Acceleration of osteogenic differentiation by sustained release of BMP2 in PLLA/graphene oxide nanofibrous scaffold

After about three decades of experience, tissue engineering has become one of the most important approaches in reconstructive medical research to treat non‐self‐healing bone injuries and lesions. Herein, nanofibrous composite scaffolds fabricated by electrospinning, which containing of poly(L‐lactic acid) (PLLA), graphene oxide (GO), and bone morphogenetic protein 2 (BMP2) for bone tissue engineering applications. After structural evaluations, adipose tissue derived mesenchymal stem cells (AT‐MSCs) were applied to monitor scaffold's biological behavior and osteoinductivity properties. All fabricated scaffolds had nanofibrous structure with interconnected pores, bead free, and well mechanical properties. But the best biological behavior including cell attachment, protein adsorption, and support cells proliferation was detected by PLLA‐GO‐BMP2 nanofibrous scaffold compared to the PLLA and PLLA‐GO. Moreover, detected ALP activity, calcium content and expression level of bone‐related gene markers in AT‐MSCs grown on PLLA‐GO‐BMP2 nanofibrous scaffold was also significantly promoted in compression with the cells grown on other scaffolds. In fact, the simultaneous presence of two factors, GO and BMP2, in the PLLA nanofibrous scaffold structure has a synergistic effect and therefore has a promising potential for tissue engineering applications in the repair of bone lesions.     

Multi-Material Scaffolds for Tissue Engineering

A trend in developing biocompatible scaffolds for tissue engineering has been to seek an ideal single material for which a given cell type will exhibit favorable behavior. While an ideal single material has proven elusive, scaffold manufacture using combinations of specialist materials can produce more versatile structures. By controlling the percentage and architecture of material components, mechanical properties, cell attachment, and proliferation may be optimized for a given function. Three specialist materials, poly-ϵ-caprolactone (PCL), fibrin, and alginate, were incorporated into multi-component scaffolds for a series of experiments testing each component with culture of fibroblasts. The rigid and formable PCL provided structure, the fibrin pore-filler allowed for cell attachment, and alginate thread provided a nutrient transfer pathway in lieu of a vascular system. The efficacy of these scaffolds was judged on fibroblast distribution and population after 7-12 days of culture.     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

3D Plotted PCL Scaffolds for Stem Cell Based Bone Tissue Engineering

The ability to control the architecture and strength of a bone tissue engineering scaffold is critical to achieve a harmony between the scaffold and the host tissue. Rapid prototyping (RP) technique is applied to tissue engineering to satisfy this need and to create a scaffold directly from the scanned and digitized image of the defect site. Design and construction of complex structures with different shapes and sizes, at micro and macro scale, with fully interconnected pore structure and appropriate mechanical properties are possible by using RP techniques. In this study, RP was used for the production of poly(ε-caprolactone) (PCL) scaffolds. Scaffolds with four different architectures were produced by using different configurations of the fibers (basic, basic-offset, crossed and crossed-offset) within the architecture of the scaffold. The structure of the prepared scaffolds were examined by scanning electron microscopy (SEM), porosity and its distribution were analyzed by micro-computed tomography (µ-CT), stiffness and modulus values were determined by dynamic mechanical analysis (DMA). It was observed that the scaffolds had very ordered structures with mean porosities about 60%, and having storage modulus values about 1 × 10⁷ Pa. These structures were then seeded with rat bone marrow origin mesenchymal stem cells (MSCs) in order to investigate the effect of scaffold structure on the cell behavior; the proliferation and differentiation of the cells on the scaffolds were studied. It was observed that cell proliferation was higher on offset scaffolds (262000 vs 235000 for basic, 287000 vs 222000 for crossed structure) and stainings for actin filaments of the cells reveal successful attachment and spreading at the surfaces of the fibers. Alkaline phosphatase (ALP) activity results were higher for the samples with lower cell proliferation, as expected. Highest MSC differentiation was observed for crossed scaffolds indicating the influence of scaffold structure on cellular activities.     

Temperature Responsive Shape‐Memory Scaffolds with Circumferentially Aligned Nanofibers for Guiding Smooth Muscle Cell Behavior

Structural simulation of the smooth muscle layer plays an important role in tissue engineering of blood vessels for the replacement of damaged arteries. However, it is difficult to construct small‐diameter tubular scaffolds to homogenously locate and align smooth muscle cells (SMCs). In this work, novel temperature responsive shape‐memory scaffolds are designed for SMC culturing. The scaffolds are composed of an outer layer of poly(lactide–glycolide–trimethylene carbonate) (PLGATMC) for programming the deformation from planar to small‐diameter tubular shape and an inner layer of aligned nanofibrous membrane of poly(lactide–glycolide)/chitosan (PLGA/CS) to regulate cell adhesion, proliferation, and morphology. The SMC behaviors and functions are dependent on the PLGA/CS ratios of membranes, and the scaffold with PLGA/CS 7:3 membrane exhibits the most suitable ability to regulate SMC behavior. The PLGA/CS@PLGATMC scaffold can be deformed into a temporary planar at 20 °C for convenient seeding and attachment of SMCs and then immediately self‐rolled into 3D tube at 37 °C. The proposed strategy offers a practical approach for the development of small‐diameter vascular scaffolds from 2D planar into 3D tubular shape by self‐rolling.     

Microrobotics and MEMS-based fabrication techniques for scaffold-based tissue engineering

Scaffold based tissue engineering strategies use cells, biomolecules and a scaffold to promote the repair and regeneration of tissues. Although scaffold-based tissue engineering approaches are being actively developed, most are still experimental, and it is not yet clear what defines an ideal scaffold/cell construct. Solid free form fabrication (SFF) techniques can precisely control matrix architecture (size, shape, interconnectivity, branching, geometry and orientation). The SFF methods enable the fabrication of scaffolds with various designs and material compositions, thus providing a control of mechanical properties, biological effects and degradation kinetics. This paper reviews the application of micro-robotics and MEMS-based fabrication techniques for scaffold design and fabrication. It also presents a novel robotic technique to fabricate scaffold/cell constructs for tissue engineering by the assembly of microscopic building blocks.     

In vitro blood compatibility and bone mineralization aspects of polymeric scaffold laden with essential oil and metallic particles for bone tissue engineering

Bone tissue engineering scaffolds necessities appropriate physicochemical and mechanical properties to support its renewal. Electrospun scaffolds have been used unequivocally in bone tissue restoration. The main intention of this research is to develop electrospun polyurethane (PU) scaffold decorated with metallic particles and essential oil with advanced properties to make them as a putative candidate. The nanocomposite scaffold exhibited appropriate wettability and suitable fiber diameter compared to the polyurethane scaffold. Interaction of the added constituents with the polyurethane was corroborated through hydrogen bonding formation. Tensile strength of the composites was enhanced compared to the polyurethane scaffold. Thermal analysis depicted the lower weight loss of the composite scaffold than the pristine PU. Blood coagulation was significantly delayed and also the composite surface rendered safe interaction with red blood cells. In vitro toxicity testing using fibroblast cells portrayed the nontoxic behavior of the fabricated material. The above-said advanced properties of the composite scaffold can be warranted for bone tissue engineering application.     

Ile‐Lys‐Val‐ala‐Val (IKVAV) peptide for neuronal tissue engineering

Despite the great advances in microsurgery, some neural injuries cannot be treated surgically. Stem cell therapy is a potential approach for treating neuroinjuries and neurodegenerative disease. Researchers have developed various bioactive scaffolds for tissue engineering, exhibiting enhanced cell viability, attachment, migration, neurite elongation, and neuronal differentiation, with the aim of developing functional tissue grafts that can be incorporated in vivo. Facilitating the appropriate interactions between the cells and extracellular matrix is crucial in scaffold design. Modification of scaffolds with biofunctional motifs such as growth factors, drugs, or peptides can improve this interaction. In this review, we focus on the laminin‐derived Ile‐Lys‐Val‐Ala‐Val peptide as a biofunctional epitope for neuronal tissue engineering. Inclusion of this bioactive peptide within a scaffold is known to enhance cell adhesion as well as neuronal differentiation in both 2‐dimensional and 3‐dimensional environments. The in vivo application of this peptide is also briefly described.     

Evaluation of 3D Printed Gelatin‐Based Scaffolds with Varying Pore Size for MSC‐Based Adipose Tissue Engineering

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion‐based 3D printing is used to fabricate photo‐crosslinkable gelatin‐based scaffolds with pore sizes in the range of 200–600 µm. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC‐based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.     

骨组织工程支架材料研究进展*

骨在组织工程中得到了非常广泛、深入的研究.支架材料与许多可降解材料一起也在进行探索性研究.用于骨组织工程的生物材料可以是三维多孔的刚硬材料,也可以是可注射材料.本文从聚合物角度综述了骨组织工程对支架材料的基本要求,用于骨组织工程的可降解生物材料、支架材料的设计和制备技术以及支架材料的表面修饰等方面的研究进展.     

Fabrication and characterization of chitosan-gelatin/single-walled carbon nanotubes electrospun composite scaffolds for cartilage tissue engineering applications

Cartilage is a connective tissue with a slow healing rate due to lack in blood circulation and slow metabolism. Designing tissue engineering scaffolds modified based on its specific features can assist its natural regeneration process. In this study, the chitosan-gelatin/single-walled carbon nanotubes functionalized by COOH (SWNTs-COOH) nanocomposite scaffolds were fabricated through electrospinning. The effect of each component and different duration of cross-linking were assessed in terms of morphology, porosity, chemical structure, thermal behavior, mechanical properties, wettability, biodegradability, and in vitro cell culture study. Adding SWNTs-COOH decreased fiber diameter, water contact angle and degradation rate while increased tensile strength, hydrophilicity, stability and cell viability, due to their high intrinsic electrical conductivity, and mechanical properties and the presence of COOH functional groups in its structure. All the sample presented a porosity percentage of more than 80%, which is essential for tissue engineering scaffolds. The presence SWNTs-COOH did not have any adverse effect on cytocompatibility. The optimal cross-linking time increased the stability of the scaffolds in PBS. It can be concluded that the chitosan-gelatin/1wt% SWNTs-COOH scaffold can be appropriate for cartilage tissue engineering applications.     

Modifying the Pores of an Inverse Opal Scaffold With Chitosan Microstructures for Truly Three‐Dimensional Cell Culture

Inverse opal scaffolds have recently emerged as a novel class of scaffolds with uniform and controllable pore sizes for tissue engineering to provide better nutrient transport, a uniform cell distribution, and an adjustable microenvironment for cell differentiation. However, when the pore size of the scaffold is much larger than the dimension of a cell, the cell actually encounters a local 2D environment and the void space associated with the pore can not be efficiently utilized. Here, we demonstrate that a truly 3D microenvironment can be created inside a pore by further functionalizing the as‐prepared inverse opal scaffold with a second polymer by freeze‐drying. The resultant inverse opal scaffold with hierarchically structured pores can enhance both cell proliferation and tissue infiltration.     

Fabricating gradient hydrogel scaffolds for 3D cell culture

Optimizing cell-material interactions is critical for maximizing regeneration in tissue engineering. Combinatorial and high-throughput (CHT) methods can be used to systematically screen tissue scaffolds to identify optimal biomaterial properties. Previous CHT platforms in tissue engineering have involved a two-dimensional (2D) cell culture format where cells were cultured on material surfaces. However, these platforms are inadequate to predict cellular response in a three-dimensional (3D) tissue scaffold. We have developed a simple CHT platform to screen cell-material interactions in 3D culture format that can be applied to screen hydrogel scaffolds. Herein we provide detailed instructions on a method to prepare gradients in elastic modulus of photopolymerizable hydrogels.     

Enhancement of biocompatibility on aligned electrospun poly(3‐hydroxybutyrate) scaffold immobilized with laminin towards murine neuroblastoma Neuro2a cell line and rat brain‐derived neural stem cells (mNSCs)

Electrospinning has been extensively used to construct tissue‐engineered scaffolds because of its ability to provide the fibrous scaffold with structurally analogous to the naturally occurring protein in the extracellular matrix of native tissues. In addition, the modification of scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation, and differentiation. In the present contribution, we prepared and investigated the potential used of aligned electrospun poly(3‐hydroxybutyrate) (PHB) scaffold immobilized with bioactive molecule to serve as nervous scaffold. Laminin was successfully immobilized on the surface using covalent binding between functional groups of modified scaffolds and protein. The ability to use for neural regeneration was evaluated in vitro towards murine neuroblastoma Neuro2a cell line and mouse brain‐derived neural stem cells. The surface modification with laminin immobilized on the PHB fibrous scaffolds supported the attachment and promoted the proliferation of Neuro2a very wells. Despite the good attachment and proliferation of Neuro2a and mouse brain‐derived neural stem cells were not able to proliferate on the neat PHB, hydrolyzed PHB and laminin immobilized on hydrolyzed PHB fibrous scaffold.     

Utilization of PVPA and its effect on the material properties and biocompatibility of PVA electrospun membrane

This study aims to investigate on the effect of the addition of polyvinylphosphonic acid (PVPA) on the material properties and biocompatibility of a polyvinyl alcohol (PVA) polymer tissue engineering scaffold fabricated by electrospinning process. Stabilization of the membranes was carried out by heat application. Fourier transform infrared spectroscopy confirmed the presence of P?O bonds and P–OH bonds and increase in the atomic percentage of phosphorus in Energy Dispersive Spectroscopy (EDS) indicated successful incorporation of PVPA to the PVA. Scanning electron microscope fiber morphology and microstructure showed that addition of PVPA reduced the average fiber diameter of the scaffold. After 1 hr of phosphate buffered saline immersion, scaffolds were observed to swell to almost 200% of their original weight. Increased tensile strength was observed at 0.1% PVPA addition but reduced values were observed when the concentration was greater than 0.1%. Cytocompatibility was examined with M3CT3‐E1 preosteoblast cells through cell viability after exposure to extract solutions and Live/Dead cell staining. Cell proliferation was quantified by MTT assay; cell adhesion was visualized by scanning electron microscope and confocal microscopy images. Bone regeneration was also observed and quantified using histomorphometric analysis and histological staining methods after implantation in rat calvarium for 4 weeks. Copyright © 2013 John Wiley & Sons, Ltd.     

A Two‐Tailed Phosphopeptide Crystallizes to Form a Lamellar Structure

The crystal structure of a designed phospholipid‐inspired amphiphilic phosphopeptide at 0.8 Å resolution is presented. The phosphorylated β‐hairpin peptide crystallizes to form a lamellar structure that is stabilized by intra‐ and intermolecular hydrogen bonding, including an extended β‐sheet structure, as well as aromatic interactions. This first reported crystal structure of a two‐tailed peptidic bilayer reveals similarities in thickness to a typical phospholipid bilayer. However, water molecules interact with the phosphopeptide in the hydrophilic region of the lattice. Additionally, solid‐state NMR was used to demonstrate correlation between the crystal structure and supramolecular nanostructures. The phosphopeptide was shown to self‐assemble into semi‐elliptical nanosheets, and solid‐state NMR provides insight into the self‐assembly mechanisms. This work brings a new dimension to the structural study of biomimetic amphiphilic peptides with determination of molecular organization at the atomic level.     

Control of Stem‐Cell Behavior by Fine Tuning the Supramolecular Assemblies of Low‐Molecular‐Weight Gelators

Controlling the behavior of stem cells through the supramolecular architecture of the extracellular matrix remains an important challenge in the culture of stem cells. Herein, we report on a new generation of low‐molecular‐weight gelators (LMWG) for the culture of isolated stem cells. The bola‐amphiphile structures derived from nucleolipids feature unique rheological and biological properties suitable for tissue engineering applications. The bola‐amphiphile‐based hydrogel scaffold exhibits the following essential properties: it is nontoxic, easy to handle, injectable, and features a biocompatible rheology. The reported glycosyl‐nucleoside bola‐amphiphiles (GNBA) are the first examples of LMWG that allow the culture of isolated stem cells in a gel matrix. The results (TEM observations and rheology) suggest that the supramolecular organizations of the matrix play a role in the behavior of stem cells in 3D environments.     

Novel 3D Neuron Regeneration Scaffolds Based on Synthetic Polypeptide Containing Neuron Cue

Neural tissue engineering has become a potential technology to restore the functionality of damaged neural tissue with the hope to cure the patients with neural disorder and to improve their quality of life. This paper reports the design and synthesis of polypeptides containing neuron stimulate, glutamic acid, for the fabrication of biomimetic 3D scaffold in neural tissue engineering application. The polypeptides are synthesized by efficient chemical reactions. Monomer γ‐benzyl glutamate‐N‐carboxyanhydride undergoes ring‐opening polymerization to form poly(γ‐benzyl‐l ‐glutamate), then hydrolyzes into poly(γ‐benzyl‐l ‐glutamate)‐r‐poly(glutamic acid) random copolymer. The glutamic acid amount is controlled by hydrolysis time. The obtained polymer molecular weight is in the range of 200 kDa for good quality of fibers. The fibrous 3D scaffolds of polypeptides are fabricated using electrospinning techniques. The scaffolds are biodegradable and biocompatible. The biocompatibility and length of neurite growth are improved with increasing amount of glutamic acid in scaffold. The 3D scaffold fabricated from aligned fibers can guide anisotropic growth of neurite along the fiber and into 3D domain. Furthermore, the length of neurite outgrowth is longer for scaffold made from aligned fibers as compared with that of isotropic fibers. This new polypeptide has potential for the application in the tissue engineering for neural regeneration.