Characterization of Chitosan Hydrogels Obtained through Phenol and Tripolyphosphate Anionic Crosslinking.

Chitosan is a deacetylated polymer of chitin that is extracted mainly from the exoskeleton of crustaceans and is the second-most abundant polymer in nature. Chitosan hydrogels are preferred for a variety of applications in bio-related fields due to their functional properties, such as antimicrobial activity and wound healing effects; however, the existing hydrogelation methods require toxic reagents and exhibit slow gelation times, which limit their application in biological fields. Therefore, a mild and rapid gelation method is necessary. We previously demonstrated that the visible light-induced gelation of chitosan obtained through phenol crosslinking (ChPh) is a rapid gelation method. To further advance this method (<10 s), we propose a dual-crosslinked chitosan hydrogel obtained by crosslinking phenol groups and crosslinking sodium tripolyphosphate (TPP) and the amino groups of chitosan. The chitosan hydrogel was prepared by immersing the ChPh hydrogel in a TPP solution after phenol crosslinking via exposure to visible light. The physicochemical properties of the dual-crosslinked hydrogels, including Young's moduli and water retentions, were subsequently investigated. Young's moduli of the dual-crosslinked hydrogels were 20 times higher than those of the hydrogels without TPP ion crosslinking. The stiffness could be manipulated by varying the immersion time, and the water retention properties of the ChPh hydrogel were improved by TPP crosslinking. Ion crosslinking could be reversed using an iron chloride solution. This method facilitates chitosan hydrogel use for various applications, particularly tissue engineering and drug delivery.

Hydrogels with Ultrasound-Treated Hyaluronic Acid Regulate CD44-Mediated Angiogenic Potential of Human Vascular Endothelial Cells In Vitro.

The development of hydrogels that allow vascular endothelial cells to form capillary-like networks is critical for advancing tissue engineering and drug discovery. In this study, we developed hydrogels composed of phenolated hyaluronic acid (HA-Ph) with an average molecular weight of 490-159 kDa via sonication in an aqueous solution. These hydrogels were synthesized by the horseradish peroxidase-catalyzed crosslinking of phenol moieties in the presence of hydrogen peroxide and phenolated gelatin. The sonication-degraded HA-Ph (198 kDa) significantly enhanced the migration ability of human umbilical vein endothelial cells (HUVECs) on cell culture plates when added to the medium compared to the original HA-Ph (490 kDa) and less-degraded HA-Ph (312-399 kDa). In addition, HUVECs cultured on these hydrogels formed networks that did not occur on hydrogels made from the original HA-Ph. CD44 expression and PI3K gene expression, both markers related to angiogenesis, were 3.5- and 1.8-fold higher, respectively, in cells cultured on sonication-degraded HA-Ph hydrogels than in those cultured on hydrogels comprising the original HA-Ph. These results highlight the potential of hydrogels containing sonication-degraded HA-Ph for tissue engineering and drug-screening applications involving human vascular endothelial cells.

3D Bioprinting of Sugar Beet Pectin through Horseradish Peroxidase-Catalyzed Cross-Linking.

Horseradish peroxidase (HRP)-mediated hydrogelation, caused by the cross-linking of phenolic groups in polymers in the presence of hydrogen peroxide (H2O2), is an effective route for bioink solidification in 3D bioprinting. Sugar beet pectin (SBP) naturally has cross-linkable phenols through the enzymatic reaction. Therefore, chemical modifications are not required, unlike the various polymers that have been used in the enzymatic cross-linking system. In this study, we report the application of SBP in extrusion-based bioprinting including HRP-mediated bioink solidification. In this system, H2O2 necessary for the solidification of inks is supplied in the gas phase. Cell-laden liver lobule-like constructs could be fabricated using bioinks consisting of 10 U/mL HRP, 4.0 and 6.0 w/v% SBP, and 6.0 × 106 cells/mL human hepatoblastoma (HepG2) cells exposed to air containing 16 ppm of H2O2 concurrently during printing and 10 min postprinting. The HepG2 cells enclosed in the printed constructs maintained their viability, metabolic activity, and hepatic functions from day 1 to day 7 of the culture, which indicates the cytocompatibility of this system. Taken together, this result demonstrates the potential of SBP and HRP cross-linking systems for 3D bioprinting, which can be applied in tissue engineering applications.

Transduction and Genome Editing of the Heart with Adeno-Associated Viral Vectors Loaded onto Electrospun Polydioxanone Nonwoven Fabrics.

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.

Phototuning of Hyaluronic-Acid-Based Hydrogel Properties to Control Network Formation in Human Vascular Endothelial Cells.

In vitro network formation by endothelial cells serves as a fundamental model for studies aimed at understanding angiogenesis. The morphogenesis of these cells to form a network is intricately regulated by the mechanical and biochemical properties of the extracellular matrix. Here the effects of modulating these properties in hydrogels derived from phenolated hyaluronic acid (HA-Ph) and phenolated gelatin (Gelatin-Ph) are presented. Visible-light irradiation in the presence of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate induces the crosslinking of these polymers, thereby forming a hydrogel and degrading HA-Ph. Human vascular endothelial cells form networks on the hydrogel prepared by visible-light irradiation for 45 min (42 W cm-2 at 450 nm) but not on the hydrogels prepared by irradiation for 15, 30, or 60 min. The irradiation time-dependent degradation of HA-Ph and the changes in the mechanical stiffness of the hydrogels, coupled with the expressions of RhoA and ß-actin genes and CD44 receptors in the cells, reveal that the network formation is synergistically influenced by the hydrogel stiffness and HA-Ph degradation. These findings highlight the potential of tailoring HA-based hydrogel properties to modulate human vascular endothelial cell responses, which is critical for advancing their application in vascular tissue engineering.

Effect of cell adhesiveness of Cell Dome shell on enclosed HeLa cells.

The Cell Dome is a dome-shaped structure (diameter: 1 mm, height: 270 µm) with cells enclosed within a cavity, covered by a hemispherical hydrogel shell, and immobilized on a glass plate. Given that the cells within Cell Dome are in contact with the inner walls of the hydrogel shell, the properties of the shell are anticipated to influence cell behavior. To date, the impact of the hydrogel shell properties on the enclosed cells has not been investigated. In this study, we explored the effects of the cell adhesiveness of hydrogel shell on the behavior of enclosed cancer cells. Hydrogel shells with varying degrees of cell adhesiveness were fabricated using aqueous solutions containing either an alginate derivative with phenolic hydroxyl moieties exclusively or a mixture of alginate and gelatin derivatives with phenolic hydroxyl moieties. Hydrogel formation was mediated by horseradish peroxidase. We used the HeLa human cervical cancer cell line, which expresses fucci2, a cell cycle marker, to observe cell behavior. Cells cultured in hydrogel shells with cell adhesiveness proliferated along the inner wall of the hydrogel shell. Conversely, cells in hydrogel shells without cell adhesiveness grew uniformly at the bottom of the cavities. Furthermore, cells in non-adhesive hydrogel shells had a higher percentage of cells in the G1/G0 phase compared to those in adhesive shells and exhibited increased resistance to mitomycin hydrochloride when the cavities became filled with cells. These results highlight the need to consider the cell adhesiveness of the hydrogel shell when selecting materials for constructing Cell Dome.

Local delivery of adeno-associated viral vectors with electrospun gelatin nanofiber mats.

Adeno-associated viral (AAV) vectors play a significant role in gene therapy, yet the typical delivery methods, like systemic and local AAV injections, often lead to unintended off-target distribution and tissue damage due to injection. In this study, we propose a localized delivery approach for AAV vectors utilizing electrospun gelatin nanofiber mats, which are cross-linked with glutaraldehyde. The AAV vectors, which encoded a green fluorescent protein (GFP), were loaded onto the mats by immersing them in a solution containing the vectors. The amount of AAV vector loaded onto the mats increased as the vector concentration in the solution increased. The loaded AAV vector was steadily released into the cell culture medium over 3 days. The mats incubated for 3 days also showed the ability to transduce into the cells cultured on them. We evaluated the effectiveness of this delivery system by attaching the mats to mouse livers. GFP expression was visible on the surface of the liver beneath the attached mats, but not in areas in direct contact with the mats. These findings suggest that the attachment of AAV vector-loaded electrospun gelatin nanofiber mats to a target site present a promising solution for localized gene delivery while reducing off-target distribution.

Horseradish Peroxidase-Mediated Bioprinting via Bioink Gelation by Alternately Extruded Support Material.

Horseradish peroxidase (HRP)-mediated extrusion bioprinting has a significant potential in tissue engineering and regenerative medicine. However, they often face challenges in terms of printing fidelity and structural integrity when using low-viscosity inks. To address this issue, a method that alternately extrudes bioinks and support material was developed in this study. The bioinks consisting of cells, HRP, and phenolated polymers, and the support material contained hydrogen peroxide (H2O2). The support material not only prevented the collapse of the constructs but also supplied H2O2 to facilitate the enzymatic reaction. 3D constructs with tall and complex shapes were successfully printed from a low-viscosity ink containing 10 U/mL HRP and 1.0% w/v phenolated hyaluronic acid (HA-Ph), with a support material containing 10 mM H2O2. Over 90% viability of mouse fibroblasts (10T1/2) was achieved following the printing process, along with a morphology and proliferation rate similar to that of nontreated cells. Furthermore, human hepatoblastoma (HepG2) cells showed an increased spheroid size over 14 days in the printed constructs. The 10T1/2 cells adhered and proliferated on the constructs printed from inks containing both phenolated gelatin and HA-Ph. These results demonstrate the great potential of this HRP-mediated extrusion bioprinting technique for tissue engineering applications.

Tuning the crosslinking and degradation of hyaluronic acid/gelatin hydrogels using hydrogen peroxide for muscle cell sheet fabrication.

Cell sheets have immense potential for medical and pharmaceutical applications including tissue regeneration, drug testing, and disease modelling. In this study, composite hydrogels were prepared from a mixture of phenolated hyaluronic acid (HA-Ph) and gelatin (Gelatin-Ph), with a controlled degree of polymer crosslinking and degradation, to fabricate muscle cell sheets from myoblasts. These hydrogels were obtained via hydrogen peroxide (H2O2)-mediated crosslinking catalysed by horseradish peroxidase (HRP) and peroxide-mediated cleavage of the polymer chains. The degrees of crosslinking and degradation were modulated by altering the exposure time to air containing H2O2. The results showed that exposing a solution of 2% w/v HA-Ph, 0.75% w/v Gelatin-Ph, and 1 unit mL-1 HRP to air with 16 ppm H2O2 for 60 min yielded a stiffer hydrogel (7.16 kPa Young's modulus) than exposure times of 15 min (0.46 kPa) and 120 min (3.98 kPa). Moreover, mouse myoblast C2C12 cells cultured on a stiff hydrogel and induced to undergo myogenic differentiation formed longer and higher-density myotubes than those on softer hydrogels. The cell sheets were readily detached within 5 min by immersing the HA-Ph/Gelatin-Ph hydrogels covered with a monolayer of cells in a medium containing hyaluronidase. Our findings demonstrate that composite hydrogels with properties tuned by controlling the exposure time to H2O2, show great promise as platforms for muscle cell sheet fabrication.

Visible light photocrosslinking of sugar beet pectin for 3D bioprinting applications.

Herein, we report the hydrogelation of sugar beet pectin (SBP) via visible light-mediated photocrosslinking and its applications in extrusion-based 3D bioprinting. Rapid hydrogelation (<15 s) was achieved by applying 405 nm visible light to an SBP solution in the presence of tris(bipyridine)ruthenium(II) chloride hexahydrate ([Ru(bpy)3]2+) and sodium persulfate (SPS). The mechanical properties of the hydrogel could be tuned by controlling the visible light irradiation time and concentrations of SBP, [Ru(bpy)3]2+, and SPS. High-fidelity 3D hydrogel constructs were fabricated by extruding inks containing 3.0 wt% SBP, 1.0 mM [Ru(bpy)3]2+, and 1.0 mM SPS. Human hepatoblastoma (HepG2) cells encapsulated in SBP hydrogels remained viable and metabolically active after 14 d of culture. Overall, this study demonstrates the feasibility of applying SBP and a visible light-mediated photocrosslinking system to the 3D bioprinting of cell-laden constructs for tissue engineering applications.

Mammalian-specific decellularized matrices derived bioink for bioengineering of liver tissue analogues: A review.

The absolute shortage of compatible liver donors and the growing number of potential recipients have led scientists to explore alternative approaches to providing tissue/ organ substitutes from bioengineered sources. Bioartificial regeneration of a fully functional tissue/organ replacement is highly dependent on the right combination of engineering tools, biological principles, and materiobiology horizons. Over the past two decades, remarkable achievements have been made in hepatic tissue engineering by converging various advanced interdisciplinary research approaches. Three-dimensional (3D) bioprinting has arisen as a promising state-of-the-art tool with strong potential to fabricate volumetric liver tissue/organ equivalents using viscosity- and degradation-controlled printable bioinks composed of hydrous microenvironments, and formulations containing living cells and associated supplements. Source of origin, biophysiochemical, or thermomechanical properties and crosslinking reaction kinetics are prerequisites for ideal bioink formulation and realizing the bioprinting process. In this review, we delve into the forecast of the potential future utility of bioprinting technology and the promise of tissue/organ- specific decellularized biomaterials as bioink substrates. Afterward, we outline various methods of decellularization, and the most relevant studies applying decellularized bioinks toward the bioengineering of in vitro liver models. Finally, the challenges and future prospects of decellularized material-based bioprinting in the direction of clinical regenerative medicine are presented to motivate further developments.

Fabrication of cell-laden microbeads and microcapsules composed of bacterial polyglucuronic acid.

In the past decades, the microencapsulation of mammalian cells into microparticles has been extensively studied for various in vitro and in vivo applications. The aim of this study was to demonstrate the viability of bacterial polyglucuronic acid (PGU), an exopolysaccharide derived from bacteria and composed of glucuronic acid units, as an effective material for cell microencapsulation. Using the method of dropping an aqueous solution of PGU-containing cells into a Ca2+-loaded solution, we produced spherical PGU microbeads with >93 % viability of the encapsulated human hepatoma HepG2 cells. Hollow-core microcapsules were formed via polyelectrolyte complex layer formation of PGU and poly-l-lysine, after which Ca2+, a cross-linker of PGU, was chelated, and this was accomplished by sequential immersion of microbeads in aqueous solutions of poly-l-lysine and sodium citrate. The encapsulated HepG2 cells proliferated and formed cell aggregates within the microparticles over a 14-day culture, with significantly larger aggregates forming within the microcapsules. Our results provide evidence for the viability of PGU for cell microencapsulation for the first time, thereby contributing to advancements in tissue engineering.

Phenol-Grafted Alginate Sulfate Hydrogel as an Injectable FGF-2 Carrier.

In the field of tissue engineering, fibroblast growth factor-2 (FGF-2) effectively regenerates damaged tissue and restores its biological function. However, FGF-2 readily diffuses and degrades under physiological conditions. Therefore, methods for the sustained and localized delivery of FGF-2 are needed. Drug delivery systems using hydrogels as carriers have attracted significant interest. Injectable hydrogels with an affinity for FGF-2 are candidates for FGF-2 delivery systems. In this study, we fabricated a hydrogel from phenol-grafted alginate sulfate (AlgS-Ph) and investigated its application to the delivery of FGF-2. The hydrogel was prepared under mild conditions via horseradish peroxidase (HRP)-mediated cross-linking. Surface plasmon resonance (SPR) measurements show that the AlgS-Ph hydrogel has an affinity for FGF-2 in accordance with its degree of sulfation. Conditions for the preparation of the AlgS-Ph hydrogel, including HRP and H2O2 concentrations, are optimized so that the hydrogel can be used as an injectable drug carrier. The hydrogel shows no cytotoxicity when using 10T1/2 cells as a model cell line. The angiogenesis assay shows that FGF-2 released from the AlgS-Ph hydrogel promotes the formation of blood vessels. These results indicate that the AlgS-Ph hydrogel is a suitable candidate for the FGF-2 carrier.

Design and Fabrication of Mature Engineered Pre-Cardiac Tissue Utilizing 3D Bioprinting Technology and Enzymatically Crosslinking Hydrogel.

The fabrication of mature engineered cardiac tissue is one of the major challenges in cardiac tissue engineering. For this purpose, we attempted to apply the 3D bioprinting approach. Aiming to construct an oriented tissue, a fine fiber-shaped scaffold with a support structure was first designed using CAD software. Then, a 3D bioprinter and cell-adhesive bio-inks were utilized to fabricate this structure. The cell-adhesive bio-inks were synthesized by combining sodium alginate and gelatin with tyramine, respectively, to form pre-gel materials that allow enzymatic crosslinking by horseradish peroxidase. By absorbance measurements, we confirmed that the tyramine modification rate of each polymer was 0.535 mmol/g-alginate and 0.219 mmol/g-gelatin. The width of the fiber-shaped scaffold was 216.8 ± 24.3 µm for the fabricated scaffold, while the design value was 200 µm. After 3D printing and adhesion-adding treatment of the scaffold with these bio-ink materials, cardiomyocytes were seeded and cultured. As a result, the cells spread onto the scaffold, and the entire pre-tissue contracted synchronously by day 6 of culture, showing a greater pulsatility than in the early days. Video analysis showed that the beating rate of pre-myocardial tissue on day 6 was 31 beats/min. In addition, we confirmed that the cardiomyocytes partially elongated along the long axis of the fiber-shaped scaffold in the pre-tissue cultured for 15 days by staining actin, suggesting the possibility of cell orientation. Furthermore, treatment with adrenaline resulted in a 7.7-fold increase in peak beating rate compared to that before treatment (from 6 beats/min to 46 beats/min), confirming the responsiveness of the pre-tissues to the drug. These results indicate that 3D bioprinting effectively produces mature cultured myocardial tissue that is oriented, contracts synchronously, and is responsive to drugs.

Human Umbilical Vein Endothelial Cells Form a Network on a Hyaluronic Acid/Gelatin Composite Hydrogel Moderately Crosslinked and Degraded by Hydrogen Peroxide.

The study of the capillary-like network formation of human umbilical vein endothelial cells (HUVECs) in vitro is important for understanding the factors that promote or inhibit angiogenesis. Here, we report the behavior of HUVECs on the composite hydrogels containing hyaluronic acid (HA) and gelatin with different degrees of degradation, inducing the different physicochemical properties of the hydrogels. The hydrogels were obtained through horseradish peroxidase (HRP)-catalyzed hydrogelation consuming hydrogen peroxide (H2O2, 16 ppm) supplied from the air, and the degradation degree was tuned by altering the exposure time to the air. The HUVECs on the composite hydrogel with intermediate stiffness (1.2 kPa) obtained through 120 min of the exposure were more elongated than those on the soft (0.4 kPa) and the stiff (2.4 kPa) composite hydrogels obtained through 15 min and 60 min of the exposure, respectively. In addition, HUVECs formed a capillary-like network only on the stiff composite hydrogel although those on the hydrogels with comparable stiffness but containing gelatin alone or alginate instead of HA did not form the network. These results show that the HA/gelatin composite hydrogels obtained through the H2O2-mediated crosslinking and degradation could be a tool for studies using HUVECs to understand the promotion and inhibition of angiogenesis.

Gelatin-Based Electrospun Nanofibers Cross-Linked Using Horseradish Peroxidase for Plasmid DNA Delivery.

The delivery of nucleic acids is indispensable for tissue engineering and gene therapy. However, the current approaches involving DNA/RNA delivery by systemic and local injections face issues such as clearance, off-target distribution, and tissue damage. In this study, we report plasmid DNA (pDNA) delivery using gelatin electrospun nanofibers obtained through horseradish peroxidase (HRP)-mediated insolubilization. The nanofibers were obtained through the electrospinning of an aqueous solution containing gelatin possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph) and HRP with subsequent HRP-mediated cross-linking of the Ph moieties by exposure to air containing 16 ppm H2O2 for 30 min. Then, Lipofectamine/pDNA complexes were immobilized on the nanofibers through immersion in the solution containing the pDNA complexes, resulting in transfection and sustained delivery of pDNA. Cells cultured on the resultant nanofibers expressed genome-editing molecules including Cas9 protein and guide RNA (gRNA), resulting in targeted gene knock-in and knock-out. These results demonstrated the potential of Gelatin-Ph nanofibers obtained through electrospinning and subsequent HRP-mediated cross-linking for gene therapy and tissue regeneration by genome editing.

Development of non-adherent cell-enclosing domes with enzymatically cross-linked hydrogel shell.

Non-adherent cells, such as hematopoietic cells and lymphocytes, are important research subjects in medical and biological fields. Therefore, a system that enables the handling of non-adherent cells in solutions in the same manner as that of adhering cells during medium exchange, exposure to chemicals, washing, and staining in imaging applications would be useful. Here, we report a 'Cell Dome' platform in which non-adherent cells can be enclosed and grown in the cavities of about 1 mm diameter and 270µm height. The domes consist of an alginate-based hydrogel shell of 90µm thickness. Cell Domes were formed on glass plates by horseradish peroxidase-mediated cross-linking. Human leukaemia cell line K562 cells enclosed in Cell Domes were stable for 29 days with every 2-3 days of medium change. The enclosed cells grew in the cavities and were stained and differentiated with reagents supplied from the surrounding medium. Additionally, K562 cells that filled the cavities (a 3D microenvironment) were more hypoxic and highly resistant to mitomycin C than those cultured in 2D. These findings demonstrate that the 'Cell Dome' may be a promising tool for conveniently culturing and evaluating non-adherent cells.

Nematode surface functionalization with hydrogel sheaths tailored in situ.

Engineering the surfaces of biological organisms allows the introduction of novel functions and enhances their native functions. However, studies on surface engineering remained limited to unicellular organisms. Herein, nematode surfaces are engineered through in situ hydrogelation mediated by horseradish peroxidase (HRP) anchored to nematode cuticles. With this method, hydrogel sheaths of approximately 10-µm thickness are fabricated from a variety of polysaccharides, proteins, and synthetic polymers. Caenorhabditis elegans and Anisakis simplex coated with a hydrogel sheath showed a negligible decrease in viability, chemotaxis and locomotion. Hydrogel sheaths containing UV-absorbable groups and catalase functioned as shields to protect nematodes from UV and hydrogen peroxide, respectively. The results also showed that hydrogel sheaths containing glucose oxidase have the potential to be used as living drug delivery systems for cancer therapy. The nematode functionalization method developed in this study has the potential to impact a wide range of fields from agriculture to medicine.

Tuning Myogenesis by Controlling Gelatin Hydrogel Properties through Hydrogen Peroxide-Mediated Cross-Linking and Degradation.

Engineering skeletal muscle tissue in vitro is important to study the mechanism of myogenesis, which is crucial for regenerating muscle cells. The physicochemical properties of the cellular microenvironment are known to govern various cell behaviours. Yet, most studies utilised synthetic materials to model the extracellular matrix that suffers from cytotoxicity to the cells. We have previously reported that the physicochemical property of hydrogels obtained from horseradish peroxidase (HRP)-catalysed cross-linking could be controlled by a simple adjustment to the exposure time to air containing H2O2. In this study, we evaluated the influence of physicochemical properties dynamics in the gelatin possessing phenol groups (Gelatin-Ph) hydrogel to regulate the myogenesis in vitro. We controlled the Young's modulus of the Gelatin-Ph hydrogel by tuning the air containing 16 ppm H2O2 exposure time for 15-60 min. Additionally, prolonged exposure to air containing H2O2 also induced Gelatin-Ph degradation. Myoblasts showed higher adhesion and myotube formation on stiff hydrogel (3.53 kPa) fabricated through 30 min of exposure to air containing H2O2 compared to those on softer hydrogel (0.77-2.79 kPa) fabricated through 15, 45, and 60 min of the exposure. These results demonstrate that the myogenesis can be tuned by changes in the physicochemical properties of Gelatin-Ph hydrogel mediated by H2O2.

Interaction of alginate with nano-hydroxyapatite-collagen using strontium provides suitable osteogenic platform.

BACKGROUND: Hydrogels based on organic/inorganic composites have been at the center of attention for the fabrication of engineered bone constructs. The establishment of a straightforward 3D microenvironment is critical to maintaining cell-to-cell interaction and cellular function, leading to appropriate regeneration. Ionic cross-linkers, Ca2+, Ba2+, and Sr2+, were used for the fabrication of Alginate-Nanohydroxyapatite-Collagen (Alg-nHA-Col) microspheres, and osteogenic properties of human osteoblasts were examined in in vitro and in vivo conditions after 21 days. RESULTS: Physicochemical properties of hydrogels illustrated that microspheres cross-linked with Sr2+ had reduced swelling, enhanced stability, and mechanical strength, as compared to the other groups. Human MG-63 osteoblasts inside Sr2+ cross-linked microspheres exhibited enhanced viability and osteogenic capacity indicated by mineralization and the increase of relevant proteins related to bone formation. PCR (Polymerase Chain Reaction) array analysis of the Wnt (Wingless-related integration site) signaling pathway revealed that Sr2+ cross-linked microspheres appropriately induced various signaling transduction pathways in human osteoblasts leading to osteogenic activity and dynamic growth. Transplantation of Sr2+ cross-linked microspheres with rat osteoblasts into cranium with critical size defect in the rat model accelerated bone formation analyzed with micro-CT and histological examination. CONCLUSION: Sr2+ cross-linked Alg-nHA-Col hydrogel can promote functionality and dynamic growth of osteoblasts.