alginate-sulfate bioconjugate was incorporated into alginate hydrogel, HGF release was sustained by a factor of 3, as compared to the release rate from non-modified hydrogel. The released factor retained activity, as shown by its induction of ERK1/2 activation and affording cytoprotection in rat neonatal cardiomyocyte cultures. In vivo, an injectable form of the affinity-binding alginate system extended by 10-fold, as compared to a saline-treated group, retention of HGF in myocardial tissue when delivered immediately after myocardial infarction. In a severe murine hindlimb ischemia model, HGF delivery from the affinity-binding system improved tissue blood perfusion and induced mature blood vessel network formation. The therapeutic efficacy of the affinity-binding system, as well as its ease of delivery by injection, provides a proof-of-concept for the potential use of this bioactive biomaterial strategy in cardiovascular repair.

Tissue engineering is an approach used to create a functional cardiac patch for the purpose of scar support after a myocardial infarct (MI). Cardiac cells, or cells of other sources, are seeded into scaffolds, which provide an artificial biomechanical support until the cells secrete extracellular matrix and regenerate into a functional tissue. In this chapter we describe the creative design of various cell microenvironments, which promote the development of a thick vascularized cardiac patch, ready to face the harsh conditions of the infarcted heart. Among these microenvironments are unique bioreactor systems that increase mass transfer through the developing cardiac tissue at the in vitro engineering stage and the use of various vascularization techniques, including the use of the body as a bioreactor to induce rapid vascularization prior to transplantation on the infarcted heart.

We report here what we believe is the first example of the encapsulation of hybridoma cells within a synthetic polymer by a simple gelation with dissolved cations in water, and at room temperature. Two lines of hybridoma cells were encapsulated within calcium cross-linked polyphosphazene gel microbeads without affecting their viability or their capability to produce antibodies. Interaction of these gel beads with the positively-charged polyelectrolyte, poly(L-lysine), of 102-kD molecular weight, produced a semipermeable membrane that was capable of retaining the cell-secreted antibodies inside the beads. Cell density increased 3.5-fold within 13 days concomitant with a 6.4-fold increase in antibody production. These synthetic membranes have the potential to aid in protein recovery schemes.

Developing advanced technologies for encouraging the ex vivo assembly of functional hepatic tissue for implantation into the human body or for in vitro drug testing is one of the challenging tasks facing tissue engineers. In the present study, we utilized a perfusion bioreactor system equipped with a novel flow-distributing mesh for online cell seeding into macroporous alginate scaffolds and cultivation of multiple constructs of the C3A human hepatocyte cell line. Optimization of the medium flow rate (100 mL/min) and perfusion duration (12 h) yielded cell constructs with high cell seeding efficiency (98% of the input cells) and cell distribution throughout the entire scaffold. Further, we show that interstitial medium flow enabled uniform cell delivery into 35 constructs lined across the bioreactor cross section. Perfusion-cultivated cell constructs revealed much greater rates of cell proliferation, albumin-specific secretion, and gene expression of the phase I enzyme, CYP3A4, and phase II enzyme, UGT2B7, than did static-cultivated constructs. Most impressive was the 50-fold increase in CYP3A4 expression of the perfused cell constructs as compared to the level in static-cultivated cell constructs. We thus believe that the hepatic tissue constructs developed herein may be used in drug discovery programs for elucidating drug metabolism and toxicity profiles and for treating failing livers.

Deciphering the cellular signals leading to cardiac muscle assembly is a major challenge in ex vivo tissue regeneration. For the first time, we demonstrate that pulsatile interstitial fluid flow in three-dimensional neonatal cardiac cell constructs can activate ERK1/2 sixfold, as compared to static-cultivated constructs. Activation of ERK1/2 was attained under physiological shear stress conditions, without activating the p38 cell death signal above its basic level. Activation of the ERK1/2 signaling cascade induced synthesis of high levels of contractile and cell-cell contact proteins by the cardiomyocytes, while its inhibition diminished the inducing effects of pulsatile flow. The pulsed medium-induced cardiac cell constructs showed improved cellularity and viability, while the regenerated cardiac tissue demonstrated some ultra-structural features of the adult myocardium. The cardiomyocytes were elongated and aligned into myofibers with defined Z-lines and multiple high-ordered sarcomeres. Numerous intercalated disks were positioned between adjacent cardiomyocytes, and deposits of collagen fibers surrounded the myofibrils. The regenerated cardiac tissue exhibited high density of connexin 43, a major protein involved in electrical cellular connections. Our research thus demonstrates that by judiciously applying fluid shear stress, cell signaling cascades can be augmented with subsequent profound effects on cardiac tissue regeneration.

Unlike heart valves or blood vessels, heart muscle has no replacement alternatives. The most challenging goal in the field of cardiovascular tissue engineering is the creation/ regeneration of an engineered heart muscle. Recent advances in methods of stem cell isolation, culture in bioreactors, and the synthesis of bioactive materials promise to create engineered cardiac tissue ex vivo. At the same time, new approaches are conceived that explore ways to induce tissue regeneration after injury. The purpose of our review is to describe the principles, status, and challenges of myocardial tissue engineering with emphasize on the concept of in situ cardiac tissue engineering and regeneration.

Cardiac tissue engineering promises to revolutionize the treatment of patients with end-stage heart failure and provide new solutions to the serious problem of heart donor shortage. By its broad definition, tissue engineering involves the construction of tissue equivalents from donor cells seeded within three-dimensional polymeric scaffolds, then culturing and implanting of the cell-seeded scaffolds to induce and direct the growth of new, healthy tissue. Here, we present an up-to-date summary of research studies in cardiac tissue engineering, with an emphasis on the critical design principles.

Although the field of tissue engineering has advanced significantly in the past decade, the inability to easily produce structured scaffolds continues to hinder its progress. We have proposed a method to create a porous scaffold utilizing directional freezing that is fast, reproducible and can be easily mass produced. Most importantly, this method creates long parallel channels within the scaffold. This should allow cells in the scaffold to grow more easily, and may aid scientists in predicting diffusion rates of nutrients and drugs throughout the scaffold. A cross-linked alginate gel was utilized in a directional freezing apparatus which incorporated a mold based on the horizontal Bridgeman design. The apparatus is designed to allow crystallization to occur in only one direction. The gel was frozen from 0°C to −40°C at a cooling rate of −18.3°C/minute. The samples were then freeze dried (leaving pores where ice dendrites had been), sectioned and viewed under a scanning electron microscope (SEM). Visual inspection revealed clear directionality present within the scaffolds. SEM photos also showed evenly spaced pores on the order of 100 μm present. A lesser magnification photo showed that the pores extended to become parallel channels producing a structured mesh that resembled an air filter. The directional freezing method is successful when used to create porous tissue engineering scaffolds, especially those with a low amount of tortuosity. By altering the cooling rate, it may be possible to create different pore distributions, thereby producing a method which can be utilized to create directional tissue engineering scaffolds quickly and effectively.Copyright © 2003 by ASME

Pre-vascularization is important for the reconstruction of dense and metabolically active myocardial tissue and its integration with the host myocardium after implantation. Herein, we demonstrate that the fabrication of micro-channels in alginate scaffold combined with the presentation of adhesion peptides and an angiogenic growth factor promote vessel-like networks in the construct, both in vitro and in vivo. Using a CO2 laser engraving system, 200 µm diameter channels were formed from top to bottom of the 2 mm thick alginate scaffold, with a channel-to-channel distance of 400 µm. Cells were seeded in a sequential manner onto the scaffolds: first, human umbilical vascular endothelial cells (HUVECs) were seeded and cultured for three days, then neonatal rat cardiomyocytes (CMs) and cardiofibroblasts were added at a final cell ratio of 50:35:15, respectively, and the constructs were cultivated for an additional seven days. A vessel-like network was formed within the cell constructs, wherein HUVECs were organized around the channels in a multilayer manner, while the CMs were located in-between the channels and exhibited the characteristic morphological features of a mature cardiac fiber. Acellular scaffolds with the affinity-bound basic fibroblast growth factor were implanted subcutaneously in mice. Increased cell penetration into the channeled scaffold and greater vessel density were found in comparison with the nonchanneled scaffolds. Our results thus point to the importance of micro-channels as a major structural promoter of vascularization in scaffolds, in conjunction with the sequential preculture of ECs and angiogenic factor presentation.

Cardiac tissue engineering offers new possibilities for the functional and structural restoration of damaged or lost heart tissue by applying cardiac patches created in vitro. Engineering such functional cardiac patches is a complex mission, involving material design on the nano- and microscale as well as the application of biological cues and stimulation patterns to promote cell survival and organization into a functional cardiac tissue. Herein, we present a novel strategy for creating a functional cardiac patch by combining the use of a macroporous alginate scaffold impregnated with magnetically responsive nanoparticles (MNPs) and the application of external magnetic stimulation. Neonatal rat cardiac cells seeded within the magnetically responsive scaffolds and stimulated by an alternating magnetic field of 5 Hz developed into matured myocardial tissue characterized by anisotropically organized striated cardiac fibers, which preserved its features for longer times than non-stimulated constructs. A greater activation of AKT phosphorylation in cardiac cell constructs after applying a short-term (20 min) external magnetic field indicated the efficacy of magnetic stimulation to actuate at a distance and provided a possible mechanism for its action. Our results point to a synergistic effect of magnetic field stimulation together with nanoparticulate features of the scaffold surface as providing the regenerating environment for cardiac cells driving their organization into functionally mature tissue.

Two approaches for cell transplantation and new tissue constructions are discussed. In one case, a novel synthetic polyphosphazene has been synthesized that can be gelled by simply adding ions to it at room temperature under aqueous conditions. This polymer has been shown to be compatible for several different cell types. Microcapsular membranes based on the complex of this polymer with poly( l -lysine) allow the inward diffusion of nutrients to nourish the encapsulated cells, but are impermeable to antibodies. In a second approach, biodegradable polyesters have been designed as scaffolds for liver cells and cartilage cells to aid in organ regeneration. Design of the polymer scaffold including the characterization of the surface chemistries for cell attachment, as well as in-vitro and in-vivo data on cell behavior are presented.