Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering

Tissue engineering is a clinically driven field and has emerged as a potential alternative to organ transplantation. The cornerstone of successful tissue engineering rests upon two essential elements: cells and scaffolds. Recently, it was found that stem cells have unique capabilities of self-renewal and multilineage differentiation to serve as a versatile cell source, while nanomaterials have lately emerged as promising candidates in producing scaffolds able to better mimic the nanostructure in natural extracellular matrix and to efficiently replace defective tissues. This article, therefore, reviews the key developments in tissue engineering, where the combination of stem cells and nanomaterial scaffolds has been utilized over the past several years. We consider the high potential, as well as the main issues related to the application of stem cells and nanomaterial scaffolds for a range of tissues including bone, cartilage, nerve, liver, eye etc. Promising in vitro results such as efficient attachment, proliferation and differentiation of stem cells have been compiled in a series of examples involving different nanomaterials. Furthermore, the merits of the marriage of stem cells and nanomaterial scaffolds are also demonstrated in vivo, providing early successes to support subsequent clinical investigations. This progress simultaneously drives mechanistic research into the mechanotransduction process responsible for the observations in order to optimize the process further. Current understanding is chiefly reported to involve the interaction of stem cells and the anchoring nanomaterial scaffolds by activating various signaling pathways. Substrate surface characteristics and scaffold bulk properties are also reported to influence not only short term stem cell adhesion, spreading and proliferation, but also longer term lineage differentiation, functionalization and viability. It is expected that the combination of stem cells and nanomaterials will develop into an important tool in tissue engineering for the innovative treatment of many diseases.     

Cytocentrifugation: a convenient and efficient method for seeding tendon-derived cells into monolayer cultures or 3-D tissue engineering scaffolds

Tendon and ligament injuries are very common, requiring some 200,000 reconstructions per year in the USA. Autografting can be used to repair these but donor tissue is limited and harvesting leads to morbidity at the graft sites. Tissue engineering has been used to grow simple tissues such as skin, cartilage and bone and due to their low vascularity and simple structure, tendons should be ideal candidates for such an approach. Scaffolds are essential for tissue engineering as they provide structure and signals that regulate growth. However, they present a physical barrier to cell seeding with the majority of the cells congregating at the scaffold surface. To address this we used centrifugation to enhance penetration of tendon-derived cells to the centres of 3-D scaffolds. The process had no apparent deleterious effects on the cells and both plating efficiency and cell distribution improved. After attachment the cells continued to proliferate and deposit a collagenous matrix. Scaffold penetration was investigated using layers of Azowipes allowing the separation and examination of individual leaves. At relatively low g-forces, cells penetrated a stack of 6 Azowipes leaving cells attached to each leaf. These data suggest that cytocentrifugation improves the penetration and homogeneity of tendon derived cells in 3-D and monolayer cultures.     

Smart scaffolds in bone tissue engineering: A systematic review of literature

AIM: To improve osteogenic differentiation and attachment of cells.METHODS: An electronic search was conducted inPub Med from January 2004 to December 2013. Studies which performed smart modifications on conventional bone scaffold materials were included. Scaffolds with controlled release or encapsulation of bioactive molecules were not included. Experiments which did not investigate response of cells toward the scaffold(cell attachment, proliferation or osteoblastic differentiation) were excluded. RESULTS: Among 1458 studies, 38 met the inclusion and exclusion criteria. The main scaffold varied extensively among the included studies. Smart modifications included addition of growth factors(group Ⅰ-11 studies), extracellular matrix-like molecules(group Ⅱ-13 studies) and nanoparticles(nano-HA)(group Ⅲ-17 studies). In all groups, surface coating was the most commonly applied approach for smart modification of scaffolds. In group I, bone morphogenetic proteins were mainly used as growth factor stabilized on polycaprolactone(PCL). In group Ⅱ, collagen 1 in combination with PCL, hydroxyapatite(HA) and tricalcium phosphate were the most frequent scaffolds used. In the third group, nano-HA with PCL and chitosan were used the most. As variable methods were used, a thorough and comprehensible compare between the results and approaches was unattainable.CONCLUSION: Regarding the variability in methodology of these in vitro studies it was demonstrated that smart modification of scaffolds can improve tissue properties.     

Chitosan and its derivatives for tissue engineering applications

Tissue engineering is an important therapeutic strategy for present and future medicine. Recently, functional biomaterial researches have been directed towards the development of improved scaffolds for regenerative medicine. Chitosan is a natural polymer from renewable resources, obtained from shell of shellfish, and the wastes of the seafood industry. It has novel properties such as biocompatibility, biodegradability, antibacterial, and wound-healing activity. Furthermore, recent studies suggested that chitosan and its derivatives are promising candidates as a supporting material for tissue engineering applications owing to their porous structure, gel forming properties, ease of chemical modification, high affinity to in vivo macromolecules, and so on. In this review, we focus on the various types of chitosan derivatives and their use in various tissue engineering applications namely, skin, bone, cartilage, liver, nerve and blood vessel.     

仿生聚己内酯支架用于乳房组织工程的可行性研究

目的探讨聚己内酯（PCL）乳房形态支架用于组织工程乳房的构建的可能性。 方法通过熔融沉积3D打印制备形态仿生的PCL支架,测量其机械性能,并使用新西兰大白兔动物模型,皮下植入该PCL支架12周和18周后,利用核磁共振成像（MRI）观察支架内部新生组织分布情况,在组织学（HE、Masson及EVG染色）上评估支架内部的脂肪、纤维及血管的分布情况,并进一步使用qRT-PCR检测了12周时PCL支架内部组织的成脂相关基因（PPAR-γ、C/EBP-β、AP-2）、炎症相关基因TNF-α及巨噬细胞标记物F4-80的表达情况,同时使用凝胶渗透色谱法分析了PCL植入体内后平均分子量的变化。2组间均数比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验,配对设计的均数比较采用配对t检验。 结果制备的PCL支架孔隙率为（85.30±1.12）%,压缩模量为（8.18±1.39）MPa,植入新西兰大白兔动物模型皮下12周后,MRI影像学显示脂肪组织已由支架周围向内部侵入,HE、Masson及EVG染色同样在该支架边缘观察到部分新生脂肪组织及血管,而支架内部则以疏松排列的纤维组织为主;与原生脂肪比较,12周PCL支架内组织的基因表达分析成脂相关基因C/EBPβ表达水平（2.32±0.28比1.00±0.02）升高,而巨噬细胞标记物F4/80表达（0.80±0.12比1.00±0.03）降低（P均< 0.01）;18周后,HE染色证实支架内部已充满脂肪组织。基因表达证实,与原生脂肪比较,支架内部组织C/EBP-β （3.30±0.63比1.00±0.02）,PPAR-γ （1.81±0.71比0.99±0.02）及AP-2表达水平（1.38±0.16比1.01±0.01）升高（P均< 0.01）;而TNF-α（0.50±0.15比1.00±0.01）及F4/80表达水平（0.52±0.09比1.00±0.03）均降低（P均< 0.001）。而植入体内PCL支架的分子量（M_n）在18周内变化不大[（65.04±2.24）kDa比（64.20±4.09） kDa]。 结论PCL支架具有较好的生物相容性,可用于组织工程乳房的构建,该新西兰大白兔动物模型的建立有利于乳房组织工程的进一步临床转化。     

Finite element method (FEM), mechanobiology and biomimetic scaffolds in bone tissue engineering

Techniques of bone reconstructive surgery are largely based on conventional, non-cell-based therapies that rely on the use of durable materials from outside the patient's body. In contrast to conventional materials, bone tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve bone tissue function. Bone tissue engineering has led to great expectations for clinical surgery or various diseases that cannot be solved with traditional devices. For example, critical-sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of bone tissue engineering is to apply engineering principles to incite and promote the natural healing process of bone which does not occur in critical-sized defects. The total market for bone tissue regeneration and repair was valued at $1.1 billion in 2007 and is projected to increase to nearly $1.6 billion by 2014.Usually, temporary biomimetic scaffolds are utilized for accommodating cell growth and bone tissue genesis. The scaffold has to promote biological processes such as the production of extra-cellular matrix and vascularisation, furthermore the scaffold has to withstand the mechanical loads acting on it and to transfer them to the natural tissues located in the vicinity. The design of a scaffold for the guided regeneration of a bony tissue requires a multidisciplinary approach. Finite element method and mechanobiology can be used in an integrated approach to find the optimal parameters governing bone scaffold performance.In this paper, a review of the studies that through a combined use of finite element method and mechano-regulation algorithms described the possible patterns of tissue differentiation in biomimetic scaffolds for bone tissue engineering is given. Firstly, the generalities of the finite element method of structural analysis are outlined; second, the issues related to the generation of a finite element model of a given anatomical site or of a bone scaffold are discussed; thirdly, the principles on which mechanobiology is based, the principal theories as well as the main applications of mechano-regulation models in bone tissue engineering are described; finally, the limitations of the mechanobiological models and the future perspectives are indicated.     

Fiber-based tissue engineering: Progress,challenges, and opportunities

Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the abovementioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice.     

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.     

Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering

The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic.     

Morphofunctional characterization of decellularized vena cava as tissue engineering scaffolds

Clinical experience for peripheral arterial disease treatment shows poor results when synthetic grafts are used to approach infrapopliteal arterial segments. However, tissue engineering may be an option to yield surrogate biocompatible neovessels. Thus, biological decellularized scaffolds could provide natural tissue architecture to use in tissue engineering, when the absence of ideal autologous veins reduces surgical options. The goal of this study was to evaluate different chemical induced decellularization protocols of the inferior vena cava of rabbits. They were decellularized with Triton X100 (TX100), sodium dodecyl sulfate (SDS) or sodium deoxycholate (DS). Afterwards, we assessed the remaining extracellular matrix (ECM) integrity, residual toxicity and the biomechanical resistance of the scaffolds. Our results showed that TX100 was not effective to remove the cells, while protocols using SDS 1% for 2 h and DS 2% for 1 h, efficiently removed the cells and were better characterized. These scaffolds preserved the original organization of ECM. In addition, the residual toxicity assessment did not reveal statistically significant changes while decellularized scaffolds retained the equivalent biomechanical properties when compared with the control. Our results concluded that protocols using SDS and DS were effective at obtaining decellularized scaffolds, which may be useful for blood vessel tissue engineering.     

Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110 μm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40 kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.     

Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.     

Evaluation of RGD- or EGF-immobilized chitosan scaffolds for chondrogenic activity

Chitosan scaffolds were prepared by freeze-drying method and modified with Arg-Gly-Asp (RGD) sequence of fibronectin or epidermal growth factor (EGF) by covalent immobilization. The results obtained from FTIR-ATR, fluorescence visualization and quantitative measurements showed that biosignal molecules, RGD and EGF, were successfully immobilized on chitosan scaffolds. ATDC5 murine chondrogenic cells were seeded on both type of scaffolds, chitosan-RGD and chitosan-EGF, and cultured for 28 days in stationary conditions. According to the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) test, considerable increase in cell proliferation was only detected on chitosan-EGF scaffolds. Biochemical analysis of the chondrocyte seeded scaffolds showed that glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA) content of the scaffolds increases with time. In conclusion, EGF-modified chitosan scaffolds (containing 1.83 microg EGF/3 mg dry scaffold) have been proposed to promote chondrogenesis and to have potential for reticular cartilage regeneration.     

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。     

Computer aided design of architecture of degradable tissue engineering scaffolds

One important factor affecting the process of tissue regeneration is scaffold stiffness loss, which should be properly balanced with the rate of tissue regeneration. The aim of the research reported here was to develop a computer tool for designing the architecture of biodegradable scaffolds fabricated by melt-dissolution deposition systems (e.g. Fused Deposition Modeling) to provide the required scaffold stiffness at each stage of degradation/regeneration. The original idea presented in the paper is that the stiffness of a tissue engineering scaffold can be controlled during degradation by means of a proper selection of the diameter of the constituent fibers and the distances between them. This idea is based on the size-effect on degradation of aliphatic polyesters. The presented computer tool combines a genetic algorithm and a diffusion-reaction model of polymer hydrolytic degradation. In particular, we show how to design the architecture of scaffolds made of poly(DL-lactide-co-glycolide) with the required Young’s modulus change during hydrolytic degradation.     

Novel chitin/nanosilica composite scaffolds for bone tissue engineering applications

Biopolymers like chitin are widely investigated as scaffolds in bone tissue engineering. Its properties like biocompatibility, biodegradability, non-toxicity, wound healing ability, antibacterial activity, hemostatic property, etc., are widely known. However, these materials are not much bioactive. Addition of material like silica can improve the bioactivity and biocompatibility of chitin. In this work, chitin composite scaffolds containing nanosilica were prepared using chitin hydrogel and their bioactivity, swelling ability and cytotoxicity was analyzed in vitro. These scaffolds were found to be bioactive in simulated body fluid (SBF) and biocompatible when tested with MG 63 cell line. These results suggest that chitin/nanosilica composite scaffolds can be useful for bone tissue engineering applications.     

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。     

Fluorometric determination of DNA in mammalian cell cultures by PicoGreen

An intercalating fluorochrome, PicoGreen, was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 91.8% of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples.     

Supercritical carbon dioxide generated vascular endothelial growth factor encapsulated poly(DL-lactic acid) scaffolds induce angiogenesis in vitro

The ability to deliver, over time, biologically active vascular endothelial growth factor-165 (VEGF) through tailored designed scaffolds offers tremendous therapeutic opportunities to tissue-engineered therapies. Porous biodegradable poly(DL-lactic) acid (PLA) scaffolds encapsulating VEGF have been generated using supercritical CO2 (scCO2) and the kinetic release and angiogenic activity of these scaffolds examined in vitro and in an ex vivo chick chorioallantoic membrane (CAM) angiogenesis model. After processing through scCO2, VEGF maintained its angiogenic activity as assessed by increased tubule formation of human umbilical vein endothelial cells (HUVEC) cultured on Matrigel (VEGF = 1937 +/- 205 microm; scCO2-VEGF = 2085 +/- 234 microm; control = 1237 +/- 179 microm). VEGF release kinetics from scCO2-VEGF incorporated PLA monolith scaffolds showed a cumulative release of VEGF (2837 +/- 761 rhog/ml) over a 21 day period in culture. In addition, VEGF encapsulated PLA scaffolds increased the blood vessel network in the CAM compared to controls; control, 24.8 +/- 9.6; VEGF/PLA, 44.1 +/- 12.1 (vessels/field). These studies demonstrate that the controlled release of growth factors encapsulated into three-dimensional PLA scaffolds can actively stimulate the rapid development of therapeutic neovascularisation to regenerate or engineer tissues.