Cell Culture Scaffolds Research Articles

Foaming of PCL-Based Composites Using scCO2-Biocompatibility and Evaluation for Biomedical Applications.

The process of foaming poly(caprolactone)-based composite materials using supercritical carbon dioxide was analyzed, especially in terms of the biocompatibility of the resultant materials. The influence of foaming process conditions and composite material properties on the functional properties of polymer solid foams, intended for artificial scaffolds for bone cell culture, was investigated. The relationship between wettability (contact angle) and water absorption rate as a result of the application of variable conditions for the production of porous structures was presented. For the evaluation of potential cytotoxicity, the MTT and PrestoBlue tests were carried out, and animal cells (mouse fibroblasts) were cultured on the materials for nine days. There was no toxic effect of composite materials made of poly(caprolactone) containing porogen particles: hydroxyapatite, crystalline nanocellulose, and graphene oxide on cells. The desired effect of the porogens used in the foaming process on the affinity of cells to the resultant material was demonstrated. The tested materials have been shown to be biocompatible and suitable for applications in biomedical engineering.

Alginate/gelatin-based hybrid hydrogels with function of injecting and encapsulating cells in situ

Multi-network hydrogels with high strength and toughness have attracted increasing attention. Herein, a hybrid hydrogel consisting of alginate, gelatin, and polyacrylamide was constructed with the combination of advantages of natural and synthetic polymers. Alginate grafted with host-guest complex of βCD/Ad-AAm was first prepared, namely Alg-βCD/Ad-AAm, then further crosslink with gelatin methacryloyl (GelMA) to form hydrogel via one-step UV light initiation. The hydrogel produced by this method has more uniform and well-crosslinked networks. The hydrogels demonstrated uniform porosity, adjustable hydrophilicity (water contact angle within 32.7–91.5°), and desired mechanical properties (maximum tensile strain of 242.8%, tensile strength of 75.9 kPa, and Young's modulus of 28.5 kPa). The hydrogel also possessed self-healing ability and pH sensitivity, showing higher mechanical tensile strength at lower pH. The temperature-adjustable viscosity of pre-gel solution (sol-gel transition point of 20.4 °C) endowed it to be 3D printed as a bioink, and the printed scaffold exhibited good resilience and toughness. Moreover, HUVEC, L929, and 3T3 cells were cultured on hydrogel surfaces for 28 days and were enveloped within the hydrogels for 3D culture, indicating excellent cytocompatibility of the hydrogels. Therefore, this hybrid hydrogel system can be used potentially in cell culture scaffold and tissue engineering.

Modern biomedical applications of graphene

Since the discovery of graphene and its derivatives in 2004, they are one of the most promising materials because of their broad potential applications in several fields, industrial, agricultural, biotechnological, biomedical, tissue engineering and many other areas. Graphene has unique two-dimensional planar structure, exceptional physical — chemical nature, great biocompatibility and novel electrical, thermal, and mechanical properties that lead to many potential applications. In this review, we present an overview of modern biomedical applications of graphene with a special attention to drug delivery, gene delivery, cancer therapy, biosensing, antibacterial materials and scaffold for cell culture.

Analysis of the aggregation mechanism of chondroitin sulfate/chitosan particles and fabrication of hydrogel cell scaffolds

In this study, the aggregation mechanism of polyion complex (PIC) particles from chitosan (CHI) and chondroitin sulfate C (CS) in phosphate-buffered saline (PBS) was analyzed, and a novel method for the fabrication of hydrogels via aggregation was developed. The PBS induced a decrease in the ζ-potential of the CS/CHI PIC particles, increase in their diameter, and aggregation in a concentration-dependent manner. The hydrogels prepared by mixing CS/CHI PIC particle dispersion and PBS showed the PIC components, with porous structure, high swelling ratio (161.4 ± 13.3%), and high storage moduli (26.2 ± 1.4 kPa). By mixing PBS with suspended adhesive cells and CS/CHI PIC particle dispersion, hydrogels with high cell-loading efficiency were successfully synthesized. The loaded cells within the hydrogels exhibited high viability, uniform distribution, and formation of cell aggregates. These results indicate that CS/CHI-based hydrogels have a potential application as three-dimensional scaffolds for cell culture in tissue engineering.

3D printed hydrogel for articular cartilage regeneration

Tissue engineering is a promising strategy for damaged cartilage tissue repair. Three-dimensional (3D) printed hydrogel exhibits great potential in cartilage tissue engineering for fabricating 3D cell culture scaffolds, owing to its similarity to the extracellular matrix (ECM). Numerous hydrogels have been tested for 3D printing in vitro articular cartilage tissues, including natural and synthetic hydrogels that mimic their in vivo counterparts. The advancement of materials science and 3D printing techniques enables a wide range of fabrication strategies that produce cartilage tissues with delicate structures and on multiple scales. Stimuli-responsive hydrogels, which rely on the external environment to transform to a desired structure or dimension, have likewise been widely studied in tissue engineering. This review summarizes the characteristics, functions, and research conducted on 3D printed hydrogels by categorizing cutting-edge hydrogel materials commonly used in cartilage tissue engineering and their complexes. The challenges and application prospects of hydrogels in cartilage tissue engineering are described. Novel composite hydrogels must be investigated to meet the requirements of native articular cartilage in the aspects of structure, scale, mechanical properties, among others. Combining stimuli-responsive hydrogels with biological scaffolds also shows great potential in various applications, including but not limited to articular cartilage, vascularization, and osteochondral repair.

Design of polysaccharidic Aloe vera gel incorporated PVA/tetracycline electrospun cell culture scaffolds for biomedical applications

In this study, hybrid nanofibrous 3D scaffolds containing Aloe vera (AV), polyvinyl alcohol (PVA) and tetracycline hydrochloride (TCH) are fabricated by electrospinning for cell culture applications. The role of polysaccharides present in AV gel is found to enhance the biocompatibility of the nanofibrous scaffolds. Different combinations of the polymers were selected to produce homogenous nanofibers with favorable mean fiber diameter and tensile strength. The surface morphology of the products was studied by SEM and it is found that the mean fiber diameter is decreased to about 188 nm upon addition of the AV component. The electrospun scaffolds were investigated by FT-IR spectroscopy to reveal the chemical structure of the samples and their crystallinity was studied by XRD. The hydrophilicity of the scaffolds was tested by optical contact angle measurements and their mechanical strength was examined by tensile strength tests. It is found that PVA is the main component contributing the mechanical stability of the scaffold structure. The fabricated scaffolds presented a more pronounced inhibitory effect against Gram-positive bacterial strains of S. aureus and B. cereus. Cell culture experiments using fibroblast L929 murine cells reveals that the AV/PVA/TCH scaffolds are promising for cell growth and the cells are capable of achieving a proper cell adhesion and proliferation. The cell viability experiment by MTT assay exhibits the contributing role of AV gel to L929 cell viability on the AV/PVA/TCH scaffolds.

Design and characterization of Persian gum/polyvinyl alcohol electrospun nanofibrous scaffolds for cell culture applications

Biocompatible electrospun nanofiber scaffolds were fabricated in this study using Persian gum (PG) and poly (vinyl alcohol) (PVA) to build an artificial extracellular matrix for cell growth. The preparation procedure involves mixing various ratios of PG/PVA to be electrospun and seeded with L929 fibroblasts. Upon addition of PG up to 60% to the solutions, a 30% decrease to around 240 μs·cm−1 is found in electrical conductivity which is in the range of semi-conductive polymers, whereas the surface tension is increased to around 3%. The fabricated scaffolds were characterized by morphological, chemical, thermal and structural analyses including SEM, FTIR spectroscopy, DSC, XRD, and tensile stress. The results showed that incorporation of 50% PG to the polymer solutions causes the formation of nanofibers with the least bead-shaped segments. All ratios of nanofibers containing PG showed significant biocompatibility with the cultured cells, which is presumably due to the radical scavenging feature of PG. The MTT and SEM analyses demonstrated that the scaffolds containing 50% PG possess the optimal cell compatibility, adhesion and proliferation properties. The fabricated PG/PVA cell culture scaffolds are potentially appropriate for wound dressing and cell culture applications in biomedicine.

Hierarchical Membranes Self‐Assembled at the Interface between Peptides and Polymer Aqueous Solutions

AbstractBiocompatible and biodegradable molecules capable of spontaneous self‐assembly into hierarchical structures have the potential to serve as building blocks for complex scaffolds for cell culture. These scaffolds are meant to mimic the natural environment required for successful cell growth. Thus, they must be precisely designed and present specific mechanical and structural properties along with required biological and chemical cues. Combining the hierarchical structure and chemical functionality of a peptide with the stability of a polymer has led to the formation of complex and stable biomaterials. Among these hybrid biomaterials, macroscopic membranes self‐assembled at the aqueous interface between self‐assembling peptides and polymer solutions have gained attention over the past decade. The goal of this mini‐review is to highlight the advantages and challenges of utilizing polymer/peptide self‐assembled macroscopic membranes toward the development of novel platforms for the study of the interaction of different cells with their environment. As most of the membranes features are determined as soon as they assemble, this review begins with a detailed description of the membranes formation mechanism, which evolved and developed over the years. The variety of membranes formed using different peptides and polymers is then described, with special focus placed on their unique structure. Then the bio‐functions of the membranes and their emerging potential for bio‐applications are presented. In the final section, the shortcomings preventing an extended use of these membranes as well as their future prospects, are discussed. This mini‐review aims to give light to an emerging niche of biomaterials capable of forming hierarchical structures utilizing natural building blocks and the naturally occurring process of self‐assembly, providing an innovative approach for creating scaffolds that mimic the natural extracellular environment.

Interdisciplinary Methods for Zoonotic Tissue Acellularization for Natural Heart Valve Substitute of Biomimetic Materials.

The goal of this work was to create a bioactive tissue-based scaffold using multi-disciplinary engineering materials and tissue engineering techniques. Materials & methods: Physical techniques such as direct laser interference lithography and proton radiation were selected as alternative methods of enzymatic and chemical decellularization to remove cells from a tissue without degradation of the extracellular matrix nor its protein structure. This study was an attempt to prepare a functional scaffold for cell culture from tissue of animal origin using new physical methods that have not been considered before. The work was carried out under full control of the histological and molecular analysis. Results & conclusions: The most important finding was that the physical methods used to obtain the decellularized tissue scaffold differed in the efficiency of cell removal from the tissue in favour of the laser method. Both the laser method and the proton method exhibited a destructive effect on tissue structure and the genetic material in cell nuclei. This effect was visible on histology images as blurred areas within the cell nucleus. The finite element 3D simulation of decellularization process of the three-layer tissue of animal origin sample reflected well the mechanical response of tissue described by hyperelastic material models and provided results comparable to the experimental ones.

Oxidized Alginate Hydrogel-Based Derivatives with Optimized Features for Cell Culture Scaffold

Oxidized Alginate Hydrogel-Based Derivatives with Optimized Features for Cell Culture Scaffold

Fiber Thickness and Porosity Control in a Biopolymer Scaffold 3D Printed through a Converted Commercial FDM Device.

3D printing has opened exciting new opportunities for the in vitro fabrication of biocompatible hybrid pseudo-tissues. Technologies based on additive manufacturing herald a near future when patients will receive therapies delivering functional tissue substitutes for the repair of their musculoskeletal tissue defects. In particular, bone tissue engineering (BTE) might extensively benefit from such an approach. However, designing an optimal 3D scaffold with adequate stiffness and biodegradability properties also guaranteeing the correct cell adhesion, proliferation, and differentiation, is still a challenge. The aim of this work was the rewiring of a commercial fuse deposition modeling (FDM) 3D printer into a 3D bioplotter, aiming at obtaining scaffold fiber thickness and porosity control during its manufacturing. Although it is well-established that FDM is a fast and low-price technology, the high temperatures required for printing lead to limitations in the biomaterials that can be used. In our hands, modifying the printing head of the FDM device with a custom-made holder has allowed to print hydrogels commonly used for embedding living cells. The results highlight a good resolution, reproducibility and repeatability of alginate/gelatin scaffolds obtained via our custom 3D bioplotter prototype, showing a viable strategy to equip a small-medium laboratory with an instrument for manufacturing good-quality 3D scaffolds for cell culture and tissue engineering applications.

Mechanical, microstructural, and rheological characterization of gelatin-dialdehyde starch hydrogels constructed by dual dynamic crosslinking

Novel gelatin/dialdehyde starch (GDS) hydrogels based on gelatin were prepared using dialdehyde starch as a macromolecular crosslinking agent. Fourier transform infrared spectroscopy, Raman spectroscopy, and X-ray diffraction showed that the dynamic hydrogel network was formed by covalent imine bonds and hydrogen bonds between dialdehyde starch and gelatin. The synergistic physical and chemical cross-linking endowed the hydrogels with excellent mechanical properties (maximum compressive stress was 0.55 MPa), fatigue resistance (they maintained their shape after ten compression cycles), toughness (54.6 kN/m2), and self-recoverability of the polymeric network. In the rheological tests, all the storage modulus were higher than loss modulus, indicating that they are elastic gels and not viscous sols. The polymer network of GDS hydrogels could recover rapidly when subjected to oscillating shear strain. In addition, with increasing dialdehyde starch concentration, the hydrogels had higher swelling stability and a denser microstructure. Given these excellent properties, the hydrogels have potential application prospects as cell culture scaffolds and carriers of bioactive substance.

Bioadaptive Porous 3D Scaffolds Comprising Cellulose and Chitosan Nanofibers Constructed by Pickering Emulsion Templating

AbstractHighly porous three‐dimensional (3D) scaffolds can mimic the lobular structure of a human liver where hepatocytes are organized. However, 3D scaffolds with uniformly porous and oriented structures are challenging to fabricate without cross‐linking agents. Herein, this work presents a Pickering emulsion‐induced interface approach to construct aligned porous scaffolds for 3D cell cultures through the combined use of surface‐carboxylated cellulose nanofibers and chitosan nanofibers as stabilizers, and freezing/lyophilization to remove the oil phase. The obtained Pickering emulsions exhibit long‐term stability and their droplet sizes are tunable from 2.7 to 10.2 µm. Assembly at the oil–water interface can be modulated by controlling the NaCl dosage and oil phase proportion, resulting in porous foams with tunable porosity and versatile architectures as an in vitro alternative to the native liver microenvironment. The foams are noncytotoxic, confirmed using mouse fibroblast NIH/3T3 cells, and the cells grow both on the surface and in the internal structure of the foam. Notably, the 3D porous scaffolds are favorable microenvironments for the formation of human liver carcinoma HepG2 spheroidal cells, which exhibit liver‐like activity. This strategy based on Pickering emulsion templating provides a new avenue for constructing bioadaptive 3D scaffolds, specifically all‐biomass porous foams, for tissue engineering.

3D printing of biocompatible low molecular weight gels: Imbricated structures with sacrificial and persistent N-alkyl-d-galactonamides

HypothesisWe have shown earlier that low molecular weight gels based on N-heptyl-d-galactonamide hydrogels can be 3D printed by solvent exchange, but they tend to dissolve in the printing bath. We wanted to explore the printing of less soluble N-alkyl-d-galactonamides with longer alkyl chains. Less soluble hydrogels could be good candidates as cell culture scaffolds. ExperimentsN-hexyl, N-octyl and N-nonyl-d-galactonamide solutions in dimethylsulfoxide are injected in a bath of water following patterns driven by a 2D drawing robot coupled to a z-platform. Solubilization of the gels with time has been determined and solubility of the gelators has been measured by NMR. Imbricated structures have been built with N-nonyl-d-galactonamide as a persistent ink and N-hexyl or N-heptyl-d-galactonamide as sacrificial inks. Human mesenchymal stem cells have been cultured on N-nonyl-d-galactonamide hydrogels prepared by cooling or by 3D printing. FindingsThe conditions for printing well-resolved 3D patterns have been determined for the three gelators. In imbricated structures, the solubilization of N-hexyl or N-heptyl-d-galactonamide occurred after a few hours or days and gave channels. Human mesenchymal stem cells grown on N-nonyl-d-galactonamide hydrogels prepared by heating–cooling, which are stable and have a fibrillar microstructure, developed properly. 3D printed hydrogels, which microstructure is made of micrometric flakes, appeared too fragile to withstand cell growth.

Kinetic Method of Producing Pores Inside Protein-Based Biomaterials without Compromising Their Structural Integrity.

Hydrogels made from globular proteins cross-linked covalently into a stable network are becoming an important type of biomaterial, with applications in artificial tissue design and cell culture scaffolds, and represent a promising system to study the mechanical and biochemical unfolding of proteins in crowded environments. Due to the small size of the globular protein domains, typically 2-5 nm, the primary network allows for a limited transfer of protein molecules and prevents the passing of particles and aggregates with dimensions over 100 nm. Here, we demonstrate a method to produce protein materials with micrometer-sized pores and increased permeability. Our approach relies on forming two competing networks: a covalent network made from cross-linked bovine serum albumin (BSA) proteins via a light-activated reaction and a physical network triggered by the aggregation of a polysaccharide, alginate, in the presence of Ca2+ ions. By fine-tuning the reaction times, we produce porous-protein hydrogels that retain the mechanical characteristics of their less-porous counterparts. We further describe a simple model to investigate the kinetic balance between the nucleation of alginate and cross-linking of BSA molecules and find the upper rate of the alginate aggregation reaction driving pore formation. By enabling a more significant permeability for protein-based materials without compromising their mechanical response, our method opens new vistas into studying protein-protein interactions and cell growth and designing novel affinity methods.

The Application of Polycaprolactone Scaffolds with Poly(ε-caprolactone)-Poly(ethylene glycol)-Poly(ε-caprolactone) Loaded on Kidney Cell Culture.

Human embryonic kidney cells are the host of adenovirus type-5 (Ad5) amplification. An Ad5-vector-based COVID-19 vaccine has been proven to be tolerated and immunogenic in healthy adults. Therefore, a rationally designed scaffold for culturing human embryonic kidney cells is useful for further studying its mechanism of action. Herein, a three-dimensional layered reticulated polycaprolactone (PCL) scaffold coated with poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) was developed to proliferate human embryonic kidney cells and to be used to amplify the Ad5 vector. The results indicate that PCEC improves the hydrophilicity and the cell culture ability of PCL cell culture scaffolds, resulting in a three times higher cell proliferation ratio of human embryonic kidney cells compared with those grown on bare PCL cell culture scaffolds. Meanwhile, the cytotoxicity test results showed that the scaffold material is noncytotoxic. This work provides an effective and scalable method for the in-depth study of adenoviruses.

Photoactuating Artificial Muscles of Motor Amphiphiles as an Extracellular Matrix Mimetic Scaffold for Mesenchymal Stem Cells.

Mimicking the native extracellular matrix (ECM) as a cell culture scaffold has long attracted scientists from the perspective of supramolecular chemistry for potential application in regenerative medicine. However, the development of the next-generation synthetic materials that mimic key aspects of ECM, with hierarchically oriented supramolecular structures, which are simultaneously highly dynamic and responsive to external stimuli, remains a major challenge. Herein, we present supramolecular assemblies formed by motor amphiphiles (MAs), which mimic the structural features of the hydrogel nature of the ECM and additionally show intrinsic dynamic behavior that allow amplifying molecular motions to macroscopic muscle-like actuating functions induced by light. The supramolecular assembly (named artificial muscle) provides an attractive approach for developing responsive ECM mimetic scaffolds for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Detailed investigations on the photoisomerization by nuclear magnetic resonance and UV–vis absorption spectroscopy, assembled structures by electron microscopy, the photoactuation process, structural order by X-ray diffraction, and cytotoxicity are presented. Artificial muscles of MAs provide fast photoactuation in water based on the hierarchically anisotropic supramolecular structures and show no cytotoxicity. Particularly important, artificial muscles of MAs with adhered hBM-MSCs still can be actuated by external light stimulation, showing their ability to convert light energy into mechanical signals in biocompatible systems. As a proof-of-concept demonstration, these results provide the potential for building photoactuating ECM mimetic scaffolds by artificial muscle-like supramolecular assemblies based on MAs and offer opportunities for signal transduction in future biohybrid systems of cells and MAs.

A loofah-inspired scaffold with enhanced mimicking mechanics and tumor cells distribution for in vitro tumor cell culture platform

Tumor cells cultured in a physiologically related three-dimensional (3D) matrix can replicate many basic characteristics of tumor tissue. Tumor tissues are harder than normal, so when using hydrogels for 3D tumor cell culture, attempts have been made to prepare hydrogel scaffolds that mimic the hardness of tumor tissues without reducing the porosity. In this study, a new 3D loofah-inspired scaffold was developed for prostate cancer cell culture. Since the loofah sponge structure of the spacer fabric, the composite scaffolds had a compression modulus similar to that of natural prostate tumor tissue at a lower hydrogel concentration (0.25% W/V), and also, endowed the scaffold with high porosity (85 ± 2.52%) for mass transfer. The results of in vitro cell experiments showed that the composite scaffold can support tumor cells to form clusters in a short time (3 days). Preliminary chemosensitivity analysis showed that the drug resistance of the composite scaffold was significantly higher than that of two-dimensional (2D) culture and COL scaffold. Therefore, the 3D tumor cell culture scaffold with bionic structures has the potential to be used as a tumor drug screening model.

Synthetic Collagen-like Polymer That Undergoes a Sol-Gel Transition Triggered by O-N Acyl Migration at Physiological pH.

We previously reported an artificial collagen gel that can be used as a cell-culture substrate by end-to-end cross-linking of collagen-like triple-helical peptides via disulfide bonds. However, the gel had to be formed a priori by polymerizing the peptide in an acidic solution containing dimethyl sulfoxide for several days, which prevented its use as an injectable gel or three-dimensional (3D) scaffold for cell culture. In this study, we developed a collagen-like peptide polymer by incorporating an O–N acyl migration-triggered triple helix formation mechanism into a collagen-like peptide, which formed a gel within 10 min. We demonstrated that the collagen-like peptide polymer can be used as a 3D cell scaffold and that the 3D structure formation of cells can be controlled by collagen-derived bioactive sequences introduced into the peptide sequence.

Combination of Polysaccharide Nanofibers Derived from Cellulose and Chitin Promotes the Adhesion, Migration and Proliferation of Mouse Fibroblast Cells.

Extracellular matrix (ECM) as a structural and biochemical scaffold to surrounding cells plays significant roles in cell adhesion, migration, proliferation and differentiation. Herein, we show the novel combination of TEMPO-oxidized cellulose nanofiber (TOCNF) and surface-N-deacetylated chitin nanofiber (SDCtNF), respectively, having carboxylate and amine groups on each crystalline surface, for mouse fibroblast cell culture. The TOCNF/SDCtNF composite scaffolds demonstrated characteristic cellular behavior, strongly depending on the molar ratios of carboxylates and amines of polysaccharide NFs. Pure TOCNF substrate exhibited good cell attachment, although intact carboxylate-free CNF made no contribution to cell adhesion. By contrast, pure SDCtNF induced crucial cell aggregation to form spheroids; nevertheless, the combination of TOCNF and SDCtNF enhanced cell attachment and subsequent proliferation. Molecular blend of carboxymethylcellulose and acid-soluble chitosan made nearly no contribution to cell culture behavior. The wound healing assay revealed that the polysaccharide combination markedly promoted skin repair for wound healing. Both of TOCNF and SDCtNF possessed rigid nanofiber nanoarchitectures with native crystalline forms and regularly-repeated functional groups, of which such structural characteristics would provide a potential for developing cell culture scaffolds having ECM functions, possibly promoting good cellular adhesion, migration and growth in the designated cellular microenvironments.