Secretome Analysis of Human Nasal Fibroblast Identifies Proteins That Promote Wound Healing.

Conditioned medium from cultured fibroblast cells is recognized to promote wound healing and growth through the secretion of enzymes, extracellular matrix proteins, and various growth factors and cytokines. The objective of this study was to profile the secreted proteins present in nasal fibroblast conditioned medium (NFCM). Nasal fibroblasts isolated from human nasal turbinates were cultured for 72 h in Defined Keratinocytes Serum Free Medium (DKSFM) or serum-free F12: Dulbecco's Modified Eagle's Medium (DMEM) to collect conditioned medium, denoted as NFCM_DKSFM and NFCM_FD, respectively. SDS-PAGE was performed to detect the presence of protein bands, followed by MALDI-TOF and mass spectrometry analysis. SignalP, SecretomeP, and TMHMM were used to identify the secreted proteins in conditioned media. PANTHER Classification System was performed to categorize the protein according to protein class, whereas STRING 10 was carried out to evaluate the predicted proteins interactions. SDS-PAGE results showed the presence of various protein with molecular weight ranging from ~10 kDa to ~260 kDa. Four protein bands were identified using MALDI-TOF. The analyses identified 104, 83, and 7 secreted proteins in NFCM_FD, NFCM_DKSFM, and DKSFM, respectively. Four protein classes involved in wound healing were identified, namely calcium-binding proteins, cell adhesion molecules, extracellular matrix proteins, and signaling molecules. STRING10 protein prediction successfully identified various pathways regulated by secretory proteins in NFCM. In conclusion, this study successfully profiled the secreted proteins of nasal fibroblasts and these proteins are predicted to play important roles in RECs wound healing through various pathways.

Approaches in Hydroxytyrosol Supplementation on Epithelial-Mesenchymal Transition in TGFß1-Induced Human Respiratory Epithelial Cells.

Hydroxytyrosol (HT) is an olive polyphenol with anti-inflammatory and antioxidant properties. This study aimed to investigate the effect of HT treatment on epithelial-mesenchymal transition (EMT) in primary human respiratory epithelial cells (RECs) isolated from human nasal turbinate. HT dose-response study and growth kinetic study on RECs was performed. Several approaches on HT treatment and TGFß1 induction with varying durations and methods was studied. RECs morphology and migration ability were evaluated. Vimentin and E-cadherin immunofluorescence staining and Western blotting [E-cadherin, vimentin, SNAIL/SLUG, AKT, phosphorylated (p)AKT, SMAD2/3 and pSMAD2/3] were performed after 72-h treatment. In silico analysis (molecular docking) of HT was performed to evaluate the potential of HT to bind with the TGFß receptor. The viability of the HT-treated RECs was concentration-dependent, where the median effective concentration (EC50) was 19.04 µg/mL. Testing of the effects of 1 and 10 µg/mL HT revealed that HT suppressed expression of the protein markers vimentin and SNAIL/SLUG while preserving E-cadherin protein expression. Supplementation with HT protected against SMAD and AKT pathway activation in the TGFß1-induced RECs. Furthermore, HT demonstrated the potential to bind with ALK5 (a TGFß receptor component) in comparison to oleuropein. TGFß1-induced EMT in RECs and HT exerted a positive effect in modulating the effects of EMT.

Effect of Different Collection Times of Dermal Fibroblast Conditioned Medium (DFCM) on In Vitro Re-Epithelialisation Process.

A key event in wound healing is re-epithelialisation, which is mainly regulated via paracrine signalling of cytokines, chemokines, and growth factors secreted by fibroblasts. Fibroblast-secreted factors can be collected from the used culture medium, known as dermal fibroblast conditioned medium (DFCM). The goal of this study was to optimise the culture condition to acquire DFCM and evaluate its effect on keratinocyte attachment, proliferation, migration, and differentiation. Confluent fibroblasts were cultured with serum-free keratinocyte-specific (DFCM-KM) and fibroblast-specific (DFCM-FM) medium at different incubation times (Days 1, 2, and 3). DFCM collected after 3 days of incubation (DFCM-KM-3 and DFCM-FM-3) contained a higher protein concentration compared to other days. Supplementation of DFCM-KM-3 enhanced keratinocyte attachment, while DFCM-FM-3 significantly increased the keratinocyte wound-healing rate, with an increment of keratinocyte area and collective cell migration, which was distinctly different from DFCM-KM-3 or control medium. Further analysis confirmed that the presence of calcium at higher concentrations in DFCM-FM facilitated the changes. The confluent dermal fibroblasts after 3 days of incubation with serum-free culture medium produced higher proteins in DFCM, resulting in enhanced in vitro re-epithelialisation. These results suggest that the delivery of DFCM could be a potential treatment strategy for wound healing.

Evaluating Feasibility of Human Tissue Engineered Respiratory Epithelium Construct as a Potential Model for Tracheal Mucosal Reconstruction.

The normal function of the airway epithelium is vital for the host's well-being. Conditions that might compromise the structure and functionality of the airway epithelium include congenital tracheal anomalies, infection, trauma and post-intubation injuries. Recently, the onset of COVID-19 and its complications in managing respiratory failure further intensified the need for tracheal tissue replacement. Thus far, plenty of naturally derived, synthetic or allogeneic materials have been studied for their applicability in tracheal tissue replacement. However, a reliable tracheal replacement material is missing. Therefore, this study used a tissue engineering approach for constructing tracheal tissue. Human respiratory epithelial cells (RECs) were isolated from nasal turbinate, and the cells were incorporated into a calcium chloride-polymerized human blood plasma to form a human tissue respiratory epithelial construct (HTREC). The quality of HTREC in vitro, focusing on the cellular proliferation, differentiation and distribution of the RECs, was examined using histological, gene expression and immunocytochemical analysis. Histological analysis showed a homogenous distribution of RECs within the HTREC, with increased proliferation of the residing RECs within 4 days of investigation. Gene expression analysis revealed a significant increase (p < 0.05) in gene expression level of proliferative and respiratory epithelial-specific markers Ki67 and MUC5B, respectively, within 4 days of investigation. Immunohistochemical analysis also confirmed the expression of Ki67 and MUC5AC markers in residing RECs within the HTREC. The findings show that calcium chloride-polymerized human blood plasma is a suitable material, which supports viability, proliferation and mucin secreting phenotype of RECs, and this suggests that HTREC can be a potential candidate for respiratory epithelial tissue reconstruction.

Perception of telemedicine among medical practitioners in Malaysia during COVID-19.

The novel Coronavirus Disease 2019 (COVID-19) has brought unprecedented changes in the way conventional health care is delivered. This study examined if clinicians' perceptions regarding telemedicine and its barriers to implementation in Malaysia have changed during this pandemic. A cross-sectional survey was conducted among Malaysian medical doctors of various specialties in four urban healthcare facilities between June 2020 and July 2020. A total of 146 (41.7%) out of 350 responses were obtained. 62% of doctors reported a reduction greater than 50% in outpatient visits during the COVID-19 pandemic. The majority of doctors either found telemedicine useful in situations similar to COVID-19 (34.2%) or that it is essential to their daily practice (42.5%). However, only 22% reported using telemedicine for consultation during the COVID-19 pandemic. 74% of doctors felt that telemedicine would only benefit up to 30% of their patient population. Significantly more female doctors (80%) felt that telemedicine would benefit their patients compared to male doctors (45.8%) (P=0.03). Physicians (51.3%) were more inclined to adopt telemedicine in comparison to surgeons (32.4%) (P=0.03). The majority cited medico-legal issues and consent (80.6%), billing and charges (66.7%) and insurance reimbursement (62.5%), technical difficulties (62.5%) as their barrier to the adoption of telemedicine. Female doctors and physicians were more willing to adopt telemedicine when compared to male doctors and surgeons. Although the COVID-19 pandemic appeared to improve the perception, significant barriers should be resolved before many can incorporate it into their practice.

Preliminary Study of In Vitro Three-Dimensional Skin Model Using an Ovine Collagen Type I Sponge Seeded with Co-Culture Skin Cells: Submerged versus Air-Liquid Interface Conditions.

Three-dimensional (3D) in vitro skin models have been widely used for cosmeceutical and pharmaceutical applications aiming to reduce animal use in experiment. This study investigate capability of ovine tendon collagen type I (OTC-I) sponge suitable platform for a 3D in vitro skin model using co-cultured skin cells (CC) containing human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) under submerged (SM) and air-liquid interface (ALI) conditions. Briefly, the extracted OTC-I was freeze-dried and crosslinked with genipin (OTC-I_GNP) and carbodiimide (OTC-I_EDC). The gross appearance, physico-chemical characteristics, biocompatibility and growth profile of seeded skin cells were assessed. The light brown and white appearance for the OTC-I_GNP scaffold and other groups were observed, respectively. The OTC-I_GNP scaffold demonstrated the highest swelling ratio (~1885%) and water uptake (94.96 ± 0.14%). The Fourier transformation infrared demonstrated amide A, B and I, II and III which represent collagen type I. The microstructure of all fabricated sponges presented a similar surface roughness with the presence of visible collagen fibers and a heterogenous porous structure. The OTC-I_EDC scaffold was more toxic and showed the lowest cell attachment and proliferation as compared to other groups. The micrographic evaluation revealed that CC potentially formed the epidermal- and dermal-like layers in both SM and ALI that prominently observed with OTC-I_GNP compared to others. In conclusion, these results suggest that OTC_GNP could be used as a 3D in vitro skin model under ALI microenvironment.

Miswak and oral health: An evidence-based review.

Poor oral health has been associated with several chronic and systemic disease. Currently, the most common method of teeth cleaning is the use of a toothbrush together with dentifrices. However, natural chewing stick such as S. persica miswak is still used in many developing countries due to their low cost and availability. The present review aims to summarize the evidences on effectiveness of miswak in promoting oral health. The search was performed using Medline via Ebscohost, Scopus and Google Scholar database to obtain relevant articles published between 2010 to May 2020 using the following set of keywords 1) Miswak OR Salvadora OR persica AND 2) dental OR caries OR plaque OR oral OR orthodontics. Isolated microbial inhibition studies were excluded from the review due to its well-established wealth of literature. Miswak was administered as ten different forms, namely mouthwash, toothpaste, chewing stick, essential oil, aqueous extract, ethanol extract, probiotic spray, dental varnish, dental cement or chewing gum. All studies reported a positive effect of miswak as an anti-plaque, anti-gingivitis, anti-cariogenic, promotion of gingival wound healing, whitening properties, orthodontic chain preservation, and biocompatibility with oral cells. Miswak in its different forms demonstrated positive effect towards oral health maintenance and management.

Development and physicochemical analysis of genipin-crosslinked gelatine sponge as a potential resorbable nasal pack.

Nasal injury following nasal surgery is an adverse consequence, and prompt treatment should be initiated. Nasal packing, either non-absorbable or absorbable, are commonly used after nasal surgery to prevent bleeding and promote wound healing. In the current study, a novel gelatine sponge crosslinked with genipin was evaluated for suitability to be used as nasal packing and compared to one of the frequently used commercial nasal packing made up of polyurethane. Gelatine at 7% and 10% (w/v) concentration were crosslinked with varying concentrations of genipin, 0.5%, 0.25%, and 0.2% (v/v). The gelatine sponges were further characterised by its water uptake ability, biodegradation, water vapour transmission rate, porosity, contact angle, chemical composition, crosslinking degree, and mechanical properties. The gelatine sponges absorbed five times more water than their dry weight and were degraded within five days. The water vapour transmission rate of the gelatine sponges was 1187.7 ± 430.2 g/(m-2 day) for 7% gelatine and 779.4 ± 375.5 g/(m-2 day) for 10% gelatine. Crosslinking of gelatine with genipin resulted in lower porosity and did not affect the wettability of gelatine sponge (contact angle: 95.3 ± 12.1° for 7% gelatine and 88.4 ± 7.2° for 10% gelatine). In terms of biodegradability, the gelatine sponges took 24-48 h to degrade completely. Genipin crosslinking improved the degradation resistance and mechanical strength of gelatine sponge. The physical and chemical properties of the gelatine sponge, i.e. biodegradability and mechanical durability, support its potential as nasal packing.

Effect of Kelulut Honey on the Cellular Dynamics of TGFß-Induced Epithelial to Mesenchymal Transition in Primary Human Keratinocytes.

Over-induction of epithelial to mesenchymal transition (EMT) by tumor growth factor beta (TGFß) in keratinocytes is a key feature in keloid scar. The present work seeks to investigate the effect of Kelulut honey (KH) on TGFß-induced EMT in human primary keratinocytes. Image analysis of the real time observation of TGFß-induced keratinocytes revealed a faster wound closure and individual migration velocity compared to the untreated control. TGFß-induced keratinocytes also have reduced circularity and display a classic EMT protein expression. Treatment of 0.0015% (v/v) KH reverses these effects. In untreated keratinocytes, KH resulted in slower initial wound closure and individual migration velocity, which sped up later on, resulting in greater wound closure at the final time point. KH treatment also led to greater directional migration compared to the control. KH treatment caused reduced circularity in keratinocytes but displayed a partial EMT protein expression. Taken together, the findings suggest the therapeutic potential of KH in preventing keloid scar by attenuating TGFß-induced EMT.

Cardioprotective Effects of Honey and Its Constituent: An Evidence-Based Review of Laboratory Studies and Clinical Trials.

Cardiovascular disease is a major public health burden worldwide. Myocardial infarction is the most common form of cardiovascular disease resulting from low blood supply to the heart. It can lead to further complications such as cardiac arrhythmia, toxic metabolite accumulation, and permanently infarcted areas. Honey is one of the most prized medicinal remedies used since ancient times. There is evidence that indicates honey can function as a cardioprotective agent in cardiovascular diseases. The present review compiles and discusses the available evidence on the effect of honey on cardiovascular diseases. Three electronic databases, namely, PubMed, Scopus, and MEDLINE via EBSCOhost, were searched between January 1959 and March 2020 to identify reports on the cardioprotective effect of honey. Based on the pre-set eligibility criteria, 25 qualified articles were selected and discussed in this review. Honey investigated in the studies included varieties according to their geological origin. Honey protects the heart via lipid metabolism improvement, antioxidative activity, blood pressure modulation, heartbeat restoration, myocardial infarct area reduction, antiaging properties, and cell apoptosis attenuation. This review establishes honey as a potential candidate to be explored further as a natural and dietary alternative to the management of cardiovascular disease.

Concentration Dependent Effect of Human Dermal Fibroblast Conditioned Medium (DFCM) from Three Various Origins on Keratinocytes Wound Healing.

Fibroblasts secrete many essential factors that can be collected from fibroblast culture medium, which is termed dermal fibroblast conditioned medium (DFCM). Fibroblasts isolated from human skin samples were cultured in vitro using the serum-free keratinocyte-specific medium (Epilife (KM1), or define keratinocytes serum-free medium, DKSFM (KM2) and serum-free fibroblast-specific medium (FM) to collect DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively). We characterised and evaluated the effects of 100-1600 µg/mL DFCM on keratinocytes based on attachment, proliferation, migration and gene expression. Supplementation with 200-400 µg/mL keratinocyte-specific DFCM-KM1 and DFCM-KM2 enhanced the attachment, proliferation and migration of sub-confluent keratinocytes, whereas 200-1600 µg/mL DFCM-FM significantly increased the healing rate in the wound healing assay, and 400-800 µg/mL DFCM-FM was suitable to enhance keratinocyte attachment and proliferation. A real-time (RT2) profiler polymerase chain reaction (PCR) array showed that 42 genes in the DFCM groups had similar fold regulation compared to the control group and most of the genes were directly involved in wound healing. In conclusion, in vitro keratinocyte re-epithelialisation is supported by the fibroblast-secreted proteins in 200-400 µg/mL DFCM-KM1 and DFCM-KM2, and 400-800 µg/mL DFCM-FM, which could be useful for treating skin injuries.

Current and Alternative Therapies for Nasal Mucosa Injury: A Review.

Nasal mucosa injury can be caused by trauma, radiotherapy, chronic infection such as sinusitis, and post sinus surgery. The rate of healing and its treatment are important in the recovery of patients especially in post sinus surgery, which introduces new injuries. In this review, the current knowledge in terms of the mechanism underlying nasal wound healing was initially discussed. The currently available treatment options for enhancement of wound healing following sinus surgery were discussed and these had included intravenous antibiotics or steroids, various nasal sprays, and nasal packing. In addition, emerging alternative therapies in nasal mucosa wound healing such as herbal medicine and the advancement of regenerative medicine therapies such as stem cells and their byproducts were also discussed. Despite the various available treatment options for wound healing in nasal mucosa, rigorous strong evidence of their efficacy is gravely warranted in order to recommend them as part of the treatment modality.

Effect of Nigella sativa and its bioactive compound on type 2 epithelial to mesenchymal transition: a systematic review.

BACKGROUND: Nigella sativa or commonly known as black seed or black cumin is one of the most ubiquitous complementary medicine. Epithelial to mesenchymal transition (EMT) of type 2 is defined by the balance between wound healing and tissue fibrosis, which is dependent to the state of inflammation. This systematic review is conducted to provide an overview regarding the reported effect of Nigella sativa and its bioactive compound on the type 2 EMT. METHODS: A search was done in EBSCOHOST, OVID and SCOPUS database to obtain potentially relevant articles that were published between 1823 and August 2019. This review includes studies that focus on the effect of Nigella sativa and its bioactive compound on the events related to type 2 EMT. RESULTS: A total of 1393 research articles were found to be potentially related to the effect of Nigella sativa and its bioactive compound, thymoquinone on Type 2 EMT. After screening was done, 22 research articles met inclusion criteria and were included in this review. Majority of the studies, reported better wound healing rate or significant prevention of tissue inflammation and organ fibrosis following Nigella sativa or thymoquinone treatments. In terms of wound healing, studies included reported progression of EMT related pathological changes after treatment with Nigella sativa or thymoquinone. Alternatively, in terms of fibrosis and inflammation, studies included reported reversal of pathological changes related to EMT after treatment with Nigella sativa or thymoquinone. CONCLUSION: Through this review, Nigella sativa and thymoquinone have been associated with events in Type 2 EMT. They have been shown to promote wound healing, attenuate tissue inflammation, and prevent organ fibrosis via regulation of the EMT process.

Modulation of Epithelial to Mesenchymal Transition Signaling Pathways by Olea Europaea and Its Active Compounds.

Epithelial-mesenchymal transition (EMT) is a significant dynamic process that causes changes in the phenotype of epithelial cells, changing them from their original phenotype to the mesenchymal cell phenotype. This event can be observed during wound healing process, fibrosis and cancer. EMT-related diseases are usually caused by inflammation that eventually leads to tissue remodeling in the damaged tissue. Prolonged inflammation causes long-term EMT activation that can lead to tissue fibrosis or cancer. Due to activation of EMT by its signaling pathway, therapeutic approaches that modulate that pathway should be explored. Olea europaea (OE) is well-known for its anti-inflammatory effects and abundant beneficial active compounds. These properties are presumed to modulate EMT events. This article reviews recent evidence of the effects of OE and its active compounds on EMT events and EMT-related diseases. Following evidence from the literature, it was shown that OE could modulate TGFß/SMAD, AKT, ERK, and Wnt/ß-catenin pathways in EMT due to a potent active compound that is present therein.

Rapid treatment of full-thickness skin loss using ovine tendon collagen type I scaffold with skin cells.

The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.

Osteogenic Induction of Wharton's Jelly-Derived Mesenchymal Stem Cell for Bone Regeneration: A Systematic Review.

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are emerging as a promising source for bone regeneration in the treatment of bone defects. Previous studies have reported the ability of WJ-MSCs to be induced into the osteogenic lineage. The purpose of this review was to systematically assess the potential of WJ-MSC differentiation into the osteogenic lineage. A comprehensive search was conducted in Medline via Ebscohost and Scopus, where relevant studies published between 1961 and 2018 were selected. The main inclusion criteria were that articles must be primary studies published in English evaluating osteogenic induction of WJ-MSCs. The literature search identified 92 related articles, but only 18 articles met the inclusion criteria. These include two animal studies, three articles containing both in vitro and in vivo assessments, and 13 articles on in vitro studies, all of which are discussed in this review. There were two types of osteogenic induction used in these studies, either chemical or physical. The studies demonstrate that WJ-MSCs are able to differentiate into osteogenic lineage and promote osteogenesis. In light of these observations, it is suggested that WJ-MSCs can be a potential source of stem cells for osteogenic induction, as an alternative to bone marrow-derived mesenchymal stem cells.

Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM).

Growth factors and extracellular matrix (ECM) proteins are involved in wound healing. Human dermal fibroblasts secrete wound-healing mediators in culture medium known as dermal fibroblast conditioned medium (DFCM). However, the composition and concentration of the secreted proteins differ with culture conditions and environmental factors. We cultured human skin fibroblasts in vitro using serum-free keratinocyte-specific media (EpiLife™ Medium [KM1] and defined keratinocyte serum-free medium [KM2]) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. We identified and compared their proteomic profiles using bicinchoninic acid assay (BCA), 1-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography MS (LC-MS/MS). DFCM-KM1 and DFCM-KM2 had higher protein concentrations than DFCM-FM but not statistically significant. MALDI-TOF/TOF MS identified the presence of fibronectin, serotransferrin, serpin and serum albumin. LC-MS/MS and bioinformatics analysis identified 59, 46 and 58 secreted proteins in DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. The most significant biological processes identified in gene ontology were cellular process, metabolic process, growth and biological regulation. STRING® analysis showed that most secretory proteins in the DFCMs were associated with biological processes (e.g. wound healing and ECM organisation), molecular function (e.g. ECM binding) and cellular component (e.g. extracellular space). ELISA confirmed the presence of fibronectin and collagen in the DFCMs. In conclusion, DFCM secretory proteins are involved in cell adhesion, attachment, proliferation and migration, which were demonstrated to have potential wound-healing effects by in vitro and in vivo studies.

The potential of Olea europaea extracts to prevent TGFß1-induced epithelial to mesenchymal transition in human nasal respiratory epithelial cells.

BACKGROUND: One of the molecular mechanisms involved in upper airway-related diseases is epithelial-to-mesenchymal transition (EMT). Olea europaea (OE) has anti-inflammatory properties and thus, great potential to prevent EMT. This study aimed to investigate the effect of OE on EMT in primary nasal human respiratory epithelial cells (RECs). METHODS: Respiratory epithelial cells were isolated and divided into four groups: control (untreated), treated with 0.05% OE (OE group), EMT induced with 5 ng/ml of transforming growth factor beta-1 (TGFß1 group) and treated with 5 ng/ml TGFß1 + 0.05% OE (TGFß1 + OE group). The effects of OE treatment on growth kinetics, morphology and protein expression in RECs were evaluated. Immunocytochemistry analysis was performed to quantitate the total percentage of E-cadherin and vimentin expression from day 1 to day 3. RESULTS: There were no significant differences between untreated RECs and OE-treated RECs in terms of their morphology, growth kinetics and protein expression. Induction with TGFß1 caused RECs to have an elongated spindle shape, a slower proliferation rate, a higher expression of vimentin and a lower expression of E-cadherin compared with the control. Cells in the TGFß1 + OE group had similar epithelial shape to untreated group however it had no significant differences in their proliferation rate when compared to TGFß1-induced RECs. Cells treated with TGFß1 + OE showed significantly reduced expression of vimentin and increased expression of E-cadherin compared with the TGFß1 group (P < 0.05). CONCLUSION: The ability of OE to inhibit EMT in RECs was shown by TGFb1-induced EMT REC morphology, growth kinetics and protein expression markers (E-cadherin and vimentin) upon treatment with OE and TGFß1. Therefore, this study could provide insight into the therapeutic potential of OE to inhibit pathological tissue remodelling and persistent inflammation.

Corneal regeneration by induced human buccal mucosa cultivated on an amniotic membrane following alkaline injury.

Various clinical disorders and injuries, such as chemical, thermal, or mechanical injuries, may lead to corneal loss that results in blindness. PURPOSE: The aims of this study were to differentiate human buccal mucosa (BMuc) into corneal epithelial-like cells, to fabricate engineered corneal tissue using buccal mucosal epithelial cells, and to reconstruct a damaged corneal epithelium in a nude rat model. Methods: BMuc were subjected to 10 d of induction factors to investigate the potential of cells to differentiate into corneal lineages. Results: Corneal stem cell markers ß1-integrin, C/EBPδ, ABCG2, p63, and CK3 were upregulated in the gene expression analysis in induced BMuc, whereas CK3 and p63 showed significant protein expression in induced BMuc compared to the uninduced cells. BMuc were then left to reach 80% confluency after differential trypsinization. The cells were harvested and cultivated on a commercially available untreated air-dried amniotic membrane (AM) in a Transwell system in induction medium. The corneal constructs were fabricated and then implanted into damaged rat corneas for up to 8 weeks. A significant improvement was detected in the treatment group at 8 weeks post-implantation, as revealed by slit lamp biomicroscopy analysis. The structure and thickness of the corneal layer were also analyzed using histological staining and time-domain optical coherence tomography scans and were found to resemble a native corneal layer. The protein expression for CK3 and p63 were continuously detected throughout the corneal epithelial layer in the corneal construct. Conclusions: In conclusion, human BMuc can be induced to express a corneal epithelial-like phenotype. The addition of BMuc improves corneal clarity, prevents vascularization, increases corneal thickness and stromal alignment, and appears to have no adverse effect on the host after implantation.

Attachment, Proliferation, and Morphological Properties of Human Dermal Fibroblasts on Ovine Tendon Collagen Scaffolds: A Comparative Study.

INTRODUCTION: Collagen type I is widely used as a biomaterial for tissue-engineered substitutes. This study aimed to fabricate different three-dimensional (3D) scaffolds using ovine tendon collagen type I (OTC-I), and compare the attachment, proliferation and morphological features of human dermal fibroblasts (HDF) on the scaffolds. METHODS: This study was conducted between the years 2014 to 2016 at the Tissue Engineering Centre, UKM Medical Centre. OTC-I was extracted from ovine tendon, and fabricated into 3D scaffolds in the form of sponge, hydrogel and film. A polystyrene surface coated with OTC-I was used as the 2D culture condition. Genipin was used to crosslink the OTC-I. A non-coated polystyrene surface was used as a control. The mechanical strength of OTC-I scaffolds was evaluated. Attachment, proliferation and morphological features of HDF were assessed and compared between conditions. RESULTS: The mechanical strength of OTC-I sponge was significantly higher than that of the other scaffolds. OTC-I scaffolds and the coated surface significantly enhanced HDF attachment and proliferation compared to the control, but no differences were observed between the scaffolds and coated surface. In contrast, the morphological features of HDF including spreading, filopodia, lamellipodia and actin cytoskeletal formation differed between conditions. CONCLUSION: OTC-I can be moulded into various scaffolds that are biocompatible and thus could be suitable as scaffolds for developing tissue substitutes for clinical applications and in vitro tissue models. However, further study is required to determine the effect of morphological properties on the functional and molecular properties of HDF.