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Biofabrication 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.

Reversible or irreversible damage causes oral mucosal defects, the treatment of which requires biomaterials with good biomechanical properties. In this study, we prepared an artificial oral mucosa tissue model using a photocured methacrylate gelatin (GelMA) hydrogel co-cultured with human gingival fibroblasts (HGFs) and human oral keratinocytes (HOKs). GelMA was fabricated via a chemical reaction between gelatin and methacrylic anhydride (MA), and its structure was confirmed via 1H NMR analysis. The culture of HOKs and HGFs was observed on the surface and in the bulk of the GelMA hydrogel, indicating that the hydrogel was biocompatible for cell adhesion and proliferation. In addition, immunofluorescent staining results indicated that HOKs form a three-dimensional cell structure while maintaining their intrinsic keratinocyte properties. Histological evaluations demonstrated that the HGF and HOK bi-layered GelMA hydrogel can be used to develop artificial oral mucosa tissue.

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13770-022-00447-3.

We developed Fenton-like reaction performing mineralized nanoparticles as novel oxidative stress amplifying anticancer agents. We fabricated Cu-doped superoxide dismutase (SOD)-loaded calcium carbonate (CaCO3)-mineralized nanoparticles (Cu/SOD-MNPs) that could simultaneously release hydrogen peroxide-generating SOD and Fenton-like reaction-inducing copper ions within cancer cells. The CaCO3 core of the Cu/SOD-MNPs effectively inhibited the release of the entrapped SOD and copper ions in physiological pH (7.4). In contrast, in acidic pH (5.0), the release of SOD and copper ions was facilitated by CaCO3 dissolution. We demonstrated that the Cu/SOD-MNPs elevated the level of highly toxic apoptosis-inducing hydroxyl radicals in the cancer cells. The Cu/SOD-MNPs showed specific toxicity for cancer cells (MCF-7 cells), whereas no or negligible toxicity was found for normal cells (NIH3T3 and HEK293 cells). This mineralized oxidative stress amplifying nanoparticle may serve as a novel cancer-specific anticancer agent.

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 (β-CD) under mild conditions. The β-CD-modified CTS (βCTS) 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 β-CD ENs. During in vitro biocompatibility assessments, the grafting of β-CD was shown to not reduce cell viability compared to pristine CTS ENs. Additionally, cells proliferated well on β-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.

Recently, human hair-derived keratin protein has been recognized as biomaterial with high potential due to its excellent bioactivity and biocompatibility. Here, we designed human hair-derived keratin-based in situ cross-linkable hydrogels that can serve as a dynamic matrix for the enhanced wound healing. We demonstrated that our developed the keratin-based hydrogels accelerated re-epithelization and wound healing process in a full-thickness animal. Also, we investigated the molecular mechanism underlying the enhanced wound healing. In conclusion, our study proposes that human hair-derived keratin-based hydrogels with excellent bioactivity have great potential for use as a wound healing material, along with its other biomedical applications.

We report on doxycycline (DOXY)-loaded calcium carbonate (CaCO3) mineralized nanoparticles (DOXY-MNPs) that can induce selective topical release of antibiotics to target oral plaque biofilms with acidic pH (ca. 4.5–5.5). The DOXY-MNPs were fabricated through a block copolymer-templated mineralization approach with simultaneous loading of an antibacterial drug, DOXY. The CaCO3 mineral core of DOXY-MNPs inhibited the leakage of entrapped DOXY in environments with normal salivary pH. However, in acidic pH, the DOXY release was triggered by the dissolution of CaCO3 mineral cores. The DOXY-MNPs showed effective antibacterial therapeutic activity to Prevotella intermedia bacterial cells with a potent treatment of biofilms.

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.

Keratin is known to be a major protein in hair, but the biological function of keratin in hair growth is unknown, which led us to conduct a pilot study to elucidate biological function of keratin in hair growth via cellular interactions with hair forming cells. Here, we show hair growth is stimulated by intradermal injection of keratin into mice, and show that outer root sheath cells undergo transforming growth factor-β2-induced apoptosis, resulting in keratin exposure. Keratin exposure appears to be critical for dermal papilla cell condensation and hair germ formation as immunodepletion and silencing keratin prevent dermal papilla cell condensation and hair germ formation. Furthermore, silencing keratin in mice resulted in a marked suppression of anagen follicle formation and hair growth. Our study imply a new finding of how to initiate hair regeneration and suggests the potent application of keratin biomaterial for the treatment of hair loss.

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 (TGFβ2)-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 TGFβ2-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.

There is a continuing attempt to study cell to cell interactions to control the growth, function, and differentiation of cells. One approach that can be utilized to assess representative cell to cell interactions is by using three-dimensional cell spheroids, also referred to as cell aggregates, generated by adhering cells to each other through cell-adhesion molecules such as cadherin. Compared with conventional two-dimensional cell monolayer, cell spheroid offers more realistic platform to predict cell behavior in high-throughput manner. To recapitulate the cell spheroid formation in vitro, microwell-mediated culture system has become a robust and efficient tool for providing uniform-sized spheroids. In this review, we first iterate the recent developments and innovations in microwell-mediated cell culture platform, focusing on formation and function of cell spheroids using various cells such as embryonic stem cells, postnatal stem cells, and somatic cells. Furthermore, the recent advancements in applications of cell spheroids generated from microwell-mediated culture system is covered in this article. The discussion on the integrative biology regarding cell to cell interaction and other biological events in cell spheroid is another focal point of this review.

Titanium implants (Ti) have been widely used in several medical fields. In clinical practice, Ti can become contaminated with bacteria through a variety of mechanisms. This contamination can lead to implant failure and serious infections. In this study, we aimed to develop a new, hybrid Ti with good biocompatibility and antibacterial properties by immobilizing ceftazidime (CFT) onto the Ti surface through polydopamine (PDA) and polyethyleneimine (PEI) chemistry. Hybrid Ti was confirmed by assessing the cell proliferation of human adipose-derived stem cells using a cell counting. The biofilm formation across the Ti surface of two bacterial strains associated with nosocomial infections, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus, was evaluated by scanning electron microscopy. The viability of the bacteria exposed to Ti surface was evaluated by cell counting. Our results clearly demonstrate that the bacterial biofilm formation as well as bacterial viability was significantly reduced on the hybrid Ti as compared to the control, Ti alone. Collectively, the Ti surface was successfully modified to form the hybrid Ti exhibiting good biocompatibility and antibacterial properties through PDA, PEI, and CFT grafting. Within the limitations of this in vitro study, we conclude that the hybrid Ti may be useful for successful implant treatment., This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2017M3A9E4048170).

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 (&lt, 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.

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.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.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.Our study suggests that 50% HTK solution containing cell culture medium represents a suitable storage medium for avulsed teeth.

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.

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.

Osteoporosis, a major skeletal disorder, is an increasing worldwide social burden among the aging society, which develops when the ratio of bone resorption regulated by osteoclasts reverses the bone formation in the bone remodeling cycle. Basically, osteoporosis is commonly treated using bone resorption inhibitors such as bisphosphonates. However, long-term intake of bisphosphonates exposes patients to traumatic side effects such as digestive disorders and medication-related osteonecrosis of the jaw (MRONJ). Therefore, an alternative solution is required in the area of bone tissue engineering. Hence, to solve this issue, in this study, we designed GNPs and functionalized GNPs’ surface through conjugation with SH-PEG-vitamin D (SPVD) via the Au-S binding system. Vitamin D-conjugated GNPs (VGNPs) were successfully synthesized and confirmed by various physicochemical analyses. These conjugated nanoparticles significantly increased cell viability and decreased tartrate-resistant acid phosphatase (TRAP) activity and actin ring formation. In addition, VGNPs suppressed the expression of genes associated with osteoclast differentiation. Finally, VGNPs also significantly inhibited the expression of reactive oxygen species (ROS). These results demonstrate the inhibitory effect of VGNPs on osteoclast differentiation and suggest that the developed nanocarriers could play a key role as a bone resorption inhibitor in the field of bone tissue engineering.

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.

Donghyun Lee,1 Dong Nyoung Heo,2 Ha Ram Nah,3 Sang Jin Lee,1 Wan-Kyu Ko,4 Jae Seo Lee,3 Ho-Jin Moon,1 Jae Beum Bang,5 Yu-Shik Hwang,6 Rui L Reis,1,7 Il Keun Kwon1 1Department of Dental Materials, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea; 2Department of Engineering Science and Mechanics, Pennsylvania State University, Pennsylvania 16802, USA; 3Department of Detistry, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea; 4Department of Neurosurgery, CHA Bundang Medical Center, CHA University, Seongnam-si, Gyeonggi-do 13496, Republic of Korea; 5Department of Dental Education, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea; 6Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea; 7The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Barco, Guimarães, Portugal Background: For effective bone regeneration, it is necessary to implant a biocompatible scaffold that is capable of inducing cell growth and continuous osteogenic stimulation at the defected site. Here, we suggest an injectable hydrogel system using enzymatic cross-linkable gelatin (Gel) and functionalized gold nanoparticles (GNPs). Methods: In this work, tyramine (Ty) was synthesized on the gelatin backbone (Gel-Ty) to enable a phenol crosslinking reaction with horseradish peroxidase (HRP). N-acetyl cysteine (NAC) was attached to the GNPs surface (G-NAC) for promoting osteo-differentiation. Results: The Gel-Ty hydrogels containing G-NAC (Gel-Ty/G-NAC) had suitable mechanical strength and biocompatibility to embed and support the growth of human adipose derived stem cells (hASCs) during a proliferation test for three days. In addition, G-NAC promoted osteo-differentiation both when it was included in Gel-Ty and when it was used directly in hASCs. The osteogenic effects were demonstrated by the alkaline phosphatase (ALP) activity test. Conclusion: These findings indicate that the phenol crosslinking reaction is suitable for injectable hydrogels for tissue regeneration and G-NAC stimulate bone regeneration. Based on our results, we suggest that Gel-Ty/G-NAC hydrogels can serve both as a biodegradable graft material for bone defect treatment and as a good template for tissue engineering applications such as drug delivery, cell delivery, and various tissue regeneration uses. Keywords: nanomaterial, gelatin, enzymatic cross-linking, osteogenesis, N-acetyl cysteine

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.

Embryoid bodies have a number of similarities with cells in gastrulation, which provides useful biological information about embryonic stem cell differentiation. Extensive research has been done to study the control of embryoid body-mediated embryonic stem cell differentiation in various research fields. Recently, microengineering technology has been used to control the size of embryoid bodies and to direct lineage specific differentiation of embryonic stem cells. However, the underlying biology of developmental events in the embryoid bodies of different sizes has not been well elucidated. In this study, embryoid bodies with different sizes were generated within microfabricated PEG microwell arrays, and a series of gene and molecular expressions related to early developmental events was investigated to further elucidate the size-mediated differentiation. The gene and molecular expression profile suggested preferential visceral endoderm formation in 450 μm embryoid bodies and preferential lateral plate mesoderm formation in 150 μm embryoid bodies. These aggregates resulted in higher cardiac differentiation in 450 μm embryoid bodies and higher endothelial differentiation in 150 μm embryoid bodies, respectively. Our findings may provide further insight for understanding embryoid body size-mediated developmental progress. Open image in new window

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 β, 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/β-catenin pathways were inhibited by repression of Hh signaling and mitochondrial biogenesis. Transfection of β-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/β-catenin signaling.

For the treatment of periodontitis, local delivery of antibiotics and their sustained release are preferable to enhance microbial susceptibility and to reduce possible side effects occurrence in systemic drug delivery. To address these issues, antibiotic loaded polymeric matrices implantable into periodontal pocket have been commercially used in dentistry and oral medicine. Recently, keratin has been drawing attention as a natural polymer for its ability to mediate cell behavior with minor or no immunogenicity enabling further process towards autologous implantation. In this study, human hair keratin was extracted with a cocktail of reducing agents, and antibiotic eluting keratin-based biofilms were fabricated. Physicochemical analysis and release test showed proper physical stability and sustained release of the loaded antibiotics. In addition, the released antibiotic suppressed the growth of various types of oral bacteria including porphyromonas gingivalis. Cellular interaction studies showed that human oral epithelial cell, human gingival fibroblast and periodontal ligament cells proliferated and guided well on biofilms. This study propose that antibiotic eluting keratin-based biofilms are provisional device for the treatment of chronic periodontitis offering advantages such as local controlled drug delivery and biocompatibility, and human hair keratin is able to be a good biomaterial for the potent applications to tissue engineering and regenerative medicine. Open image in new window

We report a theranostic nanoparticle that can express ultrasound (US) imaging and simultaneous therapeutic functions for cancer treatment. We developed doxorubicin-loaded calcium carbonate (CaCO3) hybrid nanoparticles (DOX-CaCO3-MNPs) through a block copolymer templated in situ mineralization approach. The nanoparticles exhibited strong echogenic signals at tumoral acid pH by producing carbon dioxide (CO2) bubbles and showed excellent echo persistence. In vivo results demonstrated that the DOX-CaCO3-MNPs generated CO2 bubbles at tumor tissues sufficient for echogenic reflectivity under a US field. In contrast, the DOX-CaCO3-MNPs located in the liver or tumor-free subcutaneous area did not generate the CO2 bubbles necessary for US contrast. The DOX-CaCO3-MNPs could also trigger the DOX release simultaneously with CO2 bubble generation at the acidic tumoral environment. The DOX-CaCO3-MNPs displayed effective antitumor therapeutic activity in tumor-bearing mice. The concept described in this work may serve as a useful guide for development of various theranostic nanoparticles for US imaging and therapy of various cancers.

Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite the potential biomedical applications of graphene and its derivatives, only limited information is available regarding their osteogenic activity. This study concentrates upon the effects of reduced graphene oxide (rGO)-coated hydroxyapatite (HAp) composites on osteogenic differentiation of hMSCs. The average particle sizes of HAp and rGO were 1270 ± 476 nm and 438 ± 180 nm, respectively. When coated on HAp particulates, rGO synergistically enhanced spontaneous osteogenic differentiation of hMSCs, without hampering their proliferation. This result was confirmed by determining alkaline phosphatase activity and mineralization of calcium and phosphate as early and late stage markers of osteogenic differentiation. It is suggested that rGO-coated HAp composites can be effectively utilized as dental and orthopedic bone fillers since these graphene-based particulate materials have potent effects on stimulating the spontaneous differentiation of MSCs and show superior bioactivity and osteoinductive potential.

Over the past few decades, titanium (Ti) implants have been widely used to repair fractured bones. To promote osteogenesis, immobilization of osteoinductive agents, such as recombinant human bone morphogenic protein-2 (rhBMP2), onto the Ti surface is required. In this study, we prepared rhBMP2 immobilized on glycidyl methacrylate (GMA) deposited Ti surface through initiated chemical vapor deposition (iCVD) technique. After preparation, the bio-functionalized Ti surface was characterized by physicochemical analysis. For in vitro analysis, the developed Ti was evaluated by cell proliferation, alkaline phosphatase activity, calcium deposition, and real-time polymerase chain reaction to verify their osteogenic activity against human adipose-derived stem cells (hASCs). The GMA deposited Ti surface was found to effectively immobilize a large dose of rhBMP2 as compared to untreated Ti. Additionally, rhBMP2 immobilized on Ti showed significantly enhanced osteogenic differentiation and increased calcium deposition with nontoxic cell viability. These results clearly confirm that our strategy may provide a simple, solvent-free strategy to prepare an osteoinductive Ti surface for bone tissue engineering applications.

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under in vitro conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both in vitro and in vivo. We prepared β-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR-CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR-CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR-CGNPs. In addition, the CUR-CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR-CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the in vivo results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR-CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR-CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis.

Over the past few decades, naturally derived biomaterials have been extensively used in tissue engineering and regenerative medicine owing to their biological function, structural support, excellent biocompatibility, and favorable biodegradability characteristics. Traditionally, keratin has been extracted from wool, feathers, horns, and other animal sources for industrial use, and it has also been used as a biomaterial to develop scaffolds, hydrogels, and other forms for biomedical applications. Recently, keratin extracted from human hair has emerged as a fascinating biomaterial, which as a human-derived protein, exhibits excellent biocompatibility, no immune reaction upon transplantation, good cellular interaction activity, and biodegradability. Recent development of well-defined and proficient processes for human hair keratin extraction has led to the fabrication of various types of keratin-based biomaterials, which have been employed in successful approaches for tissue regeneration. The use of human hair keratin-based biomaterials for translational biomedical applications requires better understanding of the molecular properties and biological function of keratin. This review provides a critical summary of molecular characteristics, cellular interactions, various extraction strategies, and recent advances in biomedical applications of human hair keratin that could be used to improve tissue regeneration for regenerative medicine.

Development of a vascularized tissue is one of the key challenges for the successful clinical application of tissue engineered constructs. Despite the significant efforts over the last few decades, establishing a gold standard to develop three dimensional (3D) vascularized tissues has still remained far from reality. Recent advances in the application of microfluidic platforms to the field of tissue engineering have greatly accelerated the progress toward the development of viable vascularized tissue constructs. Numerous techniques have emerged to induce the formation of vascular structure within tissues which can be broadly classified into two distinct categories, namely (1) prevascularization-based techniques and (2) vasculogenesis and angiogenesis-based techniques. This review presents an overview of the recent advancements in the vascularization techniques using both approaches for generating 3D vascular structure on microfluidic platforms.

In cartilage tissue engineering, various technical approaches using postnatal stem cells, three-dimensional (3D) scaffolds composed of synthetic or natural polymers, and culture systems have been applied to develop 3D cartilage-like tissue. In this study, scaffold-free 3D micro-cartilage-like tissue was developed via microwell-mediated cell spheroid formation and 3D dynamic chondrogenic culture in a bioreactor. First, homogenous micro-cell spheroids were generated by the self-condensation of adipose tissue-derived stem cells (ADSCs) in microfabricated poly(ethylene glycol) (PEG) hydrogel microwells. Next, chondrogenic differentiation of the micro-ADSC spheroids was induced in the presence of transforming growth factor-beta 3 under dynamic 3D culture conditions using a high aspect ratio vessel bioreactor. Several hundred viable ADSC spheroids could be generated at a time from ADSC culture in PEG microwells. The 3D dynamic chondrogenic culture of ADSCs in the bioreactor facilitated the chondrogenic mRNA expression of proteins such as sox-9, runx2, osterix, type II collagen, and aggrecan, and the well deposition of glycosaminoglycan and type II collagen, which finally generated micro-cartilage-like tissue. Therefore, the hydrogel microwell arrays could be useful for efficiently deriving initial cell condensation-mediated chondrogenic differentiation, and for developing 3D cell-based micro-cartilage-like tissue with stem cells in a controlled manner.

Since the 17th century Spica Prunella has been used as a medicinal herb. Dried and pulverized Spica Prunella samples were extracted and used in these experiments. In this study, the effects of Spica Prunella extract (SPE) on RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclastogenesis were examined. Actin ring formation, a typical marker of osteoclastogenesis, was inhibited by SPE without any toxicity. There was also a marked inhibition in the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in bone marrow-derived monocytes (BMMs). SPE also suppressed phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK) both of which are signals of the mitogen-activated protein kinases (MAPKs) signaling pathway. Additionally, SPE inhibited IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) signaling pathway, which is an important factor in osteoclastogenesis. These results indicate that SPE might suppress osteoclast differentiation by inhibiting the phosphorylation of JNK and ERK in MAPK and NF-κB signaling pathways which act as messengers in the RANKL-induced osteoclast differentiation pathway. This means that SPE could potentially have great therapeutic usage in treating bone erosive diseases such as rheumatoid arthritis or in preventing metastasis associated with bone loss.

For the final purpose of accelerating the restoration of defected bone tissue, researches for developing in vitro three dimensional (3D) mineralized tissue using various stem cells, scaffolds and culture systems have been extensively done. In this research, an integrated bioprocess to generate stem cell-based 3D mineralized construct was developed using adipose-derived stem cell (ADSC), highly porous biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres and a high-aspect ratio vessel (HARV) bioreactor system. First, ADSCs adhered uniformly on poly-L-ornithine-coated macroporous PLGA microspheres, and the ADSC/microsphere composites were cultured in the presence of osteogenic supplements in a HARV bioreactor. Alkaline phosphatase (ALPase) activity assay, immunocytochemical staining and quantitative real-time polymerase chain reaction (qPCR) analysis showed temporal increase of ALPase activity, molecular expressions of type I collagen, osteocalcin and runx2 and upregulation of runx2, Sp7, type I collagen and bone sialoprotein mRNA during 3D osteogenic culture of ADSC/microsphere composites. Finally, 3D dynamic osteogenic culture generated highly mineralized micro tissues as validated by alizarin red-S staining and SEM-EDS. The results demonstrated that cell-based 3D micro-mineralized tissue could be generated by integrated bioprocess, and potentially utilized for bone tissue regeneration. The integrated bioprocess in this study may provide an efficient and scalable culture system for application in bone tissue engineering. Open image in new window

The goal of this study was to evaluate the potential of beta-tricalcium phosphate (β-TCP) particles coated with difference concentrations (1 and 10 mM) of simvastatin acid (SVA) on bone formation in vitro. Changes in the surface morphologies and chemical compositions of SVA1-β-TCP and SVA10-β-TCP suggested that SVA was coated on their surface. These particles were further investigated by scanning electron microscopy (SEM) observa- tions and X-ray photoelectron spectroscopy (XPS) measurements. By measuring ultraviolet/visible (UV/Vis) spec- troscopy, we found that simvastatin acid (SVA) released in a sustained manner over the period of 28 days even though the initial burst happened within 1 day. These results verify that SVA1-β-TCP and SVA10-β-TCP can be use- ful as a biocompatible bone graft substitutes. Biocompatibility was evaluated by cytotoxicity tests, live/dead assay, and proliferation of preosteoblast cell-line (MC3T3-E1) cells culture. The results of assays for ALP activity, calcium deposition, and mRNA expressions of alkaline phosphatase (ALP) and osteopontin suggest that the amount of SVA plays an important role in accelerating bone formation in vitro.

This paper introduces a novel bone-regenerative scaffold that is based on the systematic combination of porous polymer microspheres, nano-hydroxyapatite, and bone morphogenetic protein-2 (BMP-2), where each component was rationally incorporated to express its intrinsic activity in bone tissue formation. Poly(lactide-co-glycolide) (PLGA) microspheres, with interconnected pore structures, were fabricated by a gas-forming method in a water-in-oil-in-water double emulsion and solvent evaporation process. Polyphosphate-functionalized nano-hydroxyapatite (PP-n-HAp) was employed as a main component and was immobilized on the pore surface of the PLGA microspheres to controllably incorporate and release BMP-2. The surface polyphosphate functionalities of PP-n-HAp enabled the stable chemical immobilization of nano-hydroxyapatite (n-HAp) on the amine-treated pore surface of the PLGA microspheres. Field-emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) confirmed the nano-level exposure of n-HAp on the pore surface of the PLGA microspheres. BMP-2 with a positive charge was bound at a high efficiency onto the anionic phosphates of surface-immobilized PP-n-HAp and was controllably released for approximately 1 month. The release rate was manipulated by adjusting the amount of loaded BMP-2. The osteogenic differentiation and proliferation of human adipose-derived stem cells (hADSCs) within the n-HAp/BMP-2-incorporated microspheres were monitored in a dynamic 3D cell culture system. Histological, immunohistochemical, and quantitative real-time polymerase chain reaction (PCR) analyses showed that the PP-n-HAp-immobilized surface promoted cell adhesion/proliferation and osteoconduction. With its osteoinductive property, the sustained release of BMP-2 further enhanced the bone tissue regenerative activity of the porous microspheres.

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.

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.

Current approaches have focused on deriving ESCs differentiation into chondrocytes from a cell source of spontaneously formed intact mesoderm in EB formation, resulting in limited yield. Our study aimed at upregulating chondrogenic differentiation of murine ESCs by enhancing mesoderm formation. Specifically, culture of mESCs with conditioned medium from a human hepatocarcinoma cell line resulted in a cell population with a gene expression pattern similar to that of primitive streak/nascent mesoderm, including up-regulation of brachyury, goosecoid, nodal, and cripto. From this cell population, reducing the embryoid body formation time resulted in enhancement of chondrogenic differentiation as evidenced by larger Alcian blue-stained cartilage nodules, higher production of sulfated glycosaminoglycan matrix, the presence of well-organised type II collagen and type II collagen, aggrecan and sox-9 gene expression. In conclusion, we present here a new approach to the generation of chondrocytes from mESCs that enhances yields and, thus, could have widespread applications in cartilage tissue engineering.