Fabrication of three-dimensional nano, micro and micro/nano scaffolds of porous poly(lactic acid) by electrospinning and comparison of cell infiltration by Z-stacking/three-dimensional projection technique

The use of electrospun extracellular matrix (ECM)-mimicking nanofibrous scaffolds for tissue engineering is limited by poor cellular infiltration. The authors hypothesised that cell penetration could be enhanced in scaffolds by using a hierarchical structure where nano fibres are combined with micron-scale fibres while preserving the overall scaffold architecture. To assess this, we fabricated electrospun porous poly(lactic acid) (PLA) scaffolds having nanoscale, microscale and combined micro/nano architecture and evaluated the structural characteristics and biological response in detail. Although the bioactivity was intermediate to that for nanofibre and microfibre scaffold, a unique result of this study was that the micro/nano combined fibrous scaffold showed improved cell infiltration and distribution than the nanofibrous scaffold. Although the cells were found to be lining the scaffold periphery in the case of nanofibrous scaffold, micro/nano scaffolds had cells dispersed throughout the scaffold. Further, as expected, the addition of nanoparticles of hydroxyapatite (nHAp) improved the bioactivity, although it did not play a significant role in cell penetration. Thus, this strategy of creating a three-dimensional (3D) micro/nano architecture that would increase the porosity of the fibrous scaffold and thereby improving the cell penetration, can be utilised for the generation of functional tissue engineered constructs in vitro.     

In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/     

Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends.

The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing failing or malfunctioning organs, by means of combining a scaffold/support material, adequate cells and bioactive molecules. Different materials have been proposed to be used as both three-dimensional porous scaffolds and hydrogel matrices for distinct tissue engineering strategies. Among them, polymers of natural origin are one of the most attractive options, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this review, the most studied and promising and recently proposed naturally derived polymers that have been suggested for tissue engineering applications are described. Different classes of such type of polymers and their blends with synthetic polymers are analysed, with special focus on polysaccharides and proteins, the systems that are more inspired by the ECM. The adaptation of conventional methods or non-conventional processing techniques for processing scaffolds from natural origin based polymers is reviewed. The use of particles, membranes and injectable systems from such kind of materials is also overviewed, especially what concerns the present status of the research that should lead towards their final application. Finally, the biological performance of tissue engineering constructs based on natural-based polymers is discussed, using several examples for different clinically relevant applications.     

The effect of process variables on the properties of melt-spun poly(lactic acid) fibres for potential use as scaffold matrix materials

Biodegradable materials in the form of fibres and yarns have attracted increasing attention due to a large surface area and various geometric possibilities in three-dimensional polymeric scaffolds for tissue engineering applications. In this study, poly(lactic acid) fibres were produced by melt spinning and subsequent solid-state drawing in order to serve as matrix materials for fibre-based scaffold architectures. The processing of both monofilament and multifilament fibres as well as draw ratios and temperatures were investigated to analyze the effect of process variables on the properties. Two different polylactides with different molecular weight were studied and characterized in terms of their tensile and thermal properties and morphology. The relevance of fibre formation, solid-state drawing and drawing temperatures was clearly supported by the results, and it was shown that the physical properties, such as crystallinity, mechanical strength and ductility can be controlled largely by the drawing process. The obtained fibres demonstrated great potential to be further processed into biotextiles (woven, knitted, or nonwoven scaffolds) using the textile technologies.     

A novel bioactive porous bredigite (Ca<Subscript>7</Subscript>MgSi<Subscript>4</Subscript>O<Subscript>16</Subscript>) scaffold with biomimetic apatite layer for bone tissue engineering

The aim of this study was to develop a novel bioactive, degradable and cytocompatible bredigite (Ca₇MgSi₄O₁₆) scaffold with biomimetic apatite layer for bone tissue engineering. Porous bredigite scaffolds were prepared using polymer sponge method. The bredigite scaffolds with biomimetic apatite layer (BTAP) were obtained by soaking bredigite scaffolds in simulated body fluid (SBF) for 10 days. The porosity and in vitro degradability of BTAP scaffolds were investigated. In addition, osteoblast-like cell morphology, proliferation and differentiation on BTAP scaffolds were evaluated and compared with β-tricalcium phosphate (β-TCP) scaffolds. The results showed that BTAP scaffolds possessed 90% of porosity. The degradation of BTAP scaffolds was comparable to that of β-TCP scaffolds. Cells on BTAP scaffolds spread well and presented a higher proliferation rate and differentiation level as compared with those on β-TCP scaffolds. Our results indicated that BTAP scaffolds were degradable and possessed the function to enhance cell proliferation and differentiation, and might be used as bone tissue engineering materials.     

Scaffold design and manufacturing: from concept to clinic

Since Robert Langer and colleagues pioneered the concept of reconstructing tissue using cells transplanted on synthetic polymer matrices in the early 1990s, research in the field of tissue engineering and regenerative medicine has exploded. This is especially true in the development of new materials and structures that serve as scaffolds for tissue reconstruction. The basic tenet of the last two decades holds scaffolds as degradable materials providing temporary function while enhancing tissue regeneration through the delivery of biologics. Although a number of new scaffolding materials and structures have been developed in research laboratories, the application of such materials practice even has been extremely limited. This paper argues that better integration of all these factors is needed to bring scaffolds from "concept to clinic". It reviews current work in all these areas and suggests where future work and funding is needed.     

Nanotechnological strategies for engineering complex tissues

Tissue engineering aims at developing functional substitutes for damaged tissues and organs. Before transplantation, cells are generally seeded on biomaterial scaffolds that recapitulate the extracellular matrix and provide cells with information that is important for tissue development. Here we review the nanocomposite nature of the extracellular matrix, describe the design considerations for different tissues and discuss the impact of nanostructures on the properties of scaffolds and their uses in monitoring the behaviour of engineered tissues. We also examine the different nanodevices used to trigger certain processes for tissue development, and offer our view on the principal challenges and prospects of applying nanotechnology in tissue engineering.     

Investigation of 2D and 3D electrospun scaffolds intended for tendon repair

Two-dimensional (2D) electrospun fibre mats have been investigated as fibrous sheets intended as biomaterials scaffolds for tissue repair. It is recognised that tissues are three-dimensional (3D) structures and that optimisation of the fabrication process should include both 2D and 3D scaffolds. Understanding the relative merits of the architecture of 2D and 3D scaffolds for tendon repair is required. This study investigated three different electrospun scaffolds based on poly(ε-caprolactone) fibres intended for repair of injured tendons, referred to as; 2D random sheet, 2D aligned sheet and 3D bundles. 2D aligned fibres and 3D bundles mimicked the parallel arrangement of collagen fibres in natural tendon and 3D bundles further replicated the tertiary layer of a tendon’s hierarchical configuration. 3D bundles demonstrated greatest tensile properties, being significantly stronger and stiffer than 2D aligned and 2D random fibres. All scaffolds supported adhesion and proliferation of tendon fibroblasts. Furthermore, 2D aligned sheets and 3D bundles allowed guidance of the cells into a parallel, longitudinal arrangement, which is similar to tendon cells in the native tissue. With their superior physical properties and ability to better replicate tendon tissue, the 3D electrospun scaffolds warrant greater investigation as synthetic grafts in tendon repair.     

Cell and biomolecule delivery for regenerative medicine

Regenerative medicine is an exciting field that aims to create regenerative alternatives to harvest tissues for transplantation. In this approach, the delivery of cells and biological molecules plays a central role. The scaffold (synthetic temporary extracellular matrix) delivers cells to the regenerative site and provides three-dimensional environments for the cells. To fulfil these functions, we design biodegradable polymer scaffolds with structural features on multiple size scales. To enhance positive cell–material interactions, we design nano-sized structural features in the scaffolds to mimic the natural extracellular matrix. We also integrate micro-sized pore networks to facilitate mass transport and neo tissue regeneration. We also design novel polymer devices and self-assembled nanospheres for biomolecule delivery to recapitulate key events in developmental and wound healing processes. Herein, we present recent work in biomedical polymer synthesis, novel processing techniques, surface engineering and biologic delivery. Examples of enhanced cellular/tissue function and regenerative outcomes of these approaches are discussed to demonstrate the excitement of the biomimetic scaffold design and biologic delivery in regenerative medicine.     

Synthesis of Photopolymerizable Hydrophilic Macromers and Evaluation of Their Applicability as Reactive Resin Components for the Fabrication of Three‐Dimensionally Structured Hydrogel Matrices by 2‐Photon‐Polymerization

Two‐photon polymerization (2‐PP) is a promising new photolithographic technique to fabricate three‐dimensional (3D), micro‐ and nano‐structured tissue engineering scaffolds from photopolymerizable monomers. Although various photo resins are known for the use in 2‐PP, there is currently a need for photo‐curable monomers processable by 2‐PP to generate biocompatible 3D‐structured hydrogel materials for soft or cartilage tissue regeneration. In the present work hydrophilic methacrylate monomers and macromers based on synthetic poly(glycerine) and poly(ethylene glycol) urethanes as well as on the biopolymers dextran and hyaluronan is prepared. The photopolymerization behavior of these substances are investigated and formed hydrogel networks are studied with regard to their mechanical properties, cytocompatibility, and hydrolytic degradation. Based on these examinations simple 3D model structures are fabricated from these photo‐curable monomers and macromers by 2‐PP. It is shown that both the synthetic monomers and the dextran methacrylate macromer are efficient 2‐PP starting materials whereas the hyaluronan methacrylate can be used for 2‐PP only in combination with suitable water‐soluble co‐monomers. No cytotoxic effects are found in preliminary chondrocyte cultivation experiments on 2‐PP‐fabricated scaffolds but initial cell adhesion on the hydrophilic scaffold surfaces is rather low and has to be further improved to apply these structures in tissue engineering.     

Design of Plasma Surface‐Activated,Electrospun Polylactide Non‐Wovens with Improved Cell Acceptance

Electrospinning is a versatile technique to generate tissue engineering matrices possessing structural features similar to the extracellular matrix. Biodegradable polylactides are well suited for processing by this technique, but their innate hydrophobicity impairs initial protein adsorption and cell adhesion. In this work, therefore, electrospun poly(L ‐lactide‐co‐D,L ‐lactide) (70/30) non‐wovens are modified with an ultrathin plasma‐polymerized allylamine (PPAAm) coating. Using scanning electron microsocopy (SEM), it is shown that the fiber structure of the non‐woven is not affected by the plasma treatment. X‐ray photoelectron spectroscopy (XPS) and contact angle measurements of PPAAm‐coated non‐wovens confirm the presence of nitrogen and oxygen‐functional groups in the coating and a hydrophilic nature of the coated non‐woven surface. Cell experiments in vitro demonstrate that the PPAAm‐coated surface promotes occupancy of the non‐woven by human MG‐63 osteoblasts accompanied by improved initial cell spreading and filopodia formation along and between the electrospun polylactide fibers. Overall, plasma‐assisted incorporation of amino groups into electrospun polylactone non‐wovens represents a promising approach to tissue engineering scaffolds with improved cell–material interfaces.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Cell and biomolecule delivery for regenerative medicine

Abstract

Regenerative medicine is an exciting field that aims to create regenerative alternatives to harvest tissues for transplantation. In this approach, the delivery of cells and biological molecules plays a central role. The scaffold (synthetic temporary extracellular matrix) delivers cells to the regenerative site and provides three-dimensional environments for the cells. To fulfil these functions, we design biodegradable polymer scaffolds with structural features on multiple size scales. To enhance positive cell–material interactions, we design nano-sized structural features in the scaffolds to mimic the natural extracellular matrix. We also integrate micro-sized pore networks to facilitate mass transport and neo tissue regeneration. We also design novel polymer devices and self-assembled nanospheres for biomolecule delivery to recapitulate key events in developmental and wound healing processes. Herein, we present recent work in biomedical polymer synthesis, novel processing techniques, surface engineering and biologic delivery. Examples of enhanced cellular/tissue function and regenerative outcomes of these approaches are discussed to demonstrate the excitement of the biomimetic scaffold design and biologic delivery in regenerative medicine.     

Hierarchically engineered fibrous scaffolds for bone regeneration

Surface properties of biomaterials play a major role in the governing of cell functionalities. It is well known that mechanical, chemical and nanotopographic cues, for example, influence cell proliferation and differentiation. Here, we present a novel coating protocol to produce hierarchically engineered fibrous scaffolds with tailorable surface characteristics, which mimic bone extracellular matrix. Based on the sol–gel method and a succession of surface treatments, hollow electrospun polylactic acid fibres were coated with a silicon–calcium–phosphate bioactive organic–inorganic glass. Compared with pure polymeric fibres that showed a completely smooth surface, the coated fibres exhibited a nanostructured topography and greater roughness. They also showed improved hydrophilic properties and a Young''s modulus sixfold higher than non-coated ones, while remaining fully flexible and easy to handle. Rat mesenchymal stem cells cultured on these fibres showed great cellular spreading and interactions with the material. This protocol can be transferred to other structures and glasses, allowing the fabrication of various materials with well-defined features. This novel approach represents therefore a valuable improvement in the production of artificial matrices able to direct stem cell fate through physical and chemical interactions.     

PLLA–collagen and PLLA–gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering

Abstract

In skin tissue engineering, a three-dimensional porous scaffold is necessary to support cell adhesion and proliferation and to guide cells moving into the repair area in the wound healing process. Structurally, the porous scaffold should have an open and interconnected porous architecture to facilitate homogenous cell distribution. Moreover, the scaffolds should be mechanically strong to protect deformation during the formation of new skin. In this study, the hybrid scaffolds were prepared by forming funnel-like collagen or gelatin sponge on a woven poly(l-lactic acid) (PLLA) mesh. The hybrid scaffolds combined the advantages of both collagen or gelatin (good cell-interactions) and PLLA mesh (high mechanical strength). The hybrid scaffolds were used to culture dermal fibroblasts for dermal tissue engineering. The funnel-like porous structure promoted homogeneous cell distribution and extracellular matrix production. The PLLA mesh reinforced the scaffold to avoid deformation. Subcutaneous implantation showed that the PLLA–collagen and PLLA–gelatin scaffolds promoted the regeneration of dermal tissue and epidermis and reduced contraction during the formation of new tissue. These results indicate that funnel-like hybrid scaffolds can be used for skin tissue regeneration.     

Manufacture and evaluation of bioactive and biodegradable materials and scaffolds for tissue engineering

For tissue regeneration and tissue engineering applications, a number of bioactive and biodegradable composites, either porous or non-porous, were fabricated. The newly developed materials included tricalcium phosphate reinforced polyhydroxybutyrate and its copolymer, poorly crystallized hydroxyapatite reinforced chitin, and plasma sprayed hydroxyapatite reinforced poly(L-lactic acid). It was shown that these new materials could be successfully produced using the manufacturing techniques adopted. In vitro experiments revealed that the incorporation of bioceramic particles in biodegradable polymers rendered the composites bioactive and significantly improved the ability of composites to induce the formation of bone-like apatite on their surfaces. Degradation of composite scaffolds in simulated body fluid was observed and could be due to the simultaneous degradation of polymer matrix and dissolution of bioceramic particles.     

In vitro investigations of a novel wound dressing concept based on biodegradable polyurethane

Non-healing and partially healing wounds are an important problem not only for the patient but also for the public health care system. Current treatment solutions are far from optimal regarding the chosen material properties as well as price and source. Biodegradable polyurethane (PUR) scaffolds have shown great promise for in vivo tissue engineering approaches, but accomplishment of the goal of scaffold degradation and new tissue formation developing in parallel has not been observed so far in skin wound repair. In this study, the mechanical properties and degradation behavior as well as the biocompatibility of a low-cost synthetic, pathogen-free, biocompatible and biodegradable extracellular matrix mimicking a PUR scaffold was evaluated in vitro. The novel PUR scaffolds were found to meet all the requirements for optimal scaffolds and wound dressings. These three-dimensional scaffolds are soft, highly porous, and form-stable and can be easily cut into any shape desired. All the material formulations investigated were found to be nontoxic. One formulation was able to be defined that supported both good fibroblast cell attachment and cell proliferation to colonize the scaffold. Tunable biodegradation velocity of the materials could be observed, and the results additionally indicated that calcium plays a crucial role in PUR degradation. Our results suggest that the PUR materials evaluated in this study are promising candidates for next-generation wound treatment systems and support the concept of using foam scaffolds for improved in vivo tissue engineering and regeneration.     

可降解水凝胶作为关节软骨修复材料的研究进展

可降解水凝胶因其良好的生物相容性和生物降解性被广泛用于关节软骨的修复和再生。本文以可降解水凝胶在软骨组织工程中的三类应用策略为主线,概述了用于原位成型可注射水凝胶的蛋白多糖类材料及纳米复合类材料;系统总结了传统工艺制造组织工程支架的优缺点及多种工艺结合的制备方法;重点归纳了近年来3D打印组织工程支架从纯软骨到骨/软骨一体化、从单层到多层的研究进展;最后分析了可降解水凝胶作为关节软骨支架材料在微观定向结构和生物活性功能化方面的局限性,并作出展望：未来开展多材料、多尺度、多诱导的高仿生梯度支架是关节软骨组织工程的一个重要研究方向。     

Gelatine/PLLA sponge-like scaffolds: morphological and biological characterization

Biodegradable synthetic polymers such as poly(lactic acid) are widely used to prepare scaffolds for cell transplantation and tissue growth, using different techniques set up for the purpose. However the poor hydrophilicity of these polymers represents the main limitation to their use as scaffolds because it causes a low affinity for the cells. An effective way to solve this problem could be represented by the addition of biopolymers that are in general highly hydrophilic. The present work concerns porous biodegradable sponge-like systems based on poly(L-lactic acid) and gelatine. Morphology and porosity characteristics of the sponges were studied by scanning electron microscopy and mercury intrusion porosimetry respectively. Blood compatibility was investigated by bovine plasma fibrinogen adsorption test and platelet adhesion test. The cell culture method was used in order to evaluate the ability of the matrices to work as scaffolds for tissue regeneration. The obtained results indicate that the sponges have interesting porous characteristics, good blood compatibility and above all good ability to support cell adhesion and growth. In fact viable and metabolically active animal cells were found inside the sponges after 8 weeks in culture. On this basis the systems produced seem to be good candidates as scaffolds for tissue regeneration.     

Effect of a synthetic link N peptide nanofiber scaffold on the matrix deposition of aggrecan and type II collagen in rabbit notochordal cells

Self-assembling peptide nanofiber scaffolds have been studied extensively as biological materials for 3-dimensional cell culture and repairing tissue defects in animals. However, few studies have applied peptide nanofiber scaffolds in the tissue engineering of intervertebral discs (IVDs). In this study, a novel functionalized peptide scaffold was specifically designed for IVD tissue engineering, and notochordal cells (NCs) as an alternative cell source for IVD degeneration were selected to investigate the bioactive scaffold material. The novel RADA16-Link N self-assembling peptide scaffold material was designed by direct coupling to a bioactive motif link N. The link N nanofiber scaffold (LN-NS) material was obtained by mixing pure RADA16-I and RADA16-Link N (1:1) designer peptide solutions. Although live/dead cell assays showed that LN-NS and RADA16-I scaffold materials were both biocompatible with NCs, the LN-NS material significantly promoted NC adhesion compared with that of the pure RADA16-I SAP scaffold material. The depositions of aggrecan and type II collagen, which are significant markers for IVD cells, were remarkably increased. Furthermore, the results indicated that the link N motif, the matrix analog of the nucleus pulposus, significantly promoted the accumulation of other extracellular matrices in vitro. We conclude that the novel LN-NS material is a promising biological scaffold material, and may have a broad range of applications in IVD tissue engineering.