Three-dimensional Hydrogel Scaffolds Research Articles

Recapitulation of physiologic and pathophysiologic pulsatile CSF flow in purpose-built high-throughput hydrocephalus bioreactors

BackgroundHydrocephalus, an accumulation of cerebrospinal fluid (CSF) in the ventricles of the brain, is often treated via a shunt system to divert the excess CSF to a different compartment; if left untreated, it can lead to serious complications and permanent brain damage. It is estimated that one in every 500 people are born with hydrocephalus. Despite more than 60 years of concerted efforts, shunts still have the highest failure rate of any neurological device requiring follow-up shunt revision surgeries and contributing to the $2 billion cost of hydrocephalus care in the US alone. The absence of a tested and validated long-term in-vitro model that can incorporate clinically relevant parameters has limited hypothesis-driven studies and, in turn, limited our progress in understanding the mechanisms of shunt obstruction in hydrocephalus. Testing clinical parameters of flow, pressure, shear, catheter material, surface modifications, and others while optimizing for minimal protein, cellular, and blood interactions has yet to be done systematically for ventricular catheters. Several studies point to the need to not only understand how cells and tissues have occluded these shunt catheters but also how to stop the likely multi-faceted failure. For instance, studies show us that tissue occluding the ventricular catheter is primarily composed of proliferating astrocytes and cells of the macrophage lineage. Cell reactivity has been observed to follow flow gradients, with elevated levels of typically pro-inflammatory interleukin-6 produced under shear stress conditions greater than 0.5 dyne/. But also, that shear can shift cellular attachment. The Automated, In vitro Model for hydrocephalus research (AIMS), presented here, improves upon our previous long-term in vitro systems with specific goals of recapitulating bulk pulsatile cerebrospinal fluid (CSF) waveforms and steady-state flow directionality relevant to ventricular catheters used in hydrocephalus.MethodsThe AIMS setup was developed to recapitulate a wide range of physiologic and pathophysiologic CSF flow patterns with varying pulse amplitude, pulsation rate, and bulk flow rate with high throughput capabilities. These variables were specified in a custom-built user interface to match clinical CSF flow measurements. In addition to flow simulation capabilities, AIMS was developed as a modular setup for chamber testing and quality control. In this study, the capacity and consistency of single inlet resin chambers (N = 40), multidirectional resin chambers (N = 5), silicone chambers (N = 40), and PETG chambers (N = 50) were investigated. The impact of the internal geometry of the chamber types on flow vectors during pulsatile physiologic and pathophysiologic flow was visualized using Computational Fluid Dynamics (CFD). Dynamic changes in ventricular volume were investigated by combining AIMS with MRI-driven silicone model of a pediatric patient’s ventricles. Parametric data were analyzed using one-way analysis of variance (ANOVA) or repeated measures ANOVA tests. Non-parametric data were analyzed using Kruskal-Wallis test. For all tests, a confidence interval was set at 0.95 (α = 0.05). In a subset of experiments, AIMS was also tested for its capability to measure the flow of florescent microspheres through the holes of unused and explanted ventricular catheters.ResultsThe analysis of peak amplitude through chambers indicated no statistically significant differences between the chamber batches. This high throughput setup was able to reproduce clinical measurements of bulk CSF flow tested in up to 50 independent pump channels such that there was no exchange of solution or flow interference between adjacent channels. Physiologic and pathophysiologic clinical measurements of CSF flow patterns were recapitulated in all four chamber types of the AIMS setup with and without augmented compliance. The AIMS setup’s automated priming feature facilitated constant fluid contact throughout the study; no leaks or ruptures were observed during short- (up to 24 h) or long-term (30 days) experiments. Finally, qualitative microscopy long-exposure image capture revealed microsphere movement under steady-state and pulsatile flow of spheres moving into the shunt catheter.ConclusionAIMS successfully simulates clinical measurements of physiologic and pathophysiologic CSF pulsation amplitude and frequency, as exemplified using clinical data of CSF exiting an externalized ventricular drain in four distinct chamber types, as well as flow patterns from a valve. This provides a promising platform for investigating the direct interaction between CSF, immune cells, and shunt hardware under relevant flow conditions when both the source of bulk flow and pulsatility are coupled. The implementation of this system in conjunction with a previously reported three-dimensional hydrogel scaffold in future work will enhance our understanding of shunt-related complications and improve treatment strategies by reducing the obstruction rate.

Synergistic Effects of Photobiomodulation and Differentiation Inducers on Osteogenic Differentiation of Adipose-Derived Stem Cells in Three-Dimensional Culture.

Osteoporosis, a common metabolic bone disorder, leads to increased fracture risk and significant morbidity, particularly in postmenopausal women and the elderly. Traditional treatments often fail to fully restore bone health and may cause side effects, prompting the exploration of regenerative therapies. Adipose-derived stem cells (ADSCs) offer potential for osteoporosis treatment, but their natural inclination toward adipogenic rather than osteogenic differentiation poses a challenge. This study investigates a novel approach combining differentiation inducers (DIs), three-dimensional (3D) hydrogel scaffolds, and photobiomodulation (PBM) to promote osteogenic differentiation of immortalised ADSCs. A dextran-based 3D hydrogel matrix, supplemented with a DI cocktail of dexamethasone, β-glycerophosphate disodium, and ascorbic acid, was used to foster osteogenesis. PBM was applied using near-infrared (825 nm), green (525 nm), and combined wavelengths at fluences of 3 J/cm2, 5 J/cm2, and 7 J/cm2 to enhance osteogenic potential. Flow cytometry identified osteoblast-specific markers, while inverted light microscopy evaluated cellular morphology. Reactive oxygen species assays measured oxidative stress, and quantitative polymerase chain reaction (qPCR) revealed upregulated gene expression linked to osteogenesis. The findings demonstrate that integrating DIs, 3D hydrogels, and PBM effectively drives osteogenic differentiation in immortalised ADSCs. The PBM enhanced osteogenic marker expression, induced morphological changes, and upregulated gene activity, presenting a promising framework for bone regeneration. Future research should assess the stability and functionality of these differentiated cells and explore their applicability in preclinical models of bone injury or degeneration. This integrative approach demonstrated specific efficacy in promoting the osteogenic differentiation of ADSCs, highlighting its potential application in developing targeted treatments for osteoporosis.

Enhancing cell proliferation in three-dimensional hydrogel scaffolds using digital light processing bioprinting technology

Enhancing cell proliferation in three-dimensional hydrogel scaffolds using digital light processing bioprinting technology

Indirect co-culture of osteoblasts and endothelial cells in vitro based on a biomimetic 3D composite hydrogel scaffold to promote the proliferation and differentiation of osteoblasts.

The field of orthopedics has long struggled with the challenge of repairing and regenerating bone defects, which involves a complex process of osteogenesis requiring coordinated interactions among different types of cells. The crucial role of endothelial cells and osteoblasts in bone vascularization and osteogenesis underscores the importance of their intimate interaction. However, efforts to bioengineer bone tissue have been impeded by the difficulty in establishing proper angiogenesis and osteogenesis in tissue structures. This study presents a novel approach to bone tissue engineering, involving a three-dimensional composite hydrogel scaffold composed of sodium alginate microspheres encapsulated in type I collagen. Using this scaffold, a three-dimensional indirect co-culture system was established for osteoblasts and endothelial cells to evaluate the osteogenic differentiation potential of osteoblasts. Results demonstrate that the non-contact co-culture system of endothelial cells and osteoblasts constructed by the composite hydrogel scaffold loaded with microspheres holds promise for bone tissue engineering. The innovative concept of an indirect co-culture system presents exciting prospects for conducting intercellular communication studies and offers a valuable in vitro tissue platform to investigate tissue regeneration.

Melt electrowriting (MEW)-PCL composite Three-Dimensional exosome hydrogel scaffold for wound healing

While mesenchymal stem cell-derived exosomes hold substantial potential in wound healing, challenges persist in terms of large-scale production and activity of 2D-culture derived exosome, as well as addressing their inactivation and loss during application. 3D exosomes can be produced more efficiently and possess higher activity. However, there lacks a delivery patch mimicking nanofibrous architecture of the extracellular matrix while facilitating the in situ delivery of exosomes, thereby minimizing dissipation of exosomes and accelerating the process of wound healing. In this study, we devised a controllable GelMA hydrogel-combined Melt Electrowriting (MEW)-PCL scaffold for in situ 3D-exosome release. We showed that biocompatible scaffolds prepared by MEW have a simulated extracellular matrix with a highly controllable arrangement of nanofibers that can support cell adhesion, proliferation and differentiation. Through cell proliferation, scratch assay, and tube formation experiments, we verified that 3D exosomes could effectively stimulate cell proliferation, migration, and tube formation, with dose-dependent effects. In vivo outcomes exhibited accelerated re-epithelialization, improved collagen maturation, and enhanced angiogenesis. Our findings suggest that 3D-cultured exosomes within the scaffold significantly enhance wound repair. This innovative delivery strategy opens up new avenues for the application of MSC-derived exosomes in wound healing.

Immune-microenvironment modulatory polyurethane-hyaluronic acid hybrid hydrogel scaffolds for diabetic wound treatment

The healing of wounds in diabetic patients is a huge challenge issue in clinical medicine due to the disordered immune. Recruiting endogenous cells to play a role in the early stage and timely reducing inflammation to promote healing in the middle or late of injuring are both prerequisites for effective treatment. Here, inspired by natural extracellular matrix, three-dimensional porous polyurethane-hyaluronic acid hybrid hydrogel scaffolds (PUHA) were prepared to repair diabetic wound through activate cell immunity by moderate foreign body reaction, provide cell adhesion growth extracellular matrix of hyaluronic acid (HA) and exhibit anti-inflammatory effect of polyurethane (PU). The interaction between PU and HA alters the compact PU hydrogel into macroporous PUHA hydrogel scaffolds with super-swelling, elastic mechanical properties, and controllable degradation, which are suitable for endogenous cells infiltration, growth and immune activation. Additionally, incorporating with RGD, PUHA hydrogel scaffolds with bioactive physicochemical features can evidently reduce the inflammation and modulate the polarization of macrophage apparently both in vitro and in vivo, mainly through downregulation of cytokine-cytokine receptor interaction genes, leading to reprogramming immune-microenvironment and rapid diabetic wound healing. This method of gathering cells initially and intervening immune-microenvironment in time provides an expected way to design biomaterials for chronic wound healing.

Pericardial Grafting of Cardiac Progenitor Cells in Self-Assembling Peptide Scaffold Improves Cardiac Function After Myocardial Infarction

Many studies have explored cardiac progenitor cell (CPC) therapy for heart disease. However, optimal scaffolds are needed to ensure the engraftment of transplanted cells. We produced a three-dimensional hydrogel scaffold (CPC-PRGmx) in which high-viability CPCs were cultured for up to 8 weeks. CPC-PRGmx contained an RGD peptide-conjugated self-assembling peptide with insulin-like growth factor-1 (IGF-1). Immediately after creating myocardial infarction (MI), we transplanted CPC-PRGmx into the pericardial space on to the surface of the MI area. Four weeks after transplantation, red fluorescent protein-expressing CPCs and in situ hybridization analysis in sex-mismatched transplantations revealed the engraftment of CPCs in the transplanted scaffold (which was cellularized with host cells). The average scar area of the CPC-PRGmx-treated group was significantly smaller than that of the non-treated group (CPC-PRGmx-treated group = 46 ± 5.1%, non-treated MI group = 59 ± 4.5%; p < 0.05). Echocardiography showed that the transplantation of CPC-PRGmx improved cardiac function and attenuated cardiac remodeling after MI. The transplantation of CPCs-PRGmx promoted angiogenesis and inhibited apoptosis, compared to the untreated MI group. CPCs-PRGmx secreted more vascular endothelial growth factor than CPCs cultured on two-dimensional dishes. Genetic fate mapping revealed that CPC-PRGmx-treated mice had more regenerated cardiomyocytes than non-treated mice in the MI area (CPC-PRGmx-treated group = 0.98 ± 0.25%, non-treated MI group = 0.25 ± 0.04%; p < 0.05). Our findings reveal the therapeutic potential of epicardial-transplanted CPC-PRGmx. Its beneficial effects may be mediated by sustainable cell viability, paracrine function, and the enhancement of de novo cardiomyogenesis.

3D-printed hyaluronic acid hydrogel scaffolds impregnated with neurotrophic factors (BDNF, GDNF) for post-traumatic brain tissue reconstruction.

Brain tissue reconstruction posttraumatic injury remains a long-standing challenge in neurotransplantology, where a tissue-engineering construct (scaffold, SC) with specific biochemical properties is deemed the most essential building block. Such three-dimensional (3D) hydrogel scaffolds can be formed using brain-abundant endogenous hyaluronic acid modified with glycidyl methacrylate by employing our proprietary photopolymerisation technique. Herein, we produced 3D hyaluronic scaffolds impregnated with neurotrophic factors (BDNF, GDNF) possessing 600 kPa Young’s moduli and 336% swelling ratios. Stringent in vitro testing of fabricated scaffolds using primary hippocampal cultures revealed lack of significant cytotoxicity: the number of viable cells in the SC+BDNF (91.67 ± 1.08%) and SC+GDNF (88.69 ± 1.2%) groups was comparable to the sham values (p > 0.05). Interestingly, BDNF-loaded scaffolds promoted the stimulation of neuronal process outgrowth during the first 3 days of cultures development (day 1: 23.34 ± 1.46 µm; day 3: 37.26 ± 1.98 µm, p < 0.05, vs. sham), whereas GDNF-loaded scaffolds increased the functional activity of neuron-glial networks of cultures at later stages of cultivation (day 14) manifested in a 1.3-fold decrease in the duration coupled with a 2.4-fold increase in the frequency of Ca2+ oscillations (p < 0.05, vs. sham). In vivo studies were carried out using C57BL/6 mice with induced traumatic brain injury, followed by surgery augmented with scaffold implantation. We found positive dynamics of the morphological changes in the treated nerve tissue in the post-traumatic period, where the GDNF-loaded scaffolds indicated more favorable regenerative potential. In comparison with controls, the physiological state of the treated mice was improved manifested by the absence of severe neurological deficit, significant changes in motor and orienting-exploratory activity, and preservation of the ability to learn and retain long-term memory. Our results suggest in favor of biocompatibility of GDNF-loaded scaffolds, which provide a platform for personalized brain implants stimulating effective morphological and functional recovery of nerve tissue after traumatic brain injury.

3D bacterial cellulose-chitosan-alginate-gelatin hydrogel scaffold for cartilage tissue engineering

Biopolymers of bacterial cellulose, chitosan, alginate and gelatin were combined to fabricate three-dimensional (3D) hydrogel scaffolds for tissue engineering applications. A suitable hydrogel material was fabricated by thoroughly mixing bacterial cellulose-chitosan slurry (BCC) with the mixed solution of alginate and gelatin (AG) at the weight ratio of BCC:AG at 80:20 and crosslinking in the aqueous solution of 1% (w/v) calcium chloride (CaCl2). Results from Fourier transform infrared (FTIR) spectroscopy showed some interactions among bacterial cellulose, chitosan, alginate and gelatin. The composite scaffolds exhibited good three-dimensional (3D) architecture with the average pore diameter of the outer surface and interior at 45.0 and 326.6 µm, respectively. The BCC-AG scaffolds presented many desirable properties, such as good compressive strength, stability and excellent biocompatibility. For the study of tissue engineered constructs, the mixture of mesenchymal stem cells (hMSCs) and the hydrogel was transferred into 24-well plate, cross-linked with 1% (w/v) CaCl2 and cultured in chondrogenic medium. The hydrogel scaffold was shown to support the cell proliferation, with the accumulation of glycosaminoglycan (GAG) and collagen at 6.26% and 6.71%ww, respectively after 42 days of incubation. The results indicated that BCC-AG scaffolds have the potential use as scaffolds for application in cartilage tissue engineering.

3D-Printed, Dual Crosslinked and Sterile Aerogel Scaffolds for Bone Tissue Engineering.

The fabrication of bioactive three-dimensional (3D) hydrogel scaffolds from biocompatible materials with a complex inner structure (mesoporous and macroporous) and highly interconnected porosity is crucial for bone tissue engineering (BTE). 3D-printing technology combined with aerogel processing allows the fabrication of functional nanostructured scaffolds from polysaccharides for BTE with personalized geometry, porosity and composition. However, these aerogels are usually fragile, with fast biodegradation rates in biological aqueous fluids, and they lack the sterility required for clinical practice. In this work, reinforced alginate-hydroxyapatite (HA) aerogel scaffolds for BTE applications were obtained by a dual strategy that combines extrusion-based 3D-printing and supercritical CO2 gel drying with an extra crosslinking step. Gel ageing in CaCl2 solutions and glutaraldehyde (GA) chemical crosslinking of aerogels were performed as intermediate and post-processing reinforcement strategies to achieve highly crosslinked aerogel scaffolds. Nitrogen adsorption–desorption (BET) and SEM analyses were performed to assess the textural parameters of the resulting alginate-HA aerogel scaffolds. The biological evaluation of the aerogel scaffolds was performed regarding cell viability, hemolytic activity and bioactivity for BTE. The impact of scCO2-based post-sterilization treatment on scaffold properties was also assessed. The obtained aerogels were dual porous, bio- and hemocompatible, as well as endowed with high bioactivity that is dependent on the HA content. This work is a step forward towards the optimization of the physicochemical performance of advanced biomaterials and their sterilization.

Low Intensity Pulsed Ultrasound for Bone Tissue Engineering.

As explained by Wolff’s law and the mechanostat hypothesis, mechanical stimulation can be used to promote bone formation. Low intensity pulsed ultrasound (LIPUS) is a source of mechanical stimulation that can activate the integrin/phosphatidylinositol 3-OH kinase/Akt pathway and upregulate osteogenic proteins through the production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). This paper analyzes the results of in vitro and in vivo studies that have evaluated the effects of LIPUS on cell behavior within three-dimensional (3D) titanium, ceramic, and hydrogel scaffolds. We focus specifically on cell morphology and attachment, cell proliferation and viability, osteogenic differentiation, mineralization, bone volume, and osseointegration. As shown by upregulated levels of alkaline phosphatase and osteocalcin, increased mineral deposition, improved cell ingrowth, greater scaffold pore occupancy by bone tissue, and superior vascularization, LIPUS generally has a positive effect and promotes bone formation within engineered scaffolds. Additionally, LIPUS can have synergistic effects by producing the piezoelectric effect and enhancing the benefits of 3D hydrogel encapsulation, growth factor delivery, and scaffold modification. Additional research should be conducted to optimize the ultrasound parameters and evaluate the effects of LIPUS with other types of scaffold materials and cell types.

Synthesis and characterization of site selective photo-crosslinkable glycidyl methacrylate functionalized gelatin-based 3D hydrogel scaffold for liver tissue engineering

The presented work outlined the development of a new biocompatible hydrogel material that has potential applications in soft tissue engineering. As a proof of concept, human hepatocytes were used to demonstrate the suitability of this material in providing conducive environment for cellular growth and functional development. Herein, a detailed synthesis of novel gelatin derivatives - photo-crosslinkable glycidyl methacrylate (GMA) functionalized gelatins (Gelatin-GMA), and preparation of three-dimensional (3D) hydrogel scaffolds for the encapsulated Huh-7.5 cells is reported. The Gelatin-GMA biopolymers were synthesized at two different pH values of 3.5 (acidic) and 10.5 (basic) where two different photo-crosslinkable polymers were formed utilizing –COOH & –OH groups in acidic pH, and –NH2 & –OH groups in basic pH. The hydrogels were prepared using an initiator (Irgacure I2959) in the presence of UV light. The Gelatin-GMA biopolymers were characterized using spectroscopic studies which confirmed the successful preparation of the polymer derivatives. Rheological measurement was carried out to characterize the mechanical properties and derive the mesh sizes of the 3D hydrogels. Subsequently, detailed in vitro hepatocyte compatibility and functionality studies were performed in the 3D cell seeded hydrogel platform. The 3D hydrogel design with larger mesh sizes utilizes the advantage of the excellent diffusion properties of porous platform, and enhanced cell-growth was observed, which in turn elicited favorable Huh-7.5 response. The hydrogels led to improved cellular functions such as differentiation, viability and proliferation. Overall, it showed that the Gelatin-GMA based hydrogels presented better results compared to control sample (GelMA) because of the higher mesh sizes in Gelatin-GMA based hydrogels. Additionally, the functional group studies of the two Gelatin-GMA samples revealed that the cell functionalities are almost unaffected even after the tripeptide - Arg-Gly-Asp (RGD) in Gelatin-GMA synthesized at pH 3.5 is no longer completely available.

Needle-injectable microcomposite cryogel scaffolds with antimicrobial properties

Porous three-dimensional hydrogel scaffolds have an exquisite ability to promote tissue repair. However, because of their high water content and invasive nature during surgical implantation, hydrogels are at an increased risk of bacterial infection. Recently, we have developed elastic biomimetic cryogels, an advanced type of polymeric hydrogel, that are syringe-deliverable through hypodermic needles. These needle-injectable cryogels have unique properties, including large and interconnected pores, mechanical robustness, and shape-memory. Like hydrogels, cryogels are also susceptible to colonization by microbial pathogens. To that end, our minimally invasive cryogels have been engineered to address this challenge. Specifically, we hybridized the cryogels with calcium peroxide microparticles to controllably produce bactericidal hydrogen peroxide. Our novel microcomposite cryogels exhibit antimicrobial properties and inhibit antibiotic-resistant bacteria (MRSA and Pseudomonas aeruginosa), the most common cause of biomaterial implant failure in modern medicine. Moreover, the cryogels showed negligible cytotoxicity toward murine fibroblasts and prevented activation of primary bone marrow-derived dendritic cells ex vivo. Finally, in vivo data suggested tissue integration, biodegradation, and minimal host inflammatory responses when the antimicrobial cryogels, even when purposely contaminated with bacteria, were subcutaneously injected in mice. Collectively, these needle-injectable microcomposite cryogels show great promise for biomedical applications, especially in tissue engineering and regenerative medicine.

Transplantation of neural stem cells encapsulated in hydrogels improve functional recovery in a cauda equina lesion model.

This study explored the therapeutic effects of transplantation of neural stem cells (NSCs) encapsulated in hydrogels in a cauda equina lesion model. NSCs were isolated from neonatal dorsal root ganglion (nDRG) and cultured in three-dimensional porous hydrogel scaffolds. Immunohistochemistry, transmission electron microscopy and TUNEL assay were performed to detect the differentiation capability, ultrastructural and pathological changes, and apoptosis of NSCs. Furthermore, the functional recovery of sensorimotor reflexes was determined using the tail-flick test. NSCs derived from DRG were able to proliferate to form neurospheres and mainly differentiate into oligodendrocytes in the three-dimensional hydrogel culture system. After transplantation of NSCs encapsulated in hydrogels, NSCs differentiated into oligodendrocytes, neurons or astrocytes in vivo. Moreover, NSCs engrafted on the hydrogels decreased apoptosis and alleviated the ultrastructural and pathological changes of injured cauda equina. Behavioral analysis showed that transplanted hydrogel-encapsulated NSCs decreased the tail-flick latency and showed a neuroprotective role on injured cauda equina. Our results indicate transplantation of hydrogel-encapsulated NSCs promotes stem cell differentiation into oligodendrocytes, neurons or astrocytes and contributes to the functional recovery of injured cauda equina, suggesting that NSCs encapsulated in hydrogels may be applied for the treatment of cauda equina injury.

Fabrication of micro-porous hyaluronic acid hydrogels through salt leaching

Development of physiologically relevant culture platforms for studying cell behavior has been of particular interest in tissue engineering as well as in development of in vitro disease models. Hyaluronic acid is a glycosaminoglycan found in the extracellular matrix, derivatives of which are commonly used to create three-dimensional biomimetic hydrogel scaffolds for the study of cell behavior in vitro. Herein, we report the fabrication of micro-porous hyaluronic acid hydrogels using a simple salt leaching technique. Porogens, specifically, salt particles of different sizes (i.e., <63 µm, 63–106 µm, 106–250 µm in diameter), were utilized to obtain a range of porous structures. The microstructure of the hydrogels was characterized through scanning electron microscopy, and rheological and swelling studies were performed to further evaluate morphological and mechanical properties. Analysis of these studies indicated that an increase in pore area corresponded to a larger porogen size, whereas porogen size had no significant effect on storage modulus or water absorption. However, porous hydrogels exhibited a greater degree of swelling and reduced storage modulus compared to nonporous hydrogels. In particular, the largest particle size of 106–250 µm resulted in a swelling ratio of 44.6 ± 2.28 and storage modulus of 54 ± 4.97 Pa versus a swelling ratio of 24.3 ± 2.30 and storage modulus of 402 ± 209 Pa noted for nonporous hyaluronic acid hydrogels, respectively. No significant difference in degradation of hydrogels was observed at later time points; however, hydrogels with the largest pore area degraded to a greater extent initially. Assessment of cellular compatibility showed no evidence of cytotoxicity for all hydrogel conditions tested. Such a system, incorporating micron-level porosity, could provide a useful tool for studying cell–matrix interactions in a physiologically relevant setting.

Effect of Pulsed Electromagnetic Fields on Human Mesenchymal Stem Cells Using 3D Magnetic Scaffolds.

Alternative bone regeneration strategies that do not rely on harvested tissue or exogenous growth factors are needed. One of the major challenges in tissue reconstruction is recreating the bone tissue microenvironment using the appropriate combination of cells, scaffold, and stimulation to direct differentiation. This study presents a bone regeneration formulation that involves the use of human adipose‐derived mesenchymal stem cells (hASCs) and a three‐dimensional (3D) hydrogel scaffold based on self‐assembled RADA16 peptides containing superparamagnetic iron oxide nanoparticles (NPs). Although superparamagnetic NPs could be used as stimulus to manipulate the cell proliferation and differentiation, in this paper their use is explored for assisting osteogenic differentiation of hASCs in conjunction with direct stimulation by extremely low‐frequency pulsed electromagnetic fields (pEMFs). Cellular morphology, proliferation, and viability, as well as alkaline phosphatase activity, calcium deposition, and osteogenic capacity were monitored for cells cultured up to 21 days in the 3D construct. The results show that the pEMFs and NPs do not have any negative effect on cell viability, but instead distinctly induced early differentiation of hASCs to an osteoblastic phenotype, when compared with cells without biophysical stimulation. This effect is attributed to synergy between the pEMFs and NPs, which may have stimulated mechanotransduction pathways, which, in turn activated biochemical signals between cells to differentiate or proliferate. This approach may offer a safe and effective option for the treatment of non‐union bone fractures. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

Poly (ethylene glycol) hydrogel scaffolds with multiscale porosity for culture of human adipose-derived stem cells

Three-dimensional (3 D) hydrogel scaffolds are an attractive option for tissue regeneration applications because they allow for cell migration, fluid exchange, and can be synthesized to closely mimic the physical properties of the extracellular matrix environment. The material properties of hydrogels play a vital role in cellular migration and differentiation. In light of this, in-depth understanding of material properties is required before such scaffolds can be used to study their influence on cells. Herein, various blends and thicknesses of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogels were synthesized, flash frozen, and dried by lyophilization to create scaffolds with multiscale porosity. Environmental scanning electron microscopy (ESEM) images demonstrated that lyophilization induced microporous voids in the PEGDMA hydrogels while swelling studies show the hydrogels retain their innate swelling properties. Change in pore size was observed between drying methods, polymer blend, and thickness when imaged in the hydrated state. Human adipose-derived stem cells (hASCs) were seeded on lyophilized and non-lyophilized hydrogels to determine if the scaffolds would support cell attachment and proliferation of a clinically relevant cell type. Cell attachment and morphology of the hASCs were evaluated using fluorescence imaging. Qualitative observations in cell attachment and morphology of hASCs on the surface of the different hydrogel spatial configurations indicate these multiscale porosity hydrogels create a suitable scaffold for hASC culture. These findings offer another factor of tunability in creating biomimetic hydrogels for various tissue engineering applications including tissue repair, regeneration, wound healing, and controlled release of growth factors.

A compartmental 3D scaffold fabrication and alignment device for neurovascular co-culture and tri-culture

Tissue engineers seek for the practical and affordable fabrication methods to be able to create the best biomimicking scaffold. The aim of the study was to create an easy-to use device to fabricate precisely designed 3D hydrogel scaffolds for co-culturing, tri-culturing and multi-culturing practices. In this study, gelatin methacryloyl (GelMA) was synthesized and textured three-dimensional hydrogel scaffolds were fabricated via photopolymerization using compartmental fabrication device. This device allowed fabricating computer aided designed (CAD) geometries precisely and encapsulating different cells in different hydrogel posts while building a whole geometry. The hydrogel texture characterization was performed using colored hydrogel solutions and it is observed that the device keep the distinct posts in contact precisely. A neural tissue model was created using neural, epithelial and endothelial cells as a proof of concept. Cell distribution was evaluated using cell trackers. The viability and the proliferation assays were also performed and the viability was above 80% while a positive proliferation was been observing. Overall findings showed that this device offers a practical way to fabricate and align precisely designed three-dimensional hydrogel scaffolds which allows modelling various types of tissues and organs and it tis promising for further applications in many fields.

Cucurbit[7]uril-Carbazole Two-Photon Photoinitiators for the Fabrication of Biocompatible Three-Dimensional Hydrogel Scaffolds by Laser Direct Writing in Aqueous Solutions.

We have introduced a novel water-soluble two-photon photoinitiator based on the host-guest interaction between 3,6-bis[2-(1-methyl-pyridinium)vinyl]-9-pentyl-carbazole diiodide (BMVPC) and cucurbit[7]uril (CB7) because most of the commercial photoinitiators have poor two-photon initiating efficiency in aqueous solutions. The binding ratio of BMVPC and CB7 was determined as 1:1 by isothermal titration calorimetry and quantum chemical calculation. The formation of the host-guest complex increases the two-photon absorption cross-section about five times, and improves the water solubility required as the photoinitiator for hydrogel fabrication. The BMVPC-CB7 inclusion complex was used as the one-component photoinitiator, and the polyethylene glycol diacrylate with promising biocompatibility was used as the hydrogel monomer to form the aqueous-phase photoresist system applied to two-photon polymerization microfabrication. A relatively low laser threshold of 4.5 mW, a high fabricating resolution of 180 nm, and the true three-dimensional (3D) fabricating capability in the aqueous solution have been obtained by using the as-prepared photoresist system. Finally, 3D engineering hydrogel scaffold microstructures with low toxicity and good biocompatibility have been fabricated and cocultured with living HeLa cells, which demonstrates the potential for further application in tissue engineering.

3D culture of murine adipose-derived multipotent mesenchymal stromal cells in hydrogel based on carbomer 974P

Actual issues during tissue regeneration are to ensure the survival of transplanted cells at the site of their application and further activity, especially in case of local pathological alterations such as inflammation and ischemia. For this purpose, the matrices that can not only fill the defects of tissues, but also be scaffolds for cells are developed.The aim of this study was to evaluate the effectiveness of 3D cultivation of murine adipose-derived multipotent mesenchymal stromal cells (MSMCs) in hydrogel based on carbomer 974P.Materials and methods. MSMCs were obtained from the adipose tissue of FVB-Cg-Tg(GFPU)5Nagy/J mice transgenic for GFP gene. The cells were phenotyped by flow cytometry and directly differentiated into osteogenic and adipogenic direction to confirm multipotent phenotype. MMSCs were cultured and directly differentiated into osteogenic direction in three-dimensional hydrogel scaffolds. For hydrogel preparation we used carbomer 974P with composition of glycerol, propylene glycol, triethylamine and agarose in original proportion.Results. The three-dimensional hydrogel based on carbomer 974P for the further engraftment with MMSCs was obtained. Modified protocols for the preparation of hydrogels based on carbomer and agarose and their rehydration by culture media for the 3D cultivation of adipose-derived MMSCs have been developed. The optimal concentration of MSMCs and the injection method for engraftment of hydrogels of the required form and size are selected. It was shown that adipose-derived MMSCs in 3D carbomer hydrogel preserve the potential of directed osteogenic differentiation.Conclusion. Three-dimensional hydrogel based on carbomer 974P is capable to support cells, provide the necessary cytoarchitectonics, maintain intercellular interactions, which can promote further long-term survival and specialization of graft.