Growth and proliferation of bone marrow mesenchymal stem cells affected by type I collagen,fibronectin and bFGF

Bone marrow mesenchymal stem cells (MSCs) can differentiate into a various type cells. It has been identified that MSCs are an attractive cell source for various tissue engineering and play a central role in the repair and regeneration of mesenchymal tissue. Expansion of MSCs in vitro is prerequisite for their applications in tissue engineering. In this study, we evaluated the effects of type I collagen (Col I), fibronectin (Fn) and basic fibroblast growth factor (bFGF) on growth and proliferation of human MSCs (hMSCs) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetra-zolium bromide (MTT) assay. Our results show that low-dose bFGF (5–20 ng/ml) has a promotion effects for growth and proliferation of hMSCs. The proliferation, however, was back to the level similar to the control one (without bFGF treatment) after exposure to high-dose bFGF (40 ng/ml). Application of Col I, coating on the silicone surface or mixed with medium directly, yielded an obvious decrease in growth and proliferation of hMSCs. Moreover, the inhibitory effects exhibited a dose-dependence manner. On the other hand, Fn coating did not promote the growth and proliferation of hMSCs, and also did not inhibit proliferation, but enhanced the adhesion of hMSCs to silicone surface. These findings indicate that Col I decreases the growth and proliferation of hMSCs and is not suitable for encouraging expansion of hMSCs in vitro. Low-dose bFGF could be preferred as medium supplementation for hMSCs expansion and Fn is a better coating material for hMSCs adhesion.     

Intervertebral disc (IVD): Structure, degeneration, repair and regeneration

Low back pain affects a large portion of the population, resulting in high care costs for therapy and treatment. One primary cause of low back pain is the degeneration of the intervertebral disc (IVD) resulting in the compression of the spinal nerves and adjacent vertebrae. Exact causes of degeneration are unknown, but it is thought that natural aging, and both biological and genetic factors may play a significant role in the degenerative process. Conventional methods to alleviate low back pain include spinal fusion and artificial disc replacement. Traditional treatments through spinal fusion may eliminate pain yet do not restore disc function and lead to further degeneration of adjacent levels by altering disc biomechanics and natural kinematics. Recently, artificial IVD replacements have started to gain interest, with two IVD implants currently approved in the United States. Although these implants facilitate the preservation of motions and disc space height, they are unable to sustain compressive forces due to their lack of elasticity. In addition, the implants may produce wear debris that can cause osteolysis and other deleterious effects. As an alternative to these conventional approaches, tissue engineered IVD constructs offer the advantage of biointegration while preserving the essential attributes of natural motion and disc space restoration. There is a great need for the development of tissue engineered scaffolds that simulate the natural 3D morphology and microenvironment of the targeted tissue. Scaffolds should facilitate biological transport to satisfy nutrition and waste removal requirements within the IVD. The discrete tissue architectures of the nucleus pulposus (NP) and annulus fibrosus (AF) have posed great challenges to IVD tissue engineering. Current attempts have not been able to satisfy the biological functions and/or mechanical properties of native tissue. Therefore, these current scaffolds are far from satisfactory. This review highlights the challenges met when tissue engineering an IVD, while offering a perspective on which biomaterials and scaffold fabrication methods have the realistic ability to serve as scaffolding constructs for IVD regeneration.     

Investigation of osteoblast-like MC3T3-E1 cells on a novel recombinant collagen-like protein surface with triple helix structure

Fol-8Col is a novel recombinant collagen-like protein incorporated with foldon sequences derived from the native T4 phage fibritin. In this paper, we examined the potential of using Fol-8Col as scaffold for bone tissue engineering. Circular dichroism (CD) spectra indicate that the triple helix structure of Fol-8Col exists at temperatures ranging from 4 to 40 °C. Lactate dehydrogenase assay results and live/death cell staining of osteoblast-like MC3T3-E1 cells, cultivated on Fol-8Col for 24 h, showed evidence of cell cytocompatibility comparable to that of native type I collagen. Attachment and spreading of osteoblast-like MC3T3-E1 cells seeded on Fol-8Col were studied by immunofluorescence staining of cell nuclei, vinculin, and F-actin. Intensive focal adhesion patches and an elongated cortical actin cytoskeleton were observed after 24 hours’ cultivation. Proliferation assays of MC3T3-E1 cells cultivated on Fol-8Col for 2 weeks revealed no consistent differences in rate and pattern compared to growth on type I collagen. Alkaline phosphatase activity assay and osteogenic gene expression, detected by RT-PCR, evaluated the osteogenic differentiation of MC3T3-E1 cells on Fol-8Col. This study shows that Fol-8Col, with a triple helix structure, has good potential for application in bone regeneration as a replacement for native collagen, thereby reducing the risk of contamination.     

Reduced graphene oxide: osteogenic potential for bone tissue engineering

Collagen (Col) type I, as the major component of the bone extracellular matrix has been broadly studied for bone tissue engineering. However,inferior mechanical properties limit its usage for load bearing applications. In this research, freeze dried Col scaffolds are coated with graphene oxide (GO) through a covalent bond of the amine Col with the graphene carboxyl groups. The prepared scaffolds were then reduced using a chemical agent. Scanning electron microscopy exhibited a porous structure for the synthesized scaffolds with an approximate pore size of 100–220 ± 12 µm, which is in the suitable range for bone tissue engineering application. Reducing the GO coating improved the compressive modulus of the Col from 250 to 970 kPa. Apatite formation was also indicated by immersing the scaffolds in simulated body fluid after five days. The cytocompatibility of the scaffolds, using human bone marrow‐derived mesenchymal stem cells, was confirmed with MTT analysis. Alkaline phosphatase assay revealed that reducing the Col–GO scaffolds can effectively activate the differentiation of hBM‐MSCs into osteoblasts after 14 days, even without the addition of an osteogenic differentiation medium. The results of this study highlight that GO and its reduced form have considerable potential as bone substitutes for orthopaedic and dental applications.Inspec keywords: molecular biophysics, tissue engineering, biochemistry, cellular biophysics, graphene, biomedical materials, bone, proteins, scanning electron microscopy, porous materials, compressive strength, biomechanicsOther keywords: human bone marrow‐derived mesenchymal stem cells, reduced graphene oxide, bone extracellular matrix, inferior mechanical properties, load bearing applications, freeze‐dried Col scaffolds, amine Col groups, graphene carboxyl groups, bone tissue engineering, collagen type I, GO‐Col scaffolds, covalent bond, scanning electron microscopy, compressive modulus, apatite formation, cytocompatibility, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide analysis, alkaline phosphatase assay, osteogenic differentiation medium, dental applications, orthopaedic applications, porous structure, time 14.0 day, CO     

Stem cell differentiation on electrospun nanofibrous substrates for vascular tissue engineering

Nanotechnology has enabled the engineering of a variety of materials to meet the current challenges and requirements in vascular tissue regeneration. In our study, poly-l-lactide (PLLA) and hybrid PLLA/collagen (PLLA/Coll) nanofibers (3:1 and 1:1) with fiber diameters of 210 to 430 nm were fabricated by electrospinning. Their morphological, chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR), and tensile instrument, respectively. Bone marrow derived mesenchymal stem cells (MSCs) seeded on electrospun nanofibers that are capable of differentiating into vascular cells have great potential for repair of the vascular system. We investigated the potential of MSCs for vascular cell differentiation in vitro on electrospun PLLA/Coll nanofibrous scaffolds using endothelial differentiation media. After 20 days of culture, MSC proliferation on PLLA/Coll(1:1) scaffolds was found 256% higher than the cell proliferation on PLLA scaffolds. SEM images showed that the MSC differentiated endothelial cells on PLLA/Coll scaffolds showed cobblestone morphology in comparison to the fibroblastic type of undifferentiated MSCs. The functionality of the cells in the presence of ‘endothelial induction media’, was further demonstrated from the immunocytochemical analysis, where the MSCs on PLLA/Coll (1:1) scaffolds differentiated to endothelial cells and expressed the endothelial cell specific proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) and Von Willebrand factor (vWF). From the results of the SEM analysis and protein expression studies, we concluded that the electrospun PLLA/Coll nanofibers could mimic the native vascular ECM environment and might be promising substrates for potential application towards vascular regeneration.     

Recreating Physiological Environments In Vitro: Design Rules for Microfluidic‐Based Vascularized Tissue Constructs

Vascularization of engineered tissue constructs remains one of the greatest unmet challenges to mimicking the native tissue microenvironment in vitro. The main obstacle is recapitulating the complexity of the physiological environment while providing simplicity in operation and manipulation of the model. Microfluidic technology has emerged as a promising tool that enables perfusion of the tissue constructs through engineered vasculatures and precise control of the vascular microenvironment cues in vitro. The tunable microenvironment includes i) biochemical cues such as coculture, supporting matrix, and growth factors and ii) engineering aspects such as vasculature engineering methods, fluid flow, and shear stress. In this systematic review, the design considerations of the microfluidic‐based in vitro model are discussed, with an emphasis on microenvironment control to enhance the development of next‐generation vascularized engineered tissues.     

Development of a novel collagen–GAG nanofibrous scaffold via electrospinning

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen–GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen–GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen–GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen–GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.     

Nanoreinforced Hydrogels for Tissue Engineering: Biomaterials that are Compatible with Load‐Bearing and Electroactive Tissues

Given their highly porous nature and excellent water retention, hydrogel‐based biomaterials can mimic critical properties of the native cellular environment. However, their potential to emulate the electromechanical milieu of native tissues or conform well with the curved topology of human organs needs to be further explored to address a broad range of physiological demands of the body. In this regard, the incorporation of nanomaterials within hydrogels has shown great promise, as a simple one‐step approach, to generate multifunctional scaffolds with previously unattainable biological, mechanical, and electrical properties. Here, recent advances in the fabrication and application of nanocomposite hydrogels in tissue engineering applications are described, with specific attention toward skeletal and electroactive tissues, such as cardiac, nerve, bone, cartilage, and skeletal muscle. Additionally, some potential uses of nanoreinforced hydrogels within the emerging disciplines of cyborganics, bionics, and soft biorobotics are highlighted.     

Anisotropic Materials for Skeletal‐Muscle‐Tissue Engineering

Repair of damaged skeletal‐muscle tissue is limited by the regenerative capacity of the native tissue. Current clinical approaches are not optimal for the treatment of large volumetric skeletal‐muscle loss. As an alternative, tissue engineering represents a promising approach for the functional restoration of damaged muscle tissue. A typical tissue‐engineering process involves the design and fabrication of a scaffold that closely mimics the native skeletal‐muscle extracellular matrix (ECM), allowing organization of cells into a physiologically relevant 3D architecture. In particular, anisotropic materials that mimic the morphology of the native skeletal‐muscle ECM, can be fabricated using various biocompatible materials to guide cell alignment, elongation, proliferation, and differentiation into myotubes. Here, an overview of fundamental concepts associated with muscle‐tissue engineering and the current status of muscle‐tissue‐engineering approaches is provided. Recent advances in the development of anisotropic scaffolds with micro‐ or nanoscale features are reviewed, and how scaffold topographical, mechanical, and biochemical cues correlate to observed cellular function and phenotype development is examined. Finally, some recent developments in both the design and utility of anisotropic materials in skeletal‐muscle‐tissue engineering are highlighted, along with their potential impact on future research and clinical applications.     

Development,optimization and characterization of a full-thickness tissue engineered human oral mucosal model for biological assessment of dental biomaterials

Restorative dental materials and oral health care products come into direct contact with oral mucosa and can cause adverse reactions. In order to obtain an accurate risk assessment, the in vitro test model must reflect the clinical situation as closely as possible. The aim of this study was to develop and optimize a three-dimensional full-thickness engineered human oral mucosal model, which can be used for biological assessment of dental materials. In this study human oral fibroblasts and keratinocytes were isolated from patients and seeded onto a number of collagen-based and synthetic scaffolds using a variety of cell seeding techniques and grown at the air/liquid interface to construct human oral mucosa equivalents. Suitability of 10 different scaffolds for engineering human oral mucosa was evaluated in terms of biocompatibility, biostability, porosity, and the ability to mimic normal human oral mucosa morphology. Finally an optimized full-thickness engineered human oral mucosa was developed and characterized using transmission electron microscopy and immunostaining. The oral mucosa reconstruct resembled native human oral mucosa and it has the potential to be used as an accurate and reproducible test model in mucotoxicity and biocompatibility evaluation of dental materials.     

Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture. Here, we printed rigid 3D filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks that could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices, and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.     

Mineralization of osteoblasts with electrospun collagen/hydroxyapatite nanofibers

Regeneration of fractured or diseased bones is the challenge faced by current technologies in tissue engineering. The major solid components of human bone consist of collagen and hydroxyapatite. Collagen (Col) and hydroxyapatite (HA) have potential in mimicking natural extracellular matrix and replacing diseased skeletal bones. More attention has been focused on HA because of its crystallographic structure similar to inorganic compound found in natural bone and extensively investigated due to its excellent biocompatibility, bioactivity and osteoconductivity properties. In the present study, electrospun nanofibrous scaffolds are fabricated with collagen (80 mg/ml) and Col/HA (1:1). The diameter of the collagen nanofibers is around 265 ± 0.64 nm and Col/HA nanofibers are 293 ± 1.45 nm. The crystalline HA (29 ± 7.5 nm) loaded into the collagen nanofibers are embedded within nanofibrous matrix of the scaffolds. Osteoblasts cultured on both scaffolds and show insignificant level of proliferation but mineralization was significantly (p < 0.001) increased to 56% in Col/HA nanofibrous scaffolds compared to collagen. Energy dispersive X-ray analysis (EDX) spectroscopy results proved the presence of higher level of calcium and phosphorous in Col/HA nanocomposites than collagen nanofibrous scaffolds grown osteoblasts. The results of the present study suggested that the designed electrospun nanofibrous scaffold (Col/HA) have potential biomaterial for bone tissue engineering.     

Fabrication and cell affinity of biomimetic structured PLGA/articular cartilage ECM composite scaffold

An ideal scaffold for cartilage tissue engineering should be biomimetic in not only mechanical property and biochemical composition, but also the morphological structure. In this research, we fabricated a composite scaffold with oriented structure to mimic cartilage physiological morphology, where natural nanofibrous articular cartilage extracellular matrix (ACECM) was used to mimic the biochemical composition, and synthetic PLGA was used to enhance the mechanical strength of ACECM. The composite scaffold has well oriented structure and more than 89% of porosity as well as about 107 μm of average pore diameter. The composite scaffold was compared with ACECM and PLGA scaffolds. Cell proliferation test showed that the number of MSCs in ACECM and composite scaffolds was noticeably bigger than that in PLGA scaffold, which was coincident with results of SEM observation and cell viability staining. The water absorption of ACECM and composite scaffolds were 22.1 and 10.2 times respectively, which was much higher than that of PLGA scaffolds (3.8 times). The compressive modulus of composite scaffold in hydrous status was 1.03 MPa, which was near 10 times higher than that of hydrous ACECM scaffold. The aforementioned results suggested that the composite scaffold has the potential for application in cartilage tissue engineering.     

Engineering of Hydrogel Materials with Perfusable Microchannels for Building Vascularized Tissues

Vascular systems are responsible for various physiological and pathological processes related to all organs in vivo, and the survival of engineered tissues for enough nutrient supply in vitro. Thus, biomimetic vascularization is highly needed for constructing both a biomimetic organ model and a reliable engineered tissue. However, many challenges remain in constructing vascularized tissues, requiring the combination of suitable biomaterials and engineering techniques. In this review, the advantages of hydrogels on building engineered vascularized tissues are discussed and recent engineering techniques for building perfusable microchannels in hydrogels are summarized, including micromolding, 3D printing, and microfluidic spinning. Furthermore, the applications of these perfusable hydrogels in manufacturing organ‐on‐a‐chip devices and transplantable engineered tissues are highlighted. Finally, current challenges in recapitulating the complexity of native vascular systems are discussed and future development of vascularized tissues is prospected.     

A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage

Tissue engineering seeks to repair or regenerate tissues through combinations of implanted cells, biomaterial scaffolds and biologically active molecules. The rapid restoration of tissue biomechanical function remains an important challenge, emphasizing the need to replicate structural and mechanical properties using novel scaffold designs. Here we present a microscale 3D weaving technique to generate anisotropic 3D woven structures as the basis for novel composite scaffolds that are consolidated with a chondrocyte-hydrogel mixture into cartilage tissue constructs. Composite scaffolds show mechanical properties of the same order of magnitude as values for native articular cartilage, as measured by compressive, tensile and shear testing. Moreover, our findings showed that porous composite scaffolds could be engineered with initial properties that reproduce the anisotropy, viscoelasticity and tension-compression nonlinearity of native articular cartilage. Such scaffolds uniquely combine the potential for load-bearing immediately after implantation in vivo with biological support for cell-based tissue regeneration without requiring cultivation in vitro.     

Construct of asymmetrical scaffold and primary cells for tissue engineered esophagus

Porous polymeric scaffolds have been widely employed as analogues of native extracellular matrix to create a living construct that would mimic the complexities of human tissue function in the field of tissue engineering. An asymmetrical porous 3-D substitute to be used as a scaffold for tissue engineered esophagus was fabricated using thermally induced phase separation (TIPS) method. The scaffold in which there are pores with 1–10 µm diameter on one side and ≥ 50–100 µm size on the other side and in the bulk was designed to mimic the mucosa constitute that is the most important functional layer of a normal esophagus. The cell and scaffold construct was evaluated using Hematoxylin and Eosin (H&E) staining as well as fluorescein diacetate (FDA) viable cell staining. It was found for the scaffold to be able to support the growth of primary esophageal epithelial cells on the side with micropores and fibroblasts in the scaffold bulk with large pores and good connectivity. A confluent layer of epithelial cells was observed throughout the surface with micropores, with multilayer of cells found at some locations. Clusters of fibroblasts were found on the other side as well as within the bulk of the scaffold.     

三维成球培养优化间充质干细胞的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)源于发育早期的中胚层,因其来源广泛、具有多向分化潜能、低免疫原性和自我更新能力,在组织工程和再生医学应用中显示出巨大的潜力,也是当前基础研究和临床研究中应用最多的一类干细胞。然而,间充质干细胞的临床应用面临许多挑战,比如治疗所需细胞数量巨大,细胞质量存在异质性,细胞体内移植后存活率低,以及二维(two-dimensional,2D)贴壁培养导致间充质干细胞特征衰减等。三维(three-dimensional,3D)成球培养可以更好地模拟体内微环境,且大量的研究证明,3D成球培养增强了间充质干细胞的细胞存活和因子分泌能力,促进了干细胞特征维持、细胞迁移和血管生成,在临床医学领域具有广阔的应用前景。基于此,综述了体外3D成球培养的方法、3D成球培养优化的间充质干细胞的生物学特性及应用,并对3D成球培养未来的研究方向进行展望。     

Fabrication of Artificial Nanobasement Membranes for Cell Compartmentalization in 3D Tissues

In recent decades, tissue engineering techniques have attracted much attention in the construction of 3D tissues or organs. However, even though precise control of cell locations in 3D has been achieved, the organized cell locations are easily destroyed because of the cell migration during the cell culture period. In human body, basement membranes (BMs) maintain the precise cell locations in 3D (compartmentalization). Constructing artificial BMs that mimic the structure and biofunctions of natural BMs remains a major challenge. Here, a nanometer‐sized artificial BM through layer‐by‐layer assembly of collagen type IV (Col‐IV) and laminin (LM), chosen because they are the main components of natural BMs, is reported. This multilayered Col‐IV/LM nanofilm imitates natural BM structure closely, showing controllable and similar components, thickness, and fibrous network. The Col‐IV/LM nanofilms have high cell adhesion properties and maintain the spreading morphology effectively. Furthermore, the barrier effect of preventing cell migration but permitting effective cell–cell crosstalk between fibroblasts and endothelial cells demonstrates the ability of Col‐IV/LM nanofilms for cell compartmentalization in 3D tissues, providing more reliable tissue models for evaluating drug efficacy, nanotoxicology, and implantation.