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.

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.

Carpal Tunnel Syndrome Caused by Lipofibromatous Hamartoma of the Median Nerve.

Lipofibromatous hamartoma (LFH) is a rare tumor of the peripheral nerves, which usually involves the median nerve. The authors reported on two rare cases of carpal tunnel syndrome due to LFH of the median nerve. A 49-year-old female patient complained of the mass and symptoms consistent with LFH. Magnetic resonance imaging (MRI) showed typical LFH findings. The symptoms were successfully ameliorated with carpal tunnel release and external neurolysis. A 37-year-old female patient complained of weakening thumb abduction and the mass where the MRI showed atypical findings. Opponensplasty and debulking operations were performed after which thumb abduction was improved; however, neurological sequelae remained. LFH of the median nerve is managed on a case-by-case basis as treatment guidelines are not very clearly defined yet. However, the less invasive treatment such as carpal tunnel release and external neurolysis than more aggressive surgical treatment should be recommended as a treatment option.

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.

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.

Cubital Tunnel Syndrome Caused by Anconeus Epitrochlearis Muscle.

OBJECTIVE: We evaluated the clinical manifestation and surgical results following operative treatment of cubital tunnel syndrome (CuTS) caused by anconeus epitrochlearis (AE) muscle. METHODS: Among 142 patients who underwent surgery for CuTS from November 2007 to October 2015, 12 were assigned to the AE group based on discovery of AE muscle; 130 patients were assigned to the other group. We analyzed retrospectively; age, sex, dominant hand, symptom duration, and weakness in hand. Severity of the disease was evaluated using the Dellon classification and postoperative symptom were evaluated using disability of arm shoulder and hand (DASH) and visual analogue scale (VAS) scores. Surgery consisted of subfascial anterior transposition following excision of AE muscle. RESULTS: AE muscle was present in 8.5% of all patients, and was more common in patients who were younger and with involvement of their dominant hand; the duration of symptom was shorter in patients with AE muscle. All patients showed postoperative improvement in symptoms according to DASH and VAS scores. CONCLUSION: The possibility of CuTS caused by AE muscle should be considered when younger patients have rapidly aggravated and activity-related cubital tunnel symptoms with a palpable mass in the cubital tunnel area. Excision of AE muscle and anterior ulnar nerve transposition may be considered effective surgical treatment.

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.

The Human Replicative Helicase, the CMG Complex, as a Target for Anti-cancer Therapy.

DNA helicases unwind or rearrange duplex DNA during replication, recombination and repair. Helicases of many pathogenic organisms such as viruses, bacteria, and protozoa have been studied as potential therapeutic targets to treat infectious diseases, and human DNA helicases as potential targets for anti-cancer therapy. DNA replication machineries perform essential tasks duplicating genome in every cell cycle, and one of the important functions of these machineries are played by DNA helicases. Replicative helicases are usually multi-subunit protein complexes, and the minimal complex active as eukaryotic replicative helicase is composed of 11 subunits, requiring a functional assembly of two subcomplexes and one protein. The hetero-hexameric MCM2-7 helicase is activated by forming a complex with Cdc45 and the hetero-tetrameric GINS complex; the Cdc45-Mcm2-7-GINS (CMG) complex. The CMG complex can be a potential target for a treatment of cancer and the feasibility of this replicative helicase as a therapeutic target has been tested recently. Several different strategies have been implemented and are under active investigations to interfere with helicase activity of the CMG complex. This review focuses on the molecular function of the CMG helicase during DNA replication and its relevance to cancers based on data published in the literature. In addition, current efforts made to identify small molecules inhibiting the CMG helicase to develop anti-cancer therapeutic strategies were summarized, with new perspectives to advance the discovery of the CMG-targeting drugs.

Effects of Osteogenic-Conditioned Medium from Human Periosteum-Derived Cells on Osteoclast Differentiation.

Stem/progenitor cell-based regenerative medicine using the osteoblast differentiation of mesenchymal stem cells (MSCs) is regarded as a promising approach for the therapeutic treatment of various bone defects. The effects of the osteogenic differentiation of stem/progenitor cells on osteoclast differentiation may have important implications for use in therapy. However, there is little data regarding the expression of osteoclastogenic proteins during osteoblastic differentiation of human periosteum-derived cells (hPDCs) and whether factors expressed during this process can modulate osteoclastogenesis. In the present study, we measured expression of RANKL in hPDCs undergoing osteoblastic differentiation and found that expression of RANKL mRNA was markedly increased in these cells in a time-dependent manner. RANKL protein expression was also significantly enhanced in osteogenic-conditioned media from hPDCs undergoing osteoblastic differentiation. We then isolated and cultured CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) mononuclear cells (MNCs) and found that these cells were well differentiated into several hematopoietic lineages. Finally, we co-cultured human trabecular bone osteoblasts (hOBs) with CD34+ HSCs and used the conditioned medium, collected from hPDCs during osteoblastic differentiation, to investigate whether factors produced during osteoblast maturation can affect osteoclast differentiation. Specifically, we measured the effect of this osteogenic-conditioned media on expression of osteoclastogenic markers and osteoclast cell number. We found that osteoclastic marker gene expression was highest in co-cultures incubated with the conditioned medium collected from hPDCs with the greatest level of osteogenic maturation. Although further study will be needed to clarify the precise mechanisms that underlie osteogenic-conditioned medium-regulated osteoclastogenesis, our results suggest that the osteogenic maturation of hPDCs could promote osteoclastic potential.

Stem Cells from Cryopreserved Human Dental Pulp Tissues Sequentially Differentiate into Definitive Endoderm and Hepatocyte-Like Cells in vitro.

We previously described a novel tissue cryopreservation protocol to enable the safe preservation of various autologous stem cell sources. The present study characterized the stem cells derived from long-term cryopreserved dental pulp tissues (hDPSCs-cryo) and analyzed their differentiation into definitive endoderm (DE) and hepatocyte-like cells (HLCs) in vitro. Human dental pulp tissues from extracted wisdom teeth were cryopreserved as per a slow freezing tissue cryopreservation protocol for at least a year. Characteristics of hDPSCs-cryo were compared to those of stem cells from fresh dental pulps (hDPSCs-fresh). hDPSCs-cryo were differentiated into DE cells in vitro with Activin A as per the Wnt3a protocol for 6 days. These cells were further differentiated into HLCs in the presence of growth factors until day 30. hDPSCs-fresh and hDPSCs-cryo displayed similar cell growth morphology, cell proliferation rates, and mesenchymal stem cell character. During differentiation into DE and HLCs in vitro, the cells flattened and became polygonal in shape, and finally adopted a hepatocyte-like shape. The differentiated DE cells at day 6 and HLCs at day 30 displayed significantly increased DE- and hepatocyte-specific markers at the mRNA and protein level, respectively. In addition, the differentiated HLCs showed detoxification and glycogen storage capacities, indicating they could share multiple functions with real hepatocytes. These data conclusively show that hPDSCs-cryo derived from long-term cryopreserved dental pulp tissues can be successfully differentiated into DE and functional hepatocytes in vitro. Thus, preservation of dental tissues could provide a valuable source of autologous stem cells for tissue engineering.

Midkine and NANOG Have Similar Immunohistochemical Expression Patterns and Contribute Equally to an Adverse Prognosis of Oral Squamous Cell Carcinoma.

To increase the overall survival rate and obtain a better prognosis for oral squamous cell carcinoma (OSCC) patients, the detection of more effective and reliable tumor prognostic markers is needed. This study is focused on the analysis of correlation between the clinicopathological features of OSCCs and the immunohistochemical (IHC) expression patterns of MIDKINE (MK) and NANOG. Sixty-two primary OSCC patients were selected and their pretreatment biopsy specimens were immunohistochemically analyzed for the MK and NANOG proteins. The IHC expression patterns, clinicopathological features, and overall survival rates were assessed to identify any correlations. MK and NANOG showed significantly similar IHC expression patterns: both demonstrated enhanced expression in histologically high-grade and clinically late-stage OSCCs. Weak or negative expression of MK and NANOG was correlated with negative neck node metastasis. Clinicopathologically, late tumor stage, neck node metastasis, high-grade tumor, and palliative treatment groups showed significantly lower overall survival rates. The enhanced expression of MK and NANOG was associated with lower overall survival rates. In particular, enhanced co-detection of MK and NANOG showed significant correlation with poor prognosis. In conclusion, enhanced IHC expression patterns of MK and NANOG in OSCC patients was significantly associated with lower overall survival rates and unfavorable clinicopathological features. These results demonstrate that analysis of IHC expression patterns of MK and NANOG in pretreatment biopsy specimens during the work-up period can provide a more definitive prognosis prediction for each OSCC patient that can help clinicians to develop a more precise individual treatment modality.

The involvement of histone methylation in osteoblastic differentiation of human periosteum-derived cells cultured in vitro under hypoxic conditions.

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia-induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum-derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis- and senescence-associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red-S-positive mineralization, and mRNA expression of osteoblast-related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein-2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein-2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.

Tumor resection from retromolar trigone, posterolateral maxilla, and anterior mandibular ramus using lower cheek flap approach: a case report and review of literature.

A surgical approach involving the retromolar trigone, posterolateral maxilla, and pterygoid region is the most challenging in the field of maxillofacial surgery. The upper cheek flap (Weber-Ferguson incision) with subciliary extension and the maxillary swing approach have been considered as alternatives; however, neither approach provides sufficient exposure of the pterygoid region and the anterior portion of the mandibular ramus. In this report, we describe two cases in which a lower cheek flap approach was used for complete tumor resection in the retromolar trigone and the anterior mandibular ramus. This approach allows full exposure of the posterolateral maxilla and the pterygoid region as well as the retromolar trigone without causing major sensory disturbances to the lower lip. A mental nerve anastomosis after tumor resection was performed in one patient and resulted in approximately 90% sensory recovery in the lower lip. The lower cheek flap approach provides adequate exposure of the posterolateral maxilla, including the pterygoid, retromolar trigone, and mandibular ramus areas. If the mental nerve can be anastomosed during flap approximation, postoperative sensory disturbances to the lower lip can be minimized.