Perfusion improves tissue architecture of engineered cardiac muscle

Cardiac muscle with a certain threshold thickness, uniformity of tissue architecture, and functionality would expand the therapeutic options currently available to patients with congenital or acquired cardiac defects. Cardiac constructs cultured in well-mixed medium had an approximately 100-microm-thick peripheral tissue-like region around a relatively cell-free interior, a structure consistent with the presence of concentration gradients within the tissue. We hypothesized that direct perfusion of cultured constructs can reduce diffusional distances for mass transport, improve control of oxygen, pH, nutrients and metabolites in the cell microenvironment, and thereby increase the thickness and spatial uniformity of engineered cardiac muscle. To test this hypothesis, constructs (9.5-mm-diameter, 2-mm-thick discs) based on neonatal rat cardiac myocytes and fibrous polyglycolic acid scaffolds were cultured either directly perfused with medium or in control spinner flasks. Perfusion improved the spatial uniformity of cell distribution and enhanced the expression of cardiac-specific markers, presumably due to the improved control of local microenvironmental conditions within the forming tissue. Medium perfusion could thus be utilized to better mimic the transport conditions within native cardiac muscle and enable in vitro engineering of cardiac constructs with clinically useful thicknesses.     

Tri-layered elastomeric scaffolds for engineering heart valve leaflets

Tissue engineered heart valves (TEHVs) that can grow and remodel have the potential to serve as permanent replacements of the current non-viable prosthetic valves particularly for pediatric patients. A major challenge in designing functional TEHVs is to mimic both structural and anisotropic mechanical characteristics of the native valve leaflets. To establish a more biomimetic model of TEHV, we fabricated tri-layered scaffolds by combining electrospinning and microfabrication techniques. These constructs were fabricated by assembling microfabricated poly(glycerol sebacate) (PGS) and fibrous PGS/poly(caprolactone) (PCL) electrospun sheets to develop elastic scaffolds with tunable anisotropic mechanical properties similar to the mechanical characteristics of the native heart valves. The engineered scaffolds supported the growth of valvular interstitial cells (VICs) and mesenchymal stem cells (MSCs) within the 3D structure and promoted the deposition of heart valve extracellular matrix (ECM). MSCs were also organized and aligned along the anisotropic axes of the engineered tri-layered scaffolds. In addition, the fabricated constructs opened and closed properly in an ex vivo model of porcine heart valve leaflet tissue replacement. The engineered tri-layered scaffolds have the potential for successful translation towards TEHV replacements.     

Macroporous elastomeric scaffolds with extensive micropores for soft tissue engineering

Macroporous scaffolds are of great value in tissue engineering. We have developed a method to fabricate macroporous scaffolds from a biocompatible and biodegradable elastomer, poly(glycerol sebacate) (PGS). This method is potentially very useful for soft tissue engineering. Our fabrication method produced macroporous scaffolds with extensive micropores. We fabricated flat scaffolds and tubular scaffolds of uniform thickness. This fabrication method demonstrated good control of variables such as pore size, porosity, and pore interconnectivity. Sodium chloride (salt) crystals, which served as solid porogens, were packed into a mold and fused in a humid chamber. PGS was cured while dispersed throughout the fused salt template. Dissolution of the salt and subsequent lyophilization produced elastomer sponges with approximately 90% porosity, interconnected macropores (75-150 microm), and extensive micropores (5-20 microm). The macropores were generated by the salt particles, while the micropores were likely generated by glycerol vapor formed during PGS curing. Such numerous micropores could facilitate cell-cell interactions and mass transport. Fibroblasts adhered to and proliferated well within the PGS scaffolds and formed three-dimensional tissue-engineered constructs within 8 days.     

Oxygen generating scaffolds for enhancing engineered tissue survival

One of the continued challenges in engineering clinically applicable tissues is the establishment of vascularization upon implantation in vivo. Although the effectiveness of an enhanced angiogenic response using various growth factors has been demonstrated in many tissue systems, the rate of angiogenesis could not be accelerated. In this study we investigated whether incorporating oxygen generating biomaterials into tissue engineered constructs would provide a sustained oxygen release over an extended period of time. We examined whether oxygen generating biomaterials are able to maintain cell viability while also maintaining structural integrity of a 3-D construct. Calcium peroxide-based oxygen generating particles were incorporated into 3-D scaffolds of Poly(d,l-lactide–co–glycolide) (PLGA). The scaffolds were designed to generate oxygen over the course of 10 days and simultaneously maintain sufficient mechanical integrity. Scaffolds containing oxygen generating materials maintained elevated levels of oxygen when incubated under hypoxic conditions. Further, these biomaterials were able to extend cell viability growth under hypoxic conditions. These findings indicate that the use of oxygen generating biomaterials may allow for increased cell survivability while neovascularization is being established after implantation. Such scaffolds may play an important role in tissue engineering where currently oxygen diffusion limits the engineering of large tissue implants.     

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.     

Bioresorbable elastomeric vascular tissue engineering scaffolds via melt spinning and electrospinning

Current surgical therapy for diseased vessels less than 6 mm in diameter involves bypass grafting with autologous arteries or veins. Although this surgical practice is common, it has significant limitations and complications, such as occlusion, intimal hyperplasia and compliance mismatch. As a result, cardiovascular biomaterials research has been motivated to develop tissue-engineered blood vessel substitutes. In this study, vascular tissue engineering scaffolds were fabricated using two different approaches, namely melt spinning and electrospinning. Small diameter tubes were fabricated from an elastomeric bioresorbable 50:50 poly(l-lactide-co-ε-caprolactone) copolymer having dimensions of 5 mm in diameter and porosity of over 75%. Scaffolds electrospun from two different solvents, acetone and 1,1,1,3,3,3-hexafluoro-2-propanol were compared in terms of their morphology, mechanical properties and cell viability. Overall, the mechanical properties of the prototype tubes exceeded the transverse tensile values of natural arteries of similar caliber. In addition to spinning the polymer separately into melt-spun and electrospun constructs, the approach in this study has successfully demonstrated that these two techniques can be combined to produce double-layered tubular scaffolds containing both melt-spun macrofibers (<200 μm in diameter) and electrospun submicron fibers (>400 nm in diameter). Since the vascular wall has a complex multilayered architecture and unique mechanical properties, there remain several significant challenges before a successful tissue-engineered artery is achieved.     

Tubular nanofiber scaffolds for tissue engineered small-diameter vascular grafts

Quick establishment of a confluent and stable endothelial cells (ECs) layer in the lumen of vascular grafts is critical for long-term patency of small-diameter vascular grafts. The objective of the study was to fabricate tubular nanofiber scaffolds, incorporate ECs onto the lumen of the scaffolds, and establish an animal model to prove the basic concept of using the scaffolds as vascular grafts. Poly(L-lactic acid)-co-poly(epsilon-caprolactone) P(LLA-CL 70:30) tubular nanofiber scaffolds were fabricated by electrospinning onto a rotating mandrel. Collagen was coated onto the scaffolds after air plasma treatment. Structure and mechanical property of the scaffolds were studied by scanning electron microscopy and tensile stress measurement, respectively. Human coronary artery endothelial cells (HCAECs) were rotationally seeded onto the lumen of the scaffolds at the speed of 6 rpm for 4 h through a customized seeding device, followed with static culture. Results showed evenly distributed and well-spread HCAECs throughout the lumen of the scaffold from 1 day onward to 10 days after seeding. Further, HCAECs maintained phenotypic expression of PECAM-1. To prove the basic concept of using the scaffolds as vascular grafts, acellular tubular P(LLA-CL) nanofiber scaffolds (inner diameter 1 mm) were implanted into rabbits to replace the inferior superficial epigastric veins. Results showed the scaffolds sustained the surgical process, kept the structure integrity, and showed the patency for 7 weeks.     

Materials and surface modification for tissue engineered vascular scaffolds

Although vascular implantation has been used as an effective treatment for cardiovascular disease for many years, off-the-shelf and regenerable vascular scaffolds are still not available. Tissue engineers have tested various materials and methods of surface modification in the attempt to develop a scaffold that is more suitable for implantation. Extracellular matrix-based natural materials and biodegradable polymers, which are the focus of this review, are considered to be suitable materials for production of tissue-engineered vascular grafts. Various methods of surface modification that have been developed will also be introduced, their impacts will be summarized and assessed, and challenges for further research will briefly be discussed.     

不同支架材料构建组织工程肌腱的性能评价

目的：总结修复肌腱损伤的主要组织工程支架材料及研究进展。 方法：由第一作者采用电子检索的方式,在CNKI数据库中检索1902-01/2010-10有关生物材料应用于组织工程肌腱支架的研究文章,关键词为“重建肌腱,生物材料,人工肌腱,组织工程,支架材料”。排除重复研究、普通综述或Meta分析类文章,筛选纳入18篇文献进行评价。 结果：来源于自然界的天然生物材料主要有蚕丝、小肠黏膜下层、胶原、衍生肌腱支架材料等,保留了组织正常的三维网架结构,组织相容性好,但力学性能较差、降解速度快。人工合成高分子材料主要为聚乳酸和聚羟基乙酸、聚乳酸-聚羟基乙酸共聚物、聚磷酸钙纤维等,但存在亲水性低、细胞黏附性能差的不足。 结论：天然及合成高分子材料作为组织工程支架材料都有各自的优缺点,绝大多数还处于研究阶段,尚未应用于临床,因此改进支架材料的性能是目前研究的主要方向之一。     

Ligament tissue engineering using synthetic biodegradable fiber scaffolds

Tissue engineering offers the possibility of replacing damaged human ligaments with engineered ligament tissues. Hence, we attempted to culture in vitro ligament tissues by seeding human anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells onto synthetic biodegradable polymer fiber scaffolds. The ACL and MCL cells readily attached to the scaffold fibers. These cells and their secreted matrix soon surrounded the scaffold fibers and bridged the gaps in between. Beginning at 2 weeks, portions of the scaffolds were completely filled with tissue matrix. By 5 weeks, the scaffolds became single bundles of tissue. Thus the cell/fiber system appears to be a viable system for culturing ligament tissues. Additionally, cell proliferation under mechanical and biochemical stimuli was studied for up to 4 days. Whereas mechanical stimulus and transforming growth factor enhanced proliferation, inflammatory agents (lipopolysaccharide and complement C5a) had a negative effect. This work can thus contribute to a sound strategy for culturing replacement ligament tissues in vitro.     

Mass transfer trends occurring in engineered ex vivo tissue scaffolds

In vivo the vasculature provides an effective delivery system for cellular nutrients; however, artificial scaffolds have no such mechanism, and the ensuing limitations in mass transfer result in limited regeneration. In these investigations, the regional mass transfer properties that occur through a model scaffold derived from the human umbilical vein (HUV) were assessed. Our aim was to define the heterogeneous behavior associated with these regional variations, and to establish if different decellularization technologies can modulate transport conditions to improve microenvironmental conditions that enhance cell integration. The effect of three decellularization methods [Triton X-100 (TX100), sodium dodecyl sulfate (SDS), and acetone/ethanol (ACE/EtOH)] on mass transfer, cellular migration, proliferation, and metabolic activity were assessed. Results show that regional variation in tissue structure and composition significantly affects both mass transfer and cell function. ACE/EtOH decellularization was shown to increase albumin mass flux through the intima and proximate-medial region (0-250 μm) when compared with sections decellularized with TX100 or SDS; although, mass flux remained constant over all regions of the full tissue thickness when using TX100. Scaffolds decellularized with TX100 were shown to promote cell migration up to 146% further relative to SDS decellularized samples. These results show that depending on scaffold derivation and expectations for cellular integration, specificities of the decellularization chemistry affect the scaffold molecular architecture resulting in variable effects on mass transfer and cellular response.     

Electrospun fine-textured scaffolds for heart tissue constructs

The structural and functional effects of fine-textured matrices with sub-micron features on the growth of cardiac myocytes were examined. Electrospinning was used to fabricate biodegradable non-woven poly(lactide)- and poly(glycolide)-based (PLGA) scaffolds for cardiac tissue engineering applications. Post-processing was applied to achieve macro-scale fiber orientation (anisotropy). In vitro studies confirmed a dose-response effect of the poly(glycolide) concentration on the degradation rate and the pH value changes. Different formulations were examined to assess scaffold effects on cell attachment, structure and function. Primary cardiomyocytes (CMs) were cultured on the electrospun scaffolds to form tissue-like constructs. Scanning electron microscopy (SEM) revealed that the fine fiber architecture of the non-woven matrix allowed the cardiomyocytes to make extensive use of provided external cues for isotropic or anisotropic growth, and to some extent to crawl inside and pull on fibers. Structural analysis by confocal microscopy indicated that cardiomyocytes had a preference for relatively hydrophobic surfaces. CMs on electrospun poly(L-lactide) (PLLA) scaffolds developed mature contractile machinery (sarcomeres). Functionality (excitability) of the engineered constructs was confirmed by optical imaging of electrical activity using voltage-sensitive dyes. We conclude that engineered cardiac tissue structure and function can be modulated by the chemistry and geometry of the provided nano- and micro-textured surfaces. Electrospinning is a versatile manufacturing technique for design of biomaterials with potentially reorganizable architecture for cell and tissue growth.     

Controlling the contractile strength of engineered cardiac muscle by hierarchal tissue architecture

The heart is a muscular organ with a wrapping, laminar structure embedded with neural and vascular networks, collagen fibrils, fibroblasts, and cardiac myocytes that facilitate contraction. We hypothesized that these non-muscle components may have functional benefit, serving as important structural alignment cues in inter- and intra-cellular organization of cardiac myocytes. Previous studies have demonstrated that alignment of engineered myocardium enhances calcium handling, but how this impacts actual force generation remains unclear. Quantitative assays are needed to determine the effect of alignment on contractile function and muscle physiology. To test this, micropatterned surfaces were used to build 2-dimensional myocardium from neonatal rat ventricular myocytes with distinct architectures: confluent isotropic (serving as the unaligned control), confluent anisotropic, and 20?μm spaced, parallel arrays of multicellular myocardial fibers. We combined image analysis of sarcomere orientation with muscular thin film contractile force assays in order to calculate the peak sarcomere-generated stress as a function of tissue architecture. Here we report that increasing peak systolic stress in engineered cardiac tissues corresponds with increasing sarcomere alignment. This change is larger than would be anticipated from enhanced calcium handling and increased uniaxial alignment alone. These results suggest that boundary conditions (heterogeneities) encoded in the extracellular space can regulate muscle tissue function, and that structural organization and cytoskeletal alignment are critically important for maximizing peak force generation.     

Microfabrication of three-dimensional engineered scaffolds

One of the principal challenges facing the field of tissue engineering over the past 2 decades has been the requirement for large-scale engineered constructs comprising precisely organized cellular microenvironments. For vital organ assist and replacement devices, microfluidic-based systems such as the microcirculation, biliary, or renal filtration and resorption systems and other functional elements containing multiple cell types must be generated to provide for viable engineered tissues and clinical benefit. Over the last several years, microfabrication technology has emerged as a versatile and powerful approach for generating precisely engineered scaffolds for engineered tissues. Fabrication process tools such as photolithography, etching, molding, and lamination have been established for applications involving a range of biocompatible and biodegradable polymeric scaffolding materials. Computational fluid dynamic designs have been used to generate scaffold designs suitable for microvasculature and a number of organ-specific constructs; these designs have been translated into 3-dimensional scaffolding using microfabrication processes. Here a brief overview of the fundamental microfabrication technologies used for tissue engineering will be presented, along with a summary of progress in a number of applications, including the liver and kidney.     

Optical mapping of impulse propagation in engineered cardiac tissue

Cardiac tissue engineering has a potential to provide functional, synchronously contractile tissue constructs for heart repair, and for studies of development and disease using in vivo-like yet controllable in vitro settings. In both cases, the utilization of bioreactors capable of providing biomimetic culture environments is instrumental for supporting cell differentiation and functional assembly. In the present study, neonatal rat heart cells were cultured on highly porous collagen scaffolds in bioreactors with electrical field stimulation. A hallmark of excitable tissues such as myocardium is the ability to propagate electrical impulses. We utilized the method of optical mapping to measure the electrical impulse propagation. The average conduction velocity recorded for the stimulated constructs (14.4 +/- 4.1 cm/s) was significantly higher than that of the nonstimulated constructs (8.6 +/- 2.3 cm/s, p = 0.003). The measured electrical propagation properties correlated to the contractile behavior and the compositions of tissue constructs. Electrical stimulation during culture significantly improved amplitude of contractions, tissue morphology, and connexin-43 expression compared to the nonsimulated controls. These data provide evidence that electrical stimulation during bioreactor cultivation can improve electrical signal propagation in engineered cardiac constructs.     

Stabilized collagen scaffolds for heart valve tissue engineering

Scaffolds for heart valve tissue engineering must function immediately after implantation but also need to tolerate cell infiltration and gradual remodeling. We hypothesized that moderately cross-linked collagen scaffolds would fulfill these requirements. To test our hypothesis, scaffolds prepared from decellularized porcine pericardium were treated with penta-galloyl glucose (PGG), a collagen-binding polyphenol, and tested for biodegradation, biaxial mechanical properties, and in vivo biocompatibility. For controls, we used un-cross-linked scaffolds and glutaraldehyde-treated scaffolds. Results confirmed complete pericardium decellularization and the ability of scaffolds to encourage fibroblast chemotaxis and to aid in creation of anatomically correct valve-shaped constructs. Glutaraldehyde cross-linking fully stabilized collagen but did not allow for tissue remodeling and calcified when implanted subdermally in rats. PGG-treated collagen was initially resistant to collagenase and then degraded gradually, indicating partial stabilization. Moreover, PGG-treated pericardium exhibited excellent biaxial mechanical properties, did not calcify in vivo, and supported infiltration by host fibroblasts and subsequent matrix remodeling. In conclusion, PGG-treated acellular pericardium is a promising scaffold for heart valve tissue engineering.     

Review: mineralization of synthetic polymer scaffolds for bone tissue engineering

It has repeatedly been shown that demineralization improves the ability of bone auto- and allografts to regenerate natural bone tissue. Conversely, much work in the field of bone tissue engineering has used composite materials consisting of a mineralized phase or materials designed to mineralize rapidly in situ. In this review, we seek to examine these disparate roles of mineralization and the underlying factors that cause this discordance and to examine methods and principles of the mineralization of synthetic polymer scaffolds. Biomimetic approaches to mineralization and phosphorus-containing materials are highlighted, and a brief section focusing on drug-delivery strategies using mineralized scaffolds is included.     

Textile-templated electrospun anisotropic scaffolds for regenerative cardiac tissue engineering

For patients with end-stage heart disease, the access to heart transplantation is limited due to the shortage of donor organs and to the potential for rejection of the donated organ. Therefore, current studies focus on bioengineering approaches for creating biomimetic cardiac patches that will assist in restoring cardiac function, by repairing and/or regenerating the intrinsically anisotropic myocardium. In this paper we present a simplified, straightforward approach for creating bioactive anisotropic cardiac patches, based on a combination of bioengineering and textile-manufacturing techniques in concert with nano-biotechnology based tissue-engineering stratagems. Using knitted conventional textiles, made of cotton or polyester yarns as template targets, we successfully electrospun anisotropic three-dimensional scaffolds from poly(lactic-co-glycolic) acid (PLGA), and thermoplastic polycarbonate-urethane (PCU, Bionate^®). The surface topography and mechanical properties of textile-templated anisotropic scaffolds significantly differed from those of scaffolds electrospun from the same materials onto conventional 2-D flat-target electrospun scaffolds. Anisotropic textile-templated scaffolds electrospun from both PLGA and PCU, supported the adhesion and proliferation of H9C2 cardiac myoblasts cell line, and guided the cardiac tissue-like anisotropic organization of these cells in vitro. All cell-seeded PCU scaffolds exhibited mechanical properties comparable to those of a human heart, but only the cells on the polyester-templated scaffolds exhibited prolonged spontaneous synchronous contractility on the entire engineered construct for 10 days in vitro at a near physiologic frequency of ∼120 bpm. Taken together, the methods described here take advantage of straightforward established textile manufacturing strategies as an efficient and cost-effective approach to engineering 3D anisotropic, elastomeric PCU scaffolds that can serve as a cardiac patch.     

Homologous structure-function relationships between native fibrocartilage and tissue engineered from MSC-seeded nanofibrous scaffolds

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of tissues to replace diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials.     

Effects of regulatory factors on engineered cardiac tissue in vitro

We tested the hypothesis that supplemental regulatory factors can improve the contractile properties and viability of cardiac tissue constructs cultured in vitro. Neonatal rat heart cells were cultured on porous collagen sponges for up to 8 days in basal medium or medium supplemented with insulin-like growth factor-I (IGF), insulin-transferrin-selenium (ITS), platelet-derived growth factor-BB (PDGF), or angiopoietin-1 (ANG). IGF and ITS enhanced contractile properties of the 8-day constructs significantly more than with unsupplemented controls according to contractile amplitude and excitation threshold, and IGF also significantly increased the amount of cardiac troponin-I and enhanced cell viability according to different assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and terminal deoxynucleotidyl transferase biotin-2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)). PDGF significantly increased the contractile amplitude of 4-day constructs and enhanced cell viability according to MTT, LDH, and TUNEL; ANG enhanced cell viability according to the LDH assay. Our results demonstrate that supplemental regulatory molecules can differentially enhance properties of cardiac tissue constructs and imply that these constructs can provide a platform for systematic in vitro studies of the effects of complex stimuli that occur in vivo to improve our basic understanding of cardiogenesis and identify underlying mechanisms that can potentially be exploited to enhance myocardial regeneration.