Identification of the MMP family as therapeutic targets and prognostic biomarkers in the microenvironment of head and neck squamous cell carcinoma.

BACKGROUND: Head and Neck Squamous Cell Carcinoma is a malignant tumor with high morbidity and mortality. The MMP family plays an important role in tumor invasion and metastasis. However, the mechanistic value of the MMP family as a therapeutic target and prognostic biomarker in HNSC has not been fully elucidated. METHODS: Oncomine, UALCAN, GEPIA, cBioportal, GeneMANIA, STRING, DAVID6.8, TRRUST, TIMER and Linkedomics were used for analysis. RESULTS: The mRNA expression levels of MMP1, MMP3, ILF3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13 and MMP16 were higher in HNSC than those in normal tissues, while the mRNA expression level of MMP15 was reduced. The relative expression levels of MMP1 and MMP14 were the highest in HNSC tissues. A significant correlation was found between the expression of MMP3, MMP11, MMP25 and the pathological stage of HNSC patients. There was no significant associations between all the MMP family members expression levels and DFS. Increased mRNA levels of MMP1, MMP8 and MMP25 were significantly associated with OS. In addition, we investigated the genetic changes of the MMP family in HNSC and found that all the MMP family members had genetic changes, most of which were amplification and depth loss. In the analysis of neighbor gene network and protein interaction, we found that the MMP family interacted with 25 neighboring genes, except for ILF3, MMP19, MMP20, MMP21, MMP23B, MMP27 and MMP28, other MMP proteins interacted with each other. Functional enrichment analysis showed that the MMP family could be present in the extracellular matrix, regulate peptidase activity, and participate in the catabolism of collagen. Meanwhile, we identified the transcription factor targets and kinase targets of the MMP family and found that ATM and ATR were the two most common kinase targets in the MMP family. We also found a significant correlation between the MMP family expression and immune cell infiltration. Cox proportional risk model analysis showed that macrophages, MMP14, MMP16, and MMP19 were significantly associated with clinical outcomes in HNSC patients. CONCLUSION: The MMP family might serve as therapeutic target and prognostic biomarker in HNSC.

The application of composite scaffold materials based on decellularized vascular matrix in tissue engineering: a review.

Decellularized vascular matrix is a natural polymeric biomaterial that comes from arteries or veins which are removed the cellular contents by physical, chemical and enzymatic means, leaving only the cytoskeletal structure and extracellular matrix to achieve cell adhesion, proliferation and differentiation and creating a suitable microenvironment for their growth. In recent years, the decellularized vascular matrix has attracted much attention in the field of tissue repair and regenerative medicine due to its remarkable cytocompatibility, biodegradability and ability to induce tissue regeneration. Firstly, this review introduces its basic properties and preparation methods; then, it focuses on the application and research of composite scaffold materials based on decellularized vascular matrix in vascular tissue engineering in terms of current in vitro and in vivo studies, and briefly outlines its applications in other tissue engineering fields; finally, it looks into the advantages and drawbacks to be overcome in the application of decellularized vascular matrix materials. In conclusion, as a new bioactive material for building engineered tissue and repairing tissue defects, decellularized vascular matrix will be widely applied in prospect.

The antioxidant peptide SS31 prevents oxidative stress, downregulates CD36 and improves renal function in diabetic nephropathy.

Background: Oxidative stress plays an independent role in the pathogenesis of diabetic nephropathy (DN). CD36, a class B scavenger receptor, mediates reactive oxygen species (ROS) production in DN. SS31 is a mitochondria-targeted antioxidant peptide that can scavenge mitochondrial ROS. The antioxidative effects of SS31 on DN and the interaction between SS31 and CD36 remain poorly understood. Herein, we examined the effects of SS31 and investigated whether SS31 treatment attenuates CD36 expression in db/db diabetic mice and high glucose (HG)-induced HK-2 cells. Methods: Eight-week-old db/m mice and db/db mice were administered with SS31 (3 mg/kg/day) for 12 weeks by intraperitoneal injection. For the in vitro studies, HG-cultured HK-2 cells were used. Biochemical parameters, body weight and histological changes in the mice were measured. The levels of oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Mn superoxide dismutase (MnSOD) and catalase (CAT), and the expression of CD36, nuclear factor-κB (NF-κB) p65 in mice and HK-2 cells were also analyzed. Results: The results showed that SS31 alleviated proteinuria, glomerular hypertrophy and tubular injury, and affected creatinine level in db/db mice. SS31 suppressed the levels of oxidative stress, NADPH oxidase subunits, CD36 and NF-κB p65, and activated MnSOD and CAT in db/db mice and HG-induced HK-2 cells. Conclusion: Taken together, these data demonstrated a renoprotective role of SS31 in DN by suppression of enhanced oxidative stress and CD36 expression.

Induced differentiation of human gingival fibroblasts into VSMC-like cells.

Vascular smooth muscle cells (VSMCs) are major component of the vascular wall, and they play an essential role in maintaining the basic physiological function and stable structure of the vascular wall. In the present study, human gingival fibroblasts (HGFs) were cultured and induced into VSMC-like cells in vitro to confirm that HGFs with properties of stem cells have the potential for differentiation. The epithelium isolated from patients was extracted from normal human gingiva consisting of epithelium and connective tissue. HGFs were first identified by morphological examination, as well as specific gene and protein expression, and then induced by 10ng/mL PDGF-BB combined with 2ng/mL of TGF-ß1 for 28 days. After induction, ICS data indicated that induced VSMC-like cells were positive for α-SMA and SM-MHC, and IFA data showed that induced cells were positive for SM22α and Cnn1. RT-PCR results demonstrated that α-SMA and SM-MHC mRNA were specifically expressed, and myofilament-like structures also appeared in induced cells. In conclusion, the data indicated that HGFs could differentiate to VSMC-like cells with typical VSMC morphologic, ultrastructural, and immunological characteristics via induction with PDGF-BB and TGF-ß1.

Human gingival fibroblasts induced and differentiated into vascular endothelial-like cells.

A novel method for repair of vascular disease, mechanical damage, and tissue rebuilding is urgently required. Vascular endothelial cells (VECs) play an essential role in vascular rebuilding and vasotransplantation. In the present study, human gingival fibroblasts (HGFs) were cultured and induced into endothelial-like cells in vitro in order to confirm that HGFs with stem cell properties possessed the potential for differentiation into endothelial-like cells. The epithelium was extracted from normal human gingiva consisting of epithelium and connective tissue, which was isolated from patients. The identification of HGFs and induced endothelial-like cells were confirmed by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunocytochemical stain (ICS), and immunofluorescence stain (ISA). The morphology of human gingival fibroblasts with 8 ng/mL VEGF165 induced for different periods of days were observed by inverted microscope. Before induction, flow cytometry analysis showed that HGFs were positive for vimentin, but negative for CD31. RT-PCR, ICS, and ISA showed vimentin, S100A4, α-SMA, collagen III, and S100A4 were specifically expressed in these fibroblast cells. After induction, ICS showed induced vascular endothelial-like cells were positive for CD34 and CD31; ISA showed cells induced were positive for vWF and E-cadherin; RT-PCR results demonstrated that tie2 was specifically expressed in the cells induced. Flow cytometry analysis of the transformation efficiency from HGFs to endothelial-like cells. In conclusion, we found that HGFs possessed capacity for being induced and differentiated into vessel endothelial-like cells with typical and specific morphological, ultrastructural, and immunological characters of endothelial-like cells by induction with VEGF.

Verification of the expression trend and interaction prediction of innate immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis.

OBJECTIVES: This study aimed to explore the expression trends of innate immune cells and immune-checkpoint molecules validated by data calculation in the process of oral mucosal carcinogenesis, as well as to explore methods of suppressing oral mucosal carcinogenesis based on immunotherapy by predicting their interactions. Me-thods 1) The cancer genome atlas (TCGA) database comprehensively scores immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis and screens out intrinsic immune cells and immune-checkpoint molecules that interfere with tumor immune escape. 2) Clinical patient blood routine data were collected for the statistical analysis of peripheral blood immune cells during the progression of oral mucosal carcinogenesis. Immune cells in peripheral blood that may affect the progression of oral mucosal carcinogenesis were screened. 3) Immunohistochemical staining was performed on intrinsic immune cells and immune-checkpoint molecules validated based on data calculation in various stages of oral mucosal carcinogenesis. 4) Special staining was used to identify innate immune cells in various stages of oral mucosal carcinogenesis based on data-calculation verification. 5) Survival analysis was conducted on intrinsic immune cells and immune-checkpoint molecules validated based on data calculation during the process of oral mucosal carcinogenesis. The association of intrinsic immune cells and immune-checkpoint molecules with the prognosis of oral squamous cell carcinoma was verified. RESULTS: The expression of monocytes and neutrophils increased during the process of oral mucosal carcinogenesis. The expression of eosinophils showed a single peak trend of up and down. The expression of mast cells decreased. In the process of oral mucosal carcinogenesis, the expression of the immune-checkpoint molecules cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death-ligand (PD-L1) increased. The expression trends of monocytes, neutrophils, and eosinophils were positively correlated with those of CTLA4 and PD-L1 immune-checkpoint molecules. The expression trend of mast cells was negatively correlated with the expression of CTLA4 and PD-L1. Monocytes, neutrophils, and eosinophils may promote tumor immune escape mediated by CTLA4 and/or PD-L1, thereby accelerating the progression of oral mucosal carcinogenesis. Mast cells may inhibit tumor immune escape mediated by CTLA4 and/or PD-L1, delaying the progression of oral mucosal carcinogenesis. CONCLUSIONS: Therefore, interference with specific immune cells in innate immunity can regulate the expression of CTLA4 and/or PD-L1 to a certain extent, inhibit tumor immune escape, and delay the progression of oral mucosal carcinogenesis.

Vascularized characteristics and functional regeneration of three-dimensional cell reconstruction of oral mucosa equivalents based on vascular homeostasis phenotypic modification.

Our prior research has effectively developed tissue-engineered vascularized oral mucosa equivalents (VOME); however, challenges such as low repeatability and stability, as well as the inability to accurately replicate the complexity of real blood vessels, were encountered. Therefore, this study aimed to screen the VOME and native oral mucosa vascular homeostasis phenotypes by tandem mass tag-tagged proteomics associated with laser capture microdissection and human angiogenesis antibody array technology. Then, lentiviruses were constructed and stably transfected with vascular endothelial-like cells (VELCs) to detect angiogenic capacity. HE, EdU Apollo tracer staining, immunofluorescence staining, scanning electron microscopy, biomechanical testing, and a small animal ultrasound imaging system were used to analyze the characteristics of vascularization homeostasis and monitor functional regeneration of the vascularized homeostatic phenotypic oral mucosal equivalents (VHPOME). The results showed that PGAM1, COL5A1, ANG, and RNH1 are potential specific angiogenesis phenotypes. High expression of PGAM1, COL5A1, and ANG and/or low expression of RNH1 can promote the angiogenesis of VOME. ANG/shRNH1 has the most significant tube-like structure-formation ability. The expression of PGAM1, COL5A1, and ANG in the VHPOME group was higher than that of the control group, and the expression of RNH1 was lower than that of the control group. COL5A1/ANG can significantly improve the mechanical properties. The blood flow signal was most significant in the ANG/shRNH1 group. PGAM1, COL5A1, ANG, shRNH1, PGAM1/ANG, COL5A1/ANG, PGAM1/shRNH1, PGAM1/shRNH1, COL5A1/shRNH1, and ANG/shRNH1 may be the targets for establishing vascularization homeostasis and functional regeneration of oral mucosal equivalent genes (groups), and ANG/shRNH1 has the most significant effect.

Significance and considerations of establishing standardized critical values for critical size defects in animal models of bone tissue regeneration.

Establishing animal models with critical size defects (CSDs) is critical for conducting experimental investigations engineering of bone tissue regeneration. Currently, a standardised protocol for establishing an animal CSDs model has not been developed. Furthermore, a consensus has not been reached regarding the critical values of CSDs. Successful establishment of animal models for CSDs is a complex process that requires researchers to meticulously consider a variety of factors such as age, species, bone defect size and anatomic location. The specific numerical values for CSDs in small animal models vary, and a clear definition of the critical value for large animal CSDs models in the literature is still lacking. This review consolidates the advancements in critical bone defects animal models by outlining the research landscape across variables, including animal species, age groups, bone defect sites, and sizes, to offer valuable guidance and a theoretical framework for the establishment of pertinent experimental animal models.

MMP7, Regulated by c-Jun, is Involved in Oral Squamous Cell Carcinoma and Associated with Cancer-Related Fibroblasts Infiltration.

OBJECTIVE: This study aimed to analyze the expression of Matrix Metalloproteinase 7 (MMP7) and molecular mechanism at the Transcription Factor (TF) level in Oral Squamous Cell Carcinoma (OSCC). METHODS: MMP7 expression was preliminarily explored in Head and Neck Squamous Cell Car-cinoma (HNSCC) in the online database, followed by functional analysis and prediction of TF of MMP7. IHC was employed to detect MMP7 levels in OSCC samples. SCC9 and 293T cells were used to explore the transcriptional and regulatory effects of predicted TF on MMP7 by reporter double luciferase assay, RT-qPCR, western blotting, and cellular immunofluorescence. Transwell and TUNEL were employed to detect the migration and apoptosis. RESULTS: MMP7 was significantly up-regulated in HNSCC and OSCC tissues. Moreover, MMP7 was positively correlated with CAFs and significantly enriched in the signaling pathway of RNA degradation. The c-Jun pathway was also up-regulated in OSCC tissues, and predicted to be optimal TF of MMP7 with positive regulatory relationship. In OSCC, silencing and over-expression of c-Jun significantly decreased and increased the level of MMP7. Meanwhile, c-Jun affected the behavior of SCC9 cells, which showed that after c-Jun gene silencing, the ability of cell migration was weakened, while apoptosis was enhanced. When c-Jun gene was overexpressed, the migration ability was enhanced, but apoptosis was not significantly affected. CONCLUSION: MMP7 has been proven to be a key protein in the development of OSCC, and has the potential to become a biological marker and therapeutic target. It has been found that c-Jun could bind to the MMP7 promoter region, and the silencing or overexpression of c-Jun can positively regulate the expression of MMP7.

Sleep and mental health during the COVID-19 pandemic: findings from an online questionnaire survey in China.

Introduction: The online study investigated the sleep, psychological conditions, and risk factors during the wave of transmission of COVID-19 since December 7, 2022. Methods: We distributed questionnaires through networking mediums to residents to gather information about COVID-19 infection, sleep, and mental status. Results: During the extraordinary period in China, 91.9% of 1094 participants claimed to be infected with COVID-19, 36.8% reported poor sleep quality, 75.9% reported anxiety, and 65.5% reported depression. In retrospect, people have experienced lower sleep quality, longer sleep latency, enhanced rising time, and decreased sleep efficiency after the infection wave. After adjusting confounding factors, the elderly, women, urban residents, people with comorbidity, anxiety, depression, stress state, and COVID-19 infection have high risks for sleep disorders during the period. Discussion: The survey indicates that sleep disturbance caused by COVID-19 involves multiple dimensions, such as physiology, psychology, and society. The COVID-19 infection-related sleep problem should be taken seriously. Apart from conventional treatment, psychological issues of insomnia can not be ignored.

Silencing CK19 regulates ferroptosis by affecting the expression of GPX4 and ACSL4 in oral squamous cell carcinoma in vivo and in vitro.

To analyze the mechanism of how interfering with the cytokeratin 19 (CK19) pathway via the ferroptosis pathway affects tumor biological behaviors in the process of oral squamous cell carcinoma (OSCC) development. TCGA was used to analyze the expression of CK19 in pan-cancer and head and neck squamous cell carcinoma (HNSC) and to explore the ferroptosis-related genes related to HNSC. The effect of silencing CK19 on the migration ability of HSC-4 cells was verified by wound healing and migration assay. HSC-4 cells with silencing of CK19 and tumor-bearing nude mouse model were constructed. RT-qPCR, immunofluorescence and western blot were used to analyze the expression of ferroptosis-related genes. CK19 is highly expressed in human OSCC and nude mice. The migration ability of cells in the CK19-silenced group was lower than that of the control group. In vivo and in vitro, CK19 was negatively correlated with the expression of ACSL4 and positively correlated with the expression of GPX4. Compared with the control group, GPX4 expression was down-regulated and ACSL4 expression was up-regulated in the CK19-silenced group. Silencing CK19 also increased intracellular Fe2+ content and MDA content. Silencing CK19 can affect the expression of GPX4 and ACSL4 to regulate ferroptosis and at the same time increase the content of MDA, Fe2+ and ROS levels, thereby activating the regulation of ferroptosis pathway in the development of OSCC.

[Effect of up-regulation of Decorin on expression of EGFR, C-Myc and p21 in nude mice with oral squamous cell carcinoma].

PURPOSE: To explore the effect of overexpression of DCN（decorin） gene on the expression of epidermal growth factor receptor (EGFR), cellular-myelocytomatosis viral oncogene (C-Myc) and cyclin dependent kinase inhibitor (p21)in tumor-bearing nude mice with oral squamous cell carcinoma（OSCC）. METHODS: The expression of DCN gene in human oral squamous cell carcinoma(HSC-3) was up-regulated by liposome transfection. Nude mice were used as the carrier of OSCC. H-E staining was used to determine the pathological grade of tumor-bearing tissues in each group. Immunohistochemistry was used to detect the expression of EGFR, C-Myc and p21 protein in tumor-bearing tissues of each group after DCN overexpression. RT-qPCR and Western blot were used to quantitatively detect the expression of EGFR, C-Myc and p21 in tumor-bearing tissues of each group after DCN overexpression, and to determine the effects of DCN overexpression on the expression of EGFR, C-Myc and p21 in tumor-bearing tissues of OSCC nude mice. SPSS 20.0 software package was used for statistical analysis. RESULTS: H-E staining showed that the animal model of OSCC was successfully constructed. The tumor-bearing tissues of nude mice in the plasmid group were significantly lighter than those in the empty vector group and non-transfected group(P＜0.05). IHC results showed that DCN, EGFR, C-Myc and p21 proteins were expressed in the tumor-bearing tissues of nude mice in each group, the expression of DCN,EGFR and C-Myc proteins in the plasmid group was significantly different from the other groups(P＜0.05).There was no significant differece in p21 protein expression in each group(P＞0.05). RT-qPCR and Western blot results showed that DCN, EGFR, C-Myc and p21 were expressed in diffrent degrees in tumor-bearing tissues of nude mice（P＜0.05）. CONCLUSIONS: DCN can inhibit the growth of tumor in OSCC nude mice. In tumor-bearing tissues of nude mice with OSCC, overexpression of DCN can down-regulate the expression of EGFR and C-Myc, and up-regulate the expression of p21.DCN may play an inhibitory role in the occurrence and development of OSCC.

CGF-HLC-I repaired the bone defect repair of the rabbits mandible through tight junction pathway.

Background: The human-like collagen I (HLC-I) combined concentrated growth factors was used to construct CGF-HLC-I composite biomaterials to repair the critical bone defect disease model of rabbit mandible. This study aimed to research the repair mechanism of CGF-HLC-I/Bio-Oss in rabbit mandibular critical bone defect, to provide a new treatment direction for clinical bone defect repair. Methods: The optimal concentration of HLC-I (0.75%) was selected in this study. Nine New Zealand white rabbits were randomly divided into 3 groups, normal control group, Bio-Gide/Bio-Oss and CGF-0.75%HLC-I/Bio-Oss group (n = 3, each group). CGF-0.75%HLC-I/Bio-Oss and Bio-Gide/Bio-Oss were implanted into rabbit mandibles, then X-ray, Micro-CT, HE and Masson staining, immunohistochemical staining and biomechanical testing were performed with the bone continuity or maturity at 4, 8 and 12 weeks after surgery. The repair mechanism was studied by bioinformatics experiments. Results: As the material degraded, the rate of new bone formation in the CGF-0.75% HLC-I/Bio-Oss group was better than that the control group by micro-CT. The biomechanical test showed that the compressive strength and elastic modulus of the CGF-0.75%HLC-I/Bio-Oss group were higher than those of the control group. HE and Masson staining showed that the bone continuity or maturity of the CGF-0.75%HLC-I/Bio-Oss group was better than that of the control group. Immunohistochemical staining showed significantly higher bone morphogenetic protein 2 (BMP2) and Runt-related transcription factor 2 (RUNX2) in the CGF-0.75%HLC-I/Bio-Oss group than the control group at 8 and 12 W and the difference gradually decreased with time. There were 131 differentially expressed proteins (DEPs) in the Bio-Gide/Bio-Oss and CGF-0.75%HLC-I/Bio-Oss groups, containing 95 up-regulated proteins and 36 down-regulated proteins. KEGG database enrichment analysis showed actinin alpha 1 (ACTN1) and myosin heavy-Chain 9 (MYH9) are the main potential differential proteins related to osteogenesis, and they are enriched in the TJs pathway. Conclusion: CGF-0.75%HLC-I/Bio-Oss materials are good biomaterials for bone regeneration which have strong osteoinductive activity. CGF-0.75%HLC-I/Bio-Oss materials can promote new bone formation, providing new ideas for the application of bone tissue engineering scaffold materials in oral clinics.

Heptamethine Cyanine-Based Application for Cancer Theranostics.

Cancer is the most common life-threatening malignant disease. The future of personalized cancer treatments relies on the development of functional agents that have tumor-targeted anticancer activities and can be detected in tumors through imaging. Cyanines, especially heptamethine cyanine (Cy7), have prospective application because of their excellent tumor-targeting capacity, high quantum yield, low tissue autofluorescence, long absorption wavelength, and low background interference. In this review, the application of Cy7 and its derivatives in tumors is comprehensively explored. Cy7 is enormously acknowledged in the field of non-invasive therapy that can "detect" and "kill" tumor cells via near-infrared fluorescence (NIRF) imaging, photothermal therapy (PTT), and photodynamic therapy (PDT). Furthermore, Cy7 is more available and has excellent properties in cancer theranostics by the presence of multifunctional nanoparticles via fulfilling multimodal imaging and combination therapy simultaneously. This review provides a comprehensive scope of Cy7's application for cancer NIRF imaging, phototherapy, nanoprobe-based combination therapy in recent years. A deeper understanding of the application of imaging and treatment underlying Cy7 in cancer may provide new strategies for drug development based on cyanine. Thus, the review will lead the way to new types with optical properties and practical transformation to clinical practice.

Identification and validation of potential novel biomarkers for oral squamous cell carcinoma.

Our study aimed to explore potential new diagnostic biomarkers in patients with oral squamous cell carcinoma (OSCC) to find new target molecules involved in the progression of OSCC. Potential novel biomarkers of OSCC were identified using a protein microarray assay. Compared with the healthy control group, there were five proteins (I309, GDF15, AXL, MMP3, and CTACK) in the serum of in situ oral cancer group. However, there were four differentially expressed proteins (MCSF, I309, MMP3, and CTACK) in the serum of the OSCC group. Receiver operating characteristic (ROC) curve analysis results suggested that these six proteins (I309, GDF15, AXL, MMP3, CTACK, and MCSF) had diagnostic value for OSCC. Based on The Cancer Genome Atlas (TCGA) database, we found that only GDF15 expression was associated with the prognosis of OSCC. Subsequently, we verified the expression levels of six proteins in HSC-3 and HaCaT cells, and the results showed that the level of these six proteins was significantly higher in HSC-3 cells than in normal HaCaT cells. Similarly, in the OSCC nude mouse model, the expression levels of these proteins were significantly upregulated in OSCC tumor tissue compared to the normal tissue. GDF15, MMP3, AXL, MCSF, I309, and CTACK may be used as biomarkers for OSCC diagnosis and provide a novel study direction for the treatment of OSCC.

[Study on expression of LIM domain only protein 1 in SD rat oral buccal mucosa carcinogenesis induced by 4-nitro-quinoline N-oxide].

OBJECTIVE: This work aimed to determine the expression changes in LIM domain only protein 1 (LMO1) in gene transcription and protein levels during oral squamous cell carcinoma (OSCC) development. METHODS: The tissues in this study were taken from our team's previous animal model building, and we performed hematoxylin-eosin (HE) staining on 49 cases. The pathological classification of the experiment group was determined on the basis of the abnormal epithelial hyperplasia degree. The expression part of LMO1 was determined by immunohistochemistry staining. The mRNA and protein LMO1 expression levels in five groups were detected by real-time fluorescent quantitative of nucleotide polymer chain reaction (RT-qPCR) and Western blot, respectively. RESULTS: HE staining determined 7 cases of the control group, 6 cases of mild epithelial dysplasia, 11 cases of moderate epithelial dysplasia, 9 cases of severe epithelial dysplasia, and 16 cases of OSCC. Immunohistochemistry results: LMO1 expression was localized in the cytoplasm, and the positive expression rates of the protein LMO1 in the control and experiment groups were 14.3% for normal buccal mucosal tissue, 33.3% for mild epithelial dysplasia, 81.8% for moderate epithelial dysplasia, 88.9% for severe epithelial dysplasia, and 93.8% for OSCC. RT-qPCR results: mRNA expression was lowest in the control group and highest in the OSCC group, the difference between the mild dysplasia and control groups was not significant (P>0.05). Pairwise comparison among other groups showed statistically significant differences (P<0.05). Western blot results: with the aggravation of the pathological degree, the protein LMO1 expression level increased gradually. The OSCC group expressed the highest LMO1 expression level. CONCLUSIONS: The oral mucosa carcinogenesis models showed abnormal the mRNA and protein LMO1 expression levels, and the mRNA and protein expression levels were positively correlated with the degree of abnormal proliferation.

Study of tissue engineered vascularised oral mucosa-like structures based on ACVM-0.25% HLC-I scaffold in vitro and in vivo.

PURPOSE: To explore the feasibility of constructing tissue-engineered vascularised oral mucosa-like structures with rabbit ACVM-0.25% HLC-I scaffold and human gingival fibroblasts (HGFs), human gingival epithelial cells (HGECs) and vascular endothelial-like cells (VEC-like cells). METHOD: Haematoxylin and Eosin (H&E) staining, immunohistochemical, immunofluorescence, 5-ethynyl-2'-deoxyuridine (EdU) staining and scanning electron microscope (SEM) were performed to detect the growth status of cells on the scaffold complex. After the scaffold complex implanted into nude mice for 28 days, tissues were harvested to observe the cell viability and morphology by the same method as above. Additionally, biomechanical experiments were used to assess the stability of composite scaffold. RESULTS: Immunofluorescence and Immunohistochemistry showed positive expression of Vimentin, S100A4 and CK, and the induced VEC-like cells had the ability to form tubule-like structures. In vitro observation results showed that HGFs, HGECs and VEC-like had good compatibility with ACVM-0.25% HLC-I and could be layered and grow in the scaffold. After implanted, the mice had no immune rejection and no obvious scar repair on the body surface. The biomfechanical test results showed that the composite scaffold has strong stability. CONCLUSION: The tissue-engineered vascularised complexes constructed by HGFs, HGECs, VEC-like cells and ACVM-0.25% HLC-I has good biocompatibility and considerable strength.

Modulation of proliferation and differentiation of gingiva­derived mesenchymal stem cells by concentrated growth factors: Potential implications in tissue engineering for dental regeneration and repair.

The aim of the present study was to evaluate the proliferation and osteogenic differentiation ability of gingiva­derived mesenchymal stem cells (GMSCs) cultured with different concentrations of concentrated growth factors (CGF). GMSCs were isolated from gingival connective tissues and characterized by flow cytometry, immunofluorescence staining and immunohistochemical staining. Cell proliferation activity was determined by the MTT assay, and the effect of CGF on MCSCs was detected with the Cell Counting Kit (CCK)­8 assay. Mineralization induction was evaluated by alkaline phosphatase (ALP)­positive cell staining and mineralized nodule formation assay. Dentin matrix acidic phosphoprotein (DMP)1, dentin sialophosphoprotein (DSPP), bone morphogenetic protein (BMP)2 and runt­related transcription factor (RUNX)2 mRNA and protein expression were evaluated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis and western blotting. The flow cytometry, immunofluorescence staining and immunohistochemical staining results indicated that the cultured cells were GMSCs. The MTT assay results revealed that the third­generation gingival stem cells exhibited the highest proliferative capacity, and the CCK­8 results indicated that 10% CGF achieved the most prominent promotion of GMSC proliferation. ALP activity analysis and mineralized nodule assay demonstrated that CGF may successfully induce osteogenic differentiation of GMSCs, whereas RT­qPCR and western blot analyses demonstrated that CGF is involved in the differentiation of GMSCs by regulating the expression of DMP1, DSPP, BMP2 and RUNX2 (P<0.05). In conclusion, CGF were demonstrated to promote the proliferation and osteogenic differentiation of GMSCs. Therefore, CGF may be applied in tissue engineering for tooth regeneration and repair.

Characterization of different biodegradable scaffolds in tissue engineering.

The aim of the present study was to compare the characteristics of acellular dermal matrix (ADM), small intestinal submucosa (SIS) and Bio­Gide scaffolds with acellular vascular matrix (ACVM)­0.25% human­like collagen I (HLC­I) scaffold in tissue engineering blood vessels. The ACVM­0.25% HLC­I scaffold was prepared and compared with ADM, SIS and Bio­Gide scaffolds via hematoxylin and eosin (H&E) staining, Masson staining and scanning electron microscope (SEM) observations. Primary human gingival fibroblasts (HGFs) were cultured and identified. Then, the experiment was established via the seeding of HGFs on different scaffolds for 1, 4 and 7 days. The compatibility of four different scaffolds with HGFs was evaluated by H&E staining, SEM observation and Cell Counting Kit­8 assay. Then, a series of experiments were conducted to evaluate water absorption capacities, mechanical abilities, the ultra­microstructure and the cytotoxicity of the four scaffolds. The ACVM­0.25% HLC­I scaffold was revealed to exhibit the best cell proliferation and good cell architecture. ADM and Bio­Gide scaffolds exhibited good mechanical stability but cell proliferation was reduced when compared with the ACVM­0.25% HLC­I scaffold. In addition, SIS scaffolds exhibited the worst cell proliferation. The ACVM­0.25% HLC­I scaffold exhibited the best water absorption, followed by the SIS and Bio­Gide scaffolds, and then the ADM scaffold. In conclusion, the ACVM­0.25% HLC­I scaffold has good mechanical properties as a tissue engineering scaffold and the present results suggest that it has better biological characterization when compared with other scaffold types.

Study of composite vascular scaffold combining with differentiated VSMC- and VEC-like cells in vitro and in vivo.

Although research into the tissue engineering of vessels has proceeded at a tremendous pace, many deficiencies still need to be resolved. A well-adopted constructed vessel requires both functional and structural properties to stimulate the native vessel and resist stress and tension in vivo. In the present study, we developed a novel three-layer composite vascular scaffold consisting of differentiated vascular smooth muscle cell-, vascular endothelial cell-like cells, and a rabbit acellular vascular matrix (ACVM)-0.25% HLC-I scaffold. HE staining, immunohistochemical assays, immunofluorescence assays (IFAs), and scanning electron microscopy were performed to monitor the growth status of cells on the scaffold material in vitro. After the vascular endothelial cell -vascular smooth muscle cell-scaffold was implanted into nude mice for three, six, and nine weeks, samples were harvested from the implanted mice and observed visually or by HE staining and IFAs for cell viability and morphology. Additionally, burst pressure resistance experiments were used to assess the maximal pressure that the engineered vessel could resist. We found that the engineered vascular endothelial cell-vascular smooth muscle cell-scaffold vessel possessed favorable biocompatibility and considerable strength, matching native vessels in vivo and in vitro, and may be significant in the future clinical implantation of tissue-engineered vasculature.