Injectable Human Hair Keratin-Fibrinogen Hydrogels for Engineering 3D Microenvironments to Accelerate Oral Tissue Regeneration.

Traumatic injury of the oral cavity is atypical and often accompanied by uncontrolled bleeding and inflammation. Injectable hydrogels have been considered to be promising candidates for the treatment of oral injuries because of their simple formulation, minimally invasive application technique, and site-specific delivery. Fibrinogen-based hydrogels have been widely explored as effective materials for wound healing in tissue engineering due to their uniqueness. Recently, an injectable foam has taken the spotlight. However, the fibrin component of this biomaterial is relatively stiff. To address these challenges, we created keratin-conjugated fibrinogen (KRT-FIB). This study aimed to develop a novel keratin biomaterial and assess cell-biomaterial interactions. Consequently, a novel injectable KRT-FIB hydrogel was optimized through rheological measurements, and its injection performance, swelling behavior, and surface morphology were investigated. We observed an excellent cell viability, proliferation, and migration/cell-cell interaction, indicating that the novel KRT-FIB-injectable hydrogel is a promising platform for oral tissue regeneration with a high clinical applicability.

Differential gene expression profiles of human periodontal ligament cells preserved in Hank's balanced salt solution and milk.

BACKGROUND/AIM: Various types of storage media have been investigated to preserve avulsed teeth. However, the efficacies of storage media mainly focus on the aspect of cell viability. The aim of this study was to evaluate and compare the gene expression profiles of human periodontal ligament cells preserved in Hank's balanced salt solution (HBSS) and milk over different storage durations. MATERIAL AND METHODS: Human periodontal ligament cells were cultured and preserved in HBSS and milk for 3 and 6 hours. Next, total RNA was isolated. QuantSeq 3' mRNA-Sequencing was used to examine differences in gene expression in HBSS- and milk-grown periodontal ligament cells. Bioinformatics analysis was also performed to predict the function of the differentially expressed genes. RESULTS: The number of differentially expressed genes shared among all groups was 101. In gene set enrichment analysis, the shared differentially expressed genes in HBSS and milk were associated with the TNF-α signaling pathway (P = 1.07E-7 ). Seven hallmark gene sets were also identified in HBSS. Moreover, hallmark gene sets associated with hypoxia (P = 7.26E-5 ) and apoptosis (P = 4.06E-4 ) were identified in HBSS. In milk, 10 hallmark gene sets along with gene sets for inflammatory response (P = 6.87E-3 ) were identified. CONCLUSIONS: Compared to those in milk, genes in HBSS were differentially expressed with increasing storage duration, suggesting that diverse and different gene expression may be involved in HBSS and milk. However, a more detailed functional analysis of these differentially expressed genes in storage solutions should be performed in the future.

Nanogels Derived from Fish Gelatin: Application to Drug Delivery System.

The gelatin extracted from mammals of porcine and bovine has been prominently used in pharmaceutical, medical, and cosmetic products. However, there have been some concerns for their usage due to religious, social and cultural objections, and animal-to-human infectious disease. Recently, gelatin from marine by-products has received growing attention as an alternative to mammalian gelatin. In this study, we demonstrate the formation of nanogels (NGs) using fish gelatin methacryloyl (GelMA) and their application possibility to the drug delivery system. The fabrication of fish GelMA NGs is carried out by crosslinking through the photopolymerization of the methacryloyl substituent present in the nanoemulsion droplets, followed by purification and redispersion. There were different characteristics depending on the aqueous phase in the emulsion and the type of solvent used in redispersion. The PBS-NGs/D.W., which was prepared using PBS for the aqueous phase and D.W. for the final dispersion solution, had a desirable particle size (<200 nm), low PdI (0.16), and high drug loading efficiency (77%). Spherical NGs particles were observed without aggregation in TEM images. In vitro release tests of doxorubicin (DOX)-GelMA NGs showed the pH-dependent release behavior of DOX. Also, the MTT experiments demonstrated that DOX-GelMA NGs effectively inhibited cell growth, while only GelMA NGs exhibit higher percentages of cell viability. Therefore, the results suggest that fish GelMA NGs have a potential for nano-carrier as fine individual particles without the aggregation and cytotoxicity to deliver small-molecule drugs.

Vitamin D-conjugated gold nanoparticles as functional carriers to enhancing osteogenic differentiation.

In an aging society, bone disorders such as osteopenia, osteoporosis, and degenerative arthritis cause serious public health problems. In order to solve these problems, researchers continue to develop therapeutic agents, increase the efficacy of developed therapeutic agents, and reduce side effects. Gold nanoparticles (GNPs) are widely used in tissue engineering applications as biosensors, drug delivery carriers, and bioactive materials. Their special surface property enables easy conjugation with ligands including functional groups such as thiols, phosphines, and amines. This creates an attractive advantage to GNPs for use in the bone tissue engineering field. However, GNPs alone are limited in their biological effects. In this study, we used thiol-PEG-vitamin D (SPVD) to conjugate vitamin D, an essential nutrient critical for maintaining normal skeletal homeostasis, to GNPs. To characterize vitamin D-conjugated GNPs (VGNPs), field emission transmission electron microscopy, energy dispersive X-ray spectroscopy, dynamic light scattering, and ultraviolet/visible absorption analysis were carried out. The developed VGNPs were well bound through the thiol groups between GNPs and vitamin D, and were fabricated in size of 60 nm. Moreover, to demonstrate VGNPs osteogenic differentiation effect, various assays were carried out through cell viability test, alkaline phosphatase assay, calcium deposition assay, real-time polymerase chain reaction, and immunofluorescence staining. As a result, the fabricated VGNPs were found to effectively enhance osteogenic differentiation of human adipose-derived stem cells (hADSCs) in vitro. Based on these results, VGNPs can be utilized as functional nanomaterials for bone regeneration in the tissue engineering field.

Graphene-Functionalized Biomimetic Scaffolds for Tissue Regeneration.

Graphene is a two-dimensional atomic layer of graphite, where carbon atoms are assembled in a honeycombed lattice structure. Recently, graphene family nanomaterials, including pristine graphene, graphene oxide and reduced graphene oxide, have increasingly attracted a great deal of interest from researchers in a variety of science, engineering and industrial fields because of their unique structural and functional features. In particular, extensive studies have been actively conducted in the biomedical and related fields, including multidisciplinary and emerging areas, as their stimulating effects on cell behaviors have been becoming an increasing concern. Herein, we are attempting to summarize some of recent findings in the fields of tissue regeneration concerning the graphene family nanomaterial-functionalized biomimetic scaffolds, and to provide the promising perspectives for the possible applications of graphene family nanomaterial.

Engineering three dimensional micro nerve tissue using postnatal stem cells from human dental apical papilla.

The in vitro generation of cell-based three dimensional (3D) nerve tissue is an attractive subject to improve graft survival and integration into host tissue for neural tissue regeneration or to model biological events in stem cell differentiation. Although 3D organotypic culture strategies are well established for 3D nerve tissue formation of pluripotent stem cells to study underlying biology in nerve development, cell-based nerve tissues have not been developed using human postnatal stem cells with therapeutic potential. Here, we established a culture strategy for the generation of in vitro cell-based 3D nerve tissue from postnatal stem cells from apical papilla (SCAPs) of teeth, which originate from neural crest-derived ectomesenchyme cells. A stem cell population capable of differentiating into neural cell lineages was generated during the ex vivo expansion of SCAPs in the presence of EGF and bFGF, and SCAPs differentiated into neural cells, showing neural cell lineage-related molecular and gene expression profiles, morphological changes and electrophysical property under neural-inductive culture conditions. Moreover, we showed the first evidence that 3D cell-based nerve-like tissue with axons and myelin structures could be generated from SCAPs via 3D organotypic culture using an integrated bioprocess composed of polyethylene glycol (PEG) microwell-mediated cell spheroid formation and subsequent dynamic culture in a high aspect ratio vessel (HARV) bioreactor. In conclusion, the culture strategy in our study provides a novel approach to develop in vitro engineered nerve tissue using SCAPs and a foundation to study biological events in the neural differentiation of postnatal stem cells. Biotechnol. Bioeng. 2017;114: 903-914. © 2016 Wiley Periodicals, Inc.

Mitochondrial function contributes to oxysterol-induced osteogenic differentiation in mouse embryonic stem cells.

Oxysterols, oxidized derivatives of cholesterol, are biologically active molecules. Specific oxysterols have potent osteogenic properties that act on osteoprogenitor cells. However, the molecular mechanisms underlying these osteoinductive effects on embryonic stem cells (ESCs) are unknown. This study investigated the effect of an oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on osteogenic differentiation of ESCs and the alterations to mitochondrial activity during differentiation. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, matrix mineralization, mRNA expression of osteogenic factors, runt-related transcription factor 2, osterix, and osteocalcin, and protein levels of collagen type IA (COLIA) and osteopontin (OPN). Treatment of cells with SS increased osteoinductive activity compared to the control group. Intracellular reactive oxygen species production, intracellular ATP content, mitochondrial membrane potential, mitochondrial mass, mitochondrial DNA copy number, and mRNA expression of peroxisome proliferator-activated receptor-γ coactivators 1α and ß, transcription factors involved in mitochondrial biogenesis, were significantly increased during osteogenesis, indicating upregulation of mitochondrial activity. Oxysterol combinations also increased protein levels of mitochondrial respiratory complexes I-V. We also found that SS treatment increased hedgehog signaling target genes, Smo and Gli1 expression. Inhibition of Hh signaling by cyclopamine suppressed mitochondrial biogenesis and ESC osteogenesis. Subsequently, oxysterol-induced Wnt/ß-catenin pathways were inhibited by repression of Hh signaling and mitochondrial biogenesis. Transfection of ß-catenin specific siRNA decreased the protein levels of COLIA and OPN, as well as ALP activity. Collectively, these data suggest that lipid-based oxysterols enhance differentiation of ESCs toward the osteogenic lineage by regulating mitochondrial activity, canonical Hh/Gli, and Wnt/ß-catenin signaling.

Femtosecond laser-patterned nanopore arrays for surface-mediated peptide treatment.

The major goal of this study was to create easy-to-use, reusable substrates capable of storing any peptides or bioactive molecules for a desired period of time until cells uptake them without the need for bioactive molecule or peptide-specific techniques. Nanopore arrays of uniform size and distribution were machined into fused silica substrates using femtosecond laser ablation and loaded with peptides by simple adsorption. The nanopore substrates were validated by examining the effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) loaded nanopores on macrophage phagocytosis and intracellular production of reactive oxygen species (ROS) with and without the pro-inflammatory lipopolysaccharide (LPS). Our results demonstrated that nanopores were generated in a uniform array fashion. Ac-SDKP peptides were stably stored in nanopores and internalized by macrophages. Significant reductions in ROS production and phagocytosis in macrophages were observed over control substrates, even in combination with LPS stimulation, indicating that loading Ac-SDKP peptides in pores significantly improved the anti-inflammatory effects. FROM THE CLINICAL EDITOR: This team of scientists intended to create easy-to-use, reusable substrates for storing peptides or bioactive molecules for a desired period of time before cellular uptake occurs, and without the need for bioactive molecule or peptide-specific techniques. They demonstrate the successful generation of nanopores in a uniform array that stably stores Ac-SDKP peptides in the nanopores. When peptides were internalized by macrophages, significant reductions in ROS production and phagocytosis were observed, indicating improved anti-inflammatory effects.

State-of-the-art techniques for promoting tissue regeneration: Combination of three-dimensional bioprinting and carbon nanomaterials.

181Biofabrication approaches, such as three-dimensional (3D) bioprinting of hydrogels, have recently garnered increasing attention, especially in the construction of 3D structures that mimic the complexity of tissues and organs with the capacity for cytocompatibility and post-printing cellular development. However, some printed gels show poor stability and maintain less shape fidelity if parameters such as polymer nature, viscosity, shear-thinning behavior, and crosslinking are affected. Therefore, researchers have incorporated various nanomaterials as bioactive fillers into polymeric hydrogels to address these limitations. Carbon-family nanomaterials (CFNs), hydroxyapatites, nanosilicates, and strontium carbonates have been incorporated into printed gels for application in various biomedical fields. In this review, following the compilation of research publications on CFNs-containing printable gels in various tissue engineering applications, we discuss the types of bioprinters, the prerequisites of bioink and biomaterial ink, as well as the progress and challenges of CFNs-containing printable gels in this field.

Antioxidants, like coenzyme Q10, selenite, and curcumin, inhibited osteoclast differentiation by suppressing reactive oxygen species generation.

Coenzyme Q10 (CoQ10), selenium, and curcumin are known to be powerful antioxidants. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts causes bone loss-related diseases. During osteoclast differentiation, the reactive oxygen species (ROS) acts as a secondary messenger on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared the relative inhibitory activities of CoQ10, sodium selenite, and curcumin on osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMMs) and RAW 264.7 cells. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. These also acted to significantly suppress the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor (OSCAR), which are genetic markers of osteoclast differentiation in a dose-dependent manner. These antioxidants also suppressed ROS-induced IκBα signaling pathways for osteoclastogenesis. Specially, curcumin displayed the highest inhibitory effect on osteoclast differentiation when concentrations were held constant. Together, CoQ10, selenite, and curcumin act as inhibitors of RANKL-induced NFATc1 which is a downstream event of NF-κB signal pathway through suppression of ROS generation, thereby suggesting their potential usefulness for the treatment of bone disease associated with excessive bone resorption.

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11.

Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 microm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 microm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.

Development of Growth Factor Releasing Hyaluronic Acid-Based Hydrogel for Pulp Regeneration: A Preliminary Study.

Growth factors play essential roles as signaling molecules in pulp regeneration. We investigated the effect of a hyaluronic acid (HA)-collagen hybrid hydrogel with controlled release of fibroblast growth factor (FGF)-2 and platelet-derived growth factor (PDGF)-BB on human pulp regeneration. The cell interaction and cytotoxicity of the HA-collagen hybrid hydrogel, the release kinetics of each growth factor, and the effects of the released growth factors on pulp cell proliferation were examined. The vitality of pulp cells was maintained. The amounts of FGF-2 and PDGF-BB released over 7 days were 68% and 50%, respectively. Groups with a different concentration of growth factor (FGF-2: 100, 200, 500, and 1000 ng/mL; PDGF-BB: 10, 50, 100, 200, and 500 ng/mL) were experimented on days 1, 3, 5, and 7. Considering FGF-2 concentration, significantly increased pulp cell proliferation was observed on days 1, 3, 5, and 7 in the 100 ng/mL group and on days 3, 5, and 7 in the 200 ng/mL group. In the case of PDGF-BB concentration, significantly increased pulp cell proliferation was observed at all four time points in the 100 ng/mL group and on days 3, 5, and 7 in the 50, 200, and 500 ng/mL groups. This indicates that the optimal concentration of FGF-2 and PDGF-BB for pulp cell proliferation was 100 ng/mL and that the HA-collagen hybrid hydrogel has potential as a controlled release delivery system for FGF-2 and PDGF-BB.

Development of a Novel Perfusion Rotating Wall Vessel Bioreactor with Ultrasound Stimulation for Mass-Production of Mineralized Tissue Constructs.

BACKGROUND: As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs. METHODS: An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model. RESULTS: Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation. CONCLUSION: This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.

Keratin-mediated hair growth and its underlying biological mechanism.

Here we show that intradermal injection of keratin promotes hair growth in mice, which results from extracellular interaction of keratin with hair forming cells. Extracellular application of keratin induces condensation of dermal papilla cells and the generation of a P-cadherin-expressing cell population (hair germ) from outer root sheath cells via keratin-mediated microenvironmental changes. Exogenous keratin-mediated hair growth is reflected by the finding that keratin exposure from transforming growth factor beta 2 (TGFß2)-induced apoptotic outer root sheath cells appears to be critical for dermal papilla cell condensation and P-cadherin-expressing hair germ formation. Immunodepletion or downregulation of keratin released from or expressed in TGFß2-induced apoptotic outer root sheath cells negatively influences dermal papilla cell condensation and hair germ formation. Our pilot study provides an evidence on initiating hair regeneration and insight into the biological function of keratin exposed from apoptotic epithelial cells in tissue regeneration and development.

Development of nanofiber coated indomethacin-eluting stent for tracheal regeneration.

In the case of tracheal rupture or stenosis, most effective way is to insert a commercially available metal stent. However, the implantation often causes a fever or a pain on the contact surface between trachea and the stent. And also the metal stent should be removed after a certain time implantation. Thus, we developed a functional tracheal drug eluting stent consisting of indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), loaded nanofibers on a bare metal stent. To control the drug release kinetics and enhancement of mucosal regeneration, gelatin and PLCL were coated layer by layer on a metal stent by an electrospinning method. Indomethacin was loaded in the gelatin layer by soaking and drying method (0.1, 0.5, and 1 wt% in ethanol for 10 min). The morphology of functional drug eluting tracheal stent was characterized by scanning electron microscope (SEM). And mechanical properties of the constructs such as air leak pressure, ultimate tensile stress, and modulus were calculated and evaluated. Drug release was performed by a high performance liquid chromatography (HPLC). Stably coated gelatin and poly(L-lactide- co-epsilon-caprolactone) (PLCL) nanofibers were observed by SEM. Bi-layered nanofibers-coated stent showed enough mechanical properties as a tracheal stent, which confirmed by a custom-designed air leak mechanical test. For indomethacin loading on a stent, stent was immersed in a series of drug solutions (different concentrations) for 10 min. At the result of HPLC, total amounts of indomethacin on a stent were approximately 77, 323, and 670 ug/stent, respectively. Time dependent drug release kinetics of the tracheal stent showed a sustained release profile regardless of indomethacin content. Thus, functionally designed nanofiber coated tracheal stent with anti-inflammatory drug may be useful for tracheal regeneration.

Poly(L-lactic acid) nanocylinders as nanofibrous structures for macroporous gelatin scaffolds.

Electrospun Nanofiber sheets have been shown to mimic the structure of extracellular matrix (ECM). Although these nanofibers have shown great potential for use as tissue engineering scaffolds, it is difficult for the electrospun nanofiber based sheets to be shaped into the desired three-dimensional structure. In this study, poly(L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, was electrospun to produce nanofibers that were treated with an amino group containing base in order to fabricate polymeric nanocylinders. The aspect ratio of the PLLA nanocylinders was tunable by varying the aminolysis time and density of the amino group containing base. The effects of changes in nanofibrous morphology of the PLLA nanocylinders/macro-porous gelatin scaffolds on cell adhesion and proliferation were evaluated. The results revealed different cell morphology, adhesion, and proliferation in the nanocylinders composite gelatin scaffold versus gelatin scaffold alone. Confocal laser scanning microscopy observation showed more spreading and a more flattened cell morphology after NIH3T3 cells were cultured on PLLA nanocylinders/gelatin scaffolds for 10 hours and 4 days. These results indicate that the gelatin/PLLA nanocylinder composite is a promising way to fabricate 3D nanofibrous scaffolds that accelerates cell adhesion and proliferation for tissue engineering.

Facile Preparation of ß-Cyclodextrin-grafted Chitosan Electrospun Nanofibrous Scaffolds as a Hydrophobic Drug Delivery Vehicle for Tissue Engineering Applications.

Despite advances in the bio-tissue engineering area, the technical basis to directly load hydrophobic drugs on chitosan (CTS) electrospun nanofibers (ENs) has not yet been fully established. In this study, we fabricated CTS ENs by using an electrospinning (ELSP) system, followed by surface modification using succinyl-beta-cyclodextrin (ß-CD) under mild conditions. The ß-CD-modified CTS (ßCTS) ENs had slightly increased hydrophobicity compared to pristine CTS ENs as well as decreased residual amine content on the surface. Through FTIR spectroscopy and thermogravimetric analysis (TGA), we characterized the surface treatment physiochemically. In the drug release test, we demonstrated the stable and sustained release of a hydrophobic drug (e.g., dexamethasone) loaded on ß-CD ENs. During in vitro biocompatibility assessments, the grafting of ß-CD was shown to not reduce cell viability compared to pristine CTS ENs. Additionally, cells proliferated well on ß-CD ENs, and this was confirmed by F-actin fluorescence staining. Overall, the material and strategies developed in this study have the potential to load a wide array of hydrophobic drugs. This could be applied as a drug carrier for a broad range of tissue engineering applications.

Photo-Cross-Linkable Human Albumin Colloidal Gels Facilitate In Vivo Vascular Integration for Regenerative Medicine.

Biodegradable cellular and acellular scaffolds have great potential to regenerate damaged tissues or organs by creating a proper extracellular matrix (ECM) capable of recruiting endogenous cells to support cellular ingrowth. However, since hydrogel-based scaffolds normally degrade through surface erosion, cell migration and ingrowth into scaffolds might be inhibited early in the implantation. This could result in insufficient de novo tissue formation in the injured area. To address these challenges, continuous and microsized strand-like networks could be incorporated into scaffolds to guide and recruit endogenous cells in rapid manner. Fabrication of such microarchitectures in scaffolds is often a laborious and time-consuming process and could compromise the structural integrity of the scaffold or impact cell viability. Here, we have developed a fast single-step approach to fabricate colloidal hydrogels, which are made up of randomly packed human serum albumin-based photo-cross-linkable microparticles with continuous internal networks of microscale voids. The human serum albumin conjugated with methacrylic groups were assembled to microsized aggregates for achieving unique porous structures inside the colloidal gels. The albumin hydrogels showed tunable mechanical properties such as elastic modulus, porosity, and biodegradability, providing a suitable ECM for various cells such as cardiomyoblasts and endothelial cells. In addition, the encapsulated cells within the hydrogel showed improved cell retention and increased survivability in vitro. Microporous structures of the colloidal gels can serve as a guide for the infiltration of host cells upon implantation, achieving rapid recruitment of hematopoietic cells and, ultimately, enhancing the tissue regeneration capacity of implanted scaffolds.

The Use of Histidine-tryptophan-ketoglutarate Solution as a New Storage Medium for the Avulsed Tooth.

INTRODUCTION: Histidine-tryptophan-ketoglutarate (HTK) is a preservation solution used for organ transplantation. The physiological pH and osmolality of this solution are known to facilitate cell proliferation and cell membrane stabilization. The purpose of the present study was to investigate the efficacy of several concentrations of HTK solution as a storage medium for avulsed teeth. METHODS: Cultured human periodontal ligament cells were stored in different concentrations of HTK solutions. After 1, 3, 6, 12, 24, 48, and 72 hours, cell viability was assessed using the Cell-Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) and LIVE/DEAD (Invitrogen, Carlsbad, CA) assay. Cell response of the most effective concentrations of HTK solution were further analyzed by gene expression profiling, and their cell viability was compared with other storage media. RESULTS: The highest cell viability was observed in 50% HTK solution in various concentrations of HTK solution (P < .05). In periodontal ligament cells stored in 50% HTK solution for 3 hours, the expression of genes related to angiogenesis, the inflammatory response, and cell proliferation was increased compared with the control. Compared with other storage media, the highest cell viability was observed in 50% HTK solution. CONCLUSIONS: Our study suggests that 50% HTK solution containing cell culture medium represents a suitable storage medium for avulsed teeth.

Cell-Laden Gelatin Methacryloyl Bioink for the Fabrication of Z-Stacked Hydrogel Scaffolds for Tissue Engineering.

Hydrogel-based scaffolds have been widely used to fabricate artificial tissues capable of replacing tissues and organs. However, several challenges inherent in fabricating tissues of large size and complex morphology using such scaffolds while ensuring cell viability remain. To address this problem, we synthesized gelatin methacryloyl (GelMA) based bioink with cells for fabricating a scaffold with superior characteristics. The bioink was grafted onto a Z-stacking bioprinter that maintained the cells at physiological temperature during the printing process, without exerting any physical pressure on the cells. Various parameters, such as the bioink composition and light exposure time, were optimized. The printing accuracy of the scaffolds was evaluated using photorheological studies. The internal morphology of the scaffolds at different time points was analyzed using electron microscopy. The Z-stacked scaffolds were fabricated using high-speed printing, with the conditions optimized to achieve high model reproducibility. Stable adhesion and high proliferation of cells encapsulated within the scaffold were confirmed. We introduced various strategies to improve the accuracy and reproducibility of Z-stack GelMA bioprinting while ensuring that the scaffolds facilitated cell adhesion, encapsulation, and proliferation. Our results demonstrate the potential of the present method for various applications in tissue engineering.