Development of a lip vermilion epithelium reconstruction model using keratinocytes from skin and oral mucosa.

Lip vermilion is unique and can be distinguished from the adjacent skin and oral mucosa. However, because of the lack of appropriate evaluation tools, skin and/or oral mucosa substitutes such as in vitro vermilion epithelial models have been used for lip product testing. We aimed to develop and characterize a lip vermilion epithelium reconstruction model (LVERM) using skin and oral keratinocytes. LVERM was manufactured by co-culturing primary skin and oral keratinocytes, using a device that allowed the separation of cell seeding, and created an intercalated cell-free zone, referred to as the vermilion part. After removing the device, LVERM construction was completed in 8 days, in a submerged condition. Subsequently, they were placed in an air-liquid interface for 7 days. To determine the epithelial characteristics of LVERM, keratin 2e (KRT2) and small proline-rich protein 3 (SPRR3) expression patterns were examined. The in vivo expression profiles of KRT2 and SPRR3 genes in vermilion were also examined. We found that a continuous multi-layered epithelium was generated in the LVERM that exhibited ortho- and para-keratinization in the skin and oral mucosa parts, respectively. Although an intermediate keratinization pattern was observed in the vermilion part, KRT2 and SPRR3 were co-expressed in the suprabasal layer, consistent with the expression pattern of a single vermilion epithelial model. Clustering analysis revealed that KRT2 and SPRR3 gene expression in vermilion was location-dependent within the sample. Therefore, LVERM can be used as an evaluation tool for lip products and has great importance in innovative approaches for cosmetic testing.

Cholesterol Is a Regulator of CAV1 Localization and Cell Migration in Oral Squamous Cell Carcinoma.

Cholesterol plays an important role in cancer progression, as it is utilized in membrane biogenesis and cell signaling. Cholesterol-lowering drugs have exhibited tumor-suppressive effects in oral squamous cell carcinoma (OSCC), suggesting that cholesterol is also essential in OSCC pathogenesis. However, the direct effects of cholesterol on OSCC cells remain unclear. Here, we investigated the role of cholesterol in OSCC with respect to caveolin-1 (CAV1), a cholesterol-binding protein involved in intracellular cholesterol transport. Cholesterol levels in OSCC cell lines were depleted using methyl-ß-cyclodextrin and increased using the methyl-ß-cyclodextrin-cholesterol complex. Functional analysis was performed using timelapse imaging, and CAV1 expression in cholesterol-manipulated cells was investigated using immunofluorescence and immunoblotting assays. CAV1 immunohistochemistry was performed on surgical OSCC samples. We observed that cholesterol addition induced polarized cell morphology, along with CAV1 localization at the trailing edge, and promoted cell migration. Moreover, CAV1 was upregulated in the lipid rafts and formed aggregates in the plasma membrane in cholesterol-added cells. High membranous CAV1 expression in tissue specimens was associated with OSCC recurrence. Therefore, cholesterol promotes the migration of OSCC cells by regulating cell polarity and CAV1 localization to the lipid raft. Furthermore, membranous CAV1 expression is a potential prognostic marker for OSCC patients.

HEATR1, a novel interactor of Pontin/Reptin, stabilizes Pontin/Reptin and promotes cell proliferation of oral squamous cell carcinoma.

Pontin and Reptin are closely related proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They form a hetero-oligomeric complex, Pontin/Reptin, which is involved in protein stability and assembly of the protein complexes as a molecular chaperone. Overexpression of Pontin and Reptin in tumor cells has been reported and is implicated in the development of various cancers. However, the molecular mechanism of Pontin/Reptin function in oral squamous cell carcinoma (OSCC) development remains unclear. Here, we identify HEAT repeat-containing protein 1 (HEATR1) as a novel binding factor of Pontin/Reptin. Functionally, HEATR1 stabilizes Pontin/Reptin and positively regulates OSCC cell proliferation by activating mTOR and pre-rRNA synthesis. We also find that HEATR1 expression is markedly upregulated in tumor region of OSCC tissue. Hence, we propose that HEATR1 is involved in the regulation of mTOR and ribosome biogenesis as a potential protein stabilizer of Pontin/Reptin in OSCC.

Identification and characterization of R2TP in the development of oral squamous cell carcinoma.

R2TP is a well-conserved molecular chaperone complex, composed of Pontin, Reptin, RPAP3, and PIH1D, in eukaryotes. Recent studies have suggested an involvement of R2TP in cancer development. However, it remains unclear if it is related to the development of oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer. Here, we identify and investigate the function of R2TP in OSCC development. Immunohistochemical analysis reveals that all of the R2TP components are strongly expressed in normal oral epithelia and OSCC tissues, where actively proliferating cells are abundant. Co-immunoprecipitation assay identifies that R2TP components form a protein complex in OSCC-derived HSC4-cells. Knockdown experiments show that all R2TP components, except for RPAP3, are required for the cell proliferation and migration of HSC-4 cells. Furthermore, we reveal that Pontin contributes to a gain-of-function (GOF) activity of mutp53-R248Q in HSC-4 cells by regulating phosphorylation levels of mutp53 at Ser15 and Ser46. To our knowledge, this study is the first to report the functional involvement of R2TP and its components in the malignant characteristics of OSCC cells.

Rac1-dependent phagocytosis of apoptotic cells by oral squamous cell carcinoma cells: A possible driving force for tumor progression.

Apoptotic cell death frequently occurs in human cancer tissues including oral squamous cell carcinoma (SCC), wherein apoptotic tumor cells are phagocytosed not only by macrophages but also by neighboring tumor cells. We previously reported that the engulfment of apoptotic SCC cells by neighboring SCC cells frequently occurs at the invading front. Therefore, we hypothesized that the phagocytosis of these apoptotic cells by tumor cells contributes to disease progression. Herein, using cultured oral SCC cells, we aimed to confirm whether tumor cells actually phagocytose apoptotic cells and to examine whether cellular activities are regulated by the phagocytosis of apoptotic cells. Co-culture experiments showed that living cells could ingest apoptotic cells into phagolysosomes. NSC23766, an inhibitor of Rac1, which is a key regulator of phagocytic cup formation in professional phagocytes, dramatically suppressed the phagocytosis of apoptotic cells by living cells. Additionally, cell migration and the secretion of DKK1, a tumor-promoting protein, were enhanced by co-culture with apoptotic cells, whereas NSC23766 inhibited these effects. These results show that tumor cells can actively phagocytose apoptotic neighbors in a Rac1-dependent manner and that such activity increases their migration. The regulation of apoptotic cell phagocytosis thus represents new directions for therapeutic intervention for oral cancer.

ROCK inhibitors enhance bone healing by promoting osteoclastic and osteoblastic differentiation.

Osteoclast and osteoblast are essential for proper bone development and remodeling as well as recovery of bone fracture. In this study, we seek chemical compounds that enhance turnover of bone metabolism for promoting bone healing. First, we screen a chemical library which includes 378 compounds by using murine pre-osteoclastic RAW264.7 cells to identify compounds that promote osteoclastic differentiation. We find that two ROCK (Rho-associated coiled-coil kinase) inhibitors, HA-1077 (Fasudil) and Y-27632, enhance osteoclastogenesis. Subsequently, we identify that these two compounds also increase osteoblastic differentiation of MC3T3-E1 cells. Finally, our in vivo experiment shows that the local administration of ROCK inhibitors accelerate the bone healing of the rat calvarial defect.

Higher Accumulation of Docosahexaenoic Acid in the Vermilion of the Human Lip than in the Skin.

The vermilion of the human lip is a unique facial area because of certain distinguishing features from the adjacent tissues such as the white lip (skin) and oral mucosa. However, the distinction in terms of molecular distribution between the vermilion and skin has remained unexplored. Therefore, we aimed to map the human lip by mass spectrometry imaging to gain understanding of the free fatty acid distribution in the vermilion. The lip specimens trimmed off during cheiloplasty were analyzed using desorption electrospray ionization-mass spectrometry imaging. Distributions of two monounsaturated fatty acids and three polyunsaturated fatty acids were observed in the human lip tissue: palmitoleic acid (POA) and oleic acid (OA) and linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA), respectively. Although POA, OA, LA, and AA were differentially distributed across the vermilion and skin, DHA showed a higher accumulation in the epithelium of the vermilion compared to that in the skin. Our results clearly demonstrated the difference in fatty acid distributions between the vermilion and skin. The highly abundant DHA in the epithelium of the vermilion may have an antioxidant role and may thus protect the lip from aging. Our findings can provide a novel strategy for treating lip disorders.

Underlying causes of Eurasian midcontinental aridity in simulations of mid-Holocene climate.

Climate model simulations uniformly show drier and warmer summers in the Eurasian midcontinent during the mid-Holocene, which is not consistent with paleoenvironmental observations. The simulated climate results from a reduction in the zonal temperature gradient, which weakens westerly flow and reduces moisture flux and precipitation in the midcontinent. As a result, sensible heating is favored over evaporation and latent heating, resulting in substantial surface-driven atmospheric warming. Thus, the discrepancy with the paleoenvironmental evidence arises initially from a problem in the simulated circulation and is exacerbated by feedback from the land surface. This region is also drier and warmer than indicated by observations in the preindustrial control simulations, and this bias arises in the same way: zonal flow and hence moisture flux into the midcontinent are too weak, and feedback from the land surface results in surface-driven warming. These analyses suggest the need to improve those aspects of climate models that affect the strength of westerly circulation.

Effects of C-xylopyranoside derivative on epithelial regeneration in an in vitro 3D oral mucosa model.

Identifying substandard tissue-engineered oral mucosa grafts with a poor epithelium before clinical use is critical to ensure quality assurance/control in regenerative medicine, leading to success of grafting. This study investigated the effects of one of the C-xylopyranoside derivatives, ß-D-xylopyranoside-n-propane-2-one (XPP), on oral epithelial regeneration. Using a three-dimensional oral mucosa model, we analyzed changes of the epithelial structure, glycosaminoglycan (GAG) synthesis, the expression levels of basement membrane zone markers, and substrates of Akt/mTOR signaling. Compared with the control, 2 mM XPP treatment increased the mean and minimal epithelial thickness, and reduced the variation of epithelial thickness. It also stimulated expressions of decorin and syndecan-1 with change of GAG amount and/or composition, and enhanced the expressions of integrin α6, CD44, and Akt/mTOR signaling substrates. These findings suggest that XPP supplementation contributes to consistent epithelial regeneration. Moreover, upregulation of those markers may play a role in increasing the quality of the oral mucosal epithelium.

Hypoxia induces an undifferentiated phenotype of oral keratinocytes in vitro.

The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G0/G1 phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated ß-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75(NTR) and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G0/G1 phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer ß-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

Downregulation of SPARC Expression Enhances the Fusion of BeWo Choriocarcinoma Cells.

Syncytiotrophoblasts, which are formed by the fusion of villous cytotrophoblasts, play an essential role in maintaining a successful pregnancy. Secreted protein acidic and rich in cysteine (SPARC) is a non-structural Ca2+-binding extracellular matrix glycoprotein involved in tissue remodeling and cell proliferation, differentiation, and migration. Previous studies have revealed that SPARC is expressed in villous and extravillous cytotrophoblasts in the first trimester and that RNA interference targeted at SPARC significantly inhibited invasion of human extravillous trophoblast HTR8/SVneo cells. However, the involvement of SPARC in cytotrophoblast fusion remains unknown. This study aimed to investigate the role of SPARC in cytotrophoblast fusion, using the BeWo choriocarcinoma cell line as a model of villous cytotrophoblasts. Immunohistochemical analysis was conducted to assess SPARC expression in normal human placentas using placental tissues obtained during the first and third trimesters of pregnancy. We investigated the effects of SPARC knockdown on trophoblast differentiation markers and cell fusion in BeWo cells using small interfering RNA. Immunohistochemical analysis revealed that SPARC expression was high in the early gestational chorionic villi and low in the late gestational chorionic villi. SPARC knockdown increased the expressions of human chorionic gonadotropin and Ovo-like transcriptional repressor 1; however, glial cells missing transcription factor 1, syncytin-1, and syncytin-2 showed no significant changes. The assessment revealed that SPARC knockdown significantly enhanced cell fusion compared to the non-silencing control. Our data suggest that SPARC plays a vital role in regulating trophoblast fusion and differentiation during placental development.

Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

The effects of carbon-ion beam irradiation on three-dimensional in vitro models of normal oral mucosa and oral cancer: development of a novel tool to evaluate cancer therapy.

Given that the original tumor microenvironment of oral cancer cannot be reproduced, predicting the therapeutic effects of irradiation using monolayer cultures and animal models of ectopic tumors is challenging. Unique properties of carbon-ion irradiation (CIR) characterized by the Bragg peak exert therapeutic effects on tumors and prevent adverse events in surrounding normal tissues. However, the underlying mechanism remains unclear. The biological effects of CIR were evaluated on three-dimensional (3D) in vitro models of normal oral mucosa (NOMM) and oral cancer (OCM3 and OCM4) consisting of HSC-3 and HSC-4 cells. A single 10- or 20-Gy dose of CIR was delivered to NOMM, OCM3, and OCM4 models. Histopathological and histomorphometric analyses and labeling indices for Ki-67, γH2AX, and TUNEL were examined after CIR. The concentrations of high mobility group box 1 (HMGB1) were measured. NOMM exhibited epithelial thinning after CIR, which could be caused by the decreased presence of Ki-67-labeled basal cells. The relative proportion of the thickness of cancer cells to the underlying stroma in cancer models decreased after CIR. This finding appeared to be supported by changes in the three labeling indices, indicating CIR-induced cancer cell death, mostly via apoptosis. Furthermore, the three indices and the HMGB1 release levels significantly differed among the OCM4 that received different doses and with different incubation times after CIR while those of the OCM3 models did not, suggesting more radiosensitivity in the OCM4. The three 3D in vitro models can be a feasible and novel tool to elucidate radiation biology.

Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology.

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.

Development of the Follow-Up Human 3D Oral Cancer Model in Cancer Treatment.

As function preservation cancer therapy, targeted radiation therapies have been developed for the quality of life of cancer patients. However, preclinical animal studies evaluating the safety and efficacy of targeted radiation therapy is challenging from the viewpoints of animal welfare and animal protection, as well as the management of animal in radiation-controlled areas under the regulations. We fabricated the human 3D oral cancer model that considers the time axis of the follow up in cancer treatment. Therefore, in this study, the 3D model with human oral cancer cells and normal oral fibroblasts was treated based on clinical protocol. After cancer treatment, the histological findings of the 3D oral cancer model indicated the clinical correlation between tumor response and surrounding normal tissue. This 3D model has potential as a tool for preclinical studies alternative to animal studies.

Predictive Ability of the Combination of White Blood Cell and C Reactive Protein Levels for Infections Following Laparoscopic Hysterectomy.

OBJECTIVE: In the present retrospective study, we evaluated potentially predictive factors and determined receiver operating characteristic (ROC) curve cut-off values for effective detection of patients at higher risk of re-hospitalization due to postoperative infection after total laparoscopic hysterectomy. MATERIALS AND METHODS: We included 168 patients who had undergone laparoscopic hysterectomy. Data were collected from medical records. Patients were classified into non-infection (n = 161) and infection (n = 7) groups based on whether they developed fever after hospital discharge. We evaluated factors conventionally known to affect postoperative infection in general, and values of white blood cell (WBC) and C-reactive protein (CRP) determined on postoperative days 1 and 3-5. RESULTS: There were significant differences in WBC 3-5 days postoperatively (WBC POD3-5) (p = 0.049), CRP 3-5 days postoperatively (CRP POD3-5) (p = 0.018) and CRP POD3-5 × WBC 1 day postoperatively (WBC POD1) (p = 0.002). Area under the ROC curves for CRP POD3-5 and CRP POD3-5 × WBC POD1 were 0.81 and 0.84, and cutoffs were 4.46 mg/dL and 46885.5, respectively. CONCLUSION: If CRP POD3-5 or CRP POD3-5 × WBC POD1 is high, the physician should be alert to postoperative infection, and the patient should be under careful management and supervision.

Recent trends and perspectives in reconstruction and regeneration of intra/extra-oral wounds using tissue-engineered oral mucosa equivalents.

Many conditions, including cancer, trauma, and congenital anomalies, can damage the oral mucosa. Multiple cultures of oral mucosal cells have been used for biocompatibility tests and oral biology studies. In recent decades, the clinical translation of tissue-engineered products has progressed significantly in developing tangible therapies and inspiring advancements in medical science. However, the reconstruction of an intraoral mucosa defect remains a significant challenge. Despite the drawbacks of donor-site morbidity and limited tissue supply, the use of autologous oral mucosa remains the gold standard for oral mucosa reconstruction and repair. Tissue engineering offers a promising solution for repairing and reconstructing oral mucosa tissues. Cell- and scaffold-based tissue engineering approaches have been employed to treat various soft tissue defects, suggesting the potential clinical use of tissue-engineered oral mucosa (TEOMs). In this review, we first cover the recent trends in the reconstruction and regeneration of extra-/intra-oral wounds using TEOMs. Next, we describe the current status and challenges of TEOMs. Finally, future strategic approaches and potential technologies to support the advancement of TEOMs for clinical use are discussed.

The EGF/EGFR axis and its downstream signaling pathways regulate the motility and proliferation of cultured oral keratinocytes.

We previously reported that the cell and colony motion of oral keratinocytes are correlated with proliferative capacity, and speculated that this may be a specific index for monitoring cell quality. However, how cell motility and proliferation are regulated by signaling pathways remains unelucidated. Here, we found that the regulation of cell motility and proliferative capacity of oral keratinocytes can be attributed to the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis. The EGFR downstream cascade involving the Src/PI3K/Akt/mTOR signaling pathway showed a major effect on cell motility and proliferative capacity in oral keratinocytes. Furthermore, both EGFR and Src attenuated E-cadherin expression. Taken together, these findings provide a potential basis for future quality control of cells for therapeutic use.

Adhesion Characteristics of Human Pancreatic Islets, Duct Epithelial Cells, and Acinar Cells to a Polymer Scaffold.

We reported in 2018 that among several extracellular matrices, fibronectin, type I collagen, type IV collagen, laminin I, fibrinogen, and bovine serum albumin, fibronectin is particularly useful for adhesion of porcine pancreatic tissue. Subsequently, we developed a technology that enables the chemical coating of the constituent motifs of fibronectin onto cell culture dishes. In this experiment, we used islets (purity ≥ 90%), duct epithelial cells (purity ≥ 60%), and acinar cells (purity ≥ 99%) isolated from human pancreas according to the Edmonton protocol published in 2000 and achieved adhesion to the constituent motifs of fibronectin. A solution including cGMP Prodo Islet Media was used as the assay solution. In islets, adhesion was enhanced with the constitutive motifs of fibronectin compared with uncoated islets. In the functional evaluation of islets, insulin mRNA expression and insulin secretion were enhanced by the constitutive motif of fibronectin compared with non-coated islets. The stimulation index was comparable between non-coated islets and fibronectin motifs. In duct epithelial cells, adhesion was mildly promoted by the fibronectin component compared with non-coated component, while in acinar cells, adhesion was inhibited by the fibronectin component compared with the non-coated component. These data suggest that the constitutive motifs of fibronectin are useful for the adhesion of islets and duct epithelial cells.

A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture.

Decellularized xenogeneic vascular grafts can be used in revascularization surgeries. We have developed decellularization methods using high hydrostatic pressure (HHP), which preserves the extracellular structure. Here, we attempted ex vivo endothelialization of HHP-decellularized xenogeneic tissues using human endothelial cells (ECs) to prevent clot formation against human blood. Slices of porcine aortic endothelium were decellularized using HHP and coated with gelatin. Human umbilical vein ECs were directly seeded and cultured under dynamic flow or static conditions for 14 days. Dynamic flow cultures tend to demonstrate higher cell coverage. We then coated the tissues with the E8 fragment of human laminin-411 (hL411), which has high affinity for ECs, and found that Dynamic/hL411showed high area coverage, almost reaching 100% (Dynamic/Gelatin vs Dynamic/hL411; 58.7 ± 11.4 vs 97.5 ± 1.9%, P = 0.0017). Immunostaining revealed sufficient endothelial cell coverage as a single cell layer in Dynamic/hL411. A clot formation assay using human whole blood showed low clot formation in Dynamic/hL411, almost similar to that in the negative control, polytetrafluoroethylene. Surface modification of HHP-decellularized xenogeneic endothelial tissues combined with dynamic culture achieved sufficient ex vivo endothelialization along with prevention of clot formation, indicating their potential for clinical use as vascular grafts in the future.