Preparation Of Scaffolds For Tissue Engineering Research Articles

Silk Fibroin Combined with Electrospinning as a Promising Strategy for Tissue Regeneration.

The development of tissue engineering scaffolds is of great significance for the repair and regeneration of damaged tissues and organs. Silk fibroin (SF) is a natural protein polymer with good biocompatibility, biodegradability, excellent physical and mechanical properties and processability, making it an ideal universal tissue engineering scaffold material. Nanofibers prepared by electrospinning have attracted extensive attention in the field of tissue engineering due to their excellent mechanical properties, high specific surface area, and similar morphology as to extracellular matrix (ECM). The combination of silk fibroin and electrospinning is a promising strategy for the preparation of tissue engineering scaffolds. In this review, the research progress of electrospun silk fibroin nanofibers in the regeneration of skin, vascular, bone, neural, tendons, cardiac, periodontal, ocular and other tissues is discussed in detail.

Polyaniline Functionalized Peptide Self-Assembled Conductive Hydrogel for 3D Cell Culture.

The functionalization of self-assembled peptide hydrogel is of great importance to broaden its applications in the field of biomedicine. In this work, conductive hydrogel is fabricated by introducing conductive polymer polyaniline into peptide self-assembled hydrogel. Compared with pure peptide formed hydrogel, the conductive hydrogel exhibits enhanced conductivity, mechanical property and stability. In addition, the hydrogel is tested to be of great injectability and 3D bio-printability and could support the viability of encapsulated cells that are sensitive to electrical signals. It should have great application prospects in the preparation of tissue engineering scaffolds.

Preparation, Properties, and Application of Graphene-Based Materials in Tissue Engineering Scaffolds.

Tissue engineering has a great application prospect as an effective treatment for tissue and organ injury, functional reduction, or loss. Bioactive tissues are reconstructed and damaged organs are repaired by the three elements, including cells, scaffold materials, and growth factors. Graphene-based composites can be used as reinforcing auxiliary materials for tissue scaffold preparation because of their large specific surface area, and good mechanical support. Tissue engineering scaffolds with graphene-based composites have been widely studied. Part of research have focused on the application of graphene-based composites in single tissue engineering. The basic principles of graphene materials used in tissue engineering are summarized in some research. Some studies emphasized the key problems and solutions urgently needed to be solved in the development of tissue engineering and discussed their application prospect. Some related studies mainly focused on the conductivity of graphene and discussed the application of electroactive scaffolds in tissue engineering. In this review, the composite materials for preparing tissue engineering scaffolds are briefly described, which emphasizes the preparation methods, biological properties, and practical applications of graphene-based composite scaffolds. The synthetic techniques, with stressing solvent casting, electrospinning, and three-dimensional printing, are introduced in detail. The mechanical, cell-oriented, and biocompatible properties of graphene-based composite scaffolds in tissue engineering are analyzed and summarized. Their applications in bone tissue engineering, nerve tissue engineering, cardiovascular tissue engineering, and other tissue engineering are summarized systematically. In addition, this work also looks forward to the difficulties and challenges in the future research, providing some references for the follow-up research of graphene-based composites in tissue engineering scaffolds. Impact statement Regeneration and repair of tissue and organ injury has become a new research hotspot in recent years. Tissue engineering scaffolds prepared with graphene-based materials have good biocompatibility, excellent mechanical properties, and strong cell orientation, which can fully induce the proliferation and differentiation of seed cells. This review briefly describes the basic materials for the preparation of tissue engineering scaffolds, and focuses on the preparation, performance, and application of graphene-based materials in tissue engineering, providing sufficient understanding of graphene applied in regenerative medicine.

Applications of 3D Bio-Printing in Tissue Engineering and Biomedicine.

In recent years, 3D bio-printing technology has developed rapidly and become an advanced bio-manufacturing technology. At present, 3D bio-printing technology has been explored in the fields of tissue engineering, drug testing and screening, regenerative medicine and clinical disease research and has achieved many research results. Among them, the application of 3D bio-printing technology in tissue engineering has been widely concerned by researchers, and it contributing many breakthroughs in the preparation of tissue engineering scaffolds. In the future, it is possible to print fully functional tissues or organs by using 3D bio-printing technology which exhibiting great potential development prospects in th applications of organ transplantation and human body implants. It is expected to solve thebiomedical problems of organ shortage and repair of damaged tissues and organs. Besides,3Dbio-printing technology will benefit human beings in more fields. Therefore, this paper reviews the current applications, research progresses and limitations of 3D bio-printing technology in biomedical and life sciences, and discusses the main printing strategies of 3D bio-printing technology. And, the research emphases, possible development trends and suggestions of the application of 3D bio-printing are summarized to provide references for the application research of 3D bio-printing.

Extraction of pectin from albedo of lemon peels for preparation of tissue engineering scaffolds

Pectin is a type of anionic polysaccharide naturally found in a number of fruits and vegetables. Although pectin is widely used for food industry, its biomedical applications such as wound dressing, drug delivery and cancer targeting have also been investigated. In our study, we combined extracted pectin (from albedo of lemon peels) with chitosan (as a natural polymer) to synthesize chitosan/pectin cryogels. The extracted pectin was subjected to qualitative and quantitative analyses. Chitosan/pectin spongy supermacroporous cryogels were produced by cryogelation method at different combinations (100:0, 80:20, 60:40 and 40:60, w/w). Polyelectrolyte interactions between pectin and chitosan and crosslinking of chitosan with glutaraldehyde were verified by using FTIR. The porosity, swelling ratio, degradation behaviors and mechanical properties of cryogels were determined. SEM analysis demonstrated the pore morphology and average pore diameters of cryogels. After all analysis, 40:60 chitosan/pectin cryogel was selected for cytotoxicity studies. Glioblastoma (U-87 MG) cell line was used to evaluate the in vitro cytotoxicity of scaffolds. MTT assay and SEM analyses demonstrated the scaffolds were nontoxic, and supported cell attachment and viability.

Facile preparation of tissue engineering scaffolds with pore size gradients using the muesli effect and their application to cell spheroid encapsulation.

Porous biodegradable scaffolds have many applications in bioengineering, ranging from cell culture and transplantation, to support structures, to induce blood vessel and tissue formation in vivo. While numerous strategies have been developed for the manufacture of porous scaffolds, it remains challenging to control the spatial organization of the pores. In this study, we introduce the use of the granular convection effect, also known as the muesli or brazil nut effect, to rapidly engineer particulate templates with a vertical size gradient. These templates can then be used to prepare scaffolds with pore size gradients. To demonstrate this approach, we prepared templates with particle size gradients, which were then infused with a prepolymer solution consisting of the pentaerythritol ethoxylate (polyol), sebacoyl chloride (acid chloride), and poly(caprolactone). Following curing, the template was dissolved to yield biodegradable polyester-ether scaffolds with pore size gradients that could be tuned depending on the size range of the particulates used. The application of these scaffolds was demonstrated using pancreatic islets, which were loaded via centrifugation and retained within the scaffold's pores without a decrease in viability. The proposed strategy provides a facile approach to prepare templates with spatially organized pores that could potentially be used for cell transplantation, or guided tissue formation.

Fabrication and In Situ Cross-Linking of Carboxylic-Acid-Functionalized Poly(Ester Amide) Scaffolds for Tissue Engineering

Three-dimensional (3D) scaffolds are important tools for tissue engineering and should ideally provide both biochemical cues and biomechanical support for cells. Poly(ester amide)s (PEAs) have emerged as promising materials for the preparation of tissue engineering scaffolds, and the pendant side chains of residues, such as l-lysine and l-aspartic acid, can provide sites for the conjugation of biochemical signals. However, it has been challenging to combine scaffold morphological stability with the presentation of reactive groups on PEA scaffolds. We describe here a new approach involving the functionalization of a l-lysine-containing PEA with maleic anhydride to simultaneously introduce cross-linkable alkenes and carboxylic acid conjugation sites. Maleic-acid-functionalized PEA was processed to form 3D scaffolds using a salt leaching method and the scaffolds were cross-linked in situ using a poly(ethylene glycol) dimethacrylate cross-linking agent by thermal free radical curing. Microcomputed tomography analysis indicated that the cross-linked scaffolds had higher polymer volume fraction, lower porosity, and smaller pore size than the non-cross-linked scaffolds, but both scaffolds exhibited high morphological stability and negligible mass loss upon incubation in phosphate-buffered saline for 5 days. The Young’s moduli of the cross-linked and non-cross-linked scaffolds were 28 and 9 kPa, respectively. Fluorescein-labeled bovine serum albumin was successfully conjugated to the scaffolds using a carbodiimide-based coupling. Finally, it was shown that the scaffolds supported the attachment and proliferation of mouse embryonic mesenchymal multipotent cells, showing their promise as platforms for tissue engineering applications.

Stem cells combined 3D electrospun nanofibrous and macrochannelled matrices: a preliminary approach in repair of rat cranial bones

Repair of cranial bone defects is an important problem in the clinical area. The use of scaffolds combined with stem cells has become a focus in the reconstruction of critical-sized bone defects. Electrospinning became a very attracting method in the preparation of tissue engineering scaffolds in the last decade, due to the unique nanofibrous structure of the electrospun matrices. However, they have a limitation for three dimensional (3D) applications, due to their two-dimensional structure and pore size which is smaller than a cellular diameter which cannot allow cell migration within the structure. In this study, electrospun poly(ε-caprolactone) (PCL) membranes were spirally wounded to prepare 3D matrices composed of nanofibers and macrochannels. Mesenchymal stromal/stem cells were injected inside the scaffolds after the constructs were implanted in the cranial bone defects in rats. New bone formation, vascularisation and intramembranous ossification of the critical size calvarial defect were accelerated by using mesenchymal stem cells combined 3D spiral-wounded electrospun matrices.

Description of D-glucosamine immobilization kinetics onto poly(lactic acid) surface via a multistep physicochemical approach for preparation of novel active biomaterials.

Poly(lactic acid) (PLA) has shown much success in the preparation of tissue engineering scaffolds as it can be fabricated with a tailored architecture. However, the PLA surface has drawbacks including the lack of biofunctional motifs which are essential for high affinity to biological cells. Therefore, this study describes a multistep physicochemical approach for the immobilization of d-glucosamine (GlcN), a naturally occurring monosaccharide having many biological functions, on the PLA surface aiming at enhancing the cell proliferation activity. In this approach, poly(acrylic acid) (PAAc) spacer arms are first introduced into the PLA surface via plasma post-irradiation grafting technique. Then, covalent coupling or physical adsorption of GlcN with/on the PAAc spacer is carried out. Factors affecting the grafting yield are controlled to produce a suitable spacer for bioimmobilization. X-ray photon spectroscopic (XPS) analyses confirm the immobilization of GlcN on the PLA surface. The XPS results reveal also that increasing the yield of grafted PAAc spacer on the PLA surface increases the amount of covalently immobilized GlcN, but actually inhibits the immobilization process using the physical adsorption method. Contact angle measurements and atomic force microscopy (AFM) show a substantial increase of surface energy and roughness of PLA surface, respectively, upon the multistep modification procedure. The cytocompatibility of the modified surfaces is assessed using a mouse embryonic fibroblast (MEF) cell line. Observation from the cell culture basically demonstrates the potential of GlcN immobilization in improving the cytocompatibility of the PLA surface. Moreover, the covalent immobilization of GlcN seems to produce more cytocompatible surfaces if compared with the physical adsorption method. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3176-3188, 2017.

Fabrication of polycaprolactone electrospun fibers with different hierarchical structures mimicking collagen fibrils for tissue engineering scaffolds

The ability to topographically mimic the surface features of collagen fibrils is an important step in the preparation of tissue engineering scaffolds. It is important to know which kinds of surface topographies of electrospun fibers are more favorable for cell growth. In this study, fibers with three kinds of hierarchical-structured surfaces were fabricated by electrospinning to mimic collagen fibrils. By combining thermally induced phase separation (TIPS) and non-solvent induced phase separation (NIPS), polycaprolactone (PCL) fibers with a porous surface were electrospun from PCL in a chloroform (CF)/dimethyl sulfoxide (DMSO) mixed solution. In addition, two additional types of fibrous membranes, with PCL fibers being the shish and decorated by PCL kebabs on the surface, were created by two different controlled homoepitaxic crystallization methods—the solution incubation method and the solvent evaporation method. It was found that the solvent evaporation method was more effective in forming kebabs and the primary optimal processing parameters were identified. The presence of pores on the fiber surfaces contributed to a much larger surface area and a higher total volume of pores. To investigate the cellular response on such scaffolds, 3T3 fibroblast cell and human umbilical vein endothelial cell (HUVEC) assays were conducted and the results indicated that both of the nanotopographies on the surfaces of the scaffolds improved cell viability and proliferation. Furthermore, the porous surface was more beneficial for enhancing cellular responses, which suggests better biocompatibility and greater potential to mimic collagen fibrils for tissue engineering application, and especially as scaffolds for endothelial layers in blood vessels.

Evaluation of Extracted β-chitosan From Loligo duvauceli for the Preparation of Tissue Engineering Scaffolds

Over the years, there has been a growing need for producing cost effective, porous scaffold biomaterials for tissue engineering. Therefore, this study focused on fabrication of β-chitosan based scaffold from the waste of the pen of locally available squid variety Loligo duvauceli (Indian Squid). The scaffold was prepared by the solvent casting method which requires less time and minimum energy. To the best of our knowledge,there are no reports on scaffold preparation from the extracted β-chitosan. The biological properties of extracted chitosan scaffolds C(L) were evaluated and compared with those of commercial chitosan scaffold C(S). The prepared scaffolds were characterized by SEM, swelling property, in vitro enzymatic degradation, and hemo, biocompatibility properties. C(L) scaffolds had an average pore size of 60 µm and the swelling ratio was 16.2 ± 2.2% with mass loss percentage of 20.8 ± 4.5% after 4 weeks of degradation. It has exhibited good hemocompatible properties and thermal stability. Mouse fibroblast 3T3 cells were able to adhere and proliferate well in the extracted β-chitosan C(L) scaffold. All these results indicated that extracted β-chitosan C(L) is a suitable alternative for biomaterials used for tissue engineering purposes.

Comparative profiling of extractable proteins in extracellular matrices of porcine cholecyst and jejunum intended for preparation of tissue engineering scaffolds.

Scaffolds prepared from cholecyst and jejunum have differential immunological potential, despite similar biocompatibility, when used as subcutaneous grafts. The reason for differential immunogenicity is probably due to differences in the nature of protein composition and biomolecules in the extracellular matrices (ECMs) of source organs that are used for preparation of the scaffolds. Against this background, the present study aims to identify the extractable proteins of ECMs derived from porcine cholecyst and jejunum. The proteins were extracted and identified through a conventional database search following sodium dodecyl sulfate-polyacrylamide gel-electrophoresis separation and mass spectroscopy. The resultant protein profile was analyzed and at least 154 proteins in cholecyst-derived extracellular matrix (CDE) and 186 proteins in jejunum-derived extracellular matrix (JDE) were identified. Both the matrices contained several extracelluar proteins including fibronectin, nidogen, decorin, and lumican that are known to participate in wound healing responses. However, the CDE had fewer cellular proteins than JDE, especially the latter contained class-I and class-II histocompatibility antigens which are incriminated as potent immunogens responsible for graft rejection. The results of the study suggested that the ECMs used for the scaffold preparation need not be "acellular" and differences in the protein composition of the ECMs might have caused the differential wound healing responses. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 489-496, 2017.

Highly adjustable biomaterial networks from three-armed biodegradable macromers

Biocompatible material platforms with adjustable properties and option for chemical modification are warranted for site-specific biomedical applications. To this end, three-armed biodegradable macromers of well-defined chemical characteristics were prepared from trivalent alcohols with different degrees of ethoxylation and different lengths of oligoester domains. A platform of 15 different macromers was established. The macromers were designed to exhibit different hydrophilicities and molecular weights and contained various types of oligoesters such as d,l-lactide, l-lactide and ε-caprolactone. Macromers chemical composition was determined and molecular weights ranged from 900 to 3000Da. Thermally induced cross-linking of methacrylated macromers was monitored by oscillation rheology. A novel variant of the solid lipid templating technique was established to fabricate macroporous tissue engineering scaffolds from these macromers. Scaffold properties were thoroughly investigated regarding mechanical properties, compositional analysis including methacrylic double bond conversion, microstructure and porosity. Material properties could be controlled by macromer chemistry. By variation of the fabrication procedure and processing parameters scaffold porosity was increased up to 88%. Basic cytocompatibility was assessed including indirect and direct contact methods. The established macromers hold promise for various biomedical purposes. Statement of SignificanceSpecific biomedical applications require tailored biomaterials with defined properties. We established a macromer platform for preparation of tissue engineering scaffolds with adjustable chemical and mechanical characteristics. Macromers were composed of trivalent core alcohols with different degrees of ethoxylation to which biodegradable domains – lactide or ε-caprolactone – were oligomerized before final methacrylation. The solid lipid templating technique was adapted to fabricate macroporous scaffolds with controlled pore structure and porosity from the developed macromers, which can also be processed by solid freeform fabrication techniques. The material platform relies on clinically established chemistries of the biodegradable domains and the macromer concept enables the fabrication of networks in which cross-polymerization kinetics, mechanical properties and surface hydrophobicity is predefined by macromer chemistry. Cytocompatibility was confirmed by indirect and direct cell contact experiments.

Influence of stepwise chondrogenesis-mimicking 3D extracellular matrix on chondrogenic differentiation of mesenchymal stem cells

Extracellular matrix (ECM) has drawn a broad attention for preparation of tissue engineering scaffolds and stem cell study. ECM scaffolds stepwise mimicking development of tissues can provide useful models to investigate the interactions between stem cells and ECM during the process of tissue development. In this study, 3D stepwise chondrogenesis-mimicking ECM scaffolds were prepared from mesenchymal stem cells (MSCs) by controlling the stages of chondrogenic differentiation. ECM scaffolds mimicking the early stage and late stage of chondrogenesis were obtained when MSCs were cultured in the chondrogenic medium for 1 and 3 w, respectively. The ECM scaffolds had different compositions as shown by immunohistochemical analysis. Stem cell (SC)-ECM scaffold was rich in collagen I and biglycan. Early stage chondrogenesis-mimicking (CE)-ECM scaffold had moderate amount of collagen II and aggrecan while late stage chondrogenesis-mimicking (CL)-ECM scaffold were rich in collagen II and aggrecan. These three ECM scaffolds had different effects on chondrogenesis of MSCs. The CE-ECM scaffold facilitated chondrogenesis, however the CL-ECM scaffolds remarkably inhibited chondrogenesis of MSCs. These ECM scaffolds not only can provide new 3D ECM models to investigate the effects of ECM on MSCs functions, but also can be used as favorable ECM scaffolds for tissue engineering.

Mathematically defined tissue engineering scaffold architectures prepared by stereolithography

The technologies employed for the preparation of conventional tissue engineering scaffolds restrict the materials choice and the extent to which the architecture can be designed. Here we show the versatility of stereolithography with respect to materials and freedom of design. Porous scaffolds are designed with computer software and built with either a poly( d, l-lactide)-based resin or a poly( d, l-lactide- co-ε-caprolactone)-based resin. Characterisation of the scaffolds by micro-computed tomography shows excellent reproduction of the designs. The mechanical properties are evaluated in compression, and show good agreement with finite element predictions. The mechanical properties of scaffolds can be controlled by the combination of material and scaffold pore architecture. The presented technology and materials enable an accurate preparation of tissue engineering scaffolds with a large freedom of design, and properties ranging from rigid and strong to highly flexible and elastic.

A poly( d, l-lactide) resin for the preparation of tissue engineering scaffolds by stereolithography

Porous polylactide constructs were prepared by stereolithography, for the first time without the use of reactive diluents. Star-shaped poly( d, l-lactide) oligomers with 2, 3 and 6 arms were synthesised, end-functionalised with methacryloyl chloride and photo-crosslinked in the presence of ethyl lactate as a non-reactive diluent. The molecular weights of the arms of the macromers were 0.2, 0.6, 1.1 and 5 kg/mol, allowing variation of the crosslink density of the resulting networks. Networks prepared from macromers of which the molecular weight per arm was 0.6 kg/mol or higher had good mechanical properties, similar to linear high-molecular weight poly( d, l-lactide). A resin based on a 2-armed poly( d, l-lactide) macromer with a molecular weight of 0.6 kg/mol per arm (75 wt%), ethyl lactate (19 wt%), photo-initiator (6 wt%), inhibitor and dye was prepared. Using this resin, films and computer-designed porous constructs were accurately fabricated by stereolithography. Pre-osteoblasts showed good adherence to these photo-crosslinked networks. The proliferation rate on these materials was comparable to that on high-molecular weight poly( d, l-lactide) and tissue culture polystyrene.

Fumaric Acid Monoethyl Ester-Functionalized Poly(d,l-lactide)/N-vinyl-2-pyrrolidone Resins for the Preparation of Tissue Engineering Scaffolds by Stereolithography

Polymer networks were prepared by photocross-linking fumaric acid monoethyl ester (FAME) functionalized, three-armed poly(D,L-lactide) oligomers using N-vinyl-2-pyrrolidone (NVP) as diluent and comonomer. The use of NVP together with FAME-functionalized oligomers resulted in copolymerization at high rates, and networks with gel contents in excess of 90% were obtained. The hydrophilicity of the poly(D,L-lactide) networks increases with increasing amounts of NVP, networks containing 50 wt % of NVP absorbed 40% of water. As the amount of NVP was increased from 30 to 50 wt %, the Young's modulus after equilibration in water decreased from 0.8 to 0.2 GPa, as opposed to an increase from 1.5 to 2.1 GPa in the dry state. Mouse preosteoblasts readily adhered and spread onto all prepared networks. Using stereolithography, porous structures with a well-defined gyroid architecture were prepared from these novel materials. This allows the preparation of tissue engineering scaffolds with optimized pore architecture and tunable material properties.

In vivo performance of simvastatin‐loaded electrospun spiral‐wound polycaprolactone scaffolds in reconstruction of cranial bone defects in the rat model

Reconstruction of large bone defects is still a major problem. Tissue-engineering approaches have become a focus in regeneration of bone. In particular, critical-sized defects do not ossify spontaneously. The use of electrospinning is attracting increasing attention in the preparation of tissue-engineering scaffolds. Recently, acellular scaffolds carrying bioactive agents have been used as scaffolds in "in situ" tissue engineering for soft and hard tissue repair. Poly(epsilon-caprolactone) (PCL) with two different molecular weights were synthesized, and the blends of these two were electrospun into nonwoven membranes composed of nanofibers/micropores. To stimulate bone formation, an active drug, "simvastatin" was loaded either after the membranes were formed or during electrospinning. The matrices were then spiral-wound to produce scaffolds with 3D-structures having both macro- and microchannels. Eight-millimeter diameter critical size cranial defects were created in rats. Scaffolds with or without simvastatin were then implanted into these defects. Samples from the implant sites were removed after 1, 3, and 6 months postimplantation. Bone regeneration and tissue response were followed by X-ray microcomputed tomography and histological analysis. These in vivo results exhibited osseous tissue integration within the implant and mineralized bone restoration of the calvarium. Both microCT and histological data clearly demonstrated that the more successful results were observed with the "simvastatin-containing PCL scaffolds," in which simvastatin was incorporated into the PCL scaffolds during electrospinning. For these samples, bone mineralization was quite significant when compared with the other groups.