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.

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.

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.

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.

Reduced graphene oxide-coated hydroxyapatite composites stimulate spontaneous osteogenic differentiation of human mesenchymal stem cells.

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.

Mycoplasma detection and elimination are necessary for the application of stem cell from human dental apical papilla to tissue engineering and regenerative medicine.

BACKGROUND: Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access. However, almost human oral tissues have been reported to be infected by mycoplasma which gives rise to oral cavity in teeth, and mycoplasma contamination of ex-vivo cultured stem cells from such dental tissues and its effect on stem cell culture has received little attention. RESULTS: In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy. The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining. Such contaminated mycoplasma could be successfully eliminated using elimination kit, and proliferation test showed decreased proliferation activity in mycoplasma-contaminated cells. After elimination of contaminated mycoplasma, stem cells from apical papilla showed osteogenic and neural lineage differentiation under certain culture conditions. CONCLUSION: Our study proposes that the evaluation of mycoplasma contamination and elimination process might be required in the use of stem cells from apical papilla for their potent applications to tissue engineering and regenerative medicine.

Development of collagenase-resistant collagen and its interaction with adult human dermal fibroblasts.

Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary source in biomedical application. Collagen has been widely used in the crosslinked form to extend the durability of collagen. The chemical treatment influences the structural integrity of collagen molecule resulting in the loss of triple helical characteristic. The structural characteristic of collagen is importantly related to its biological function for the interaction with cell. In this study, structural stability of collagen was enhanced thought EGCG treatment, resulting in high resistance against degradation by bacterial collagenase and MMP-1, which is confirmed by collagen zymography. The triple helical structure of EGCG-treated collagen could be maintained at 37 degrees C in comparison with collagen, which confirmed by CD spectra analysis, and EGCG-treated collagen showed high free-radical scavenging activity. Also, with fibroblasts culture on EGCG-treated collagen, the structural stability of EGCG-treated collagen provided a favorable support for cell function in cell adhesion and actin filament expression. These observations underscore the need for native, triple helical collagen conformation as a prerequisite for integrin-mediated cell adhesion and functions. According to this experiment, EGCG-treated collagen assumes to provide a practical benefit to resist the degradation by collagenase retaining its structural characteristic, and can be a suitable biomaterial for biomedical application.

Polymeric stent materials dysregulate macrophage and endothelial cell functions: implications for coronary artery stent.

BACKGROUND: Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells. METHODS: Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry. RESULTS: We demonstrated that poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule (VCAM)-1 along with decreased nitric oxide production, indicating that ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced the release of elastase or elastase-like protease, which further accelerated polymer degradation. CONCLUSIONS: This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate that polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis.

Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson's disease?

Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson's disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups (PBS injection) demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [(18)F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase (dopaminergic neuronal marker) staining and G protein-activated inward rectifier potassium channel 2 (A9 dopaminergic neuronal marker) were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stem cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.

Osteoblastic/cementoblastic and neural differentiation of dental stem cells and their applications to tissue engineering and regenerative medicine.

Recently, dental stem and progenitor cells have been harvested from periodontal tissues such as dental pulp, periodontal ligament, follicle, and papilla. These cells have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and multilineage differentiation capacity. These dental stem and progenitor cells are known to be derived from ectomesenchymal origin formed during tooth development. A great deal of research has been accomplished for directing osteoblastic/cementoblastic differentiation and neural differentiation from dental stem cells. To differentiate dental stem cells for use in tissue engineering and regenerative medicine, there needs to be efficient in vitro differentiation toward the osteoblastic/cementoblastic and neural lineage with well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source. This review focuses on the multilineage differentiation capacity, especially into osteoblastic/cementoblastic lineage and neural lineages, of dental stem cells such as dental pulp stem cells (DPSC), dental follicle stem cells (DFSC), periodontal ligament stem cells (PDLSC), and dental papilla stem cells (DPPSC). It also covers various experimental strategies that could be used to direct lineage-specific differentiation, and their potential applications in tissue engineering and regenerative medicine.

Stem cells in bone tissue engineering.

Bone tissue engineering has been one of the most promising areas of research, providing a potential clinical application to cure bone defects. Recently, various stem cells including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose tissue-derived stem cells (ADSCs), muscle-derived stem cells (MDSCs) and dental pulp stem cells (DPSCs) have received extensive attention in the field of bone tissue engineering due to their distinct biological capability to differentiate into osteogenic lineages. The application of these stem cells to bone tissue engineering requires inducing in vitro differentiation of these cells into bone forming cells, osteoblasts. For this purpose, efficient in vitro differentiation towards osteogenic lineage requires the development of well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source for application to bone tissue engineering therapies. This review provides a critical examination of the various experimental strategies that could be used to direct the differentiation of ESC, BM-MSC, UCB-MSC, ADSC, MDSC and DPSC towards osteogenic lineages and their potential applications in tissue engineering, particularly in the regeneration of bone.