Development and in vitro evaluation of mucoadhesive patches of methotrexate for targeted delivery in oral cancer.

The present study focused on the development of a mucoadhesive patch of methotrexate (MTX) for targeted delivery in oral cancer. Initially, MTX-loaded liposomes were prepared using the thin film hydration method, and had a mean diameter of 105.7-137.4 nm and percentage entrapment efficiency of 54.6±3.5. These liposomes were cast in optimized mucoadhesive film. The film was characterized by its release pattern, thickness, weight and percentage swelling index and the sustained release profile of the optimized film was evaluated. The developed liposomes and liposomes cast in the film formulation were evaluated for cytotoxicity in HSC-3 cells using an MTT assay, and a significant decrease in the half maximal inhibitory concentration of MTX was identified with the MTX-entrapped liposomal film, M-LP-F7. The results of the mitochondria-dependent intrinsic pathway demonstrated that there was significant mitochondrial membrane potential disruption with M-LP-F7 compared with the plain drug. M-LP-F7 increased the rate of apoptosis in HSC-3 cells by almost 3-fold. Elevated levels of reactive oxygen species provided evidence that M-LP-F7 exerts a pro-oxidant effect in HSC-3 cells.

Effects of GPNMB on proliferation and odontoblastic differentiation of human dental pulp cells.

Glycoprotein (transmembrane) nonmetastatic melanoma protein b (GPNMB) plays crucial roles in odontogenesis. However, the role of GPNMB in human dental pulp cells (hDPCs) is still unclear. Therefore, in this study, we investigated the expression and function of the GPNMB in odontoblastic differentiation of hDPCs. Cells were cultured in odontoblast differentiation-inducing medium; the expression of the GPNMB was assessed by reverse transcriptase polymerase chain reaction and Western blot analysis. We performed gene knockdown of GPNMB in hDPCs using lentivirus-mediated small interfering RNA (siRNA)-GPNMB. The proliferation of cells was measured by the MTT assay, and the differentiation of cells was detected with alkaline phosphatase (ALP) activity assay, qRT-PCR and Western blot were used to determine the expression levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). The expression level of GPNMB was significantly increased during odontoblastic differentiation of hDPCs. Suppression of GPNMB expression by siRNA-GPNMB obviously promoted the proliferation of hDPCs. Furthermore, siRNA-GPNMB significantly inhibited the activity of ALP and expression levels of DSPP and DMP-1 during odontoblastic differentiation of hDPCs. Our results show that GPNMB plays an important role in regulating the expression of key pluripotency genes in hDPCs and modifying odontogenic differentiation.

Magnetic resonance imaging of temporomandibular joint: morphometric study of asymptomatic volunteers.

BACKGROUND: The aims of this study were to determine the best suited magnetic resonance imaging scanning plane, scanning sequence, and imaging modality for the evaluation of the temporomandibular joint (TMJ) and quantitatively assess the relationship of articular disk position to condyle position. METHODS: One hundred four TMJs in 52 symptom-free heads were examined by magnetic resonance imaging. The best scanning plane, scanning sequence, and scanning parameter were determined according to the imaging time and image quality. Bilateral symmetry of the articular disk and mandibular condyle was measured by using the automatic measurement of 3.0-T GE Excite Signa MR scanner. RESULTS: Fast spin-echo sequence, oblique sagittal imaging plane, and proton density imaging were the best suited scanning sequence, scanning planes, and imaging modality, respectively. The thicknesses of the anterior and posterior bands and for the intermediate zone were not statistically different for both sides. The posterior band of the disk was found to originate in an area adjacent to the 12-o'clock position of the condyle (± 5 degrees), whereas the anterior band of the disk originated adjacent to 1-o'clock position (28 ± 6 degrees). The anteroposterior diameter and mediolateral diameter of the condylar processes were not statistically different for both sides. The axial condylar angle between the plane of the greatest mediolateral diameter of the condylar processes and the midsagittal plane were also not statistically different for both sides. CONCLUSIONS: The magnetic resonance images can depict clearly major regional anatomic structures and position in the TMJ, which can be used in the early diagnosis for the TMJ disorder.

The clinical significance of CDK1 expression in oral squamous cell carcinoma

China OBJECTIVES: To evaluate the clinical significance of cyclin-dependent kinase 1 (CDK1) in 77 oral squamous cell carcinomas (OSCC) using immunohistochemical methods. Study DESIGN: Immunohistochemical expression of CDK1 was compared with various clinic pathological features in 77 OSCC and 60 controlled epithelia adjacent to the tumours. In addition, correlation of CDK1 expression and prognostic and the 5-year accumulative survival rate of OSCC were investigated. RESULTS: The CDK1 protein was expressed in 52 cases of 77 tumor tissues (67.5%), compared with 21 cases of 60controlled (35.0%). The expression of CDK1 was significantly correlated with the histological grade of OSCC(P<0.05). The CDK1 protein was over-expressed in recurrent tumors or in those with lymph node metastasis. Statistical analysis showed a significant reduction in the 5-year accumulative survival rate in CDK1 positive cases compared with CDK1 negative cases (P<0.05). Namely, the CDK1 positive patients had poor prognosis. CONCLUSIONS: The expression of CDK1 might serve as malignant degree and prognostic markers for the survival of OSCC

The clinical significance of CDK1 expression in oral squamous cell carcinoma.

OBJECTIVES: To evaluate the clinical significance of cyclin-dependent kinase 1 (CDK1) in 77 oral squamous cell carcinomas (OSCC) using immunohistochemical methods. STUDY DESIGN: Immunohistochemical expression of CDK1 was compared with various clinicopathological features in 77 OSCC and 60 controlled epithelia adjacent to the tumours. In addition, correlation of CDK1 expression and prognostic and the 5-year accumulative survival rate of OSCC were investigated. RESULTS: The CDK1 protein was expressed in 52 cases of 77 tumor tissues (67.5%), compared with 21 cases of 60 controlled (35.0%). The expression of CDK1 was significantly correlated with the histological grade of OSCC (P<0.05). The CDK1 protein was over-expressed in recurrent tumors or in those with lymph node metastasis. Statistical analysis showed a significant reduction in the 5-year accumulative survival rate in CDK1 positive cases compared with CDK1 negative cases (P<0.05). Namely, the CDK1 positive patients had poor prognosis. CONCLUSIONS: The expression of CDK1 might serve as malignant degree and prognostic markers for the survival of OSCC.

[Expression and significance of P75 neurotrophin receptor in oral squamous cell carcinoma].

PURPOSE: To investigate the expression of P75NTR in oral squamous cell carcinoma and evaluate the possibilities of P75NTR as a prognostic indicator of oral squamous cell carcinoma as well as a marker of cancer stem cell. METHODS: SP immunohistochemical method was used to detect the expressions of tongue squamous cell carcinoma line Tca8113 and 43 surgically resected squamous cell carcinoma tissues. Five peri-carcinoma tissues and 8 normal tissues were selected as control. SPSS16.0 software package was applied for X(2) test. RESULTS: P75NTR was positive in the Tca8113 cell line and located in the cell membrane. Thirty-three cases of the 43 OSCC patients showed positive expression of P75NTR. Para-carcinoma tissues and normal tissues showed negative expression of P75NTR. Positive P75NTR located in the cell membrane and dispersed in the cancer nest, and had a trend of cluster distribution. The expression of P75NTR had a relationship with tumor clinical stage(P<0.05) and lymph node metastasis(P<0.05). CONCLUSIONS: The expression of P75NTR can predict the prognosis of oral squamous cell carcinoma, yet it can not be taken as a marker of cancer stem cell alone. Further study should be taken to investigate the role and mechanism of P75NTR in the development of tumor.

[Influence of p75 neurotrophin receptor knockout on the regeneration of facial nerves after crush injury in mouse].

OBJECTIVE: To investigate the role of p75 neurotrophin receptor (p75NTR) in the regeneration of facial nerve crush injury. METHODS: In p75NTR knockout mice and wild type mice, the regenerating fibres in the facial nerve were also labelled by an anterograde tracer cholera toxin B (CTB). The next day after injury of facial nerve, CTB was injected into the trunk of the nerve in the proximal side of the crush, and then anterograde tracing and immunohistochemistry were used to examine the regeneration of axons after facial nerve crush injury. In p75NTR knockout mice and wild type mice, the facial nerves on one side were crushed and regenerating neurons in the facial nerve nucleus were labelled by Fast Blue. The facial nerve trunk was cut in the bifurcated region in the 4th day after injury and the stump was inserted into a small polymer tube containing Fast Blue. Retrograde tracing and labling motoneuron counting were used to examine the survival of motoneurons in the facial nerve nucleus after facial nerve crush injury. RESULTS: The results showed that the axonal growth of injured axons in the facial nerve of p75NTR knockout mice was significantly retarded. The number of regenerated neurons in the facial nerve nucleus in p75NTR knockout mice was significantly reduced (P < 0.05). Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR knockout mice (P < 0.01). CONCLUSION: p75NTR plays an important role in the regeneration of injured peripheral nerves after injury.

Deletion of p75NTR impairs regeneration of peripheral nerves in mice.

AIMS: After peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves. MAIN METHODS: In p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry. KEY FINDINGS: The results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice. SIGNIFICANCE: Our data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury.

BACKGROUND: The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons. METHODOLOGY/PRINCIPAL FINDINGS: The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions. CONCLUSIONS/SIGNIFICANCE: Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.

[Ectopic bone induction in vivo after transplantation of skeletal satellite cells from green fluorescence protein transgenic mouse transfected by adenoviral vectors encoding bone morphogenetic protein-2].

OBJECTIVE: To observe the ability of induced ectopic bone using skeletal muscles satellite cells (SMSCs) from newborn green fluorescence protein (GFP) transgenic mice mediated by Ad-BMP2. METHODS: Transplantation of SMSCs transduced with Ad-BMP2 into back lamb muscles of subfascia in wildtype 129sv mice with a complex of collagen scaffords, then the tissue histologic examination, X ray plain film, fluorescence microscopy were used. RESULTS: Transplantation of SMSCs transfected with Ad-BMP2 into back lamb muscles of subfascia generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks and mature bone formation 4 weeks after transplantation. SMSCs non-transfected with Ad-BMP2 failed to induce ectopic bone formation. CONCLUSION: SMSCs retain differentiation potentitality into osteoblasts in response to Ad-BMP2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.

[Isolation, identification, culture and bionomics of skeletal muscles satellite cells from green fluorescent protein transgenic mouse in vitro].

OBJECTIVE: To investigate the green fluorescent protein (GFP) expression and the bionomics of skeletal muscles satellite cells (SMSCs) in vitro in GFP transgenic mouse. METHODS: The newborn transgenic mice were acquired to separate skeletal muscles satellite cells with enzyme digestion method. Cells were cultured and subcultured in vitro. Morphological observation, growth curve were investigated to evaluate the proliferation and differentiation characteristics of skeletal muscles satellite cells, fluorescence microscope was used to observe the GFP expression. The cells were identified by immunocytochemical stain. In the basis of identification of anti-sarcometric actin anti-body, the combination of anti-desmin antibody and DAPI (4, 6-diamidino-2-phenylindole) were used to detect the purification of skeletal muscles satellite cells. RESULTS: Immunocytofluorescence suggested the good retain of GFP fluorescence in skeletal muscles satellite cells. The cells showed strong proliferative ability and they were positive with immunocytochemical stain of anti-sarcometric actin antibody and anti-desmin antibody. The combination of anti-desmin and DAPI stain can be used to determine the purification of SMSCs. CONCLUSION: Skeletal muscles satellite cells cultured in vitro showed strong proliferation and differentiation ability. They are fit to construct the cell bank of tissure engineering and to be a useful tool to explore cells fate after transplantation since these cells retain the expression of GFP.

[Correlation between periodontal disease and coronary atherosclerotic heart disease].

OBJECTIVE: To investigate the correlation between coronary atherosclerotic heart disease (CAD) and periodontal disease (PD). METHOD: Forty-five patients with CAD (CAD group) and 40 patients without CAD (control group) were compared with their pathological changes of periodontal tissues and inflammatory markers [high sensitive C reactive protein (hsCRP), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha)]. RESULTS: Univariate analysis showed that the prevalence of PD was 84.44% in CAD group and 22.50% in control group (P < 0.01). The levels of hsCRP, IL-1beta, and TNF-alpha were (5.75 +/- 1.26) mg/L, (10.32 +/- 2.96) ng/L, and (9.17 +/- 2.14) ng/L in CAD group and (1.13 +/- 0.73) mg/ L, (2.87 +/- 1.45) ng/L, and (5.84 +/- 1.96) ng/L in control group (P < 0.01). Gingival index and plaque index were statistically different between two both groups (P < 0.01). Logistic regression analysis showed that in addition to pulse pressure and low density lipoprotein cholesterol, periodontal disease index was a higher risk factor of CAD. Its relative risk was 1.217 (95% CI was 1.120-1.805, P < 0.05). CONCLUSION: PD can cause CAD. The improvement of public oral health plays an important role in the prevention and treatment of CAD.