Cell Scaffolds For Tissue Engineering Research Articles

Nanofabrication Technologies to Control Cell and Tissue Function in Three-Dimension.

In the 2000s, advances in cellular micropatterning using microfabrication contributed to the development of cell-based biosensors for the functional evaluation of newly synthesized drugs, resulting in a revolutionary evolution in drug screening. To this end, it is essential to utilize cell patterning to control the morphology of adherent cells and to understand contact and paracrine-mediated interactions between heterogeneous cells. This suggests that the regulation of the cellular environment by means of microfabricated synthetic surfaces is not only a valuable endeavor for basic research in biology and histology, but is also highly useful to engineer artificial cell scaffolds for tissue regeneration. This review particularly focuses on surface engineering techniques for the cellular micropatterning of three-dimensional (3D) spheroids. To establish cell microarrays, composed of a cell adhesive region surrounded by a cell non-adherent surface, it is quite important to control a protein-repellent surface in the micro-scale. Thus, this review is focused on the surface chemistries of the biologically inspired micropatterning of two-dimensional non-fouling characters. As cells are formed into spheroids, their survival, functions, and engraftment in the transplanted site are significantly improved compared to single-cell transplantation. To improve the therapeutic effect of cell spheroids even further, various biomaterials (e.g., fibers and hydrogels) have been developed for spheroid engineering. These biomaterials not only can control the overall spheroid formation (e.g., size, shape, aggregation speed, and degree of compaction), but also can regulate cell-to-cell and cell-to-matrix interactions in spheroids. These important approaches to cell engineering result in their applications to tissue regeneration, where the cell-biomaterial composite is injected into diseased area. This approach allows the operating surgeon to implant the cell and polymer combinations with minimum invasiveness. The polymers utilized in hydrogels are structurally similar to components of the extracellular matrix in vivo, and are considered biocompatible. This review will provide an overview of the critical design to make hydrogels when used as cell scaffolds for tissue engineering. In addition, the new strategy of injectable hydrogel will be discussed as future directions.

Thiol-ene-mediated degradable POSS-PEG/PEG hybrid hydrogels as potential cell scaffolds in tissue engineering

Tissue engineering provides therapeutic strategies for the loss or failure of human organs and tissues. As a three-dimensional biomaterial that simulates extracellular matrix (ECM) in tissue engineering, hydrogels have attracted much attention and been used as cell scaffolds. Herein, three POSS-PEG/PEG hydrogels with different solid contents were designed and developed through thiol-ene click reaction between a star-shaped cross-linker allyl-terminated poly(ethylene glycol)-functionalized polyhedral oligosiloxane (POSS-PEG-Allyl) and poly(ethylene glycol)-dithiol (SH-PEG-SH). The chemical structures, microscopic morphologies, element distribution mapping, rheological properties, swelling properties, in vitro degradation and cytocompatibility of the hydrogels were further investigated. The results showed that the hybrid hydrogels contain a large amount of water, which can create a suitable water environment for cell growth. The mechanical strength of the hybrid hydrogels is improved by introducing POSS nanoparticles to the hydrogel system, and the ester groups in the cross-linker allow the hydrogel network can be biodegrade by hydrolytic cleavage. The mechanical strength and the degradation rate can be adjusted by the solid content of the hybrid hydrogels. Besides, the hybrid hydrogel has excellent cell compatibility and porous internal structure, which provide conditions for cell growth and proliferation. Therefore, it can be considered that the hybrid hydrogel has great application potential as a cell scaffold material.

Construction of 3D bioprinting of HAP/collagen scaffold in gelation bath for bone tissue engineering.

Reconstruction of bone defects remains a clinical challenge, and 3D bioprinting is a fabrication technology to treat it via tissue engineering. Collagen is currently the most popular cell scaffold for tissue engineering; however, a shortage of printability and low mechanical strength limited its application via 3D bioprinting. In the study, aiding with a gelatin support bath, a collagen-based scaffold was fabricated via 3D printing, where hydroxyapatite (HAP) and bone marrow mesenchymal stem cells (BMSCs) were added to mimic the composition of bone. The results showed that the blend of HAP and collagen showed suitable rheological performance for 3D extrusion printing and enhanced the composite scaffold's strength. The gelatin support bath could effectively support the HAP/collagen scaffold's dimension with designed patterns at room temperature. BMSCs in/on the scaffold kept living and proliferating, and there was a high alkaline phosphate expression. The printed collagen-based scaffold with biocompatibility, mechanical properties and bioactivity provides a new way for bone tissue engineering via 3D bioprinting.

Mechanical properties of collagen fibrils determined by buckling analysis

The mechanical properties of biological nanofibers such as collagen fibrils are important in many applications, ranging from tissue-engineering to cancer treatment. However, mechanical testing is not straightforward at the nanometer scale. Here, we use the theory of column-buckling to determine the bending properties of individual collagen fibrils. To achieve this, fibrils were deposited on a manually pre-stretched foil, which was then released with the fibrils attached. Atomic Force Microscopy (AFM) imaging was used to determine the tensile modulus by measuring the buckling-wavelength and the radius for each fibril. Comparison with data obtained by AFM nanoindentation and other, more sophisticated methods, shows that our results are in very good agreement. The great advantage of this simple approach is that it can be used to quickly determine mechanical properties without force or stress-strain measurements, which are challenging to obtain accurately and at high throughput at the nanoscale. The method could be applied to any nanofibers, not just collagen fibrils. Statement of significanceCollagen fibrils are the main constituent of the extracellular matrix, and alterations of their mechanical properties can have significant effects on cell adhesion and motility. This has, ultimately, implications in age-related diseases and cancer. Furthermore, tuning the mechanical properties of collagen fibrils could be an important tool in the design of artificial cell scaffolds in tissue-engineering. For these reasons, it is important to have methods that can be used to determine the mechanical properties of fibrils at the single-fibril level and, therefore, at the nanometer scale. The method presented here has the advantage of being easy to use and avoids some of the fundamental issues of more established methods.

Temperature-Responsive Aldehyde Hydrogels with Injectable, Self-Healing, and Tunable Mechanical Properties.

Injectable and self-healing hydrogels with exemplary biocompatibility and tunable mechanical properties are urgently needed due to their significant advantages for tissue engineering applications. Here, we report a new temperature-responsive aldehyde hydrogel with dual physical-cross-linked networks and injectable and self-healing properties prepared from an ABA-type triblock copolymer, poly{[FPMA(4-formylphenyl methacrylate)-co-DEGMA[di(ethylene glycol) methyl ether methacrylate]-b-MPC(2-methacryloyloxyethyl phosphorylcholine)-b-(FPMA-co-DEGMA)}. The thermoresponsive poly(DEGMA) segments drive the dehydration and hydrophobic interaction, enabling polymer chain winding as the first cross-linking network, when the temperature is raised above the critical gelation temperature. Meanwhile, the benzaldehyde groups offer physical interactions, including hydrogen bonding and hydrophobic and π-π stacking interactions as the second cross-linking network. When increasing the benzaldehyde content in the triblock copolymers from 0 to 8.2 mol %, the critical gelation temperature of the resulted hydrogels dropped from 35.5 to 19.9 °C and the mechanical modulus increased from 21 to 1411 Pa. Owing to the physical-cross-linked networks, the hydrogel demonstrated excellent injectability and self-healing properties. The cell viabilities tested from MTT assays toward both normal lung fibroblast cells (MRC-5) and cancerous cervical (HeLa) cells were found to be 100 and 101%, respectively, for varying polymer concentrations up to 1 mg/mL. The 3D cell encapsulation of the hydrogels was evaluated by a cytotoxicity Live/Dead assay, showing 92% cell viability. With these attractive physiochemical and biological properties, this temperature-responsive aldehyde hydrogel can be a promising candidate as a cell scaffold for tissue engineering.

Fabrication of Injectable Chitosan-Chondroitin Sulfate Hydrogel Embedding Kartogenin-Loaded Microspheres as an Ultrasound-Triggered Drug Delivery System for Cartilage Tissue Engineering.

Ultrasound-responsive microspheres (MPs) derived from natural polysaccharides and injectable hydrogels have been widely investigated as a biocompatible, biodegradable, and controllable drug delivery system and cell scaffolds for tissue engineering. In this study, kartogenin (KGN) loaded poly (lactide-co-glycolic acid) (PLGA) MPs (MPs@KGN) were fabricated by premix membrane emulsification (PME) method which were sonicated by an ultrasound transducer. Furthermore, carboxymethyl chitosan-oxidized chondroitin sulfate (CMC-OCS) hydrogel were prepared via the Schiff’ base reaction-embedded MPs to produce a CMC-OCS/MPs scaffold. In the current work, morphology, mechanical property, porosity determination, swelling property, in vitro degradation, KGN release from scaffolds, cytotoxicity, and cell bioactivity were investigated. The results showed that MPs presented an obvious collapse after ultrasound treatment. The embedded PLGA MPs could enhance the compressive elastic modulus of soft CMC-OCS hydrogel. The cumulative release KGN from MPs exhibited a slow rate which would display an appropriate collapse after ultrasound, allowing KGN to maintain a continuous concentration for at least 28 days. Moreover, the composite CMC-OCS@MPs scaffolds exhibited faster gelation, lower swelling ratio, and lower in vitro degradation. CCK-8 and LIVE/DEAD staining showed these scaffolds did not influence rabbit bone marrow mesenchymal stem cells (rBMMSCs) proliferation. Then these scaffolds were cultured with rBMMSCs for 2 weeks, and the immunofluorescent staining of collagen II (COL-2) showed that CMC-OCS hydrogel embedded with MPs@KGN (CMC-OCS@MPs@KGN) with ultrasound had the ability to increase the COL-2 synthesis. Overall, due to the improved mechanical property and the ability of sustained KGN release, this injectable hydrogel with ultrasound-responsive property is a promising system for cartilage tissue engineering.

Construction and characterization of degradable fish scales for enhancing cellular adhesion and potential using as tissue engineering scaffolds

As a framework for tissue engineering regeneration, the characteristics of cell scaffold materials directly affect cell adhesion, migration and metabolism. In this study, we have fabricated decellularized and decalcified fish scale-derived scaffolds and determined its basic physicochemical properties to serve as cell scaffolds in tissue engineering. Scanning electron microscopy (SEM) results showed that there were radial grooves and ring ridges on the surface of the scale-derived scaffolds, which could simulate three-dimensional microenvironment for cells culture. Similarity to the bone extracellular matrix, the main components of the fish scales were hydroxyapatite (HA) and type I collagen fibers, which were conducive to cells spreading and proliferation. Moreover, for culturing L929 cells and rat bone marrow mesenchymal stem cells (BMSCs), the fish scales as cell scaffolds exhibited high cytocompatibility to enhance cells adhesion and proliferation, and also displayed the ability to guide cells migration along the ridge channels. Accordingly, the results suggested that the fish scale-derived scaffolds had a great potential as a natural extracellular matrix for tissue engineering.

Effects of phosphate ion concentration on in-vitro fibrillogenesis of sturgeon type I collagen

Nonmammalian collagens have attracted significant attention owing to their potential for use as a source of cell scaffolds for tissue engineering. Since the morphology of collagen fibrils controls cell proliferation and differentiation, its regulation is essential for fabricating scaffolds with desirable characteristics. In this study, we evaluated the effects of the phosphate ion (Pi) concentration on the characteristics of fibrils formed from swim bladder type I collagen (SBC) and skin type I collagen (SC) from the Bester sturgeon. An increase in the Pi concentration decreased the fibril formation rate, promoted the formation of thick fibrils, and increased the thermal stability of the fibrils for both SBC and SC. However, the SBC and SC fibrils exhibited different fibril formation rates, degrees of fibrillogenesis, morphologies, and denaturation temperatures for the same reaction conditions. Finally, by regulating the Pi concentration, various types of SBC and SC fibrils could be coated on cell culture wells, and fibroblasts could be cultured on them. The results showed that thin fibrils enhance fibroblast extension and proliferation, whereas thick fibrils restrain fibroblast extension but orient them in the same direction. The results of this study suggest that SBC fibrils, which exhibit diverse morphologies, are suitable for use as a novel scaffold material, whose characteristics can be tailored readily by varying the Pi concentration.

In Situ Forming and Reversibly Cross-Linkable Hydrogels Based on Copolypept(o)ides and Polysaccharides.

The emerging tide of hydrogels in biomedical fields drives them to possess good biocompatibility, tunable mechanical properties, and fast gelation process. Herein, a composite hydrogel containing copolypept(o)ides and functional polysaccharides was constructed through dynamic acylhydrazone linkages. First, a series of peptide-peptoid copolymers were synthesized by ring-opening polymerization of sarcosine (Sar) and l-glutamic acid γ-benzyl ester (BLG) N-carboxyanhydrides (NCAs). The benzyl groups of BLG units were substituted with hydrazide groups through ester-amide exchange aminolysis reaction. The statistical copolymer of poly(sarcosine-co-glutamate-hydrazide) (P(Sar-co-GH)) was chosen as an optimized precursor due to its excellent water solubility and gel-forming ability with aldehyde-modified sodium alginate (OSA). Moreover, cellulose nanocrystals (CNCs) were prepared as nanofillers to reinforce the P(Sar-co-GH)-OSA hydrogel. We demonstrated that the copolymer sequences and composition contents made a difference to the properties of the formed hydrogels by variation of the cross-linking density. The dynamic acylhydrazone bonds endowed hydrogels with pH responsiveness and reversible networks. The NIH/3T3 cells encapsulated in the hydrogels maintained high viability and proliferation abilities, indicating that the nanocomposite hydrogels could be explored to fabricate a customized responsive drug delivery system or cell scaffolds for tissue engineering.

Frontispiece: Advances in Cell Scaffolds for Tissue Engineering: The Value of Liquid Crystalline Elastomers

Frontispiece: Advances in Cell Scaffolds for Tissue Engineering: The Value of Liquid Crystalline Elastomers

Advances in Cell Scaffolds for Tissue Engineering: The Value of Liquid Crystalline Elastomers.

Recent discoveries evidenced that many cells organize into well-aligned nematic domains, showing also their topological defects and suggesting the liquid crystalline order to be necessary for some biological functions. These evidences were described as the basis for the development of a new area of research in which polymeric liquid crystals were developed to exploit and promote cell adhesion and proliferation towards tissue regeneration. To address the requirements of tissue engineering, new biocompatible materials must be designed and synthesized to support cell adherence and growth together with nutrient transport under physiological condition. This Minireview presents a journey that, starting from the first discovery of liquid crystalline phases in biological (natural) materials with different structures and physical-chemical properties, will inform readers of the very recent application of liquid crystal polymeric materials as functional cell scaffolds to address current tissue engineering issues.

Stem Cell Scaffolds for Tissue Engineering

Stem Cell Scaffolds for Tissue Engineering

Highly Ordered 1D Fullerene Crystals for Concurrent Control of Macroscopic Cellular Orientation and Differentiation toward Large-Scale Tissue Engineering.

A highly aligned 1D fullerene whisker (FW) scaffold in a centimeter area is fabricated by interfacial alignment. The resulting aligned FW scaffold enables concurrent control over cellular orientation and differentiation to muscle cells. This aligned FW scaffold is made by a facile method, and hence the substrate is a promising alternative to other cell scaffolds for tissue engineering.

Fabrication of thermoplastic ductile films of chitin butyrate/poly(ɛ-caprolactone) blends and their cytocompatibility

We fabricate thermoplastic films of chitin burtyrate (ChB)/poly(ɛ-caprolactone) (PCL) blends with different degree of miscibility (miscible (M), partially miscible (PM), and immiscible (IM)), and examined the feasibility as a cell scaffold system through evaluating mechanical properties and cytocompatibility. We found a remediation of the brittleness and an increase in ductility of ChB by blending PCL for the M and PM blends. The blend films were subjected to alkaline hydrolysis (2-M NaOH/37°C/48h) with expectation of the improvement of the surface hydrophilicity and cell accessibility. ATR-FTIR spectroscopy of the alkaline-treated PM and IM films revealed that PCL component and ester side-chains of acyl chitin were selectively removed from the surface domain. L929 fibroblast cells well adhered and proliferated on these films. Therefore, the materials possess a great potential for the utilization as a thermoplastic cell scaffold in tissue engineering by adequate selection of the degree of miscibility and post treatment.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)-based scaffolds for tissue engineering.

Development and selection of an ideal scaffold is of importance for tissue engineering. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible bioresorbable copolymer that belongs to the polyhydroxyalkanoate family. Because of its good biocompatibility, PHBHHx has been widely used as a cell scaffold for tissue engineering. This review focuses on the utilization of PHBHHx-based scaffolds in tissue engineering. Advances in the preparation, modification, and application of PHBHHx scaffolds are discussed.

Dextran Acetate-Based Sponge as Cell Scaffold for Tissue Engineering

Dextran Acetate-Based Sponge as Cell Scaffold for Tissue Engineering

Redox‐Responsive Degradable PEG Cryogels as Potential Cell Scaffolds in Tissue Engineering

A Michael addition strategy involving the reaction between a maleimide double bond and amine groups is investigated for the synthesis of cryogels at subzero temperature. Low-molecular-weight PEG-based building blocks with amine end groups and disulfide-containing building blocks with maleimide end groups are combined to synthesize redox-responsive PEG cryogels. The cryogels exhibit an interconnected macroporous morphology, a high compressive modulus and gelation yields of around 95%. While the cryogels are stable under physiological conditions, complete dissolution of the cryogels into water-soluble products is obtained in the presence of a reducing agent (glutathione) in the medium. Cell seeding experiments and toxicologic analysis demonstrate their potential as scaffolds in tissue engineering.

Bio rapid prototyping by extruding/aspirating/refilling thermoreversible hydrogel

This paper reports a method for rapid prototyping of cell tissues, which is based on a system that extrudes, aspirates and refills a mixture of cells and thermoreversible hydrogel as a scaffold. In the extruding mode, a cell-mixed scaffold solution in the sol state is extruded from a cooled micronozzle into a temperature-controlled substrate, which keeps the scaffold in the gel state. In the aspiration mode, the opposite process is performed by Bernoulli suction. In the refilling mode, the solution is extruded into a groove created in the aspiration mode. The minimum width of extruded hydrogel pattern is 114 ± 15 µm by employing a nozzle of diameter 100 µm, and that of aspirated groove was 355 ± 10 µm using a 500 µm-diameter nozzle. Gum arabic is mixed with the scaffold solution to avoid peeling-off of the gel pattern from the substrate. Patterning of Sf-9 cell tissue is demonstrated, and the stability of the patterned cell is investigated. This system offers a procedure for rapid prototyping and local modification of cell scaffolds for tissue engineering.

Cell adhesion and tissue response to hydroxyapatite nanocrystal-coated poly(l-lactic acid) fabric

Cell adhesion and tissue response to poly(l-lactic acid) (PLLA) fabric coated with nanosized hydroxyapatite (HAp) crystals were studied. The HAp nanocrystals were prepared by the wet chemical process followed by calcination at 800 degrees C with an anti-sintering agent to prevent calcination-induced sintering. After the PLLA fabric was hydrolyzed with an alkaline aqueous solution, the HAp nanocrystals were coated via ionic interaction between the calcium ions on the HAp and the carboxyl groups on the alkali-treated PLLA. The PLLA surface uniformly coated with the HAp nanocrystals was observed by scanning electron microscope. The ionic interaction between the HAp and the PLLA was estimated by FT-IR. Improved cell adhesion to the HAp nanocrystal-coated surface was demonstrated by in vitro testing using a mouse fibroblast cell line L929. Furthermore, reduced inflammatory response to the HAp nanocrystal-coated PLLA fabric (as compared with a non-treated one) was confirmed by a subcutaneous implantation test with rats. Thus the HAp nanocrystal-coated PLLA developed has possible efficacy as an implant material in the fields of general and orthopedic surgery, and as a cell scaffold in tissue engineering.

Stochastic Analysis of Stepwise Fluorescence Quenching Reactions on Single-Walled Carbon Nanotubes: Single Molecule Sensors

The 1D quantum confinement of photogenerated excitons in single-walled carbon nanotubes (SWNT) can amplify the detection of molecular adsorption to where single-molecule discrimination is realizable, even from within living cells and tissues. Toward this aim, we present a type 1 collagen film, similar to those used as 3D cell scaffolds for tissue engineering, containing embedded SWNT capable of reporting single-molecule adsorption of quenching molecules. We utilize hidden Markov modeling to link single-molecule adsorption events to rate constants for H2O2, H+, and Fe(CN)6(3-). Among the three kinds of reactant molecules studied, H2O2 has the highest quenching equilibrium constant of 1.59 at 20 microM, whereas H+ is so insensitive that a similar equilibrium constant is achieved only with a concentration of 0.1 M (pH 1). The results were self-consistent because reverse (unquenching) rate constants (600 micros(-1) for H2O2, 1130 micros(-1) for H+ and 4000 micros(-1) for Fe(CN)6(3-)) were observed to be concentration-independent and the forward (quenching) rate constants varied monotonically with concentration. The quenching rate constants also increased with an increase in the redox potential of the quencher, indicating that electron transfer increases the adsorption equilibrium constant on the nanotube surface and, hence, the dwell time of the quencher. These developments provide the material, analytical, and mechanistic groundwork for SWNT to function as single-molecule stochastic biosensors.