Porous Hydrogel Scaffolds Research Articles

Crystal Transduction 3D Printing of Bio‐Hydrogels with High Fidelity and Order Micro Pores

Abstract3D printing of bio‐hydrogel scaffolds are widely used in tissue regeneration. However, due to the ultra‐soft properties of bio‐hydrogels, it is hard to print them precisely. Here, crystal transduction 3D printing with high fidelity is proposed to address this challenge. A phase‐transition bio‐inks system with beeswax is developed for crystal transduction, which can accelerate energy consumption and induce soft bio‐inks to quickly harden during printing. Interestingly, an interconnected porous hydrogel scaffold can be obtained after washing the crystal beeswax. The porous hydrogel scaffold demonstrated excellent biocompatibility and cell proliferation effect in vitro and is free from defense responses and immunogenicity in vivo. Muscle analog porous scaffolds constructed by high‐fidelity 3D printing significantly improve the tissue function recovery of rats with muscle defects, compared with the conventional printed hydrogel with a non‐matched shape. These structure‐performance design rules create exciting opportunities to customize 3D‐printed porous hydrogel scaffolds with high fidelity.

Gelatin-Based Polymers Can Be Processed to Highly Resilient Biocompatible Porous Hydrogel Scaffolds for Soft Tissue Regeneration Applications

Tissue regeneration relies on the mechanical properties of the surrounding environment, and it has already been shown that mechanostimulation is highly dependent on the stiffness of the native biological tissue. The main advantage of injectable hydrogels in medical applications is their ability to be delivered through minimally invasive techniques. Natural polymer-based hydrogels have been widely used in biomedical applications, due to their high biocompatibility, low immunogenicity, and similarity to soft tissues. However, the crucial combination of low stiffness with high resilience has not been achieved for natural polymers. The current study focuses on the development of novel gelatin-based injectable hydrogels for soft tissue regeneration applications, elucidating the effects of the formulation parameters on the resilience, microstructure, biocompatibility, and mechanical properties. Non-foamed hydrogels demonstrated resilience of at least 95%, while porous hydrogels maintained resilience above 90%, allowing them to withstand mechanical stresses and dynamic conditions within the body. The adjustable modulus of these hydrogels provides the necessary flexibility to mimic the mechanical properties of soft and very soft tissues, without compromising resilience. Environmental Scanning Electron Microscopy (ESEM) observations of the porous hydrogels indicated round interconnected pore structures, desired for cell migration and nutrient flow. Biocompatibility tests on fibroblasts and pre-adipocytes confirmed high biocompatibility, both directly and indirectly. In summary, structuring these new hydrogels for achieving adjustable stiffness, along with the excellent resilience and biocompatibility, is expected to enable this new technology to fit various soft tissue regeneration applications.

The Porous SilMA Hydrogel Scaffolds Carrying Dual-Sensitive Paclitaxel Nanoparticles Promote Neuronal Differentiation for Spinal Cord Injury Repair.

In the intricate pathological milieu post-spinal cord injury (SCI), neural stem cells (NSCs) frequently differentiate into astrocytes rather than neurons, significantly limiting nerve repair. Hence, the utilization of biocompatible hydrogel scaffolds in conjunction with exogenous factors to foster the differentiation of NSCs into neurons has the potential for SCI repair. In this study, we engineered a 3D-printed porous SilMA hydrogel scaffold (SM) supplemented with pH-/temperature-responsive paclitaxel nanoparticles (PTX-NPs). We analyzed the biocompatibility of a specific concentration of PTX-NPs and its effect on NSC differentiation. We also established an SCI model to explore the ability of composite scaffolds for in vivo nerve repair. The physical adsorption of an optimal PTX-NPs dosage can simultaneously achieve pH/temperature-responsive release and commendable biocompatibility, primarily reflected in cell viability, morphology, and proliferation. An appropriate PTX-NPs concentration can steer NSC differentiation towards neurons over astrocytes, a phenomenon that is also efficacious in simulated injury settings. Immunoblotting analysis confirmed that PTX-NPs-induced NSC differentiation occurred via the MAPK/ERK signaling cascade. The repair of hemisected SCI in rats demonstrated that the composite scaffold augmented neuronal regeneration at the injury site, curtailed astrocyte and fibrotic scar production, and enhanced motor function recovery in rat hind limbs. The scaffold's porous architecture serves as a cellular and drug carrier, providing a favorable microenvironment for nerve regeneration. These findings corroborate that this strategy amplifies neuronal expression within the injury milieu, significantly aiding in SCI repair.

Preliminary study on the preparation of antler powder/chitosan/β-glycerophosphate sodium/polyvinyl alcohol porous hydrogel scaffolds and their osteogenic effects.

Introduction: The production of bone-like structural scaffolds through bone tissue engineering technology is a promising method for bone regeneration to repair bone defects. Deer antler, an easily harvested and abundantly sourced initial bone tissue structure, resembles the composition and structure of human cancellous bone and can serve as a new material for allogeneic bone transplantation. Methods: This study involved the preparation and characterization of antler powder/chitosan/β-glycerophosphate sodium/polyvinyl alcohol (AP/CS/β-GP/PVA) porous hydrogel scaffolds to verify their material properties and osteogenic mechanisms. The microstructure, hydrophilicity, and mechanical properties of the scaffolds were studied using Scanning Electron Microscopy (SEM), contact angle measurement, and a universal material testing machine. The interactions between the various components were investigated using Fourier-Transform Infrared Spectroscopy (FTIR). Biocompatibility, osteogenic properties, and expression of osteogenesis-related proteins of the scaffolds were evaluated through Cell Counting Kit-8 (CCK-8) assays, alkaline phosphatase staining, Alizarin Red staining, live/dead cell staining, and Western blot analysis. Results: The results showed that as the content of deer antler powder increased, both the hydrophilicity and mechanical properties of the scaffold materials improved, while the porosity slightly decreased with an increase in deer antler powder content. Cell culture experiments demonstrated that scaffolds with a higher proportion of deer antler powder were beneficial for the proliferation and differentiation of mouse pre-osteoblast (MC3T3-E1) cells, with the scaffolds containing 10% and 8% deer antler powder showing the best effects. The upregulation of RUNX2, OCN, OSX, and OPN protein expression may promote differentiation. Discussion: Therefore, the AP/CS/β-GP/PVA hydrogel scaffolds have the potential to become a promising biomaterial for bone tissue engineering.

Magnesium-Doped Nano-Hydroxyapatite/Polyvinyl Alcohol/Chitosan Composite Hydrogel: Preparation and Characterization.

Polyvinyl alcohol/Chitosan hydrogel is often employed as a carrier because it is non-toxic, biodegradable, and has a three-dimensional network structure. Meanwhile, Magnesium-doped nano-hydroxyapatite(Mg-nHA) demonstrated high characterization to promote the osteogenic differentiation of bone marrow derived mesenchymal stem cell(BMSCs). Therefore, in order to develop a porous hydrogel scaffold for the application of bone tissue engineering, an appropriate-type Mg-nHA hydrogel scaffold was developed and evaluated. A composite hydrogel containing magnesium-doped nano-hydroxyapatite (Mg-nHA/PVA/CS) was developed using a magnetic stirring-ion exchange method and cyclic freeze-thaw method design, with polyvinyl alcohol and chitosan as the main components. Fourier transform infrared spectra (FTIR), electron energy dispersive spectroscopy (EDS), X-ray photoelectron spectrometer (XPS) and scanning electron microscopy (SEM) were employed to analyze the chemical structure, porosity, and elemental composition of each hydrogels. The equilibrium swelling degree, moisture content, pH change, potential for biomineralization, biocompatibility, the osteogenic potential and magnesium ion release rate of the composite hydrogel were also evaluated. SEM analysis revealed a well-defined 3D spatial structure of micropores in the synthesised hydrogel. FTIR analysis showed that doping nanoparticles had little effect on the hydrogel's structure and both the 5% Mg-nHA/PVA/CS and 10% Mg-nHA/PVA/CS groups promoted amide bond formation. EDS observation indicated that the new material exhibited favourable biomineralization ability, with optimal performance seen in the 5% Mg-nHA/PVA/CS group. The composite hydrogel not only displayed favourable water content, enhanced biocompatibility, and porosity (similar to human cancellous bone), but also maintained an equilibrium swelling degree and released magnesium ions that created an alkaline environment around it. Additionally, it facilitated the proliferation of bone marrow mesenchymal stem cells and their osteogenic differentiation. The Mg-nHA/PVA/CS hydrogel demonstrates significant potential for application in the field of bone repair, making it an excellent composite material for bone tissue engineering.

Novel 3D-printing bilayer GelMA-based hydrogel containing BP, β-TCP and exosomes for cartilage–bone integrated repair

The integrated repair of cartilage and bone involves the migration and differentiation of cells, which has always been a difficult problem to be solved. We utilize the natural biomaterial gelatin to construct gelatin methacryloyl (GelMA), a hydrogel scaffold with high cell affinity. GelMA is mixed with different components to print a bi-layer porous hydrogel scaffold with different modulus and composition in upper and lower layers through three-dimensional (3D) printing technology. The upper scaffold adds black phosphorus (BP) and human umbilical cord mesenchymal stem cells (hUMSCs) exosomes (exos) in GelMA, which has a relatively lower elastic modulus and is conducive to the differentiation of BMSCs into cartilage. In the lower scaffold, in addition to BP and hUMSCs exos, β-tricalcium phosphate (β-TCP), which has osteoconductive and osteoinductive effects, is added to GelMA. The addition of β-TCP significantly enhances the elastic modulus of the hydrogel scaffold, which is conducive to the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs). In vitro experiments have confirmed that the bi-layer scaffolds can promote osteogenesis and chondrogenic differentiation respectively. And in the rabbit cartilage–bone injury model, MRI and micro-CT results show that the 3D printed bi-layer GelMA composite scaffold has a repair effect close to normal tissue.

Porous Poly(2-hydroxyethyl methacrylate) Hydrogel Scaffolds for Tissue Engineering: Influence of Crosslinking Systems and Silk Sericin Concentration on Scaffold Properties.

Tailored porous structures of poly(2-hydroxyethyl methacrylate) (PHEMA) and silk sericin (SS) were used to create porous hydrogel scaffolds using two distinct crosslinking systems. These structures were designed to closely mimic the porous nature of the native extracellular matrix. Conventional free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) was performed in the presence of different concentrations of SS (1.25, 2.50, 5.00% w/v) with two crosslinking systems. A chemical crosslinking system with N'N-methylene bisacrylamide (MBAAm) and a physical crosslinking system with dimethylurea (DMU) were used: C-PHEMA/SS (crosslinked using MBAAm) and C-PHEMA/pC-SS (crosslinked using MBAAm and DMU). The focus of this study was on investigating the impact of these crosslinking methods on various properties of the scaffolds, including pore size, pore characteristics, polymerization time, morphology, molecular interaction, in vitro degradation, thermal properties, and in vitro cytotoxicity. The various crosslinked networks were found to appreciably influence the properties of the scaffolds, especially the pore sizes, in which smaller sizes and higher numbers of pores with high regularity were seen in C-PHEMA/1.25 pC-SS (17 ± 2 μm) than in C-PHEMA/1.25 SS (34 ± 3 μm). Semi-interpenetrating networks were created by crosslinking PHEMA-MBAAm-PHEMA while incorporating free protein molecules of SS within the networks. The additional crosslinking step involving DMU occurred through hydrogen bonding of the -C=O and -N-H groups with the SS, resulting in the simultaneous incorporation of DMU and SS within the PHEMA networks. As a consequence of this process, the scaffold C-PHEMA/pC-SS exhibited smaller pore sizes compared to scaffolds without DMU crosslinking. Moreover, the incorporation of higher loadings of SS led to even smaller pore sizes. Additionally, the gelation time of C-PHEMA/pC-SS was delayed due to the presence of DMU in the crosslinking system. Both porous hydrogel scaffolds, C-PHEMA/pC-SS and PHEMA, were found to be non-cytotoxic to the normal human skin dermal fibroblast cell line (NHDF cells). This promising result indicates that these hydrogel scaffolds have potential for use in tissue engineering applications.

Simulating the mechanical stimulation of cells on a porous hydrogel scaffold using an FSI model to predict cell differentiation.

3D-structured hydrogel scaffolds are frequently used in tissue engineering applications as they can provide a supportive and biocompatible environment for the growth and regeneration of new tissue. Hydrogel scaffolds seeded with human mesenchymal stem cells (MSCs) can be mechanically stimulated in bioreactors to promote the formation of cartilage or bone tissue. Although in vitro and in vivo experiments are necessary to understand the biological response of cells and tissues to mechanical stimulation, in silico methods are cost-effective and powerful approaches that can support these experimental investigations. In this study, we simulated the fluid-structure interaction (FSI) to predict cell differentiation on the entire surface of a 3D-structured hydrogel scaffold seeded with cells due to dynamic compressive load stimulation. The computational FSI model made it possible to simultaneously investigate the influence of both mechanical deformation and flow of the culture medium on the cells on the scaffold surface during stimulation. The transient one-way FSI model thus opens up significantly more possibilities for predicting cell differentiation in mechanically stimulated scaffolds than previous static microscale computational approaches used in mechanobiology. In a first parameter study, the impact of the amplitude of a sinusoidal compression ranging from 1% to 10% on the phenotype of cells seeded on a porous hydrogel scaffold was analyzed. The simulation results show that the number of cells differentiating into bone tissue gradually decreases with increasing compression amplitude, while differentiation into cartilage cells initially multiplied with increasing compression amplitude in the range of 2% up to 7% and then decreased. Fibrous cell differentiation was predicted from a compression of 5% and increased moderately up to a compression of 10%. At high compression amplitudes of 9% and 10%, negligible areas on the scaffold surface experienced high stimuli where no cell differentiation could occur. In summary, this study shows that simulation of the FSI system is a versatile approach in computational mechanobiology that can be used to study the effects of, for example, different scaffold designs and stimulation parameters on cell differentiation in mechanically stimulated 3D-structured scaffolds.

3D printed alginate/gelatin-based porous hydrogel scaffolds to improve diabetic wound healing

Diabetic wounds are difficult to heal due to hampered vascularization and tissue regeneration. Wound dressings need frequent replacement and show limited effects on guiding tissue repair. Here, bio-inks containing sodium alginate (SA), oxidized sodium alginate (OSA), gelatin (Gel) and CaCO3 microspheres were prepared. To increase the printability, all bio-inks were pre-crosslinked with calcium ions (Ca2+). Porous hydrogel scaffolds with 4.43 ± 0.14 μm2 pore area and 184 ± 25 μm line diameter were fabricated via 3D printing. After lyophilization and swelling in PBS (pH 6.4), SA/OSA/Gel scaffold showed more excellent structural stability than SA and SA/Gel, which was attributed to the Schiff base reaction between OSA and Gel. NIH-3T3 cells on SA/OSA/Gel proliferated faster and showed better spreading morphology than those on SA and SA/Gel. After placed onto full-thickness wounds on SD rat back, SA/OSA/Gel scaffold guided tissue growth, integrated well with the regenerated tissue and accelerated wound healing, which promoted angiogenesis and showed 93.0 ± 2.5% of collagen deposition between degraded fragments of hydrogel scaffold. Taken together, porous hydrogel scaffolds fabricated via 3D printing bio-inks composed of SA, OSA, Gel and CaCO3 provide a potential strategy to improve diabetic wound healing.

Perfusion of MC3T3E1 Preosteoblast Spheroids within Polysaccharide-Based Hydrogel Scaffolds: An Experimental and Numerical Study at the Bioreactor Scale.

The traditional 3D culture systems in vitro lack the biological and mechanical spatiotemporal stimuli characteristic to native tissue development. In our study, we combined porous polysaccharide-based hydrogel scaffolds with a bioreactor-type perfusion device that generates favorable mechanical stresses while enhancing nutrient transfers. MC3T3E1 mouse osteoblasts were seeded in the scaffolds and cultivated for 3 weeks under dynamic conditions at a perfusion rate of 10 mL min-1. The spatial distribution of the cells labeled with superparamagnetic iron oxide nanoparticles was visualized by MRI. Confocal microscopy was used to assess cell numbers, their distribution inside the scaffolds, cell viability, and proliferation. The oxygen diffusion coefficient in the hydrogel was measured experimentally. Numerical simulations of the flow and oxygen transport within the bioreactor were performed using a lattice Boltzmann method with a two-relaxation time scheme. Last, the influence of cell density and spheroid size on cell oxygenation was investigated. The cells spontaneously organized into spheroids with a diameter of 30-100 μm. Cell viability remained unchanged under dynamic conditions but decreased under static culture. The cell proliferation (Ki67 expression) in spheroids was not observed. The flow simulation showed that the local fluid velocity reached 27 mm s-1 at the height where the cross-sectional area of the flow was the smallest. The shear stress exerted by the fluid on the scaffolds may locally rise to 100 mPa, compared with the average value of 25 mPa. The oxygen diffusion coefficient in the hydrogel was 1.6×10-9 m2 s-1. The simulation of oxygen transport and consumption confirmed that the cells in spheroids did not suffer from hypoxia when the bioreactor was perfused at 10 mL min-1, and suggested the existence of optimal spheroid size and spacing for appropriate oxygenation. Collectively, these findings enabled us to define the optimal conditions inside the bioreactor for an efficient in vitro cell organization and survival in spheroids, which are paramount to future applications with organoids.

Tough PEG‐only hydrogels with complex 3D structure enabled by digital light processing of “all‐PEG” resins

AbstractDigital light processing (DLP) of structurally complex poly(ethylene glycol) (PEG) hydrogels with high mechanical toughness represents a long‐standing challenge in the field of 3D printing. Here, we report a 3D printing approach for the high‐resolution manufacturing of structurally complex and mechanically strong PEG hydrogels via heat‐assisted DLP. Instead of using aqueous solutions of photo‐crosslinkable monomers, PEG macromonomer melts were first printed in the absence of water, resulting in bulk PEG networks. Then, post‐printing swelling of the printed networks was achieved in water, producing high‐fidelity 3D hydrogels with complex structures. By employing a dual‐macromonomer resin containing a PEG‐based four‐arm macrophotoinitiator, “all‐PEG” hydrogel constructs were produced with compressive toughness up to 1.3 MJ m−3. By this approach, porous 3D hydrogel scaffolds with trabecular‐like architecture were fabricated, and the scaffold surface supported cell attachment and the formation of a monolayer mimicking bone‐lining cells. This study highlights the promises of heat‐assisted DLP of PEG photopolymers for hydrogel fabrication, which may accelerate the development of 3D tissue‐like constructs for regenerative medicine.

Bone Marrow Adipocytes Contribute to Tumor Microenvironment-Driven Chemoresistance via Sequestration of Doxorubicin.

Chemoresistance is a significant problem in the effective treatment of bone metastasis. Adipocytes are a major stromal cell type in the bone marrow and may play a crucial role in developing microenvironment-driven chemoresistance. However, detailed investigation remains challenging due to the anatomical inaccessibility and intrinsic tissue complexity of the bone marrow microenvironment. In this study, we developed 2D and 3D in vitro models of bone marrow adipocytes to examine the mechanisms underlying adipocyte-induced chemoresistance. We first established a protocol for the rapid and robust differentiation of human bone marrow stromal cells (hBMSCs) into mature adipocytes in 2D tissue culture plastic using rosiglitazone (10 μM), a PPARγ agonist. Next, we created a 3D adipocyte culture model by inducing aggregation of hBMSCs and adipogenesis to create adipocyte spheroids in porous hydrogel scaffolds that mimic bone marrow sinusoids. Simulated chemotherapy treatment with doxorubicin (2.5 μM) demonstrated that mature adipocytes sequester doxorubicin in lipid droplets, resulting in reduced cytotoxicity. Lastly, we performed direct coculture of human multiple myeloma cells (MM1.S) with the established 3D adipocyte model in the presence of doxorubicin. This resulted in significantly accelerated multiple myeloma proliferation following doxorubicin treatment. Our findings suggest that the sequestration of hydrophobic chemotherapeutics by mature adipocytes represents a potent mechanism of bone marrow microenvironment-driven chemoresistance.

Injectable 3D‐Printed Porous Scaffolds for Adipose Stem Cell Delivery and Endometrial Regeneration

AbstractStem‐cell‐based therapeutic strategies are promising in the clinical treatment of intrauterine adhesions (IUAs), while endometrial regeneration still hardly restores the structure and function of the endometrium because of the inadequate microenvironment for the grafted stem cells and subsequent limited therapeutic efficiency. Herein, an injectable porous hydrogel scaffold (PH scaffold) with customizable shapes is presented by using a microfluidic‐based 3D printing technique for adipose‐derived stem cells (ADSCs) delivery to enhance endometrial regeneration. These scaffolds display a controllable interconnected porous structure, which not only facilitates the encapsulation of ADSCs within the scaffold but also supports the recovery to their original shapes after injection. Furthermore, the cell viability of the laden ADSCs is well‐maintained post‐injection, exhibiting promotive effects on cell migration, proliferation, and tube formation. Based on these features, an ADSCs‐laden PH scaffold with a hollow endometrium‐mimicking morphology is designed and in situ injected into the damaged endometrium in rats of IUAs. These results show that the ADSCs‐laden PH scaffolds can enhance functional endometrial regeneration by suppressing the inflammatory response, promoting cell proliferation, and improving vascularization. Thus, it is believed that such unique 3D‐printed porous scaffolds are promising candidates for cell delivery, which also provides a minimally‐invasive and effective strategy for endometrial regeneration.

Conductive Hydrogel Scaffolds for the 3D Localization and Orientation of Fibroblasts.

Dermal wounds and their healing are a collection of complex, multistep processes which are poorly recapitulated by existing 2D in vitro platforms. Biomaterial scaffolds that support the 3D growth of cell cultures can better resemble the native dermal environment, while bioelectronics has been used as a tool to modulate cell proliferation, differentiation, and migration. A porous conductive hydrogel scaffold which mimics the properties of dermis, while promoting the viability and growth of fibroblasts is described. As these scaffolds are also electrically conductive, the application of exogenous electrical stimulation directs the migration of cells across and/or through the material. The mechanical properties of the scaffold, as well as the amplitude and/or duration of the electrical pulses, are independently tunable and further influence the resulting fibroblast networks. This biomaterial platform may enable better recapitulation of wound healing and can be utilized to develop and screen therapeutic interventions.

A physically cross-linked double network polysaccharides/Ca2+ hydrogel scaffold for skeletal muscle tissue engineering

The current therapy for muscle injury is facing challenges in tissue self-regeneration and function restoration, which declines steeply with age. Tissue engineering is a promising strategy for treating skeletal muscular disorders. Natural polysaccharides have great potential to support cell growth and attachment as tissue-engineered scaffolds. A three-dimensional (3D) porous hydrogel scaffold has been prepared based on the double network of natural polysaccharide sodium alginate (SA), chitosan (CS) and Ca2+ ions through the freeze-drying process. The physical, mechanical, and biological properties of the CS/SA/Ca2+ hydrogel scaffold can be controlled by adjusting the SA/CS ratio. The physically cross-linked double network hydrogel showed good biosafety, high stability and satisfactory degradation rate. When the CS/SA molar ratio was 1:1.2 (H-1.2 group), the pore size and compression modulus of the double network hydrogel were 104.06 ± 2.64 µm and 40.00 ± 2.52 kPa, respectively. The results of in vitro evaluation showed that the 3D structure of the CS/SA/Ca2+ double network hydrogel could well support adhesion and proliferation of C2C12 murine myoblasts and porcine skeletal muscle stem cells. The best cell growth effect was achieved in the H-1.2 group. The physically cross-linked CS/SA/Ca2+ double network hydrogel can be a promising printable material as the cell scaffold in applications of skeletal muscle tissue engineering.

Potential of an Aligned Porous Hydrogel Scaffold Combined with Periodontal Ligament Stem Cells or Gingival Mesenchymal Stem Cells to Promote Tissue Regeneration in Rat Periodontal Defects.

Periodontal tissue regeneration is a major challenge in tissue engineering due to its regenerated environment complexity. It aims to regenerate not only the supporting alveolar bone and cementum around teeth but also the key connecting periodontal ligament. Herein, a constructed aligned porous hydrogel scaffold carrying cells based on chitosan (CHI) and oxidized chondroitin sulfate (OCS) treated with a freeze-casting technique was fabricated, which aimed to induce the arrangement of periodontal tissue regeneration. The microscopic morphology and physical and chemical properties of the hydrogel scaffold were evaluated. The biocompatibilities with periodontal ligament stem cells (PDLSCs) or gingival-derived mesenchymal stem cells (GMSCs) were verified, respectively, by Live/Dead staining and CCK8 in vitro. Furthermore, the regeneration effect of the aligned porous hydrogel scaffold combined with PDLSCs and GMSCs was evaluated in vivo. The biocompatibility experiments showed no statistical significance between the hydrogel culture group and blank control (P > 0.05). In a rat periodontal defect model, PDLSC and GMSC hydrogel experimental groups showed more pronounced bone tissue repair than the blank control (P < 0.05) in micro-CT. In addition, there was more tissue repair (P < 0.05) of PDLSC and GMSC hydrogel groups from histological staining images. Higher expressions of OPN, Runx-2, and COL-I were detected in both of the above groups via immunohistochemistry staining. More importantly, the group with the aligned porous hydrogel induced more order periodontal ligament formation than that with the ordinary hydrogel in Masson's trichrome analysis. Collectively, it is expected to promote periodontal tissue regeneration utilizing an aligned porous hydrogel scaffold combined with PDLSCs and GMSCs (CHI-OCS-PDLSC/GMSC composite), which provides an alternative possibility for clinical application.

3D printed structured porous hydrogel promotes osteogenic differentiation of BMSCs

Hydrogel is a suitable material for three-dimensional cell culture, and is rich in water content and can better simulate a natural extracellular matrix. However, the relatively compact pores in the interior often inhibit the growth and differentiation of containing cells. Additionally, the impact of the pore structure on the differentiation of cells remains to be unknown. In this work, porous hydrogel scaffolds with medium pore sizes (100–1000 μm) were prepared using 3D printing technology, and the hydrogel scaffolds without pores and non-printed hydrogel scaffolds were designed as control to investigate the effect of 3D printed porous structures on osteogenic differentiation of BMSCs within hydrogel scaffolds, both in vitro and in vivo. The pore structure of 3D printed structured porous scaffold was explicitly inspected by SEM. The water content and expansion rate of hydrogel did not change, despite its porous structure. The BMSCs were evenly distributed across the porous scaffold and maintained high activity, thus benefiting from the sufficient circulation and diffusion of oxygen and nutrients due to the regular pores. In vitro, the osteogenic differentiation of BMSCs in the structured porous hydrogels was more prominent compared to the non-porous groups. Moreover, the mineralization of the hydrogel scaffolds was also enhanced. After ectopic implantation in the posterior gluteal muscle punch, the structured porous hydrogel exhibited relatively good mineralization and ectopic osteogenesis by the three-dimensional reconstruction of micro-CT, collagen staining, and immunological analysis. The 3D printing-based approach provides a facile strategy for the preparation of hydrogels with intermediate pore structures. This can promote the viability and osteogenic differentiation of BMSCs in hydrogel scaffolds.

Three-dimensional highly porous hydrogel scaffold for neural circuit dissection and modulation.

Biomimetic brain structures and artificial neural networks have provided a simplified strategy for quantitatively investigating the complex structural and functional characteristics of highly interconnected neural networks. To achieve this, three-dimensional (3D) cell culture approaches have attracted much attention, which can mimic cell-cell interactions at the organism level and help better understand the function of specific neurons and neuronal networks than traditional two-dimensional cell culture methods. However, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. To resolve this, 3D hydrogel scaffolds can be fabricated via an innovative thermal treatment followed by an esterification process. A highly porous microstructure was formed within the bulk hydrogel scaffold, which showed a high porosity of 91% and a low Young's modulus of 6.11 kPa. Due to the merits of the fabricated hydrogel scaffolds, we constructed 3D neural networks and detected spontaneous action potentials in vitro. We successfully induced seizure-like waveforms in 3D cultured neurons and suppressed hyperactivated discharges by selectively activating γ-aminobutyric acid-ergic (GABAergic) interneurons. These results prove the advantages of our hydrogel scaffolds and demonstrate their application potential in the accurate dissection of neural circuits, which may help develop effective treatments for various neurological disorders. STATEMENT OF SIGNIFICANCE: While 3D cell culture approaches have attracted much attention and offer more advantages than two-dimensional cell culture methods, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. Herein, we developed a simplified and low-cost strategy for fabricating highly porous and cytocompatible hydrogel scaffolds for the construction of three-dimensional (3D) neural networks in vitro. The cultured 3D neural networks can mimic the in vivo connection among different neuron subgroups and help accurately dissect and manipulate the structure and function of specific neural circuits.

Alginate/Gelatin Hydrogel Scaffold Containing nCeO2 as a Potential Osteogenic Nanomaterial for Bone Tissue Engineering.

Clinicians frequently face difficulties when trying to fix bone abnormalities. Gelatin-Alginate (GA) is frequently employed as a carrier because it is non-toxic, biodegradable, and has a three-dimensional network structure. Meanwhile, cerium oxide nanoparticles (nCeO2) demonstrated high antioxidant enzyme simulation activity. Therefore, in order to develop a porous hydrogel scaffold for the application of bone tissue engineering, an appropriate-type GA-nCeO2 hydrogel scaffold was developed and evaluated. GA-nCeO2 hydrogel scaffold was prepared by the lyophilized method and characterized. The surface morphology and cell adhesion of the scaffold were observed by the scanning electron microscope. CCK8 and live-dead staining methods were used to evaluate its biological safety and cell proliferation. Then the osteogenic differentiation in early and late stages was discussed. The expression of osteogenic genes was also detected by RT-PCR. Finally, a bone defect model was made in SD rats, and bone formation in vivo was detected. The results showed that GA-nCeO2 hydrogel scaffold exhibited a typical three-dimensional porous structure with a mean pore ratio of 70.61 ± 1.94%. The GA-nCeO2 hydrogel was successfully endowed with simulated enzyme activity including superoxide dismutase (SOD) and catalase (CAT) after the addition of nCeO2. Osteoblasts demonstrated superior cell proliferation and adhesion on composite scaffolds, and both mineralization test and gene expression demonstrated the strong osteogenic potential of GA-nCeO2 hydrogel. The outcomes of hematoxylin and eosin (H&E) staining and Masson trichrome staining in the femoral defect model of SD rats further supported the scaffold's favorable biocompatibility and bone-promoting capacity. Due to its favorable safety, degradability, and bone formation property, GA-nCeO2 hydrogel was anticipated to be used as a potential bone defect healing material.

Porous hydrogel scaffolds integrating Prussian Blue nanoparticles: A versatile strategy for electrochemical (bio)sensing

Hydrogels have emerged as promising porous soft materials from which to develop a wide range of biosensing platforms due to the possibility to easily engineer their properties and to effectively immobilize enzymes and nanomaterials in their matrix. Despite their attractive properties, the ability to form stable hydrogel films integrating well–dispersed catalytic nanomaterials and enzymes on electrode surfaces is still required to enable their implementation into electrochemical biosensors. Here, we report a facile approach to prepare hydrogel films embedding Prussian Blue nanoparticles (PBNPs) and different enzymes on electrode surfaces for electrochemical biosensing. A one-pot strategy was employed for the synthesis, under mild conditions, of PBNPs and the simultaneous immobilization of enzymes in a highly functionalized carboxymethyl cellulose matrix, yielding homogeneous hydrogel composites. Highly porous hydrogel films were successfully prepared by drop casting the hydrogel composites on the surface of flexible screen–printed electrodes (SPEs). Due to the high loading of well–dispersed PBNPs, the hydrogel films demonstrated good electrocatalytic activity for both the oxidation of NADH and the reduction of hydrogen peroxide resulting in high detection sensitivity. Two amperometric biosensors for the rapid detection of glucose and ethanol in serum were realized by employing hydrogel composites integrating glucose oxidase and alcohol dehydrogenase. By combining the unique features of hydrogels on flexible electrochemical strips, our approach holds great promise for the development of portable electrochemical (bio)sensors, which are easy to fabricate and versatile for the detection of a variety of analytes.