Three-dimensional Cell Scaffolds Research Articles

Tissue-Adhesive Hydrogel Spray System for Live Cell Immobilization on Biological Surfaces.

Gelatin hydrogels are used as three-dimensional cell scaffolds and can be prepared using various methods. One widely accepted approach involves crosslinking gelatin amino groups with poly(ethylene glycol) (PEG) modified with N-hydroxysuccinimide ester (PEG-NHS). This method enables the encapsulation of live cells within the hydrogels and also facilitates the adhesion of the hydrogel to biological tissues by crosslinking their surface amino groups. Consequently, these hydrogels are valuable tools for immobilizing cells that secrete beneficial substances in vivo. However, the application of gelatin hydrogels is limited due to the requirement for several minutes to solidify under conditions of neutral pH and polymer concentrations suitable for live cells. This limitation makes it impractical for use with biological tissues, which have complex shapes or inclined surfaces, restricting its application to semi-closed spaces. In this study, we propose a tissue-adhesive hydrogel that can be sprayed and immobilized with live cells on biological tissue surfaces. This hydrogel system combines two components: (1) gelatin/PEG-NHS hydrogels and (2) instantaneously solidifying PEG hydrogels. The sprayed hydrogel solidified within 5 s after dispensing while maintaining the adhesive properties of the PEG-NHS component. The resulting hydrogels exhibited protein permeability, and the viability of encapsulated human mesenchymal stem/stromal cells (hMSCs) remained above 90% for at least 7 days. This developed hydrogel system represents a promising approach for immobilizing live cells on tissue surfaces with complex shapes.

High-Throughput Dispensing of Viscous Solutions for Biomedical Applications

Cells cultured in three-dimensional scaffolds express a phenotype closer to in vivo cells than cells cultured in two-dimensional containers. Natural polymers are suitable materials to make three-dimensional scaffolds to develop disease models for high-throughput drug screening owing to their excellent biocompatibility. However, natural polymer solutions have a range of viscosities, and none of the currently available liquid dispensers are capable of dispensing highly viscous polymer solutions. Here, we report the development of an automated scaffold dispensing system for rapid, reliable, and homogeneous creation of scaffolds in well-plate formats. We employ computer-controlled solenoid valves to regulate air pressure impinging upon a syringe barrel filled with scaffold solution to be dispensed. Automated dispensing of scaffold solution is achieved via a programmable software interface that coordinates solution extrusion and the movement of a dispensing head. We show that our pneumatically actuated dispensing system can evenly distribute high-viscosity, chitosan-based polymer solutions into 96- and 384-well plates to yield highly uniform three-dimensional scaffolds after lyophilization. We provide a proof-of-concept demonstration of high-throughput drug screening by culturing glioblastoma cells in scaffolds and exposing them to temozolomide. This work introduces a device that can hasten the creation of three-dimensional cell scaffolds and their application to high-throughput testing.

Effectiveness and cytotoxicity of two desensitizing agents: a dentin permeability measurement and dentin barrier testing in vitro study

BackgroundWhen evaluating the efficacy and safety of various desensitizing products in vitro, their mechanism of action and clinical utility should be considered during test model selection. This study aimed to evaluate the effects of two desensitizers, an in-office use material and an at-home use material, on dentin specimen permeability, and their dentin barrier cytotoxicity with appropriate test models.MethodsTwo materials, GLUMA desensitizer (GLU) containing glutaraldehyde and remineralizing and desensitizing gel (RD) containing sodium fluoride and fumed silica, were selected. Human dentin specimens were divided into three groups (n = 6): in groups 1 and 2, GLU was applied, and in group 3, RD was applied and immersed in artificial saliva (AS) for 24 h. Dentin specimen permeability before and after each treatment/post-treatment was measured using a hydraulic device under a pressure of 20 cm H2O. The perfusion fluid was deionized water, except in group 2 where 2% bovine serum albumin (BSA) was used. The representative specimens before and after treatment from each group were investigated using scanning electron microscopy. To measure cytotoxicity, test materials were applied to the occlusal surfaces of human dentin disks under which three-dimensional cell scaffolds were placed. After 24-h contact within the test device, cell viability was measured via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.ResultsGLU significantly reduced the dentin permeability and occluded the dentinal tubules when 2% BSA was used as perfusion fluid. RD significantly reduced dentin permeability and occluded the tubules, but permeability rebounded after AS immersion. GLU significantly decreased cell viability, but RD was non-cytotoxic.ConclusionsIn vitro GLU application induced effective dentinal tubule occlusion only following the introduction of simulated dentinal fluid. RD provided effective tubule occlusion, but its full remineralization potential was not realized after a short period of immersion in AS. GLU may harm the pulp, whereas RD is sufficiently biocompatible.

The Preparation of Novel P(OEGMA-co-MEO2MA) Microgels-Based Thermosensitive Hydrogel and Its Application in Three-Dimensional Cell Scaffold.

Thermosensitive hydrogel scaffolds have attracted particular attention in three-dimensional (3D) cell culture. It is very necessary to develop a type of thermosensitive hydrogel material with low shrinkage, and excellent biocompatibility and biodegradability. Here, five types of thermosensitive microgels with different volume phase transition temperature (VPTT) or particle sizes were first synthesized using 2-methyl-2-propenoic acid-2-(2-methoxyethoxy) ethyl ester (MEO2MA) and oligoethylene glycol methyl ether methacrylate (OEGMA) as thermosensitive monomers by free radical polymerization. Their VPTT and particle sizes were investigated by a nanometer particle size meter and an ultraviolet spectrophotometer. The feasibility of using these P(OEGMA-co-MEO2MA) microgels to construct thermosensitive hydrogel by means of the thermal induction method is discussed for the first time. The prepared thermosensitive hydrogel with the optimum performance was screened for in situ embedding and three-dimensional (3D) culture of MCF-7 breast cancer cells. The experimental results of AO/EB and MTT methods indicate that the pioneering scaffold material has prominent biocompatibility, and cells grow rapidly in the 3D scaffold and maintain high proliferative capacity. At the same time, there is also a tendency to aggregate to form multicellular spheres. Therefore, this original P(OEGMA-co-MEO2MA) thermosensitive hydrogel can serve as a highly biocompatible and easily functionalized 3D cell culture platform with great potential in the biomedical area.

Peptide- and Protein-Graphene Oxide Conjugate Materials for Controlling Mesenchymal Stem Cell Fate

Stem cells hold great potential in tissue engineering due to their prolonged self-renewal and ability to differentiate into a variety of cell lines. Leveraging stem cells in regenerative healing requires the ability to support stem cell vitality, localize and retain the stem cells at the site where they are needed, and control their differentiation into specified cell types. To address these needs, graphene oxide (GO), a two-dimensional nanocarbon material, has been widely investigated due to its unique structural characteristics that interface with biomolecules and impart cytocompatibility. To expand on the utility of GO in tissue engineering applications, researchers have incorporated GO with peptides and proteins to provide an additional level of control over stem cell fate. This review focuses on how peptide- and protein-GO conjugates are utilized for stem cell instruction, growth, delivery, and retention in the following ways: GO as a delivery vehicle for stem cell-inducing proteins; peptide- and protein-GO conjugates as two- or three-dimensional cell scaffolds; and GO as an additive in peptide- and protein-based hydrogels. These technologies highlight the value that peptide- and protein-GO conjugates possess in addressing the challenges associated with stem cell therapies. Future directions for the continued research of peptide- and protein-GO conjugates are projected as we look to further advance the field of regenerative tissue engineering using these powerful hybrid materials. Stem cell therapies can be used to replace or rebuild damaged tissue through natural healing pathways. However, using stem cells in tissue engineering requires novel technologies that control stem cell fate. Hybrid materials comprised of graphene oxide (GO) and proteins represent a promising new technology with the ability to interface with stem cells. In these systems, the GO acts as a scaffold for cell growth and supports the protein which, in turn, supplies the appropriate chemical cues to direct stem cell fate toward regenerative healing.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Engineered Three-Dimensional Scaffolds Modulating Fate of Breast Cancer Cells Using Stiffness and Morphology Related Cell Adhesion.

Goal: Artificially engineering the tumor microenvironment in vitro as a vital tool for understanding the mechanism of tumor progression. In this study, we developed three-dimensional cell scaffold systems with different topographical features and mechanical properties but similar surface chemistry. The cell behavior was modulated by the topography and mechanical properties of the scaffold. Methods: Adenocarcinoma (MCF7), triple-negative (MDA-MB-231) and premalignant (MCF10AneoT) breast cancer cells were seeded on the scaffold systems. The cell viability, cell-cell interaction and cell-matrix interactions were analyzed. The preferential growth and alignment of specific population of cells were demonstrated. Results: Among the different scaffolds, triple-negative breast cancer cells preferred honeycomb scaffolds while adenocarcinoma cells favored mesh scaffolds and premalignant cells preferred the aligned scaffolds. Conclusions: The 3D model system developed here can be used to support growth of only specific cell populations or for the growth of tumors. This model can be used for understanding the topographical and mechanical features affecting tumorigenesis, cancer cell growth and migration behavior of malignant and metastatic cancer cells.

Chitosan/gellan gum ratio content into blends modulates the scaffolding capacity of hydrogels on bone mesenchymal stem cells

Here, we have demonstrated the production and characterization of hydrogel scaffolds based on chitosan/gellan gum (CS/GG) assemblies, without any covalent and metallic crosslinking agents, conventionally used to yield non-soluble polysaccharide-based materials. The polyelectrolyte complexes (physical hydrogels called as PECs) are characterized by Fourier-transform infrared spectroscopy, wide-angle X-ray scattering, and scanning electron microscopy. Hydrogels containing chitosan (CS) excesses (ranging from 60 to 80 wt%) were created. Durable polysaccharide-based scaffolds with structural homogeneity and interconnecting pore networks are developed by modulating the CS/GG weight ratio. The CS/GG hydrogel prepared at 80/20 CS/GG weight ratio (sample CS/GG80-20) is cytocompatible, supporting the attachment, growth, and spreading of bone marrow mesenchymal stem cells (BMSCs) after nine days of cell culture. The cytocompatibility is correlated to the swelling capacity of the PEC in PBS buffer (pH 7.4). By controlling the CS content, we can tune the water uptake of the material, enhancing the capacity to serve as a three-dimensional cell scaffold for BMSCs. This work presents for the first time that CS/GG hydrogels can be applied as scaffolds for tissue engineering purposes.

Appllication of human acellular dermal matrix in surgical treatment of genitourinary disease

Humanacellular dermal matrix (HADM) is widely used in the field of burn wound repair and tissue engineering plastic surgery. HADM is manufactored by physical and chemical decellular process to remove the antigenic components that might cause immune rejection in dermis.The extracellular matrix of three-dimensional cell scaffold structure with collagen fibers had been used for wound repair and tissue regeneration, while HADM characterized with low absorption rate after implantation and strong ability to induce angiogenesis in host tissue. Studies reported that after the HADM was implanted into the patient, the host cells, such as fibroblasts and myofibroblasts, as well as lymphocytes, macrophages, granulocytes and mast cells, rapidly infiltrated the graft. The connective tissue and neovascularization were then formed within the HADM three-dimensional cell scaffold, the lymphatic system also appears after vascular reconstruction. Traditional urethral reconstruction using autologous skin flaps has some defects, such as complexity of the technology, risk of necrosis of the skin flaps after transplantation, and failure to achieve functional repair of the urethral epithelium. It has been reported that using HADM to reconstruct the urethra in patients with urethral stricture, hypospadias and bladder-vaginal fistula, showed promising results. Others have reported the experience of using HADM to repair and reconstruct congenital classic bladder exstrophy. HADM has also been used for tissue repair in patients with penile skin defect caused by Fonier's gangrene and hidradenitis suppurativa, and implanted under Bucks' fascia to enlarge the penis. The report of HADM implantation for treating premature ejaculation also deserves attention. Researchers found that HADM implantation can form a tissue barrier between the skin and corpus cavernosum, which can effectively reduce penile sensitivity and treat premature ejaculation. The safety and effectiveness of HADM implantation in the treatment of premature ejaculation need to be further standardized by data from multi-center, large-sample clinical studies. In summary, HADM is the extracellular matrix and three-dimensional cell scaffold of human dermis. As a new type of tissue repair material, new blood vessels are formed actively after implantation, which shows good histocompatibility. HADM has shown increasingly broad application prospects in treatment of genitourinary diseases including penis, urethra and bladder diseases. HADM has also been used in the treatment of premature ejaculation in recent clinical studies, and its long-term safety and efficacy need to be further investigated.

Secondary Photocrosslinking of Click Hydrogels To Probe Myoblast Mechanotransduction in Three Dimensions.

Muscle cells sense the mechanical properties of their microenvironment, and these properties can change in response to injury or disease. Hydrogels with dynamic material properties can be used to study the effect of such varying mechanical signals. Here, we report the ability of azadibenzocyclooctyne to undergo a cytocompatible, photoinitiated crosslinking reaction. This reaction is exploited as a strategy for on-demand stiffening of three-dimensional cell scaffolds formed through an initial strain-promoted azide-alkyne cycloaddition. Myoblasts encapsulated in these networks respond to increased matrix stiffness through decreased cell spreading and nuclear localization of Yes-associated protein 1 (YAP). However, when the photocrosslinking reaction is delayed to allow cell spreading, elongated myoblasts display increased YAP nuclear localization.

Liquid Crystal Elastomer Microspheres as Three-Dimensional Cell Scaffolds Supporting the Attachment and Proliferation of Myoblasts

We report that liquid crystal elastomers (LCEs), often portrayed as artificial muscles, serve as scaffolds for skeletal muscle cell. A simultaneous microemulsion photopolymerization and cross-linking results in nematic LCE microspheres 10-30 μm in diameter that when conjoined form a LCE construct that serves as the first proof-of-concept for responsive LCE muscle cell scaffolds. Confocal microscopy experiments clearly established that LCEs with a globular, porous morphology permit both attachment and proliferation of C2C12 myoblasts, while the nonporous elastomer morphology, prepared in the absence of a microemulsion, does not. In addition, cytotoxicity and proliferation assays confirm that the liquid crystal elastomer materials are biocompatible promoting cellular proliferation without any inherent cytotoxicity.

Monolithic cryogels with open porous structure and unique double-continuous macroporous networks

A novel approach to create macroporous cryogels (or gels synthesized at sub-zero temperatures) with increased mechanical strength is presented. The cryogels with unique double-continuous macroporous networks were prepared via sequential freezing–thawing, when the new cryogel was synthesized inside the interconnected macropores of the preformed or primary cryogel. The prepared double-continuous macroporous cryogels (DM cryogels) have continuously interlacing networks of primary cryogel with the newly synthesized cryogels visualized by confocal laser scanning microscopy and scanning electron microscopy. The open structures of the DM cryogels with up to 150-μm-sized interconnected pores and tissue-like elasticity make them attractive as three-dimensional cell scaffolds for tissue-engineering applications. In particular, a method of preparation of macroporous cryogels with open porous structure and/or gradient porosity, increased mechanical strength and increased content of functional groups is shown.

Fabrication of three-dimensional cell scaffolds with spatial gradients of biomolecules

Multicellular processes of development and tissue regeneration are often sophisticatedly regulated by the spatial arrangement of extracellular matrix and signaling molecules in a timeand concentration-dependent manner. Concentration gradients of biomolecules such as growth factors and transcriptional factors play a pivotal role in the in vivo induction and formation of tissues and organs with complex structural architecture. It is therefore conceivable that a three-dimensional bioengineered scaffold with a spatial gradient of biomolecules could allow cells to induce regeneration of tissue with the natural morphological structure. This mini review article overviews fabrication techniques for functional gradient materials, and several concrete examples are introduced to emphasize importance of gradient materials in tissue engineering. Rec.9/28/2006, Acc.11/26/2006, pp102-106

Impregnation of Plasmid DNA into Three-Dimensional Scaffolds and Medium Perfusion Enhancein VitroDNA Expression of Mesenchymal Stem Cells

This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.

Inverted-Colloidal-Crystal Hydrogel Matrices as Three-Dimensional Cell Scaffolds

Successful engineering of functional tissues requires the development of three-dimensional (3D) scaffolds that can provide an optimum microenvironment for tissue growth and regeneration. A new class of 3D scaffolds with a high degree of organization and unique topography is fabricated from polyacrylamide hydrogel. The hydrogel matrix is molded by inverted colloidal crystals made from 104 μm poly(methyl methacrylate) spheres. The topography of the scaffold can be described as hexagonally packed 97 μm spherical cavities interconnected by a network of channels. The scale of the long-range ordering of the cavities exceeds several millimeters. In contrast to analogous material in the bulk, hydrogel shaped as an inverted opal exhibits much higher swelling ratios; its swelling kinetics is an order of magnitude faster as well. The engineered scaffold possesses desirable mechanical and optical properties that can facilitate tissue regeneration while allowing for continuous high-resolution optical monitoring of cell proliferation and cell–cell interaction within the scaffold. The scaffold biocompatibility as well as cellular growth and infiltration within the scaffold were observed for two distinct human cell lines which were seeded on the scaffold and were tracked microscopically up to a depth of 250 μm within the scaffold for a duration of up to five weeks. Ease of production, a unique 3D structure, biocompatibility, and optical transparency make this new type of hydrogel scaffold suitable for most challenging tasks in tissue engineering.

Imaging magnetically labeled cells with magnetomotive optical coherence tomography

We introduce a novel contrast mechanism for optical coherence tomography (OCT) whereby the optical scattering of magnetically labeled cells is modified by means of an externally applied magnetic field. This modification is made through the addition of a small electromagnet to the imaging arm of a conventional OCT interferometer. We measure the magnetomotive OCT signal by differencing pairs of axial scans (A-scans) acquired with the magnetic field on and off. Magnetomotive contrast is demonstrated in bulk three-dimensional cell scaffolds containing macrophages labeled with microparticles of iron oxide, demonstrating magnetic-specific contrast over a dynamic range of 30 dB.