Scaffolds For Tissue Regeneration Applications Research Articles

Biocomposite Scaffolds for Tissue Engineering: Materials, Fabrication Techniques and Future Directions

Tissue engineering is an interdisciplinary field that combines materials, methods, and biological molecules to engineer newly formed tissues to replace or restore functional organs. Biomaterials-based scaffolds play a crucial role in developing new tissue by interacting with human cells. Tissue engineering scaffolds with ideal characteristics, namely, nontoxicity, biodegradability, and appropriate mechanical and surface properties, are vital for tissue regeneration applications. However, current biocomposite scaffolds face significant limitations, particularly in achieving structural durability, controlled degradation rates, and effective cellular integration. These qualities are essential for maintaining long-term functionality in vivo. Although commonly utilized biomaterials can provide physical and chemical properties needed for tissue regeneration, inadequate biomimetic properties, as well as insufficient interactions of cells-scaffolds interaction, still need to be improved for the application of tissue engineering in vivo. It is impossible to achieve some essential features using a single material, so combining two or more materials may accomplish the requirements. In order to achieve a proper scaffold design, a suitable fabrication technique and combination of biomaterials with controlled micro or nanostructures are needed to achieve the proper biological responses. This review emphasizes advancements in scaffold durability, biocompatibility, and cellular responsiveness. It focuses on natural and synthetic polymer combinations and innovative fabrication techniques. Developing stimulus-responsive 3D scaffolds is critical, as these scaffolds enhance cell adhesion and promote functional tissue formation while maintaining structural integrity over time. This review also highlights the natural polymers, smart materials, and recent advanced techniques currently used to create emerging scaffolds for tissue regeneration applications.

Development of Scaffolds from Bio-Based Natural Materials for Tissue Regeneration Applications: A Review.

Tissue damage and organ failure are major problems that many people face worldwide. Most of them benefit from treatment related to modern technology's tissue regeneration process. Tissue engineering is one of the booming fields widely used to replace damaged tissue. Scaffold is a base material in which cells and growth factors are embedded to construct a substitute tissue. Various materials have been used to develop scaffolds. Bio-based natural materials are biocompatible, safe, and do not release toxic compounds during biodegradation. Therefore, it is highly recommendable to fabricate scaffolds using such materials. To date, there have been no singular materials that fulfill all the features of the scaffold. Hence, combining two or more materials is encouraged to obtain the desired characteristics. To design a reliable scaffold by combining different materials, there is a need to choose a good fabrication technique. In this review article, the bio-based natural materials and fine fabrication techniques that are currently used in developing scaffolds for tissue regeneration applications, along with the number of articles published on each material, are briefly discussed. It is envisaged to gain explicit knowledge of developing scaffolds from bio-based natural materials for tissue regeneration applications.

Enzymatic co-crosslinking of star-shaped poly(ethylene glycol) tyramine and hyaluronic acid tyramine conjugates provides elastic biocompatible and biodegradable hydrogels.

A combination of the viscoelastic properties of hyaluronic acid (HA) and the elastic properties of star shaped 8-arm poly(ethylene glycol) (8-arm PEG) was used to design in-situ forming hydrogels. Hydrogels were prepared by the enzymatic crosslinking of a partially tyramine modified 8-arm PEG and a tyramine conjugated HA using horseradish peroxidase in the presence of hydrogen peroxide. Hydrogels of the homopolymer conjugates and mixtures thereof were rapidly formed within seconds under physiological conditions at low polymer and enzyme concentrations. Elastic hydrogels with high gel content (≥95%) and high storage moduli (up to 22.4 kPa) were obtained. An in vitro study in the presence of hyaluronidase (100 U/mL) revealed that with increasing PEG content the degradation time of the hybrid hydrogels increased up to several weeks, whereas hydrogels composed of only hyaluronic acid degraded within 2 weeks. Human mesenchymal stem cells (hMSCs) incorporated in the hybrid hydrogels remained viable as shown by a PrestoBlue and a live-dead assay, confirming the biocompatibility of the constructs. The production of an extracellular matrix by re-differentiation of encapsulated human chondrocytes was followed over a period of 28 days. Gene expression indicated that these highly elastic hydrogels induced an enhanced production of collagen type II. At low PEG-TA/HA-TA ratios a higher expression of SOX 9 and ACAN was observed. These results indicate that by modulating the ratio of PEG/HA, injectable hydrogels can be prepared applicable as scaffolds for tissue regeneration applications.

Layered Double Hydroxide as a Potent Non-viral Vector for Nucleic Acid Delivery Using Gene-Activated Scaffolds for Tissue Regeneration Applications.

Nonviral vectors offer a safe alternative to viral vectors for gene therapy applications, albeit typically exhibiting lower transfection efficiencies. As a result, there remains a significant need for the development of a nonviral delivery system with low cytotoxicity and high transfection efficacy as a tool for safe and transient gene delivery. This study assesses MgAl-NO3 layered double hydroxide (LDH) as a nonviral vector to deliver nucleic acids (pDNA, miRNA and siRNA) to mesenchymal stromal cells (MSCs) in 2D culture and using a 3D tissue engineering scaffold approach. Nanoparticles were formulated by complexing LDH with pDNA, microRNA (miRNA) mimics and inhibitors, and siRNA at varying mass ratios of LDH:nucleic acid. In 2D monolayer, pDNA delivery demonstrated significant cytotoxicity issues, and low cellular transfection was deemed to be a result of the poor physicochemical properties of the LDH–pDNA nanoparticles. However, the lower mass ratios required to successfully complex with miRNA and siRNA cargo allowed for efficient delivery to MSCs. Furthermore, incorporation of LDH–miRNA nanoparticles into collagen-nanohydroxyapatite scaffolds resulted in successful overexpression of miRNA in MSCs, demonstrating the development of an efficacious miRNA delivery platform for gene therapy applications in regenerative medicine.

Fabrication of a thick three-dimensional scaffold with an open cellular-like structure using airbrushing and thermal cross-linking of molded short nanofibers

Nanoscale fibers mimicking the extracellular matrix of natural tissue can be produced by conventional electrospinning, but this approach results in two-dimensional thin dense fibrous mats which can hinder effective cell infiltration. The aim of the present study was to design a thick, three-dimensional (3D) cylindrical scaffold with an open pore structure assembled from short polycaprolactone (PCL) fibers using a facile airbrushing approach. In addition, magnesium particles were incorporated into the PCL solution to both enhance the mechanical properties of the scaffold and stimulate cellular activity following cell seeding. Separated short composite airbrushed fibers were assembled into a 3D cylindrical structure by cold-press molding and thermal cross-linking. The microstructure, chemical composition, porosity and thermal properties were subsequently investigated, along with changes in mechanical performance following immersion in PBS for 60 d. The results showed that the assembled 3D fibrous 10 mm thick cylindrical matrix had an interconnected fibrous network structure with 31.5%–60% porosity. Encapsulation of the Mg particles into the 3D assembled fibrous scaffold enhanced the mechanical properties of the plain PCL scaffolds. The results also demonstrated controlled release of Mg ions into the PBS media for up to 60 d, as evaluated by changes in Mg ion concentration and pH of the media. In addition, the 3D fibrous assembled matrix was shown to support human osteoblast-like cell adhesion, proliferation and penetration. The results suggest that this novel fabrication method of biodegradable thick 3D scaffolds with an open pore structure is promising for the production of a new generation of 3D scaffolds for tissue regeneration applications.

Electrospun and Electrosprayed Scaffolds for Tissue Engineering.

Electrospinning and electrospraying technologies provide an accessible and universal synthesis method for the continuous preparation of nanostructured materials. This chapter introduces recent uses of electrospun and electrosprayed scaffolds for tissue regeneration applications. More recent in vitro and in vivo of electrospun fibers are also discussed in relation to soft and hard tissue engineering applications. The focus is made on the bone, vascular, skin, neural and soft tissue regeneration. An introduction is presented regarding the production of biomaterials made by synthetic and natural polymers and inorganic and metallic materials for use in the production of scaffolds for regenerative medicine. For this proposal, the following techniques are discussed: electrospraying, co-axial and emulsion electrospinning and bio-electrospraying. Tissue engineering is an exciting and rapidly developing field for the understanding of how to regenerate the human body.

Invitro skin models and tissue engineering protocols for skin graft applications.

In this review, we present a brief introduction of the skin structure, a concise compilation of skin-related disorders, and a thorough discussion of different invitro skin models, artificial skin substitutes, skin grafts, and dermal tissue engineering protocols. The advantages of the development of invitro skin disorder models, such as UV radiation and the prototype model, melanoma model, wound healing model, psoriasis model, and full-thickness model are also discussed. Different typesof skin grafts including allografts, autografts, allogeneic, and xenogeneic are described in detail with their associated applications. We also discuss different tissue engineering protocols for the design of various typesof skin substitutes and their commercial outcomes. Brief highlights are given of the new generation three-dimensional printed scaffolds for tissue regeneration applications.

Antibacterial and conductive injectable hydrogels based on quaternized chitosan-graft-polyaniline/oxidized dextran for tissue engineering

Biomaterials with injectability, conductivity and antibacterial effect simultaneously have been rarely reported. Herein, we developed a new series of in situ forming antibacterial conductive degradable hydrogels using quaternized chitosan (QCS) grafted polyaniline with oxidized dextran as crosslinker. The chemical structures, morphologies, electrochemical property, conductivity, swelling ratio, rheological property, in vitro biodegradation and gelation time of hydrogels were characterized. Injectability was verified by in vivo subcutaneous injection on a Sprague Dawley rat. The antibacterial activity of the hydrogels was firstly evaluated employing antibacterial assay using Escherichia coli and Staphylococcus aureus in vitro. The hydrogels containing polyaniline showed enhanced antibacterial activity compared to QCS hydrogel, especially for hydrogels with 3wt% polyaniline showing 95kill% and 90kill% for E. coli and S. aureus, respectively. Compared with QCS hydrogel, the hydrogels with 3wt% polyaniline still showed enhanced antibacterial activity for E. coli in vivo. The adipose-derived mesenchymal stem cells (ADMSCs) were used to evaluate the cytotoxicity of the hydrogels and hydrogels with polyaniline showed better cytocompatibility than QCS hydrogel. The electroactive hydrogels could significantly enhance the proliferation of C2C12 myoblasts compared to QCS hydrogel. This work opens the way to fabricate in situ forming antibacterial and electroactive degradable hydrogels as a new class of bioactive scaffolds for tissue regeneration applications.

3D Printing of Scaffolds for Tissue Regeneration Applications.

The current need for organ and tissue replacement, repair, and regeneration for patients is continually growing such that supply is not meeting demand primarily due to a paucity of donors as well as biocompatibility issues leading to immune rejection of the transplant. In order to overcome these drawbacks, scientists have investigated the use of scaffolds as an alternative to transplantation. These scaffolds are designed to mimic the extracellular matrix (ECM) by providing structural support as well as promoting attachment, proliferation, and differentiation with the ultimate goal of yielding functional tissues or organs. Initial attempts at developing scaffolds were problematic and subsequently inspired an interest in 3D printing as a mode for generating scaffolds. Utilizing three-dimensional printing (3DP) technologies, ECM-like scaffolds can be produced with a high degree of complexity, where fine details can be included at a micrometer level. In this Review, the criteria for printing viable and functional scaffolds, scaffolding materials, and 3DP technologies used to print scaffolds for tissue engineering are discussed. Creating biofunctional scaffolds could potentially help to meet the demand by patients for tissues and organs without having to wait or rely on donors for transplantation.

Cryogenically fabricated three-dimensional chitosan scaffolds with pore size-controlled structures for biomedical applications

A cryogenic plotting system and manufacturing stage was designed to fabricate three-dimensional (3D) chitosan scaffolds for tissue regeneration applications. The combination of cryogenics and freeze-drying yielded highly porous and structurally stable 3D chitosan scaffolds. The pore size of the chitosan scaffold, which varied from 90 to 400μm, was reproducibly dependent on processing parameters. Crosslinked chitosan scaffolds exhibited rough surfaces and were stable with regard to shapeability and pore size. During the process, changes in the dimensions of the final scaffold, due to shrinkage and swelling, were within ±10% of the dimensions specified in the initial design. MG-63 cells were cultured on the fabricated scaffolds to evaluate the structures as substrates for tissue regeneration. Cell proliferation on the structured scaffolds was higher than on conventional spongy-type chitosan scaffold. In particular, scaffolds with 200-μm mean pores exhibited the highest rate of cell proliferation.

Role of electrospun fibre diameter and corresponding specific surface area (SSA) on cell attachment

In order to develop scaffolds for tissue regeneration applications, it is important to develop an understanding of the kinetics of cell attachment as a function of scaffold geometry. In the present study, we investigated how the specific surface area of electrospun scaffolds affected cell attachment and spreading. Number of cells attached to the scaffold was measured by the relative intensity of a metabolic dye (MTS) and cell spreading was analysed for individual cells by measuring the area of projected F-actin cytoskeleton. We varied the fibre diameter to obtain a specific surface area distribution in the range 2.24-18.79 microm(-1). In addition, we had one case where the scaffolds had beads in them and therefore had non-uniform fibres. For each of these different geometries, we varied the cell-seeding density (0.5-1 x 10(5)) and the serum concentration (0-12%) over the first 8 h in an electrospun polycaprolactone NIH 3T3 fibroblast system. Cells on beaded scaffolds showed the lowest attachment and almost no F-actin spreading in all experiments indicating uniform fibre diameter is essential for electrospun scaffolds. For the uniform fibre scaffolds, cell attachment was a function of scaffold specific surface area (SSA) (18.79-2.24 microm(-1)) and followed two distinct trends: when scaffold SSA was < 7.13 microm(-1), cell adhesion rate remained largely unchanged; however, for SSA > 7.13 microm(-1) there was a significant increase in cellular attachment rate with increasing SSA. This indicated that nanofibrous scaffolds increased cellular adhesion compared to microfibrous scaffolds. This phenomenon is true for serum concentrations of 7.5% and higher. For 5% and lower serum concentration, cell attachment is low and higher SSA fails to make a significant improvement in cell attachment. When cell attachment was investigated at a single-cell level by measuring the projected actin area, a similar trend was noted where the effect of higher SSA led to higher projected area for cells at 8 h. These results indicate that uniform electrospun scaffolds with SSA provide a faster cell attachment compared to lower SSA and beaded scaffolds. These results indicate that continuous electrospun nanofibrous scaffolds may be a good substrate for rapid tissue regeneration.

HRP-Loaded Bioresorbable Microspheres: Effect of Copolymer Composition and Molecular Weight on Microstructure and Release Profile

Poly(DL-lactic-co-glycolic acid) microspheres are prepared using a double-emulsion technique and are loaded with the model enzyme horseradish peroxidase (HRP). These microspheres can be used alone or as coatings for bioresorbable fibers that may be used as scaffolds for tissue regeneration applications. The present study focuses on the effect of the copolymer's composition and initial molecular weight on the microsphere structure, encapsulation efficiency, and cumulative protein release for 12 weeks. The release profiles generally exhibits an initial burst effect accompanied by slow release over an extended period of time, during which diffusion rather than degradation controlled HRP release from these structures. An increase in the initial molecular weight or in the copolymer's lactic acid content results in larger microspheres with smoother surfaces, and a decrease in the burst release and in the total HRP release. Molecular weight is found to have a stronger effect than copolymer composition. We demonstrate that it is possible to obtain versatile release profiles, which can be tailored for specific applications by choosing the right initial molecular weight and copolymer composition.

Novel composite fiber structures to provide drug/protein delivery for medical implants and tissue regeneration

A novel class of bioresorbable composite (core/shell) fiber structures loaded with bioactive agents was developed and studied. These unique polymeric structures are designed to combine good mechanical properties with a desired controlled release profile, in order to serve as scaffolds for tissue regeneration applications and as basic elements of medical implants. These core/shell fiber structures were formed by “coating” core polymer fibers with drug/protein-containing poly( dl-lactic-co-glycolic acid) porous structures. The shell preparation (“coating”) was performed by the freeze-drying of water-in-oil emulsions. Both water soluble and water insoluble agents can be incorporated in these structures and their activity is preserved, since the fiber fabrication requires neither high temperatures nor harsh solvents in the vicinity of the bioactive agents. Examples for release profiles of protein (horseradish peroxidase) and drug (paclitaxel) are presented. We have demonstrated that appropriate selection of the emulsion’s parameters can yield a variety of new core/shell fiber structures with desirable drug/protein release behavior. This will lead to the engineering of new implants and scaffolds, and will advance the field of tissue regeneration and medical implants.

Novel bioresorbabale composite fiber structures loaded with proteins for tissue regeneration applications: Microstructure and protein release

Novel bioresorbable core/shell composite fiber structures loaded with proteins were developed and studied. These unique polymeric structures are designed to combine good mechanical properties with a desired controlled protein-release profile, to serve as scaffolds for tissue regeneration applications. Core/shell fiber structures were formed by "coating" poly(L-lactic acid) fibers with protein-containing poly(DL-lactic-co-glycolic acid) porous structures. Shell preparation (coating) was performed by the freeze-drying of water in oil emulsions. The present study focused on the effect of the emulsion's formulation on the porous shell structure and on the resulting cumulative protein release from the composite fibers for 90 days. Horseradish peroxidase (HRP) was used as the protein source. The release profiles usually exhibited an initial burst effect, accompanied by a decrease in release rate with time, as is typical for diffusion-controlled systems. The HRP content and the emulsion's organic:aqueous phase ratio exhibited significant effects on both the shell microstructure and the HRP release profile from the composite fibers, whereas the polymer content of the emulsion's organic phase only affected these fiber characteristics in certain cases. Proper selection of the emulsion's parameters can yield core/shell fiber structures with the desired protein release behavior and other useful physical properties.

Microsphere‐based bioresorbable structures loaded with proteins for tissue regeneration applications

Novel bioresorbable fiber/microsphere composite structures loaded with proteins were developed and studied. These unique polymeric structures are designed to combine good mechanical properties with a desired controlled protein-release profile, in order to serve as scaffolds for tissue regeneration applications. The composite fiber structures were formed by "coating" poly(L-lactic acid) fibers with protein-containing poly(DL-lactic-co-glycolic acid) microspheres. The microspheres were prepared by a double emulsion technique and were loaded with the model enzyme horseradish peroxidase (HRP). The present study focused on the effect of the double emulsion's composition and processing conditions on the microsphere structure and on the resulting cumulative protein release for 90 days. The release profiles generally exhibited an initial burst effect, a lag period and an increased release rate after 2 months. HRP release from these structures was governed by diffusion, rather than by degradation. A decrease in the emulsion's mixing rate significantly improved the release profile through unique matrix-like structures. The initial burst effect can be reduced by decreasing the internal phase quantity and its protein content, or by adding a surfactant to the internal emulsion. Proper selection of the double emulsion formulation and processing conditions can yield fiber microsphere structures with the desired protein release behavior.