Biodegradable nanomats produced by electrospinning: expanding multifunctionality and potential for tissue engineering

With increasing interest in nanotechnology, development of nanofibers (n-fibers) by using the technique of electrospinning is gaining new momentum. Among important potential applications of n-fiber-based structures, scaffolds for tissue-engineering represent an advancing front. Nanoscaffolds (n-scaffolds) are closer to natural extracellular matrix (ECM) and its nanoscale fibrous structure. Although the technique of electrospinning is relatively old, various improvements have been made in the last decades to explore the spinning of submicron fibers from biodegradable polymers and to develop also multifunctional drug-releasing and bioactive scaffolds. Various factors can affect the properties of resulting nanostructures that can be classified into three main categories, namely: (1) Substrate related, (2) Apparatus related, and (3) Environment related factors. Developed n-scaffolds were tested for their cytocompatibility using different cell models and were seeded with cells for to develop tissue engineering constructs. Most importantly, studies have looked at the potential of using n-scaffolds for the development of blood vessels. There is a large area ahead for further applications and development of the field. For instance, multifunctional scaffolds that can be used as controlled delivery system do have a potential and have yet to be investigated for engineering of various tissues. So far, in vivo data on n-scaffolds are scarce, but in future reports are expected to emerge. With the convergence of the fields of nanotechnology, drug release and tissue engineering, new solutions could be found for the current limitations of tissue engineering scaffolds, which may enhance their functionality upon in vivo implantation. In this paper electrospinning process, factors affecting it, used polymers, developed n-scaffolds and their characterization are reviewed with focus on application in tissue engineering.     

Biodegradable nanomats produced by electrospinning: expanding multifunctionality and potential for tissue engineering

With increasing interest in nanotechnology, development of nanofibers (n-fibers) by using the technique of electrospinning is gaining new momentum. Among important potential applications of n-fiber-based structures, scaffolds for tissue-engineering represent an advancing front. Nanoscaffolds (n-scaffolds) are closer to natural extracellular matrix (ECM) and its nanoscale fibrous structure. Although the technique of electrospinning is relatively old, various improvements have been made in the last decades to explore the spinning of submicron fibers from biodegradable polymers and to develop also multifunctional drug-releasing and bioactive scaffolds. Various factors can affect the properties of resulting nanostructures that can be classified into three main categories, namely: (1) Substrate related, (2) Apparatus related, and (3) Environment related factors. Developed n-scaffolds were tested for their cytocompatibility using different cell models and were seeded with cells for to develop tissue engineering constructs. Most importantly, studies have looked at the potential of using n-scaffolds for the development of blood vessels. There is a large area ahead for further applications and development of the field. For instance, multifunctional scaffolds that can be used as controlled delivery system do have a potential and have yet to be investigated for engineering of various tissues. So far, in vivo data on n-scaffolds are scarce, but in future reports are expected to emerge. With the convergence of the fields of nanotechnology, drug release and tissue engineering, new solutions could be found for the current limitations of tissue engineering scaffolds, which may enhance their functionality upon in vivo implantation. In this paper electrospinning process, factors affecting it, used polymers, developed n-scaffolds and their characterization are reviewed with focus on application in tissue engineering.     

Nano- and micro-fiber combined scaffolds: A new architecture for bone tissue engineering

One possible interesting way of designing a scaffold for bone tissue engineering is to base it on trying to mimic the biophysical structure of natural extracellular matrix (ECM). This work was developed in order to produce scaffolds for supporting bone cells. Nano and micro fiber combined scaffolds were originally produced from starch based biomaterials by means of a fiber bonding and a electrospinning, two step methodology. The cell culture studies with SaOs-2 human osteoblast-like cell line and rat bone marrow stromal cells demonstrated that presence of nanofibers influenced cell shape and cytoskeletal organization of the cells on the nano/micro combined scaffolds. Moreover, cell viability and Alkaline Phosphatase (ALP) activity for both cell types was found to be higher in nano/micro combined scaffolds than in control scaffolds based on fiber meshes without nanofibers. Consequently, the developed structures are believed have a great potential on the 3D organization and guidance of cells that is provided for engineering of 3-dimensional bone tissues.     

Cellular materials as porous scaffolds for tissue engineering

A major goal of tissue engineering is to synthesize or regenerate tissues and organs. Today, this is done by providing a synthetic porous scaffold, or matrix, which mimics the body's own extracellular matrix, onto which cells attach, multiply, migrate and function. Porous scaffolds are currently being developed for regeneration of skin, cartilage, bone, nerve and liver. The microstructures of many porous scaffolds ressemble that of an engineering foam. In this paper, we describe the microstructural requirements for porous scaffolds, review several processes for making them and show typical microstructures. Clinical studies have found that a collagen-based scaffold for skin regeneration reduces wound contraction during the healing process, reducing scar formation. The process of wound contraction is not well understood. Here, we describe the measurement of contraction of collagen-based scaffolds by fibroblasts in vitro using a cell force monitor.     

Degradable polyester scaffolds with controlled surface chemistry combining minimal protein adsorption with specific bioactivation

Advanced biomaterials and scaffolds for tissue engineering place high demands on materials and exceed the passive biocompatibility requirements previously considered acceptable for biomedical implants. Together with degradability, the activation of specific cell–material interactions and a three-dimensional environment that mimics the extracellular matrix are core challenges and prerequisites for the organization of living cells to functional tissue. Moreover, although bioactive signalling combined with minimization of non-specific protein adsorption is an advanced modification technique for flat surfaces, it is usually not accomplished for three-dimensional fibrous scaffolds used in tissue engineering. Here, we present a one-step preparation of fully synthetic, bioactive and degradable extracellular matrix-mimetic scaffolds by electrospinning, using poly(D,L-lactide-co-glycolide) as the matrix polymer. Addition of a functional, amphiphilic macromolecule based on star-shaped poly(ethylene oxide) transforms current biomedically used degradable polyesters into hydrophilic fibres, which causes the suppression of non-specific protein adsorption on the fibres’ surface. The subsequent covalent attachment of cell-adhesion-mediating peptides to the hydrophilic fibres promotes specific bioactivation and enables adhesion of cells through exclusive recognition of the immobilized binding motifs. This approach permits synthetic materials to directly control cell behaviour, for example, resembling the binding of cells to fibronectin immobilized on collagen fibres in the extracellular matrix of connective tissue.     

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.     

Chitosan particles agglomerated scaffolds for cartilage and osteochondral tissue engineering approaches with adipose tissue derived stem cells

It is well accepted that natural tissue regeneration is unlikely to occur if the cells are not supplied with an extracellular matrix (ECM) substitute. With this goal, several different methodologies have been used to produce a variety of 3D scaffolds as artificial ECM substitutes suitable for bone and cartilage tissue engineering. Furthermore, osteochondral tissue engineering presents new challenges since the combination of scaffolding and co-culture requirements from both bone and cartilage applications is required in order to achieve a successful osteochondral construct. In this paper, an innovative processing route based on a chitosan particles aggregation methodology for the production of cartilage and osteochondral tissue engineering scaffolds is reported. An extensive characterization is presented including a morphological evaluation using Micro-Computed Tomography (μCT) and 3D virtual models built with an image processing software. Mechanical and water uptake characterizations were also carried out, evidencing the potential of the developed scaffolds for the proposed applications. Cytotoxicity tests show that the developed chitosan particles agglomerated scaffolds do not exert toxic effects on cells. Furthermore, osteochondral bilayered scaffolds could also be developed. Preliminary seeding of mesenchymal stem cells isolated from human adipose tissue was performed aiming at developing solutions for chondrogenic and osteogenic differentiation for osteochondral tissue engineering applications. An erratum to this article is available at .     

Assembly of cells and vesicles for organ engineering

Abstract

The development of materials and technologies for the assembly of cells and/or vesicles is a key for the next generation of tissue engineering. Since the introduction of the tissue engineering concept in 1993, various types of scaffolds have been developed for the regeneration of connective tissues in vitro and in vivo. Cartilage, bone and skin have been successfully regenerated in vitro, and these regenerated tissues have been applied clinically. However, organs such as the liver and pancreas constitute numerous cell types, contain small amounts of extracellular matrix, and are highly vascularized. Therefore, organ engineering will require the assembly of cells and/or vesicles. In particular, adhesion between cells/vesicles will be required for regeneration of organs in vitro. This review introduces and discusses the key technologies and materials for the assembly of cells/vesicles for organ regeneration.     

Nanostructured polymeric scaffolds for orthopaedic regenerative engineering

Successful regeneration necessitates the development of three-dimensional (3-D) tissue-inducing scaffolds that mimic the hierarchical architecture of native tissue extracellular matrix (ECM). Cells in nature recognize and interact with the surface topography they are exposed to via ECM proteins. The interaction of cells with nanotopographical features such as pores, ridges, groves, fibers, nodes, and their combinations has proven to be an important signaling modality in controlling cellular processes. Integrating nanotopographical cues is especially important in engineering complex tissues that have multiple cell types and require precisely defined cell-cell and cell-matrix interactions on the nanoscale. Thus, in a regenerative engineering approach, nanoscale materials/scaffolds play a paramount role in controlling cell fate and the consequent regenerative capacity. Advances in nanotechnology have generated a new toolbox for the fabrication of tissue-specific nanostructured scaffolds. For example, biodegradable polymers such as polyesters, polyphosphazenes, polymer blends and composites can be electrospun into ECM-mimicking matrices composed of nanofibers, which provide high surface area for cell attachment, growth, and differentiation. This review provides the fundamental guidelines for the design and development of nanostructured scaffolds for the regeneration of various tissue types in human upper and lower extremities such as skin, ligament, tendon, and bone. Examples focusing on the collective work of our laboratory in those areas are discussed to demonstrate the regenerative efficacy of this approach. Furthermore, preliminary strategies and significant challenges to integrate these individual tissues into one complex organ through regenerative engineering-based integrated graft systems are also discussed.     

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.     

纳米纤维组织工程支架及其纳米效应研究进展

综述了纳米纤维组织工程支架的最新研究进展,评述了其制备技术、特点及其存在的纳米效应.指出天然细胞外基质为三维纳米纤维结构,其纤维连续,直径为纳米级,包含比例确定的纳米与微米空间,且与细胞存在纳米水平的相互作用;然而,目前的人工纤维支架,其纤维直径多为微米或数百纳米,或者缺乏纳米空间.采用生物纳米技术获得的细菌纤维素纳米纤维,其直径小于10 nm,自身呈三维网状结构,富含纳米空间,是构建新一代纳米组织工程支架的理想材料.     

A cultured living bone equivalent enhances bone formation when compared to a cell seeding approach

The development of cell therapy methods to confer osteogenic potential to synthetic bone replacement materials has become common during the last years. At present, in the bone tissue engineering field, two different approaches use patient own cultured osteogenic cells in combination with a scaffold material to engineer autologous osteogenic grafts. One of the approaches consists of seeding cells on a suitable biomaterial, after which the construct is ready for implantation. In the other approach, the seeded cells are further cultured on the scaffold to obtain in vitro formed bone (extracellular matrix and cells), prior to implantation. In the present study, we investigated the in vivo osteogenic potential of both methods through the implantation of porous hydroxyapatite (HA) scaffolds coated with a layer of in vitro formed bone and porous HA scaffolds seeded with osteogenic cells. Results showed that as early as 2 days after implantation, de novo bone tissue was formed on scaffolds in which an in vitro bone-like tissue was cultured, while it was only detected on the cell seeded implants from 4 days onwards. In addition, after 4 days of implantation statistical analysis revealed a significantly higher amount of bone in the bone-like tissue containing scaffolds as compared to cell seeded ones.     

Growth of fibroblast and vascular smooth muscle cells in fibroin/collagen scaffold

In our recent study, a novel fibroin/collagen scaffold with improved mechanical properties and controllable porous structure was prepared through freeze–drying method. In this research, the cyto-compatibility was further studied, using fibroblast and vascular smooth muscle cells (VSMC) as the model cells. MTT results indicated that the growth of fibroblast and VSMC both further improved in the fibroin/collagen scaffold than in pure fibroin scaffolds. The confocal and SEM results showed that fibroblast cells and VSMCs had better adhesion and spreading properties in the fibroin/collagen scaffolds. Although further studies, such as the extracellular matrix production and the functional gene expression, are necessary to clarify the biocompatibility of the fibroin/collagen scaffolds, the present results indicate that the fibroin/collagen scaffold is a new scaffold material suitable for tissue engineering. On the other hand, the mild and all-aqueous preparation processes also make it possible to embed different growth factors inside the scaffolds to maximize cell functions and the formation of specific tissues.     

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.     

Nanofibrous scaffold engineering using electrospinning

Scaffold plays a critical role in tissue engineering where it provides necessary structural support for the cells to accommodate and to guide their growth in the three dimensional space into a specific tissue. Therefore, engineering scaffolds favorable for cell/tissue growth is of great importance and a pre-requisite for scaffold-based tissue engineering. Electrospinning is a versatile method that has been recently adapted in engineering nano-fibrous scaffolds that mimic the structural features of biological extracellular matrix (ECM). It offers many advantages over conventional scaffold methodologies, for example, capable of producing ultra-fine fibers with high porosity, high spatial orientation, high aspect ratio, and high surface area, which are highly required for the initial cell attachment, tissue formation, and continued function. Considering these astonishing merits, this article emphasis on nano-fibrous scaffold engineering by electrospinning.     

Growth of outgrowth endothelial cells on aligned PLLA nanofibrous scaffolds

Tissue engineering holds great promise in providing vascular grafts as substitutes for damaged small-diameter blood vessels. Two of the key factors in vascular tissue engineering are biocompatible scaffolds that mimic the effects of extracellular matrix and the source of seeding cells. Synthetic poly-l-lactic acid (PLLA) nanofibers has been shown to be excellent scaffolds for tissue engineering. Outgrowth endothelial cells (OECs) isolated from human peripheral blood could also be expanded in vitro and stably maintain the differentiated phenotypes and could be used as the seeding cells for engineering autologous vascular crafts. Here we tested the possibility of combining these two together. We found that PLLA nanofibers are not only biocompatible with OECs originally isolated from rabbit peripheral blood, the aligned PLLA fibers actually promoted and guided their sustained proliferation. These results suggest that aligned PLLA could be excellent both as the scaffolds and as a promoter of cell growth during vascular tissue engineering.     

Development of a novel collagen–GAG nanofibrous scaffold via electrospinning

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

Tuning of Cell–Biomaterial Anchorage for Tissue Regeneration

Which mechanisms mediate cell attachment to biomaterials? What role does the surface charge or wettability play on cell–material anchorage? What are the currently investigated strategies to modify cell–matrix adherence spatiotemporally? Considering the development of scaffolds made of biocompatible materials to temporarily replace the structure and/or function of the extracellular matrix, focus is given to the analysis of the specific (i.e., cell adhesive peptide sequences) and unspecific (i.e., surface charge, wettability) mechanisms mediating cell‐matrix interactions. Furthermore, because natural tissue regeneration is characterized by the dynamic attachment/detachment of different cell populations, the design of advanced scaffolds for tissue engineering, based in the spatiotemporal tuning of cell–matrix anchorage is discussed.     

Biomanufacturing for tissue engineering: Present and future trends

Tissue engineering, often referred to as regenerative medicine and reparative medicine, is an interdisciplinary field that necessitates the combined effort of cell biologists, engineers, material scientists, mathematicians, geneticists, and clinicians toward the development of biological substitutes that restore, maintain, or improve tissue function. It has emerged as a rapidly expanding approach to address the organ shortage problem and comprises tissue regeneration and organ substitution. Cells placed on/or within constructs is the most common strategy in tissue engineering. Successful cell seeding depends on fast attachment of cell to scaffolds, high cell survival and uniform cell distribution. The seeding time is strongly dependent on the scaffold material and architecture. Scaffolds provide an initial biochemical substrate for the novel tissue until cells can produce their own extra-cellular matrix (ECM). Thus scaffolds not only define the 3D space for the formation of new tissues, but also serve to provide tissues with appropriate functions. These scaffolds are often critical, both in vivo (within the body) or in vitro (outside the body) mimicking in vivo conditions. Additive fabrication processes represent a new group of non-conventional fabrication techniques recently introduced in the biomedical engineering field. In tissue engineering, additive fabrication processes have been used to produce scaffolds with customised external shape and predefined internal morphology, allowing good control of pore size and pore distribution. This article provides a comprehensive state-of-the-art review of the application of biomanufacturing additive processes in the field of tissue engineering. New and moving trends in biomanufacturing technologies and the concept of direct cell-printing technologies are also discussed.     

Osteoblast behaviour on in situ photopolymerizable three-dimensional scaffolds based on D, L-lactide, ε-caprolactone and trimethylene carbonate

Polymer networks formed by photocrosslinking of multifunctional oligomers have great potential as injectable and in situ forming materials for bone tissue engineering. Porous scaffolds varying in polyester type and crosslinking density were prepared from methacrylate-endcapped oligomers based on D,L-lactide, ε -caprolactone and trimethylene carbonate: LA/CL-hexanediol, LA/CL-dipentaerythritol and LA/TMC-HXD. The biocompatibility and bone formation were related with the degradation time and mechanical properties. The viability of fibroblasts was evaluated after incubation with extraction medium by MTT-assay. All scaffolds showed a good biocompatibility. Rat bone marrow cells were cultured on the scaffolds for 21 days and were able to attach and differentiate on the scaffolds. The cells expressed high alkaline phosphatase activity, have formed a mineralized extracellular matrix and secreted osteocalcin. TEM of the polymer interface revealed osteoblasts which secreted an extracellular matrix containing matrix vesicles loaded with apatite crystals. LA/TMC-HXD, LA/CL-HXD and LA/CL-DPENT had a 50% mass loss at 3,5 months respectively 6 and 7, 5 months. The mechanical properties improve by increasing the branching of the precursor methacrylates (by replacing HXD by DPENT) but do not depend on their chemical composition. Hence, scaffolds with high elastic properties and variable degradation time can be obtained, which are promising for bone tissue engineering. Author to whom all correspondence should be addressed.