Biomaterial aided differentiation and maturation of induced pluripotent stem cells

Engineering/reprogramming differentiated adult somatic cells to gain the ability to differentiate into any type of cell lineage are called as induced pluripotent stem cells (iPSCs). Offering unlimited self-renewal and differentiation potential, these iPSC are aspired to meet the growing demands in the field of regenerative medicine, tissue engineering, disease modeling, nanotechnology, and drug discovery. Biomaterial fabrication with the rapid evolution of technology increased their versatility and utility in regenerative medicine and tissue engineering, revolutionizing the stem cell biology research with the property to guide the process of proliferation, differentiation, and morphogenesis. Combining traditional culture platforms of iPSC with biomaterials aids to overcome the limitations associated with derivation, proliferation, and maturation, thereby could improve the clinical translation of iPSC. The present review discusses in brief about the reprogramming techniques for the derivation iPSC and details on several biomaterial guided differentiation of iPSC to different cell types with specific relevance to tissue engineering/regenerative medicine.     

3D Non-Woven Polyvinylidene Fluoride Scaffolds: Fibre Cross Section and Texturizing Patterns Have Impact on Growth of Mesenchymal Stromal Cells

Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds.     

聚己内酯静电纺丝纳米纤维支架用于小鼠诱导多能干细胞培养

干细胞联合生物支架材料体外构建功能性组织与器官,成为当前组织再生研究的重要策略,而探求具有良好生物相容性的支架材料是其关键.本研究采用扫描电镜、噻唑蓝（MTT）法、荧光显微染色等方法检测小鼠诱导多能干细胞（murine induced pluripotent stem cells, miPSCs）在聚己内酯（poly ε-caprolactone, PCL）静电纺丝纳米纤维支架上的粘附、增殖等生物学特性,探究聚己内酯纳米纤维支架与miPSCs的生物相容性. 结果显示,miPSC在PCL纳米纤维支架上具有良好粘附性并呈集落样生长,其增殖能力及干性标记物（Oct4-GFP+）的表达均不亚于标准对照组;扫描电镜显示,miPSC在PCL纳米纤维支架材料上呈现出绒毛状突起的表面结构.上述结果表明,PCL纳米纤维支架可促进miPSCs的粘附、自我增殖以及干性维持,两者具有良好的生物相容性,为下一步联合生物支架材料与干细胞构建功能性组织奠定了基础.     

微环境对细胞的影响以及仿生学在组织工程支架中的应用

微环境影响着细胞的增殖、迁移、分化以及细胞功能,细胞微环境影响细胞命运的因素包括细胞之间相互作用、细胞与细胞外基质相互作用、可溶性信号分子以及缺氧和营养对细胞的影响。组织工程支架的制备就是要利用仿生学原理最大程度模拟细胞微环境,从而应用于细胞行为研究以及临床治疗。全面了解细胞微环境对细胞的影响因素是制备组织工程支架的重要条件,而组织工程支架的研究也进一步推动了细胞微环境对细胞影响的认识。组织工程支架研究在组织工程研究中仍具有广阔前景,新的制备工艺也在组织工程支架研究中发挥着巨大推动作用。     

Nanoscale engineering of extracellular matrix-mimetic bioadhesive surfaces and implants for tissue engineering

Background

The goal of tissue engineering is to restore tissue function using biomimetic scaffolds which direct desired cell fates such as attachment, proliferation and differentiation. Cell behavior in vivo is determined by a complex interaction of cells with extracellular biosignals, many of which exist on a nanoscale. Therefore, recent efforts in tissue engineering biomaterial development have focused on incorporating extracellular matrix- (ECM) derived peptides or proteins into biomaterials in order to mimic natural ECM. Concurrent advances in nanotechnology have also made it possible to manipulate protein and peptide presentation on surfaces on a nanoscale level.

Scope of Review

This review discusses protein and peptide nanopatterning techniques and examples of how nanoscale engineering of bioadhesive materials may enhance outcomes for regenerative medicine.

Major Conclusions

Synergy between ECM-mimetic tissue engineering and nanotechnology fields can be found in three major strategies: (1) Mimicking nanoscale orientation of ECM peptide domains to maintain native bioactivity, (2) Presenting adhesive peptides at unnaturally high densities, and (3) Engineering multivalent ECM-derived peptide constructs.

General Significance

Combining bioadhesion and nanopatterning technologies to allow nanoscale control of adhesive motifs on the cell–material interface may result in exciting advances in tissue engineering.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

MWCNTs enhance hBMSCs spreading but delay their proliferation in the direction of differentiation acceleration

Investigating the ability of films of pristine multiwalled nanotubes (MWCNTs) to influence human mesenchymal stem cells’ proliferation, morphology, and differentiation into osteoblasts, we concluded to the following: A. MWCNTs delay the proliferation of hBMS cells but increase their differentiation. The enhancement of the differentiation markers could be a result of decreased proliferation and maturation of the extracellular matrix B. Cell spread on MWCNTs toward a polygonal shape with many thin filopodia to attach to the surfaces. Spreading may be critical in supporting osteogenic differentiation in pre-osteoblastic progenitors, being related with cytoskeletal tension. C. hBMS cells prefer MWCNTs than tissue plastic to attach and grow, being non-toxic to these cells. MWCNTs can be regarded as osteoinductive biomaterial topographies for bone regenerative engineering.     

Designing biomaterials for in situ periodontal tissue regeneration

The regeneration of periodontal tissue poses a significant challenge to biomaterial scientists, tissue engineers and periodontal clinicians. Recent advances in this field have shifted the focus from the attempt to recreate tissue replacements/constructs ex vivo to the development of biofunctionalized biomaterials that incorporate and release regulatory signals in a precise and near-physiological fashion to achieve in situ regeneration. The molecular and physical information coded within the biomaterials define a local biochemical and mechanical niche with complex and dynamic regulation that establishes key interactions with host endogenous cells and, hence, may help to unlock latent regenerative pathways in the body by instructing cell homing and regulating cell proliferation/differentiation. In the future, these innovative principles and biomaterial devices promise to have a profound impact on periodontal reconstructive therapy and are also likely to reconcile the clinical and commercial pressures on other tissue engineering endeavors.     

Preparation and Characterization of Electrospun PLCL/Poloxamer Nanofibers and Dextran/Gelatin Hydrogels for Skin Tissue Engineering

In this study, two different biomaterials were fabricated and their potential use as a bilayer scaffold for skin tissue engineering applications was assessed. The upper layer biomaterial was a Poly(ε-caprolactone-co-lactide)/Poloxamer (PLCL/Poloxamer) nanofiber membrane fabricated using electrospinning technology. The PLCL/Poloxamer nanofibers (PLCL/Poloxamer, 9/1) exhibited strong mechanical properties (stress/strain values of 9.37±0.38 MPa/187.43±10.66%) and good biocompatibility to support adipose-derived stem cells proliferation. The lower layer biomaterial was a hydrogel composed of 10% dextran and 20% gelatin without the addition of a chemical crosslinking agent. The 5/5 dextran/gelatin hydrogel displayed high swelling property, good compressive strength, capacity to present more than 3 weeks and was able to support cells proliferation. A bilayer scaffold was fabricated using these two materials by underlaying the nanofibers and casting hydrogel to mimic the structure and biological function of native skin tissue. The upper layer membrane provided mechanical support in the scaffold and the lower layer hydrogel provided adequate space to allow cells to proliferate and generate extracellular matrix. The biocompatibility of bilayer scaffold was preliminarily investigated to assess the potential cytotoxicity. The results show that cell viability had not been affected when cocultured with bilayer scaffold. As a consequence, the bilayer scaffold composed of PLCL/Poloxamer nanofibers and dextran/gelatin hydrogels is biocompatible and possesses its potentially high application prospect in the field of skin tissue engineering.     

Ammonia plasma treatment of polystyrene surfaces enhances proliferation of primary human mesenchymal stem cells and human endothelial cells

The control of surface properties is a substantial step in the development and improvement of biomaterials for clinical applications as well as for their use in tissue engineering. Interaction of the substrate surface with the biochemical or biological environment is crucial for the outcome of the applied biomaterial and therefore should meet specific requirements regarding the chemical composition, wettability, elasticity, and charge. In this study, we examined the effect of chemical groups introduced by low pressure plasma treatments of polystyrene surfaces on the cell behavior of primary human mesenchymal stem cells (hMSCs) and dermal microvascular endothelial cells (hDMECs). X-ray photoelectron spectroscopy analysis and contact angle measurements were employed to evaluate ammonia-, carbon dioxide-, and acrylic acid-plasma modifications to substrate surfaces. HMSCs and hDMECs were analyzed simultaneously to identify the most suitable surface functionalization for each cell type. Significantly higher cell proliferation was detected on ammonia plasma-treated surfaces. Cell-material interaction could be shown on all created interfaces as well as the expression of typical cell markers. Hence, the applied plasma treatment presents a suitable tool to improve culture condition on polystyrene for two important cell types (hMSCs and hDMECs) in the field of tissue engineering.     

The Inhibitory Effect of an RGD-Human Chitin-Binding Domain Fusion Protein on the Adhesion of Fibroblasts to Reacetylated Chitosan Films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.     

Multiwalled carbon nanotubes enhance human bone marrow mesenchymal stem cells’ spreading but delay their proliferation in the direction of differentiation acceleration

Investigating the ability of films of pristine (purified, without any functionalization) multiwalled carbon nanotubes (MWCNTs) to influence human bone marrow mesenchymal stem cells’ (hBMSCs) proliferation, morphology, and differentiation into osteoblasts, we concluded to the following: A. MWCNTs delay the proliferation of hBMSCs but increase their differentiation. The enhancement of the differentiation markers could be a result of decreased proliferation and maturation of the extracellular matrix B. Cell spread on MWCNTs toward a polygonal shape with many thin filopodia to attach to the surfaces. Spreading may be critical in supporting osteogenic differentiation in pre-osteoblastic progenitors, being related with cytoskeletal tension. C. hBMSCs prefer MWCNTs than tissue plastic to attach and grow, being non-toxic to these cells. MWCNTs can be regarded as osteoinductive biomaterial topographies for bone regenerative engineering.     

Growth factor modulation of the extracellular matrix

Two cell culture models were utilized to characterize the actions of peptide growth factors on the composition of the extracellular matrix of embryonic mesenchymal tissue. To model the three-dimensional architecture of mesenchymal tissue, chick embryonic mesenchymal cells were maintained in organ culture as adherent cell populations in small three-dimensional tissue spheroids and as sparse populations of cells embedded in a mesh of hydrated native collagen fibrils. Cell proliferation was stimulated by a variety of growth factors. All of the growth factors that elicited a mitogenic response in both of these culture systems also stimulated the deposition of an abundant fibronectin-containing extracellular matrix that colocalized with the regions of active cell proliferation. The suggestion that the matrigenic actions of growth factors for intact mesenchymal tissue are an integral part of mitogenic signaling is supported by the observation that surfaces derivatized with ProNectin, an artificial mimic of the RGD attachment domain of fibronectin, stimulated the proliferation of embryonic mesenchyme in the absence of exogenous growth factors. All of the growth factors that activated proliferation and fibronectin matrix accumulation stimulated the transformation of the mesenchymal cells into myofibroblasts that displayed the marker alpha-smooth muscle actin.     

柴胡解毒汤对人肝星状细胞LX-2增殖及凋亡的影响

目的:观察柴胡解毒汤含药血清对人肝星状细胞LX-2增殖、凋亡情况的影响,探究其抗肝纤维化作用的可能机制。方法:将Wistar大鼠分为实验组与对照组,分别以柴胡解毒汤、生理盐水灌胃5天后取血制备大鼠含药血清与正常血清。同时复苏人肝星状细胞LX-2后进行细胞培养和细胞传代。当肝星状细胞数量达到预定值时,将肝星状细胞分为对照组和药物组。观察LX-2细胞在与含药血清孵育24 h、36 h、48 h、72 h后,用CCK-8比色法测定细胞的增殖抑制率。用流式细胞术(Annexin V-FITC,PI染色法)检测细胞凋亡的概况。结果:柴胡解毒汤含药血清在给药24 h后可抑制LX-2细胞的增殖,36 h、48 h、72 h的增殖抑制率分别为0.37%、0.46%、0.44%,并可诱导LX-2细胞发生凋亡,48 h、72 h凋亡概率分别为(9.80±0.95)%、(36.40±5.09)%,差异具有统计学意义(P0.05)。结论:柴胡解毒汤具有抑制LX-2细胞增殖、诱导LX-2细胞发生凋亡的能力,这可能是柴胡解毒汤抗纤维化作用的机制之一。     

Adipose-derived stem cells: isolation, expansion and differentiation

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.     

Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system

To meet specific requirements of developing tissues urgently needed in tissue engineering, biomaterial research and drug toxicity testing, a versatile perfusion culture system was developed. First an individual biomaterial is selected and then mounted in a MINUSHEET^® tissue carrier. After sterilization the assembly is transferred by fine forceps to a 24 well culture plate for seeding cells or mounting tissue on it. To support spatial (3D) development a carrier can be placed in various types of perfusion culture containers. In the basic version a constant flow of culture medium provides contained tissue with always fresh nutrition and respiratory gas. For example, epithelia can be transferred to a gradient container, where they are exposed to different fluids at the luminal and basal side. To observe development of tissue under the microscope, in a different type of container a transparent lid and base are integrated. Finally, stem/progenitor cells are incubated in a container filled by an artificial interstitium to support spatial development. In the past years the described system was applied in numerous own and external investigations. To present an actual overview of resulting experimental data, the present paper was written.     

Mathematical modelling of human mesenchymal stem cell proliferation and differentiation inside artificial porous scaffolds

We present a mathematical model for the proliferation and differentiation of human mesenchymal stem cells grown inside artificial porous scaffolds under different oxygen concentrations. The values of parameters in the model are determined by comparison of the model solutions to published experimental data, complemented with a sensitivity analysis of the fitted parameters. It is shown that a simple hypothesis whereby the secretion of extra-cellular matrix (ECM) is oxygen dependent and that ECM itself stimulates cell proliferation is sufficient to explain the experimental data, which under conditions of low oxygen reveals increased total cell proliferation, upregulation of the numbers of undifferentiated cells, and extended lag phase. These results may help further to understand how cells proliferate inside artificial materials and are of importance to the field of tissue engineering.     

Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

骨髓基质细胞与交联明胶-聚羟基丁酸酯膜的生物相容性研究

目的研究生物材料交联明胶-聚羟基丁酸酯膜与骨髓基质细胞的生物相容性,探讨新型材料在骨组织工程中的应用前景。方法体外培养兔骨髓基质细胞,分别接种于G-PHB(交联明胶-聚羟基丁酸酯)、PHB(聚羟基丁酸酯)和G(交联明胶)材料膜片。采用MTT法检测细胞增殖活性,体视学方法检测细胞粘附能力,荧光双染法检测细胞完整性,扫描电镜观察细胞-材料界面。结果MTT检测发现G-PHB组增殖活性最强,而且表现为最佳的细胞粘附特性,与对照组比较差异有显著性意义。各组细胞完整性分析没有发现显著性差异。扫描电镜观察显示,G-PHB组细胞粘附及铺展良好,优于其他各组。结论交联后的生物降解膜材料G-PHB与BMSCs细胞的体外相容性明显优于单纯膜材料PHB和明胶,在骨组织工程学领域具有良好的研究价值和应用潜力。