Polyphenols in bee products and prevention of cell senescence.

Sustaining the continuity of cells and their homeostasis throughout the lifespan is compulsory for the survival of an organism. Cellular senescence is one of mechanisms involved in cell homeostasis and survival, and plays both important and detrimental roles in the maintenance of malfunctioned and normal cells. However, when exposed to various insults (genetic, metabolic and environmental), the cells undergo oxidative stress which may induce premature senescence, or so-called stress-induced premature senescence. Many age-related diseases are associated with premature senescence. Hence, there is growing interest in the intake of natural sources such as dietary food, which has protective functions on human health and diseases as well as on premature senescence. There are many natural food sources which have beneficial effects on delaying cell senescence, of which bee products are one of them. Bee products (honey, propolis, royal jelly, bee pollen, bee bread, venom and wax) are rich in polyphenols, a compound that exerts powerful antioxidant actions against oxidative stress and is able to delay premature senescence that is linked to ageing. This review describes the factors triggering senescence, the biomarkers involved and the prevention of senescence by the polyphenols present in bee products. Thus, it is hoped that this will provide new insights into the clinical management of age-related diseases.

Team Science Approaches to Unravel Monogenic Parkinson's Disease on a Global Scale.

BACKGROUND: Until recently, about three-quarters of all monogenic Parkinson's disease (PD) studies were performed in European/White ancestry, thereby severely limiting our insights into genotype-phenotype relationships at a global scale. OBJECTIVE: To identify the multi-ancestry spectrum of monogenic PD. METHODS: The first systematic approach to embrace monogenic PD worldwide, The Michael J. Fox Foundation Global Monogenic PD Project, contacted authors of publications reporting individuals carrying pathogenic variants in known PD-causing genes. In contrast, the Global Parkinson's Genetics Program's Monogenic Network took a different approach by targeting PD centers underrepresented or not yet represented in the medical literature. RESULTS: In this article, we describe combining both efforts in a merger project resulting in a global monogenic PD cohort with the buildup of a sustainable infrastructure to identify the multi-ancestry spectrum of monogenic PD and enable studies of factors modifying penetrance and expressivity of monogenic PD. CONCLUSIONS: This effort demonstrates the value of future research based on team science approaches to generate comprehensive and globally relevant results. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Understanding monogenic Parkinson's disease at a global scale.

Until recently, about three-quarters of all monogenic Parkinson's disease (PD) studies were performed in European/White ancestry, thereby severely limiting our insights into genotype-phenotype relationships at global scale. The first systematic approach to embrace monogenic PD worldwide, The Michael J. Fox Foundation Global Monogenic PD (MJFF GMPD) Project, contacted authors of publications reporting individuals carrying pathogenic variants in known PD-causing genes. In contrast, the Global Parkinson's Genetics Program's (GP2) Monogenic Network took a different approach by targeting PD centers not yet represented in the medical literature. Here, we describe combining both efforts in a "merger project" resulting in a global monogenic PD cohort with build-up of a sustainable infrastructure to identify the multi-ancestry spectrum of monogenic PD and enable studies of factors modifying penetrance and expression of monogenic PD. This effort demonstrates the value of future research based on team science approaches to generate comprehensive and globally relevant results.

Loss-of-Function Variant in the SMPD1 Gene in Progressive Supranuclear Palsy-Richardson Syndrome Patients of Chinese Ancestry.

Lysosomal dysfunction plays an important role in neurodegenerative diseases, including Parkinson's disease (PD) and possibly Parkinson-plus syndromes such as progressive supranuclear palsy (PSP). This role is exemplified by the involvement of variants in the GBA1 gene, which results in a deficiency of the lysosomal enzyme glucocerebrosidase and is the most frequently identified genetic factor underlying PD worldwide. Pathogenic variants in the SMPD1 gene are a recessive cause of Niemann-Pick disease types A and B. Here, we provide the first report on an association between a loss-of-function variant in the SMPD1 gene present in a heterozygous state (p.Pro332Arg/p.P332R, which is known to result in reduced lysosomal acid sphingomyelinase activity), with PSP-Richardson syndrome in three unrelated patients of Chinese ancestry.

Correction: Evaluation of the osteogenic potential of demineralized and decellularized bovine bone granules following implantation in rat calvaria critical-size defect model.

[This corrects the article DOI: 10.1371/journal.pone.0294291.].

Exploring the Properties of Deer Antler Velvet (DAV) and its Potential Use in Dentistry: A Narrative Review

@#For centuries, deer antler velvet (DAV) has been a staple in traditional medicine due to its numerous health benefits, including immune stimulation, anti-ageing, wound healing, and anti-osteoporosis properties. Researchers attribute the therapeutic advantages of DAV to various compounds in its extract, ranging from growth hormones to minerals and amino acids. Recently, there has been renewed interest in these natural products for their potential use in treating chronic diseases such as bone health and development, bacterial infections, and sports supplementation to increase athletic performance. The available literature supports that DAV significantly affects bone development, chondrogenesis, and antimicrobial properties, making it a valuable resource in medical and dental settings. This review aims to provide an in-depth overview of the potential applications of DAV in modern medicine and dentistry.

Modulation Agents of Wound Healing in Ocular Surgeries

@#Wound healing is a complex process that includes haemostasis and inflammation, followed by a proliferation period and repair and finally remodelling. Ocular surgeries, particularly in glaucoma cases, aim at minimal fibrosis to preserve the function of trabeculectomy as an alternative pathway for aqueous drainage. Hence, it is important to find an agent to modulate the wound healing process. This review presents compilation of wound modulation agents that have been tested in vitro, in vivo, or clinically on patients undergoing ocular surgeries, particularly for glaucoma. We identified agents into four groups, mostly for glaucoma filtration operations: anti-metabolites, anti-growth factors, mechanical barriers and rho kinases. The effect of these agents is highlighted in this review. In conclusion, despite recognized drawbacks of antimetabolites, they are still regarded as the gold standard and the most efficient treatment as anti-scarring agents use in ocular surgeries. More studies are needed to inquire agents that efficient yet has minimal adverse effects both in short and long term.

Loss-of-Function Variant in the SMPD1 Gene in Progressive Supranuclear Palsy-Richardson Syndrome Patients of Chinese Ancestry

Lysosomal dysfunction plays an important role in neurodegenerative diseases, including Parkinson’s disease (PD) and possibly Parkinson-plus syndromes such as progressive supranuclear palsy (PSP). This role is exemplified by the involvement of variants in the GBA1 gene, which results in a deficiency of the lysosomal enzyme glucocerebrosidase and is the most frequently identified genetic factor underlying PD worldwide. Pathogenic variants in the SMPD1 gene are a recessive cause of Niemann–Pick disease types A and B. Here, we provide the first report on an association between a loss-of-function variant in the SMPD1 gene present in a heterozygous state (p.Pro332Arg/p.P332R, which is known to result in reduced lysosomal acid sphingomyelinase activity), with PSP-Richardson syndrome in three unrelated patients of Chinese ancestry.

Evaluation of the osteogenic potential of demineralized and decellularized bovine bone granules following implantation in rat calvaria critical-size defect model.

The aim of this study was to compare the ability of demineralized (DMB) and decellularized (DCC) bovine bone granules to support bone regeneration in rat calvaria critical-size defects. DMB and DCC were prepared using a previously published method. The granule size used ranged between 500 and 750 µm. A total of forty-eight Sprague-Dawley rats were divided into two groups (n = 24). A pair of 5 mm diameter defects were created on the calvaria of the rats in the right and left parietal bone in both groups. Group A animals received DMB granules and Group B received DCC granules in the right parietal defect side while the left parietal untreated defect acted as sham surgery for both groups. Four animals per group were euthanized in a CO2 chamber at day 7, 14 and 21 post-surgery and the calvaria implantation site biopsy harvested was subjected to osteogenic gene expression analysis. Another four animals per group were euthanized at days 15, 30 and 60 post surgery and the calvaria implantation site biopsy harvested was subjected to histological, immunohistochemistry, RAMAN spectroscopy and Micro-CT analysis at the mentioned time points. Statistical analysis was conducted using t-tests and ANOVA. Histomorphometry showed significantly higher new bone formation in the DCC sites (p<0.05) compared to DMB. Both DMB and DCC implantation sites showed distinct staining for osteocalcin and osteopontin proteins compared to their respective sham sites. By day 21 after implantation, DCC sites demonstrated significantly elevated mRNA levels of osteonectin (p<0.001), osteopontin (p<0.001), osteocalcin (p<0.0001), ALP (p<0.01), and BMP-2 (p<0.001) compared to DMB. However, VEGF expression showed no significant differences at this time point between the two groups. Micro-CT analysis also showed enhanced defect closure and higher bone density in DCC implanted sites while RAMAN spectra demonstrated increased abundance of collagen and bone minerals, especially, PO43- ions than DMB. In conclusion, both DMB and DCC granules demonstrated favorable osteogenic potential in critical-sized defects, with DCC exhibited superior osteoconductive, osteoinductive and osteogenesis properties.

Evaluation of decellularization process for developing osteogenic bovine cancellous bone scaffolds in-vitro.

Current immunological issues in bone grafting regarding the transfer of xenogeneic donor bone cells into the recipient are challenging the industry to produce safer acellular natural matrices for bone regeneration. The aim of this study was to investigate the efficacy of a novel decellularization technique for producing bovine cancellous bone scaffold and compare its physicochemical, mechanical, and biological characteristics with demineralized cancellous bone scaffold in an in-vitro study. Cancellous bone blocks were harvested from a bovine femoral head (18-24 months old) subjected to physical cleansing and chemical defatting, and further processed in two ways. Group I was subjected to demineralization, while Group II underwent decellularization through physical, chemical, and enzymatic treatments. Both were then freeze-dried, and gamma radiated, finally producing a demineralized bovine cancellous bone (DMB) scaffold and decellularized bovine cancellous bone (DCC) scaffold. Both DMB and DCC scaffolds were subjected to histological evaluation, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), fourier-transform infrared spectroscopy (FTIR), quantification of lipid, collagen, and residual nucleic acid content, and mechanical testing. The osteogenic potential was investigated through the recellularization of scaffolds with human osteoblast cell seeding and examined for cell attachment, proliferation, and mineralization by Alizarin staining and gene expression. DCC produced a complete acellular extracellular matrix (ECM) with the absence of nucleic acid content, wider pores with extensive interconnectivity and partially retaining collagen fibrils. DCC demonstrated a higher cell proliferation rate, upregulation of osteogenic differentiation markers, and substantial mineralized nodules production. Our findings suggest that the decellularization technique produced an acellular DCC scaffold with minimal damage to ECM and possesses osteogenic potential through the mechanisms of osteoconduction, osteoinduction, and osteogenesis in-vitro.

Salivary aquaporin-3 as a screening biomarker for xerostomia in patients with periodontal disease and the effects of xerostomia on oral health-related quality of life.

Xerostomia is a subjective condition of dryness of the oral cavity that may lead to several oral problems deteriorating oral health-related quality of life. This study aimed to (1) determine the prevalence of xerostomia, (2) compare the general health status, unstimulated salivary flow rate, and oral health-related quality of life in xerostomics and non-xerostomics, and (3) investigate the potential of salivary aquaporin-3 (AQP-3) as a screening biomarker for xerostomia in patients with periodontal disease. Demographics and systemic health data were collected from 109 healthy participants, 20 to 55 years old, with Community Periodontal Index (CPI) score ≥ 3. For subjective assessment of xerostomia, Shortened Xerostomia Inventory (SXI) was used. For objective assessment of xerostomia, unstimulated salivary flow rate was measured. Shortened Oral Health Impact Profile (S-OHIP) was utilized for oral health-related quality of life assessment. The collected saliva samples were processed and stored at -80°C. Quantification of salivary AQP-3 protein was done with enzyme-linked immunosorbent assay. Xerostomia was reported in 78% of the subjects based on SXI score. Median concentration of AQP-3 was significantly higher in xerostomics compared to non-xerostomics, p = 0.001. Moreover, oral health-related quality of life was significantly poor in xerostomics compared to non-xerostomics, p = 0.002. Furthermore, there were significant correlations between AQP-3 and SXI (r = 0.21, p = 0.025), AQP-3 and S-OHIP (r = 0.2, p = 0.042), S-OHIP and SXI (r = 0.37, p < 0.001), unstimulated salivary flow rate and random blood glucose level (r = 0.32, p = 0.001), and body mass index and mean arterial pressure (r = 0.44, p < 0.001). Regression analysis showed that body mass index, CPI score 3, and salivary AQP-3 were suitable predictors for presence of xerostomia. AQP-3 could be a potential screening biomarker for xerostomia in patients with periodontal disease for its early identification may help improve oral health-related quality of life of the individuals.

Dental Anomalies and Muscle Segment Homeobox1 Gene Polymorphism in Nonsyndromic Cleft Lip with or without Palate Children

@#This study aims to determine the prevalence of dental anomalies and MSX1 gene 799G>T polymorphism and its association with non-syndromic cleft lip with or without palate (NSCLP) attending Hospital Universiti Sains Malaysia. Clinical and radiological assessments on 37 NSCL±P patients and 80 non-cleft children were done to detect dental anomalies. The buccal cells were collected and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was used to identify polymorphism. NSCL±P was higher among males (54%) and mostly unilateral cleft lip and palate (51.3%). The prevalence of dental anomalies in morphology in NSCL±P was 18.9% (95% CI: 5.7%, 32.2%) and non-cleft was 6.3% (95% CI: 0.8%, 11.7%). Hypodontia in NSCLP was 75% (95% CI: 61.2%, 90.2%) and non-cleft was 7.5% (95% CI: 1.6%, 13.4%). There was a significant association between NSCLP and anomalies in morphology (P= 0.04; OR=3.5)) and number (P< 0.01; OR= 40). There was an absence of rare 799G>T polymorphism in all NSCL±P and non-cleft children indicating that all samples contain common 799G polymorphism. In conclusion, the prevalence of dental anomalies in morphology and number was significantly higher in NSCL±P compared to non-cleft children. However, it was not significantly associated with MSX1 799G>T polymorphism.

Long-term treatment of dentine with triple antibiotic paste promotes stem cell viability and attachment.

Objective: Successful regenerative endodontic procedures in dental treatment are critically associated with complete disinfection of the root canal and require irrigants and medicaments. One factor for consideration is the biocompatibility of the medicament as this can affect the intracanal dentine and subsequently the dental stem cell viability required for the repair of the dentine-pulp complex. This in vitro study investigated the effect of a 4-week treatment of calcium hydroxide [Ca(OH)2] and triple antibiotic paste (TAP) on the irrigated radicular dentine by analysing dentine interaction with dental stem cells. Methods: TAP consists of metronidazole, ciprofloxacin and minocycline. Dentine chips were prepared and treated with either Ca(OH)2 or TAP for 4-weeks, irrigated by 1.5% sodium hypochlorite (NaOCl), rinsed with saline, followed by 17% ethylenediaminetetraacetic acid (EDTA). Dental pulp stem cells (DPSCs) cultured on the surface of the dentine chips were analysed on days 1, 3 and 7 of cell seeding for PrestoBlue viability assays, 6-diamidino-2 phenylindole (DAPI) staining and scanning electron microscopy (SEM). An independent t-test (SPSS software version 24.0) was used to statistically analyse the PrestoBlue assay data. Results: DPSCs grown from dentine treated with TAP showed significantly higher cell viability than the Ca(OH)2 and control groups (p < 0.05). DAPI staining of the seeded DPSCs on the treated dental chips complemented the findings of the viability assay. SEM studies also revealed improvements in the cell spreading and attachment of DPSCs grown on TAP-treated dentine compared with Ca(OH)2. Conclusion: The treatment of dentine with TAP for 4 weeks provided a better microenvironment for the viability and attachment of DPSCs when compared to Ca(OH)2.

Angiogenic and Migratory Gene Expression Analysis of Stem Cells From Exfoliated Deciduous Teeth for Wound Repair Application.

BACKGROUND: The migration and differentiation of stem cells take place during the reparative phase of the healing cascade. Chemokine ligands and receptors are the key players in the homing process during the early stage of capillary morphogenesis. Stem cells from exfoliated deciduous teeth are known to possess a huge potential benefit for tissue regeneration. However, the gene expression of SHED engaging in angiogenesis and migratory activity during tissue healing is not fully understood. This study aims to assess the gene expression of SHED following in-vitro angiogenesis and migratory induction protocol. METHODS: Scratch test assay was conducted following an angiogenic induction of SHED by supplementation of EGM-2 and VEGF. For the detection of migratory cell markers, angiogenic markers, and stem cell markers, RNA samples were extracted on days 1, 3, 7, 10, and 14 after the angiogenic induction in a transwell chamber, followed by RT-PCR analysis. RESULTS: The findings suggested that SHED formed endothelial cells at higher capacity under an immature state with higher seeding density. SHED undergoing angiogenesis and migratory activity showed elevated IL-8, CCR1, CXCR4, and CCL28 expression. CCR1 expression significantly increased in the A+M+ group (p<0.05). CONCLUSION: The gene expression of these chemokines, particularly CCR1, which closely represent cellular migration, suggests the potential use of SHED for cell-based therapy to enhance tissue repair.

Effectiveness of Self-Assembling Peptide (P11-4) in Dental Hard Tissue Conditions: A Comprehensive Review.

The limitations on the use of fluoride therapy in dental caries prevention has necessitated the development of newer preventive agents. This review focusses on the recent and significant studies on P11-4 peptide with an emphasis on different applications in dental hard tissue conditions. The self-assembling peptide P11-4 diffuses into the subsurface lesion assembles into aggregates throughout the lesion, supporting the nucleation of de novo hydroxyapatite nanocrystals, resulting in increased mineral density. P11-4 treated teeth shows more remarkable changes in the lesion area between the first and second weeks. The biomimetic remineralisation facilitated in conjunction with fluoride application is an effective and non-invasive treatment for early carious lesions. Despite, some studies have reported that the P11-4 group had the least amount of remineralised enamel microhardness and a significantly lower mean calcium/phosphate weight percentage ratio than the others. In addition, when compared to a low-viscosity resin, self-assembling peptides could neither inhibit nor mask the lesions significantly. Moreover, when it is combined with other agents, better results can be achieved, allowing more effective biomimetic remineralisation. Other applications discussed include treatment of dental erosion, tooth whitening and dentinal caries. However, the evidence on its true clinical potential in varied dental diseases still remains under-explored, which calls for future cohort studies on its in vivo efficacy.

Tannic acid enhances cisplatin effect on cell proliferation and apoptosis of human osteosarcoma cell line (U2OS).

BACKGROUND: The increase in cases of chemoresistance of cisplatin for osteosarcoma treatment has called for the need to establish a new treatment regime. Tannic acid (TA) possesses a potent antiproliferative effect against various cancers. Therefore, this study investigated the effect of TA combined with cisplatin on human osteosarcoma cell lines (U2OS). METHODS: MTT assay was used to determine the half-maximal inhibitory concentration (IC50), while the combination index (CI) value was utilized to analyze the interaction within each combination. The antiproliferative effect of the treatment was evaluated by trypan blue exclusion assay. The morphological changes of cells were observed under a phase-contrast inverted microscope. The nuclear morphology and percentage of apoptosis cells were evaluated by using the Hoechst 33258 staining and annexin V/PI assay, respectively. RESULTS: The U2OS cells showed cytotoxic effect when treated with TA and cisplatin, with IC50 at 4.47 µg/mL and 16.25 µg/mL, respectively. The TA demonstrated no significant inhibition effect on the normal human fetal osteoblast cells (hFOB 1.19); yet, interestingly, a potent proliferative effect was indicated. Synergistic interaction was triggered when TA was combined with cisplatin at percentage ratios of 90:10 and 85:15. Meanwhile, antagonistic interaction was induced in the combination at percentage ratios of 75:25 and 50:50. On the other hand, a significant antiproliferative effect with prominent morphological alteration was detected in the cells treated with a combination of TA and cisplatin at the percentage ratio of 90:10. Additionally, combination-treated cells demonstrated the highest percentage of apoptosis cells, with distinct chromosomal condensation, nuclear fragmentation, reduction of nuclear volume, and notable apoptotic body. CONCLUSION: Therefore, there is a high potential for the inclusion of TA in the cisplatin-based chemotherapeutic regimen of osteosarcoma.

Amniotic membrane matrix effects on calcineurin-NFAT-related gene expressions of SHED treated with VEGF for endothelial differentiation.

The nuclear factor of activated T-cell (NFAT) signaling pathway is involved in angiogenesis following initiation by vascular endothelial growth factor (VEGF). A number of angiogenic genes have been associated with calcineurin in the NFAT pathway, forming a calcineurin-NFAT pathway. This study aims to investigate the involvement of four angiogenic genes within the calcineurin-NFAT pathway in the endothelial-like differentiation of stem cells from human exfoliated deciduous teeth (SHED) cultured on a human amniotic membrane (HAM) induced by VEGF. SHED were induced with VEGF for 24 h, then cultured on the stromal side of HAM. The cells were then further induced with VEGF until days 1 and 14. To understand the role of calcineurin, its potent inhibitor, cyclosporin A (CsA), was added into the culture. Results from SEM and H&E analyses showed SHED grew on HAM surface. Gene expression study of Cox-2 showed a drastically reduced expression with CsA treatment indicating Cox-2 involvement in the calcineurin-NFAT pathway. Meanwhile, IL-8 was probably controlled by another pathway as it showed no CsA inhibition. In contrast, high expression of ICAM-1 and RCAN1.4 by VEGF and CsA implied that these genes were not controlled by the calcineurin-NFAT-dependent pathway. In conclusion, the results of this study suggest the involvement of Cox-2 in the calcineurin-NFAT-dependent pathway while RCAN1.4 was controlled by NFAT molecule in endothelial-like differentiation of SHED cultured on HAM with VEGF induction.

Identification of Risk Loci for Parkinson Disease in Asians and Comparison of Risk Between Asians and Europeans: A Genome-Wide Association Study.

Importance: Large-scale genome-wide association studies in the European population have identified 90 risk variants associated with Parkinson disease (PD); however, there are limited studies in the largest population worldwide (ie, Asian). Objectives: To identify novel genome-wide significant loci for PD in Asian individuals and to compare genetic risk between Asian and European cohorts. Design Setting, and Participants: Genome-wide association data generated from PD cases and controls in an Asian population (ie, Singapore/Malaysia, Hong Kong, Taiwan, mainland China, and South Korea) were collected from January 1, 2016, to December 31, 2018, as part of an ongoing study. Results were combined with inverse variance meta-analysis, and replication of top loci in European and Japanese samples was performed. Discovery samples of 31 575 individuals passing quality control of 35 994 recruited were used, with a greater than 90% participation rate. A replication cohort of 1 926 361 European-ancestry and 3509 Japanese samples was analyzed. Parkinson disease was diagnosed using UK Parkinson's Disease Society Brain Bank Criteria. Main Outcomes and Measures: Genotypes of common variants, association with disease status, and polygenic risk scores. Results: Of 31 575 samples identified, 6724 PD cases (mean [SD] age, 64.3 [10] years; age at onset, 58.8 [10.6] years; 3472 [53.2%] men) and 24 851 controls (age, 59.4 [11.4] years; 11 030 [45.0%] men) were analyzed in the discovery study. Eleven genome-wide significant loci were identified; 2 of these loci were novel (SV2C and WBSCR17) and 9 were previously found in Europeans. Replication in European-ancestry and Japanese samples showed robust association for SV2C (rs246814; odds ratio, 1.16; 95% CI, 1.11-1.21; P = 1.17 × 10-10 in meta-analysis of discovery and replication samples) but showed potential genetic heterogeneity at WBSCR17 (rs9638616; I2=67.1%; P = 3.40 × 10-3 for hetereogeneity). Polygenic risk score models including variants at these 11 loci were associated with a significant improvement in area under the curve over the model based on 78 European loci alone (63.1% vs 60.2%; P = 6.81 × 10-12). Conclusions and Relevance: This study identified 2 apparently novel gene loci and found 9 previously identified European loci to be associated with PD in this large, meta-genome-wide association study in a worldwide population of Asian individuals and reports similarities and differences in genetic risk factors between Asian and European individuals in the risk for PD. These findings may lead to improved stratification of Asian patients and controls based on polygenic risk scores. Our findings have potential academic and clinical importance for risk stratification and precision medicine in Asia.

Amniotic Membrane Enhance the Effect of Vascular Endothelial Growth Factor on the Angiogenic Marker Expression of Stem Cells from Human Exfoliated Deciduous Teeth.

Previously, it was reported that human amniotic membrane (AM) induced stem cells from human deciduous exfoliated teeth (SHED) endothelial-like-cell differentiation. This interesting effect of AM matrix on SHED demands further elucidation. Objective of this in vitro work was to study the effect of 24-h VEGF induced on SHED endothelial differentiation when seeded on acellular stromal side (SS) of AM matrix. Stemness of SHED was identified by flow cytometry. Cell attachment and morphological changes towards the matrix was observed by scanning electron microscopy. Protein expression of endothelial marker was examined by Western blot. The expression of stem cells and endothelial-specific gene markers of VEGF-induced SHED cultured on human AM was inspected via reverse transcriptase-polymerase chain reaction. Results showed SHED at both passages retain stemness property. Ang-1 protein was expressed in SHED. Cells treated with VEGF and cultured on AM transformed attached well to AM. VEGF-induced SHED expressed both stem cell and endothelial-specific markers throughout the treatments and timeline. Interestingly, prolonged VEGF treatment increased the expression of Cox-2 and VE-Cadherin genes in all treated groups when compared to SHED. It was concluded that the VEGF-induced SHED showed better expression of endothelial-specific markers when cultured on SS of AM, with prolonged VEGF treatment.