Fiber alignment and coculture with fibroblasts improves the differentiated phenotype of murine embryonic stem cell-derived cardiomyocytes for cardiac tissue engineering

Embryonic stem cells (ESCs) are an important source of cardiomyocytes for regenerating injured myocardium. The successful use of ESC-derived cardiomyocytes in cardiac tissue engineering requires an understanding of the important scaffold properties and culture conditions to promote cell attachment, differentiation, organization, and contractile function. The goal of this work was to investigate how scaffold architecture and coculture with fibroblasts influences the differentiated phenotype of murine ESC-derived cardiomyocytes (mESCDCs). Electrospinning was used to process an elastomeric biodegradable polyurethane (PU) into aligned or unaligned fibrous scaffolds. Bioreactor produced mESCDCs were seeded onto the PU scaffolds either on their own or after pre-seeding the scaffolds with mouse embryonic fibroblasts (MEFs). Viable mESCDCs attached to the PU scaffolds and were functionally contractile in all conditions tested. Importantly, the aligned scaffolds led to the anisotropic organization of rod-shaped cells, improved sarcomere organization, and increased mESCDC aspect ratio (length-to-diameter ratio) when compared to cells on the unaligned scaffolds. In addition, pre-seeding the scaffolds with MEFs improved mESCDC sarcomere formation compared to mESCDCs cultured alone. These results suggest that both fiber alignment and pre-treatment of scaffolds with fibroblasts improve the differentiation of mESCDCs and are important parameters for developing engineered myocardial tissue constructs using ESC-derived cardiac cells.     

A novel composite scaffold for cardiac tissue engineering

Summary One approach to the engineering of functional cardiac tissue for basic studies and potential clinical use involves bioreactor cultivation of dissociated cells on a biomaterial scaffold. Our objective was to develop a scaffold that is (1) highly porous with large intereconnected pores (to facilitate mass transport), (2) hydrophilic (to enhance cell attachment), (3) structurally stable (to withstand the shearing forces during bioreactor cultivation), (4) degradable (to provide ultimate biocompatibility of the tissue graft), and (5) elastic (to enable transmission of contractile forces). The scaffold of choice was made as a composite of poly(Dl-lactide-co-caprolactone), poly(Dl-lactide-co-glycolide) (PLGA), and type I collagen, with open interconnected pores and the average void volume of 80±5%. Neonatal rat heart cells suspended in Matrigel were seeded into the scaffold at a physiologically high density (1.35×10⁸ cells/cm³) and cultivated for 8 d in cartridges perfused with culture medium or in orbitally mixed dishes (25 rpm); collagen sponge (Ultrafoam^⋆m) and PLGA sponge served as controls. Construct cellularity, presence of cardiac markers, and contractile properties were markedly improved in composite scaffolds as compared with both controls.     

Biomimetic approach to cardiac tissue engineering

Here, we review an approach to tissue engineering of functional myocardium that is biomimetic in nature, as it involves the use of culture systems designed to recapitulate some aspects of the actual in vivo environment. To mimic the capillary network, subpopulations of neonatal rat heart cells were cultured on a highly porous elastomer scaffold with a parallel array of channels perfused with culture medium. To mimic oxygen supply by haemoglobin, the culture medium was supplemented with a perfluorocarbon (PFC) emulsion. Constructs cultivated in the presence of PFC contained higher amounts of DNA and cardiac markers and had significantly better contractile properties than control constructs cultured without PFC. To induce synchronous contractions of cultured constructs, electrical signals mimicking those in native heart were applied. Over only 8 days of cultivation, electrical stimulation induced cell alignment and coupling, markedly increased the amplitude of synchronous construct contractions and resulted in a remarkable level of ultrastructural organization. The biomimetic approach is discussed in the overall context of cardiac tissue engineering, and the possibility to engineer functional human cardiac grafts based on human stem cells.     

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.     

Injectable cardiac tissue engineering for the treatment of myocardial infarction

Heart disease is a leading cause of morbidity and mortality worldwide. Myocardial infarction leads to permanent loss of cardiac tissue and ultimately heart failure. However, current therapies could only stall the progression of the disease. Thus, new therapies are needed to regenerate damaged hearts to overcome poor prognosis of patients with heart failure. The shortage of heart donors is also a factor for innovating new therapies. Although the cardiac performance by cell-based therapy has improved, unsatisfactory cell retention and transplant survival still plague this technique. Because biomaterials can improve the cell retention, survival and differentiation, cardiac tissue engineering is now being explored as an approach to support cell-based therapies and enhance their efficacy for cardiac disease. In the last decade, cardiac tissue engineering has made considerable progress. Among different kinds of approaches in the cardiac tissue engineering, the approach of injectable cardiac tissue engineering is more minimally invasive than that of in vitro engineered tissue or epicardial patch implantation. It is therefore clinically appealing. In this review, we strive to describe the major progress in the flied of injectable cardiac tissue engineering, including seeding cell sources, biomaterials and novel findings in preclinical studies and clinical applications. The remaining problems will also be discussed.     

Polyethylenimine: A new differentiation factor to endothelial/cardiac tissue

Among different tissues, endothelial/cardiac types require specific factors to promote myocardial regeneration after occurred injuries. Herein, cardiac stem cells (CSCs) as the major cell population that involved in cardiovascular repair were selected to study the role of polyethyleneimine (PEI) agent on endothelial differentiation. After preparation of electrospun network of PEI with polyacrylonitrile, the related characterizations were carried out including scanning electron microscope (SEM), field-emission SEM, water contact angle, Fourier transform infrared spectroscopy and mechanical properties. Also, the release kinetic of the corresponding agent was studied up to 7 days. The cell differentiation studies were done in the following with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, Real-time polymerase chain reaction and immunostaining method. The whole obtained results approved the higher differentiation of CSCs into endothelial/cardiac cells. Finally, it is recommended that the PEI delivering increases the healing potency of CSCs and accordingly the regeneration speed of damaged cardiovascular tissue would be improved.     

Biomimetic and bioactive nanofibrous scaffolds from electrospun composite nanofibers

Electrospinning is an enabling technology that can architecturally (in terms of geometry, morphology or topography) and biochemically fabricate engineered cellular scaffolds that mimic the native extracellular matrix (ECM). This is especially important and forms one of the essential paradigms in the area of tissue engineering. While biomimesis of the physical dimensions of native ECM's major constituents (eg, collagen) is no longer a fabrication-related challenge in tissue engineering research, conveying bioactivity to electrospun nanofibrous structures will determine the efficiency of utilizing electrospun nanofibers for regenerating biologically functional tissues. This can certainly be achieved through developing composite nanofibers. This article gives a brief overview on the current development and application status of employing electrospun composite nanofibers for constructing biomimetic and bioactive tissue scaffolds. Considering that composites consist of at least two material components and phases, this review details three different configurations of nanofibrous composite structures by using hybridizing basic binary material systems as example. These are components blended composite nanofiber, core-shell structured composite nanofiber, and nanofibrous mingled structure.     

Nanoparticles for bone tissue engineering

Tissue engineering (TE) envisions the creation of functional substitutes for damaged tissues through integrated solutions, where medical, biological, and engineering principles are combined. Bone regeneration is one of the areas in which designing a model that mimics all tissue properties is still a challenge. The hierarchical structure and high vascularization of bone hampers a TE approach, especially in large bone defects. Nanotechnology can open up a new era for TE, allowing the creation of nanostructures that are comparable in size to those appearing in natural bone. Therefore, nanoengineered systems are now able to more closely mimic the structures observed in naturally occurring systems, and it is also possible to combine several approaches ‐ such as drug delivery and cell labeling ‐ within a single system. This review aims to cover the most recent developments on the use of different nanoparticles for bone TE, with emphasis on their application for scaffolds improvement; drug and gene delivery carriers, and labeling techniques. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:590–611, 2017     

Cryogenic 3D printing for tissue engineering

We describe a new cryogenic 3D printing technology for freezing hydrogels, with a potential impact to tissue engineering. We show that complex frozen hydrogel structures can be generated when the 3D object is printed immersed in a liquid coolant (liquid nitrogen), whose upper surface is maintained at the same level as the highest deposited layer of the object. This novel approach ensures that the process of freezing is controlled precisely, and that already printed frozen layers remain at a constant temperature. We describe the device and present results which illustrate the potential of the new technology.     

Electrospun composite scaffolds containing poly(octanediol-co-citrate) for cardiac tissue engineering

A biocompatible and elastomeric nanofibrous scaffold is electrospun from a blend of poly(1,8-octanediol-co-citrate) [POC] and poly(L-lactic acid) -co-poly-(3-caprolactone) [PLCL] for application as a bioengineered patch for cardiac tissue engineering. The characterization of the scaffolds was carried out by Fourier transform infra red spectroscopy, scanning electron microscopy (SEM), and tensile measurement. The mechanical properties of the scaffolds are studied with regard to the percentage of POC incorporated with PLCL and the results of the study showed that the mechanical property and degradation behavior of the composites can be tuned with respect to the concentration of POC blended with PLCL. The composite scaffolds with POC: PLCL weight ratio of 40:60 [POC/PLCL4060] was found to have a tensile strength of 1.04 ± 0.11 MPa and Young's Modulus of 0.51 ± 0.10 MPa, comparable to the native cardiac tissue. The proliferation of cardiac myoblast cells on the electrospun POC/PLCL scaffolds was found to increase from Days 2 to 8, with the increasing concentration of POC in the composite. The morphology and cytoskeletal observation of the cells also demonstrated the biocompatibility of the POC containing scaffolds. Electrospun POC/PLCL4060 nanofibers are promising elastomeric substrates that might provide the necessary mechanical cues to cardiac muscle cells for regeneration of the heart.     

Cryopreservation of hMSCs seeded silk nanofibers based tissue engineered constructs

Long term cryopreservation of tissue engineering constructs is of paramount importance to meet off-the shelf requirements for medical applications. In the present study, the effect of cryopreservation using natural osmolytes such as trehalose and ectoin with and without conventional Me₂SO on the cryopreservation of tissue engineered constructs (TECs) was evaluated. MSCs derived from umbilical cord were seeded on electrospun nanofibrous silk fibroin scaffolds and cultured to develop TECs. TECs were subjected to controlled rate freezing using nine different freezing solutions. Among these, freezing medium consisting of natural osmolytes like trehalose (40 mM), ectoin (40 mM), catalase (100 μg) as antioxidant and Me₂SO (2.5%) was found to be the most effective. Optimality of the chosen cryoprotectants was confirmed by cell viability (PI live/dead staining), cell proliferation (MTT assay), microstructure analysis (SEM), membrane integrity (confocal microscopy) and in vitro osteogenic differentiation (ALP assay, RT-PCR and histology) study carried out with post-thaw cryopreserved TECs. The mechanical integrity of the cryopreserved scaffold was found to be unaltered.     

生物可降解材料构建组织工程软骨的研究进展

关节软骨修复困难,目前临床上治疗关节软骨损伤难以达到满意的效果。组织工程学的兴起为其提供了新的选择。本文介绍了组织工程软骨的发展历史,重点叙述了各种天然支架材料、人工合成材料、复合材料及纳米材料在软骨组织工程中的应用及其优势。目前应用的天然材料存在力学强度差及免疫源性的不足;人工合成材料降解速率快,降解产物具有细胞毒性,有待进一步完善。表面修饰等技术的应用在一定程度上克服了某些材料的不足;复合材料综合了数种材料的优点,是今后材料技术发展的方向;纳米技术的出现使新合成的材料成为纳米量级,具有了普通材料无可比拟的优势,这为组织工程材料的发展提供了新的思路。本文还对组织工程支架材料存在的问题、下一步的发展方向和前瞻性研究做了介绍。     

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% ± 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% ± 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

应用于组织工程支架制备的电纺技术

组织工程技术为修复病损的组织和器官提供了一种新的途径,在组织工程中,细胞支架起着支撑细胞生长、引导组织再生、控制组织结构和释放活性因子等作用。针对电纺技术的新发展和细胞支架的新理念,综述了国内外利用电纺技术制备细胞支架的工艺条件、制备方法、组织细胞培养等方面的研究进展,并结合作者所在研究团队的研究工作提出了对未来电纺技术在组织工程中应用的研究重点和发展方向的认识。     

Preparation and characterization of poly (lactic-co-glycolic acid) nanofibers containing simvastatin coated with hyaluronic acid for using in periodontal tissue engineering

Periodontal diseases can lead to soft tissue defects. Tissue engineering can provide functional replacements for damaged tissues. Recently, electrospun nanofibers have attracted great interest for tissue engineering and drug delivery applications. This has been revealed that statins exhibit positive impacts on the proliferation and regeneration of periodontal tissues. Electrospun simvastatin loaded poly (lactic-co-glycolic acid) (SIM-PLGA-NF) were prepared using electrospinning technique. Optimal conditions for preparation of SIM-PLGA-NF (PLGA concentration of 30 wt%, voltage of 15 kV, and flow rate of 1.5 ml h⁻¹) were identified using a 2³ factorial design. The optimized SIM-PLGA-NFs (diameter of 640.2 ± 32.5 nm and simvastatin entrapment efficacy of 99.6 ± 1.5%) were surface modified with 1% w/v hyaluronic acid solution (1%HA- SIM-PLGA-NF) to improve their compatibility with fibroblasts and potential application as a periodontal tissue engineering scaffold. HA-SIM-PLGA NFs were analyzed using SEM, FTIR, and XRD. 1%HA-SIM-PLGA-NF had uniform, bead-free and interwoven morphology, which is similar to the extracellular matrix. The mechanical performance of SIM-PLGA-NFs and release profile of simvastatin from these nanofibers have been also greatly improved after coating with HA. In vitro cellular tests showed that the proliferation, adhesion, and differentiation of fibroblast cells positively enhanced on the surface of 1%HA- SIM-PLGA-NF. These results demonstrate the potential application of 1%HA-SIM-PLGA-NFs as a scaffold for periodontal tissue engineering.     

Design considerations and challenges for mechanical stretch bioreactors in tissue engineering

With the increase in average life expectancy and growing aging population, lack of functional grafts for replacement surgeries has become a severe problem. Engineered tissues are a promising alternative to this problem because they can mimic the physiological function of the native tissues and be cultured on demand. Cyclic stretch is important for developing many engineered tissues such as hearts, heart valves, muscles, and bones. Thus a variety of stretch bioreactors and corresponding scaffolds have been designed and tested to study the underlying mechanism of tissue formation and to optimize the mechanical conditions applied to the engineered tissues. In this review, we look at various designs of stretch bioreactors and common scaffolds and offer insights for future improvements in tissue engineering applications. First, we summarize the requirements and common configuration of stretch bioreactors. Next, we present the features of different actuating and motion transforming systems and their applications. Since most bioreactors must measure detailed distributions of loads and deformations on engineered tissues, techniques with high accuracy, precision, and frequency have been developed. We also cover the key points in designing culture chambers, nutrition exchanging systems, and regimens used for specific tissues. Since scaffolds are essential for providing biophysical microenvironments for residing cells, we discuss materials and technologies used in fabricating scaffolds to mimic anisotropic native tissues, including decellularized tissues, hydrogels, biocompatible polymers, electrospinning, and 3D bioprinting techniques. Finally, we present the potential future directions for improving stretch bioreactors and scaffolds. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:543–553, 2016     

Cardiospheres and tissue engineering for myocardial regeneration: potential for clinical application

Tissue engineering is an increasingly expanding area of research in the cardiovascular field that involves engineering, chemistry, biology and medicine. Cardiac tissue engineering (CTE) aims to regenerate myocardial damage by combining cells, matrix, biological active molecules and physiological stimuli. The rationale behind CTE applications is that in order to regenerate the ventricular wall after a myocardial infarction it is necessary to combine procedures that regenerate both cardiomyocytes and the extracellular matrix. The application of (stem) cells together with a matrix could represent an environment protected from the inflammatory and pro-apoptotic signals, a stemness/survival reservoir slowly releasing cells and factors promoting tissue regeneration and angiogenesis. This review will focus on the applications and advantages that CTE application could offer compared to conventional cell therapy.