Surface-modified 3D scaffolds for tissue engineering

The aim of this work was to use sol–gel processing to develop bioactive materials to serve as scaffolds for tissue engineering that will allow the incorporation and release of proteins to stimulate cell function and tissue growth. We obtained organofunctionalized silica with large content of amine and mercaptan groups (up to 25%). The developed method can allow the incorporation and delivery of proteins at a controlled rate. We also produced bioactive foams with binary SiO₂–CaO and ternary SiO₂–CaO–P₂O₅ compositions. In order to enhance peptide–material surface properties, the bioactive foams were modified with amine and mercaptan groups. These materials exhibit a highly interconnected macroporous network and high surface area. These textural features together with the incorporation of organic functionally groups may enable them to be used as scaffolds for the engineering of soft tissue.     

Ultrafine fibrous gelatin scaffolds with deep cell infiltration mimicking 3D ECMs for soft tissue repair

In this research, ultrafine fibrous scaffolds with deep cell infiltration and sufficient water stability have been developed from gelatin, aiming to mimic the extracellular matrices (ECMs) as three dimensional (3D) stromas for soft tissue repair. The ultrafine fibrous scaffolds produced from the current technologies of electrospinning and phase separation are either lack of 3D oriented fibrous structure or too compact to be penetrated by cells. Whilst electrospun scaffolds are able to emulate two dimensional (2D) ECMs, they cannot mimic the 3D ECM stroma. In this work, ultralow concentration phase separation (ULCPS) has been developed to fabricate gelatin scaffolds with 3D randomly oriented ultrafine fibers and loose structures. Besides, a non-toxic citric acid crosslinking system has been established for the ULCPS method. This system could endow the scaffolds with sufficient water stability, while maintain the fibrous structures of scaffolds. Comparing with electrospun scaffolds, the ULCPS scaffolds showed improved cytocompatibility and more importantly, cell infiltration. This research has proved the possibility of using gelatin ULCPS scaffolds as the substitutes of 3D ECMs.     

Fabrication of nano-structured electrospun collagen scaffold intended for nerve tissue engineering

Nerve tissue engineering is one of the most promising methods in nerve tissue regeneration. The development of blended collagen and glycosaminoglycan scaffolds can potentially be used in many soft tissue engineering applications. In this study an attempt was made to develop two types of random and aligned electrospun, nanofibrous scaffold using collagen and a common type of glycosaminoglycan. Ion chromatography test, MTT and attachment assays were conducted respectively to trace the release of glycosaminoglycan, and to investigate the biocompatibility of the scaffold. Cell cultural tests showed that the scaffold acted as a positive factor to support connective tissue cell outgrowth. The positive effect of fiber orientation on cell outgrowth organization was traced through SEM images. Porosity percentage calculation and tensile strength measurement of the webs specified analogous properties to the native neural matrix tissue. These results suggested that nanostructured porous collagen-glycosaminoglycan scaffold is a potential cell carrier in nerve tissue engineering.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

3D bioprinting of multicellular scaffolds for osteochondral regeneration

Regeneration of osteochondral tissue is of great potentialities in repairing severe osteochondral defects. However, anisotropic physiological characteristics and tissue linage difference make the regeneration of osteochondral tissue remain a huge challenge. Herein, a multicellular system based on a bilayered co-culture scaffold mimicking osteochondral tissues was successfully developed for an alternative of osteochondral regeneration via a 3D bioprinting strategy. The dual function of integrally repairing both cartilage and bone could be achieved by designing multiple-cells distribution and a cell-induced bioink containing bioceramic particles. As an important bioactive agent, the Li-Mg-Si bioceramics-containing bioink exhibited the function of simultaneously stimulating multiple cells for differentiation towards specific directions. The 3D bioprinted co-culture scaffolds showed the capacity for osteochondral tissue regeneration by inducing osteogenic and chondrogenic differentiation in vitro and accelerating the repair of severe osteochondral defects in vivo. This study offers a potential strategy for complex tissue reconstruction through bioprinting multiple tissue cells in combination of bioceramics-stimulating bioinks.     

Electrospinning nanofibers to ID,2D,and 3D scaffolds and their biomedical applications

Electrospinning is a popular and effective method of producing porous nanofibers with a large surface area,superior physical and chemical properties,and a controllable pore size.Owing to these properties,electrospun nanofibers can mimic the extracellular matrix and some human tissue structures,based on the fiber configuration.Consequently,the application of electrospun nanofibers as biomaterials,varying from two-dimensional(2D)wound dressings to three-dimensional(3D)tissue engineering scaffolds,has increased rapidly in recent years.Nanofibers can either be uniform fiber strands or coaxial drug carriers,and their overall structure varies from random mesh-like mats to aligned or gradient scaffolds.In addition,the pore size of the fibers can be adjusted or the fibers can be loaded with disparate medicines to provide different functions.This review discusses the various structures and applications of 2D fiber mats and 3D nanofibrous scaffolds made up of different one-dimensional(1D)fibers in tissue engineering.In particular,we focus on the improvements made in recent years,especially in the fields of wound healing,angiogenesis,and tissue regeneration.     

Fabrication and characterization of electrospun osteon mimicking scaffolds for bone tissue engineering

Skeletal loss and bone deficiencies are a major worldwide problem with over 600,000 procedures performed in the US alone annually, making bone one of the most transplanted tissues, second to blood only. Bone is a composite tissue composed of organic matrix, inorganic bone mineral, and water. Structurally bone is organized into two distinct types: trabecular (or cancellous) and cortical (or compact) bones. Trabecular bone is characterized by an extensive interconnected network of pores. Cortical bone is composed of tightly packed units, called osteons, oriented parallel along to the axis of the bone. While the majority of scaffolds attempt to replicate the structure of the trabecular bone, fewer attempts have been made to create scaffolds to mimic the structure of cortical bone. The aim of this study was to develop a technique to fabricate scaffolds that mimic the organization of an osteon, the structural unit of cortical bone. We successfully built a rotating stage for PGA fibers and utilized it for collecting electrospun nanofibers and creating scaffolds. Resulting scaffolds consisted of concentric layers of electrospun PLLA or gelatin/PLLA nanofibers wrapped around PGA microfiber core with diameters that ranged from 200 to 600 μm. Scaffolds were mineralized by incubation in 10× simulated body fluid, and scaffolds composed of 10%gelatin/PLLA had significantly higher amounts of calcium phosphate. The electrospun scaffolds also supported cellular attachment and proliferation of MC3T3 cells over the period of 28 days.     

Elastin and collagen enhances electrospun aligned polyurethane as scaffolds for vascular graft

Mismatch in mechanical properties between synthetic vascular graft and arteries contribute to graft failure. The viscoelastic properties of arteries are conferred by elastin and collagen. In this study, the mechanical properties and cellular interactions of aligned nanofibrous polyurethane (PU) scaffolds blended with elastin, collagen or a mixture of both proteins were examined. Elastin softened PU to a peak stress and strain of 7.86 MPa and 112.28 % respectively, which are similar to those observed in blood vessels. Collagen-blended PU increased in peak stress to 28.14 MPa. The growth of smooth muscle cells (SMCs) on both collagen-blended and elastin/collagen-blended scaffold increased by 283 and 224 % respectively when compared to PU. Smooth muscle myosin staining indicated that the cells are contractile SMCs which are favored in vascular tissue engineering. Elastin and collagen are beneficial for creating compliant synthetic vascular grafts as elastin provided the necessary viscoelastic properties while collagen enhanced the cellular interactions.     

新型骨-软骨一体化修复支架材料的制备

利用可降解聚合物微球的相互粘结制备了一种新型的组织工程支架材料, 可用于软骨和软骨下骨损伤的修复。采用光学显微镜、 扫描电镜对支架的表面形貌、 内部结构进行了表征, 同时研究了支架材料的力学性能, 此外还研究了微球的粒径对支架材料孔隙率的影响。结果显示, 该材料在结构上分为乳酸-羟基乙酸共聚物(PLGA)层和PLGA/生物活性玻璃(BG)层; 材料的孔隙三维连通、 分布均匀; 采用粒径为150~200μm微球所制备的支架孔隙率为(53.37±4.39)%, 在10%的应变下材料压缩强度便已达到了0.9MPa, 显示了较强的力学性能; 随着微球粒径的变小, 材料孔隙率逐渐增大。这种微球支架在骨-软骨组织缺损修复方面有着很大的研究价值和应用价值。      

Quantifying the 3D macrostructure of tissue scaffolds

The need to shift from tissue replacement to tissue regeneration has led to the development of tissue engineering and in situ tissue regeneration. Both of these strategies often employ the use of scaffolds--templates that allow cells to attach and then guide the new tissue growth. There are many design criteria for an ideal scaffold. These criteria vary depending on the tissue type and location in the body. In any application of a scaffold it is vital to be able to characterise the scaffold before it goes into in vitro testing. In vitro testing allows the cell response to be investigated before its in vivo performance is assessed. A full characterisation of events in vitro and in vivo, in three dimensions (3D), is necessary if a scaffold's performance and effectiveness is to be fully quantified. This paper focuses on porous scaffolds for bone regeneration, suggests appropriate design criteria for a bone regenerating scaffold and then reviews techniques for obtaining the vitally important quantification of its pore structure. The techniques discussed will include newly developed methods of quantifying X-ray microtomography (microCT) images in 3D and for predicting the scaffolds mechanical properties and the likely paths of fluid flow (and hence potential cell migration). The complications in investigating scaffold performance in vitro are then discussed. Finally, the use of microCT for imaging scaffolds for in vivo tests is reviewed.     

Migration of intervertebral disc cells into dense collagen scaffolds intended for functional replacement

Invasion of cells from surrounding tissues is a crucial step for regeneration when using a-cellular scaffolds as a replacement of the nucleus pulposus (NP). The aim of current study was to assess whether NP and surrounding annulus fibrosus (AF) cells are capable of migrating into dense collagen scaffolds. We seeded freshly harvested caprine NP and AF cells onto scaffolds consisting of 1.5 and 3.0% type I collagen matrices, prepared by plastic compression, to assess cell invasion. The migration distance appeared both time and density dependent and was higher for NP (25%) compared to AF (10%) cells after 4 weeks. Migration distance was not enhanced by Hst-2, a peptide derived from saliva known to enhance fibroblast migration, and this was confirmed in a scratch assay. In conclusion, we revealed invasion of cells into dense collagen scaffolds and therewith encouraging first steps towards the use of a-cellular scaffolds for NP replacement.     

Release of lysozyme from electrospun PVA/lysozyme-gelatin scaffolds

This article describes an electrospinning process in fabricating ultra fine fibers with core-shell structure. A biodegradable polymer, poly(vinyl alcohol) (PVA), was used as the shell; lysozyme was a kind of antioxidant; and gelatin were used as the core.Morphology and microstructure of the ultra fine fibers were characterized by scanning electron microscope (SEM), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) analysis. As a comparison, composite nanofiber PVA/lysozyme-gelatin blend was prepared by a normal electrospinning process. In vitro drug release behaviors of the nanofibrous membranes were determined in phosphatebuffered saline (PBS) solution. It was found that core-shell nanofibers PVA/lysozyme-gelatin obviously exhibit higher initial release rates compared to that of PVA/lysozymegelatin blend nanofibers. The current method may find wide application in controlled release of bioactive proteins and tissue engineering.     

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solution's properties (surface tension, electrical conductivity and viscosity) and scaffold's properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation.     

Release of lysozyme from electrospun PVA/lysozyme-gelatin scaffolds

This article describes an electrospinning process in fabricating ultra fine fibers with core-shell structure. A biodegradable polymer, poly(vinyl alcohol) (PVA), was used as the shell; lysozyme was a kind of antioxidant; and gelatin were used as the core. Morphology and microstructure of the ultra fine fibers were characterized by scanning electron microscope (SEM), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) analysis. As a comparison, composite nanofiber PVA/lysozyme-gelatin blend was prepared by a normal electrospinning process. In vitro drug release behaviors of the nanofibrous membranes were determined in phosphate-buffered saline (PBS) solution. It was found that core-shell nanofibers PVA/lysozyme-gelatin obviously exhibit higher initial release rates compared to that of PVA/lysozyme-gelatin blend nanofibers. The current method may find wide application in controlled release of bioactive proteins and tissue engineering.     

Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

The electrospun scaffolds are potential application in vascular tissue engineering since they can mimic the nano-sized dimension of natural extracellular matrix (ECM). We prepared a fibrous scaffold from polycarbonateurethane (PCU) by electrospinning technology. In order to improve the hydrophilicity and hemocompatibility of the fibrous scaffold, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto the fiber surface by surface-initiated atom transfer radical polymerization (SI-ATRP) method. Although SI-ATRP has been developed and used for surface modification for many years, there are only few studies about the modification of electrospun fiber by this method. The modified fibrous scaffolds were characterized by SEM, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). The scaffold morphology showed no significant difference when PEGMA was grafted onto the scaffold surface. Based on the water contact angle measurement, the surface hydrophilicity of the scaffold surface was improved significantly after grafting hydrophilic PEGMA (P = 0.0012). The modified surface showed effective resistance for platelet adhesion compared with the unmodified surface. Activated partial thromboplastin time (APTT) of the PCU-g-PEGMA scaffold was much longer than that of the unmodified PCU scaffold. The cyto-compatibility of electrospun nanofibrous scaffolds was tested by human umbilical vein endothelial cells (HUVECs). The images of 7-day cultured cells on the scaffold surface were observed by SEM. The modified scaffolds showed high tendency to induce cell adhesion. Moreover, the cells reached out pseudopodia along the fibrous direction and formed a continuous monolayer. Hemolysis test showed that the grafted chains of PEGMA reduced blood coagulation. These results indicated that the modified electrospun nanofibrous scaffolds were potential application as artificial blood vessels.