Sphingosine-1-phosphate Treatment Improves Cryopreservation Efficiency in Human Mesenchymal Stem Cells.

The actin cytoskeleton plays a crucial role not only in maintaining cell shape and viability but also in homing/engraftment properties of mesenchymal stem cells (MSCs), a valuable source of cell therapy. Therefore, during the cryopreservation process of MSCs, protecting the actin cytoskeleton from the freezing/thawing stress is critical in maintaining their functionality and therapeutic potential. In this study, the safety and cryoprotective potential of sphingosine-1-phosphate (S1P), which has a stabilizing effect on actin cytoskeleton, on dental pulp-derived MSCs (DP-MSCs) was investigated. Our results demonstrated that S1P treatment did not adversely affect viability and stemness of DP-MSCs. Furthermore, S1P pretreatment enhanced cell viability and proliferation properties of post-freeze/thaw DP-MSCs, protecting them against damage to the actin cytoskeleton and adhesion ability as well. These findings suggest that a new cryopreservation method using S1P pretreatment can enhance the overall quality of cryopreserved MSCs by stabilizing the actin cytoskeleton and making them more suitable for various applications in regenerative medicine and cell therapy.

A Case Report of Primary Extranodal Diffuse Large B-Cell Lymphoma Involving the Masseter Muscle: Histological-Radiological Correlation.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non- Hodgkin's lymphoma (NHL). However, the primary skeletal muscle involvement of DLBCL is extremely rare, comprising less than 1% of all the extranodal lymphoma. To date, only 8 cases of extranodal NHL involving the masticator muscles have been reported in the literature. CASE PRESENTATION: A 70-year-old male presented with a rapid progression of painless facial swelling in the left cheek. CT, MRI and US findings demonstrated a well-defined, soft tissue mass in the left masseter muscle. The histopathological diagnosis was DLBCL by US-guided core needle biopsy. The patient received three cycles of chemotherapy. CONCLUSION: Because of its rarity, primary muscular DLBCL must be considered in differential diagnosis with all possible causes of intramuscular masses. Even the integration of multiple imaging methods does not lead to a definitive diagnosis, the biopsy is the only possibility for an early diagnosis. Therefore, clinical awareness and high suspicion of this disease are important for early diagnosis and proper treatment.

Scaffold-free 3D culturing enhance pluripotency, immunomodulatory factors, and differentiation potential of Wharton's jelly-mesenchymal stem cells.

Mesenchymal stem cells (MSCs) show a decline in pluripotency and differentiation with increased cell culture passages in 2D cultures. The 2D monolayer culture fails to correctly imitate the architecture and microenvironments of in-vivo cell models. Alternatively, 3D culture may improve the simulations of in-vivo cell microenvironments with wide applications in cell culture and drug discovery. In the present study, we compared various 3D culturing techniques such as 3D micro-well (3D-S), hanging drop (HD), and ultra-low attachment (ULA) plate-based spheroid culture to study their effect on morphology, viability, pluripotency, cell surface markers, immunomodulatory factors, and differentiation capabilities of Wharton's jelly-mesenchymal stem cells (WJ-MSCs). The cell morphology, viability, and senescence of 3D cultured WJ-MSCs were comparable to cells in 2D culture. The expression of pluripotency markers (OCT4, SOX2, and NANOG) was enhanced upto 2-8 fold in 3D cultured WJ-MSCs when compared to 2D culture. Moreover, the immunomodulatory factors (IDO, IL-10, LIF, ANG1, and VEGF) were significantly elevated in ULA based 3D cultured WJ-MSCs. Furthermore, significant enhancement in the differentiation potential of WJ-MSCs towards adipocyte (ADP and C/EBP-α), osteocyte (OPN and RUNX2), and definitive endodermal (SOX17, FOXA2, and CXCR4) lineages in 3D culture conditions were observed. Additionally, the osteogenic and adipogenic differentiation potential of WJ-MSCs over the time points 7 days, 14 days, and 28 days was also significantly increased in 3D culture groups. Our study demonstrates that stemness properties of WJ-MSCs were significantly enhanced in 3D cultures and ULA-based culture outperformed other methods with high pluripotency gene expression and enhanced differentiation potential. This study indicates the efficacy of 3D cultures to bridge the gap between 2D cell culture and animal models in regenerative medicine.

Complications According to the Handling of the Pterygoid Plate During Bimaxillary Surgery.

BACKGROUND: During bimaxillary surgery, manipulation of the pterygoid plate is required to facilitate movement of the maxilla. This study examined the complications that occurred after handling the pterygoid plate during a Le Fort I osteotomy. PATIENTS AND METHODS: This study compared and analyzed complications according to the pterygoid plate handling method in 80 patients who underwent bimaxillary surgery at Pusan National University Dental Hospital from December 2015 to July 2020. The pterygoid plate was fractured or removed intentionally only if it interfered with the maxilla. Otherwise, it was not treated. The complications during surgery and the follow-up period were investigated. RESULTS: Fourteen patients experienced complications, of which excessive bleeding, hearing problems, and nonunion were encountered in 10, 2, and 2 patients, respectively. Of the 10 patients with excessive bleeding patients, the pterygoid plate was manipulated in 8 patients, which was controlled during surgery. Two patients complained of hearing loss with ear congestion immediately after surgery; both patients improved spontaneously within 1 month. Two nonunion patients underwent plate refixation at least 6 months postoperatively, and normal healing was achieved afterward. CONCLUSIONS: Fracture and removal of the pterygoid plate during orthognathic surgery did not significantly affect the occurrence of complications during and after surgery.

Delayed Soft Tissue Changes after Clockwise Rotation of the Maxillo-Mandibular Complex.

OBJECTIVE: The purpose of this study was to evaluate delayed soft tissue changes of the maxilla-mandibular complex MMC using three-dimensional (3D) cone-beam computed tomography after clockwise repositioning orthognathic surgery. METHODS: This study included 21 patients that underwent maxilla-mandibular complex clockwise rotational orthognathic surgery by 1 doctor from January 2015 to June 2019. Radiographic images (panorama, lateral cephalogram, posteroanterior view, and conebeam computed tomography) were taken and 3D analysis was performed using the Invivo 5 (Anatomage Inc, Santa Clara, CA) to acquire 3D images before surgery, immediately after surgery, at 6 months after surgery and 21 months after surgery. The 9 soft tissue landmarks were measured and compared in terms of postoperative changes in transverse, vertical, and anteroposterior directions. The points were at the outer commissure of the eye fissure (Exocathion; Exc_r, Exc_l), at the midline of both the nasal root and the nasofrontal suture, analogous to bony N (soft tissue nasion; N), the most prominent point on the nasal tip (Pronasale; Prn), the most lateral point in the curved baseline of each ala, indicating the facial insertion of the nasal wing base (Alare curvature; Ac_r, Ac_l), the most lateral point on the soft tissue contour of each mandibular angle (Soft tissue Gonion; Go_r, Go_l), and the most inferior midpoint on the soft tissue contour of the chin (soft tissue menton; Me). RESULTS: The most prominent point of the nasal tip (Prn) moved 1.36 mm upward and 1.55 mm forward in the vertical and anteroposterior planes immediately after surgery. However, there were no significant changes in Ac_r and Ac_l even immediately after surgery. Both soft tissue gonions shifted downward and forward between immediately after surgery and 6 months after surgery. However, no significant change was observed in the value of any of the 9 soft tissue points between 6 months and 21 months after surgery ( P value < 0.05). CONCLUSIONS: No significant changes were observed between 6 and 21 months after surgery, which suggests no delayed soft tissue changes occur in surgically treated patients after the resolution of surgically-related facial edema and swelling and postsurgical remodeling of hard tissue in overlying soft tissue.

Comparison of Pluripotency, Differentiation, and Mitochondrial Metabolism Capacity in Three-Dimensional Spheroid Formation of Dental Pulp-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are valuable candidates in tissue engineering and stem cell-based therapy. Traditionally, MSCs derived from various tissues have been successfully expanded in vitro using adherent culture plates commonly called as monolayer two-dimensional (2D) cultures. Recently, many studies demonstrated that stemness and multilineage differentiation potential could be enhanced to greater extent when MSCs are cultured as suspended aggregates by means of three-dimensional (3D) culturing techniques. However, there are limited reports on changed mitochondrial metabolism on 3D spheroid formation of MSCs. Therefore, the present study was aimed at investigating the stemness, differentiation potential, and mitochondrial metabolism capacity of 3D dental pulp-derived MSC (DPSC) spheroids in comparison to monolayer cultured DPSCs. We isolated dental pulp-derived MSCs (DPSCs) and successfully developed a 3D culture system which facilitated the formation of MSC spheroids. The cell aggregation was observed after 2 hours, and spheroids were formed after 24 hours and remained in shape for 72 hours. After spheroid formation, the levels of pluripotent markers increased along with enhancement in adipogenic and osteogenic potential compared to 2D cultured control cells. However, decreased proliferative capacity, cell cycle arrest, and elevated apoptosis rate were observed with the time course of the 3D culture except for the initial 24-hour aggregation. Furthermore, oxygen consumption rates of living cells decreased with the time course of the aggregation except for the initial 24 hours. Overall, our study indicated that the short-term 3D culture of MSCs could be a suitable alternative to culture the cells.

Prediction of African Swine Fever Virus Inhibitors by Molecular Docking-Driven Machine Learning Models.

African swine fever virus (ASFV) causes a highly contagious and severe hemorrhagic viral disease with high mortality in domestic pigs of all ages. Although the virus is harmless to humans, the ongoing ASFV epidemic could have severe economic consequences for global food security. Recent studies have found a few antiviral agents that can inhibit ASFV infections. However, currently, there are no vaccines or antiviral drugs. Hence, there is an urgent need to identify new drugs to treat ASFV. Based on the structural information data on the targets of ASFV, we used molecular docking and machine learning models to identify novel antiviral agents. We confirmed that compounds with high affinity present in the region of interest belonged to subsets in the chemical space using principal component analysis and k-means clustering in molecular docking studies of FDA-approved drugs. These methods predicted pentagastrin as a potential antiviral drug against ASFVs. Finally, it was also observed that the compound had an inhibitory effect on AsfvPolX activity. Results from the present study suggest that molecular docking and machine learning models can play an important role in identifying potential antiviral drugs against ASFVs.

Zinc Sulfate Stimulates Osteogenic Phenotypes in Periosteum-Derived Cells and Co-Cultures of Periosteum-Derived Cells and THP-1 Cells.

Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.

Human Dental Pulp-Derived Mesenchymal Stem Cell Potential to Differentiate into Smooth Muscle-Like Cells In Vitro.

Previous studies have shown that mesenchymal stem cells (MSCs) derived from various tissue sources can be differentiated into smooth muscle-like cells (SMLCs) in vitro. In this paper, dental pulp-derived mesenchymal stem cells (DPSCs) were evaluated for their differentiation ability towards smooth muscle-like cells (SMLCs) under the effect of widely used cytokines (TGF-ß1 and PDGF-BB) with special focus on different culturing environments. For this purpose, both the commercially used culturing plates (Norm-c) and 0.1% gelatin-precoated (Gel-c) plates were used. Isolated cells displayed plastic adherence, pluripotency and cell surface marker profiling, and adipogenic and osteogenic differentiation potential with lineage specific marker expression. Differentiated cells induced under different culturing plates showed successful differentiation into SMLCs by positively expressing smooth muscle cell (SMC) specific markers both at the mRNA and protein levels. Gelatin coating could substantially enhance DPSC differentiation potential than Norm-c-induced cells. However, the absence of mature marker MHY-11 by immunostaining results from all treatment groups further indicated the development of immature and synthetic SMLCs. Finally, it was concluded that DPSC differentiation ability into SMLCs can be enhanced under cytokine treatment as well as by altering the cellular niche by precoating the culturing plates with suitable substrates. However, to get fully functional, contractile, and mature SMLCs, still many different cytokine cocktail combinations and more suitable coating substrates will be needed.

Evaluation of odonto/osteogenic differentiation potential from different regions derived dental tissue stem cells and effect of 17ß-estradiol on efficiency.

BACKGROUND: The dentin is a tissue, which is formed by odontoblasts at the pulp interface of the teeth that supports the enamel. Odontoblasts, the cranial neural crest cells are derived from ectodermal mesenchymal stem cells (MSCs) and are long and polarized cells. They are present at the outer surface of dentin and play a prominent role about dentin formation. Recently, attention has been focused on induction of odontoblast using various type of MSCs and effects of the 17ß-estradiol supplementation. In this study, we establish an efficient odonto/osteoblast differentiation protocol using 17ß-estradiol supplementation while comparing the odonto/osteoblast ability of various dental MSCs. METHODS: Same donor derived four types of dental MSCs namely dental pulp stem cells (DPSCs), stem cells from apical papilla (SCAP), dental follicle stem cells (DFSCs), and periodontal ligament stem cells (PDLSCs) were evaluated for their stemness characteristics and potency towards odonto/osteoblast (Induced odonto/osteoblast) differentiation. Then 17ß-estradiol supplementation of 0 and 10 µM was applied to the odonto/osteoblast differentiation media for 14 days respectively. Furthermore, mRNA and protein levels of odonto/osteoblast markers were evaluated. RESULTS: All of the experimental groups displayed stemness characteristics by showing adipocyte and chondrocyte differentiation abilities, expression for cell surface markers and cell proliferation capacity without any significant differences. Moreover, all dental derived MSCs were shown to have odonto/osteoblast differentiation ability when cultured under specific conditions and also showed positive expression for odontoblast markers at both mRNA and protein level. Among all, DPSCs revealed the higher differentiation potential than other dental MSCs. Furthermore, odonto/osteoblast differentiation potential was enhanced by supplementing the differentiation media with 17ß-estradiol (E2). CONCLUSIONS: Thus, DPSCs possess higher odonto/osteogenic potential than the SCAPs, DFSCs, PDLSCs and their differentiation capacity can by further enhanced under E2 supplementation.

Parthenolide Has Negative Effects on In Vitro Enhanced Osteogenic Phenotypes by Inflammatory Cytokine TNF-α via Inhibiting JNK Signaling.

Nuclear factor kappa B (NF-κB) regulates inflammatory gene expression and represents a likely target for novel disease treatment approaches, including skeletal disorders. Several plant-derived sesquiterpene lactones can inhibit the activation of NF-κB. Parthenolide (PTL) is an abundant sesquiterpene lactone, found in Mexican Indian Asteraceae family plants, with reported anti-inflammatory activity, through the inhibition of a common step in the NF-κB activation pathway. This study examined the effects of PTL on the enhanced, in vitro, osteogenic phenotypes of human periosteum-derived cells (hPDCs), mediated by the inflammatory cytokine tumor necrosis factor (TNF)-α. PTL had no significant effects on hPDC viability or osteoblastic activities, whereas TNF-α had positive effects on the in vitro osteoblastic differentiation of hPDCs. c-Jun N-terminal kinase (JNK) signaling played an important role in the enhanced osteoblastic differentiation of TNF-α-treated hPDCs. Treatment with 1 µM PTL did not affect TNF-α-treated hPDCs; however, 5 and 10 µM PTL treatment decreased the histochemical detection and activity of alkaline phosphatase (ALP), alizarin red-positive mineralization, and the expression of ALP and osteocalcin mRNA. JNK phosphorylation decreased significantly in TNF-α-treated hPDCs pretreated with PTL. These results suggested that PTL exerts negative effects on the increased osteoblastic differentiation of TNF-α-treated hPDCs by inhibiting JNK signaling.

Delay of cell growth and loss of stemness by inhibition of reverse transcription in human mesenchymal stem cells derived from dental tissue.

The present study investigated the cellular properties in the dental tissue-derived mesenchymal stem cells (DSCs) exposed to nevirapine (NVP), an inhibitor of reverse transcriptase (RTase). After a prolonged exposure of DSCs for 2 weeks, the population doubling time (PDT) was significantly (P < .05) increased by delayed cell growth in the DSCs treated with 250 and 500 µM NVP, compared with untreated DSCs. Furthermore, the G1 phase of cell cycle with high activity of senescence-associated ß-galactosidase was also significantly (P < .05) increased in the 250 µM NVP-treated DSCs, compared with untreated DSCs. The level of telomerase activity was unchanged between control and treatment. However, following the treatment of NVP, negative surface markers for mesenchymal stem cells (MSCs), such as CD34 and CD45, were significantly (P < .05) increased, while positive surface markers for MSCs, such as CD90 and CD105, were significantly (P < .05) decreased in the NVP-treated DSCs than those of untreated DSCs. Furthermore, the differentiation capacity into mesodermal lineage was gradually decreased, and a significant (P < .05) decrease of expression level of NANOG, OCT-4 and SOX-2 transcripts was observed in the DSCs treated with NVP, compared with untreated control DSCs. Taken together, the present results have revealed that inhibition of RTase by NVP induces delayed cell growth and loss of stemness.

Natural Products as Sources of Novel Drug Candidates for the Pharmacological Management of Osteoarthritis: A Narrative Review.

Osteoarthritis is a chronic degenerative articular disorder. Formation of bone spurs, synovial inflammation, loss of cartilage, and underlying bone restructuring have been reported to be the main pathologic characteristics of osteoarthritis symptoms. The onset and progression of osteoarthritis are attributed to various inflammatory cytokines in joint tissues and fluids that are produced by chondrocytes and/or interact with chondrocytes, as well as to low-grade inflammation in intra-articular tissues. Disruption of the equilibrium between the synthesis and degradation of the cartilage of the joint is the major cause of osteoarthritis. Hence, developing a promising pharmacological tool to restore the equilibrium between the synthesis and degradation of osteoarthritic joint cartilage can be a useful strategy for effectively managing osteoarthritis. In this review, we provide an overview of the research results pertaining to the search for a novel candidate agent for osteoarthritis management via restoration of the equilibrium between cartilage synthesis and degradation. We especially focused on investigations of medicinal plants and natural products derived from them to shed light on the potential pharmacotherapy of osteoarthritis.

Three-Dimensional Spheroid Formation of Cryopreserved Human Dental Follicle-Derived Stem Cells Enhances Pluripotency and Osteogenic Induction Properties.

Background: Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, three-dimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional mono-layer cultured MSCs. Methods: In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties. Results: In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis. Conclusion: To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.

Comparative analysis of three different protocols for cholinergic neuron differentiation in vitro using mesenchymal stem cells from human dental pulp.

A decrease in the activity of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis in the cholinergic neurons cause neurological disorders involving a decline in cognitive abilities, such as Alzheimer's disease. Mesenchymal stem cells (MSCs) can be used as an efficient therapeutic agents due to their neuronal differentiation potential. Different source derived MSCs may have different differentiation potential under different inductions. Various in vitro protocols have been developed to differentiate MSCs into specific neurons but the comparative effect of different protocols utilizing same source derived MSCs, is not known. To address this issue, dental pulp derived MSCs (DPSCs) were differentiated into cholinergic neurons using three different protocols. In protocol I, DPSCs were pre-induced with serum-free ADMEM containing 1 mM of ß-mercaptoethanol for 24 h and then incubated with 100 ng/ml nerve growth factor (NGF) for 6 days. Under protocol II, DPSCs were cultured in serum-free ADMEM containing 15 µg/ml of D609 (tricyclodecan-9-yl-xanthogenate) for 4 days. Under protocol III, the DPSCs were cultured in serum-free ADMEM containing 10 ng/ml of basic fibroblast growth factor (bFGF), 50 µM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 µM of retinoic acid (RA) for 7 days. The DPSCs were successfully trans-differentiated under all the protocols, exhibited neuron-like morphologies with upregulated cholinergic neuron-specific markers such as ChAT, HB9, ISL1, BETA-3, and MAP2 both at mRNA and protein levels in comparison to untreated cells. However, protocol III-induced cells showed the highest expression of the cholinergic markers and secreted the highest level of acetylcholine.

Development of bone regeneration strategies using human periosteum-derived osteoblasts and oxygen-releasing microparticles in mandibular osteomyelitis model of miniature pig.

Hypoxia and limited vascularization inhibit bone growth and recovery after surgical debridement to treat osteomyelitis. Similarly, despite significant efforts to create functional tissue-engineered organs, clinical success is often hindered by insufficient oxygen diffusion and poor vascularization. To overcome these shortcomings, we previously used the oxygen carrier perfluorooctane (PFO) to develop PFO emulsion-loaded hollow microparticles (PFO-HPs). PFO-HPs act as a local oxygen source that increase cell viability and maintains the osteogenic differentiation potency of human periosteum-derived cells (hPDCs) under hypoxic conditions. In the present study, we used a miniature pig model of mandibular osteomyelitis to investigate bone regeneration using hPDCs seeded on PFO-HPs (hPDCs/PFO-HP) or hPDCs seeded on phosphate-buffered saline (PBS)-HPs (hPDCs/PBS-HP). Osteomyelitis is characterized by a series of microbial invasion, vascular disruption, bony necrosis, and sequestrum formation due to impaired host defense response. Sequential plain radiography, computed tomography (CT), and 3D reconstructed CT images revealed new bone formation was more advanced in defects that had been implanted with the hPDCs/PFO-HPs than in defects implanted with the hPDCs/PBS-HP. Thus, PFO-HPs are a promising tissue engineering approach to repair challenging bone defects and regenerate structurally organized bone tissue with 3D architecture.

In vitro differentiation of single donor derived human dental mesenchymal stem cells into pancreatic ß cell-like cells.

The present study was carried out to investigate and compare the in vitro differentiation potential of mesenchymal stem cells (MSCs) isolated from human dental tissues (pulp, papilla, and follicle) of the same donor. MSCs were isolated from dental tissues (pulp, papilla, and follicle) following digestion method and were analyzed for the expression of pluripotent markers and cell surface markers. All three types of MSCs were evaluated for their potential to differentiate into mesenchymal lineages. Further, the MSCs were differentiated into pancreatic ß cell-like cells using multistep protocol and characterized for the expression of pancreatic lineage specific markers. Functional properties of differentiated pancreatic ß cell-like cells were assessed by dithizone staining and glucose challenge test. All three types of MSCs showed fibroblast-like morphology upon culture and expressed pluripotent, and mesenchymal cell surface markers. These MSCs were successfully differentiated into mesenchymal lineages and transdifferentiated into pancreatic ß cell-like cells. Among them, dental follicle derived MSCs exhibits higher transdifferentiation potency toward pancreatic lineage as evaluated by the expression of pancreatic lineage specific markers both at mRNA and protein level, and secreted higher insulin upon glucose challenge. Additionally, follicle-derived MSCs showed higher dithizone staining upon differentiation. All three types of MSCs from a single donor possess similar cellular properties and can differentiate into pancreatic lineage. However, dental follicle derived MSCs showed higher potency toward pancreatic lineage than pulp and papilla derived MSCs, suggesting their potential application in future stem cell based therapy for the treatment of diabetes.

Open reduction and internal fixation for Mason type III radial head fractures: Is it different from that for Mason type II fractures?

PURPOSE: The objective of this study was to investigate whether the outcomes of Mason type III radial head fractures (RHFs) treated by open reduction and internal fixation (ORIF) were comparable to those of Mason type II RHFs treated by ORIF. METHODS: A total of 87 surgically treated RHF patients were reviewed. Their fractures were Mason type II in 39 patients (all treated by ORIF) and Mason type III in 48 patients (40 treated by ORIF, 7 by radial head arthroplasty, and 1 by resection). Although ORIF was preferred for Mason type III RHFs in our series, an arthroplasty was performed when the fracture accompanied severe associated injuries or multiple traumas. Radiological and functional outcomes were evaluated and complications were reviewed. RESULTS: When Mason type II and Mason type III in general were compared, QuickDASH score, a shortened version of the Disabilities of the Arm, Shoulder and Hand (DASH) score, and forearm rotation were significantly worse in Mason type III. However, when comparing Mason type II and Mason type III treated by ORIF in which the proportion of associated injuries were not significantly different, there was no significant difference in QuickDASH score, range of extension/flexion, or complication rate. Forearm rotations were significantly more limited in Mason type III treated by ORIF (7° for pronation and 7° for supination), and Mason type had an independent effect on forearm rotations in multivariate analysis. CONCLUSION: ORIF for Mason type III fractures with low level of associated injury can be as good as that for Mason type II fractures, except for less forearm rotation.

The Role of Human Umbilical Vein Endothelial Cells in Osteogenic Differentiation of Dental Follicle-Derived Stem Cells in In Vitro Co-cultures.

Angiogenesis and vascularization are essential for the growth and survival of most tissues. Engineered bone tissue requires an active blood vessel network for survival and integration with mature host tissue. Angiogenesis also has an effect on cell growth and differentiation in vitro. However, the effect of angiogenic factors on osteoprogenitor cell differentiation remains unclear. We studied the effects of human umbilical vein endothelial cells (HUVECs) on osteogenic differentiation of dental follicle-derived stem cells (DFSCs) in vitro by co-culturing DFSCs and HUVECs. Cell viability, based on metabolic activity and DNA content, was highest for co-cultures with a DFSC/HUVEC ratio of 50:50 in a 1:1 mixture of mesenchymal stem cell growth medium and endothelial cell growth medium. Osteoblastic and angiogenic phenotypes were enhanced in co-cultures with a DFSC/HUVEC ratio of 50:50 compared with DFSC monocultures. Increased expression of angiogenic phenotypes and vascular endothelial growth factor (VEGF) levels were observed over time in both 50:50 DFSC/HUVEC co-cultures and DFSC monocultures during culture period. Our results showed that increased angiogenic activity in DFSC/HUVEC co-cultures may stimulate osteoblast maturation of DFSCs. Therefore, the secretion of angiogenic factors from HUVECs may play a role in the osteogenic differentiation of DFSCs.

Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo.

The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves.