Collagen and fibrin are widely used in tissue engineering due to their excellent biocompatibility and bioactivities that support in vivo tissue formation. These two hydrogels naturally present in different wound healing stages with different regulatory effects on cells, and both of them are mechanically weak in the reconstructed hydrogels. We conducted a comparative study by the growth of rat dermal fibroblasts or dermal fibroblasts and epidermal keratinocytes together in collagen and fibrin constructs respectively with and without the reinforcement of electrospun poly(lactic acid) nanofiber mesh. Cell proliferation, gel contraction and elastic modulus of the constructs were measured on the same gels at multiple time points during the 22 day culturing period using multiple non-destructive techniques. The results demonstrated considerably different cellular activities within the two types of constructs. Co-culturing keratinocytes with fibroblasts in the collagen constructs reduced the fibroblast proliferation, collagen contraction and mechanical strength at late culture point regardless of the presence of nanofibers. Co-culturing keratinocytes with fibroblasts in the fibrin constructs promoted fibroblast proliferation but exerted no influence on fibrin contraction and mechanical strength. The presence of nanofibers in the collagen and fibrin constructs played a favorable role on the fibroblast proliferation when keratinocytes were absent. Thus, this study exhibited new evidence of the strong cross-talk between keratinocytes and fibroblasts, which can be used to control fibroblast proliferation and construct contraction. This cross-talk activity is extracellular matrix-dependent in terms of the fibrous network morphology, density and strength.
In this day and age, the expectation of cosmetic products to effectively slow down skin photoaging is constantly increasing. However, the detrimental effects of UVB on the skin are not easy to tackle as UVB dysregulates a wide range of molecular changes on the cellular level. In our research, irradiated keratinocyte cells not only experienced a compromise in their redox system, but processes from RNA translation to protein synthesis and folding were also affected. Aside from this, proteins involved in various other processes like DNA repair and maintenance, glycolysis, cell growth, proliferation, and migration were affected while the cells approached imminent cell death. Additionally, the collagen degradation pathway was also activated by UVB irradiation through the upregulation of inflammatory and collagen degrading markers. Nevertheless, with the treatment of Swietenia macrophylla (S. macrophylla) seed extract and fractions, the dysregulation of many genes and proteins by UVB was reversed. The reversal effects were particularly promising with the S. macrophylla hexane fraction (SMHF) and S. macrophylla ethyl acetate fraction (SMEAF). SMHF was able to oppose the detrimental effects of UVB in several different processes such as the redox system, DNA repair and maintenance, RNA transcription to translation, protein maintenance and synthesis, cell growth, migration and proliferation, and cell glycolysis, while SMEAF successfully suppressed markers related to skin inflammation, collagen degradation, and cell apoptosis. Thus, in summary, our research not only provided a deeper insight into the molecular changes within irradiated keratinocytes, but also serves as a model platform for future cosmetic research to build upon. Subsequently, both SMHF and SMEAF also displayed potential photoprotective properties that warrant further fractionation and in vivo clinical trials to investigate and obtain potential novel bioactive compounds against photoaging.
Chitosan (beta-1, 4-D-glucosamine) is a deacetylated form of chitin with excellent biological properties in wound management. The natural properties of chitosan have the physical and chemical limitations to be widely used in biomedical fields. The improvement of the physical and chemical properties of chitosan with some additional chemicals will alter its biocompatibility. Therefore, the biological attribute of the modified chitosan must be evaluated. In this study, the cytotoxicity of oligo-chitosan (OC) and N, O- carboxymethyl-chitosan (NO-CMC) derivatives (O-C 1%, O-C 5%, NO-CMC 1% and NO-CMC 5%) was evaluated using primary normal human epidermal keratinocyte (pNHEK) cultures as an in vitro toxicology model at standardized cell passages (fourth passages). 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl tetrazolium bromide (MTT) was used as a cell viability assay. The O-C 1% is one of the most compatible chitosan derivatives because it steadily sustained >70% of viable cells until 72 hr post-treatment. This was followed by O-C 5%, NO-CMC 5% and NO-CMC 1%. Therefore, oligo-chitosan had the ideal properties of a biocompatible material compared to N, O- carboxymethyl-chitosan in this study.
In this study, we developed positively charged liquid crystalline nanoparticles (LCN) coated with chitosan (CHI) to enhance the skin permeation and distribution of 5α-reductase inhibitors for the treatment of androgenetic alopecia. LCN and surface-modified LCN (CHI-LCN) were prepared by ultrasonication method, and their physicochemical properties were characterized. In vitro and in vivo skin permeation and retention were studied using porcine abdominal skin and mice skin using the Franz diffusion cell. Skin distribution and cellular uptake of LCN and CHI-LCN were also investigated. The particle size and surface charge were 244.9 ± 2.1 nm and -19.2 ± 1.1 mV, respectively, for LCNs and 300.0 ± 7.6 nm and 24.7 ± 2.4 mV, respectively, for CHI-LCN. The permeation of 5α-reductase inhibitors was significantly greater with CHI-LCN compared with LCN, whereas there was no significant difference observed in the skin distribution. In fluorescence studies, fluorescence intensity was higher for CHI-LCNs throughout the skin, whereas more intense fluorescence was seen only in the epidermis layer for LCN. CHI-LCN showed greater cellular uptake than LCN, resulting in internalization of 98.5 ± 1.9% of nanoparticles into human keratinocyte cells. In conclusion, surface modification of LCN with CHI is a promising strategy for increasing skin permeation of 5α-reductase inhibitors for topical delivery.
The healing of wounds, including those from burns, currently exerts a burden on healthcare systems worldwide. Hydrogels are widely used as wound dressings and in the field of tissue engineering. The popularity of bacterial cellulose-based hydrogels has increased owing to their biocompatibility. Previous study demonstrated that bacterial cellulose/acrylic acid (BC/AA) hydrogel increased the healing rate of burn wound. This in vivo study using athymic mice has extended the use of BC/AA hydrogel by the addition of human epidermal keratinocytes and human dermal fibroblasts. The results showed that hydrogel loaded with cells produces the greatest acceleration on burn wound healing, followed by treatment with hydrogel alone, compared with the untreated group. The percentage wound reduction on day 13 in the mice treated with hydrogel loaded with cells (77.34 ± 6.21%) was significantly higher than that in the control-treated mice (64.79 ± 6.84%). Histological analysis, the expression of collagen type I via immunohistochemistry, and transmission electron microscopy indicated a greater deposition of collagen in the mice treated with hydrogel loaded with cells than in the mice administered other treatments. Therefore, the BC/AA hydrogel has promising application as a wound dressing and a cell carrier.
Galectin-3 (Gal-3) is a carbohydrate-binding protein, that promotes angiogenesis through mediating angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). There is strong evidence confirming FGF involvement in tumor growth and progression by disrupting cell proliferation and angiogenesis. In this study, we investigated the effect of β-cyclodextrin:everolimus:FGF-7 inclusion complex (Complex) on Caco-2 cell migration, cell motility and colony formation. In addition, we examined the inhibitory effect of the Complex on the circulating proteins; Gal-3 and FGF-7. Swiss Target Prediction concluded that Gal-3 and FGF are possible targets for β-CD. Results of the chemotaxis cell migration assay on Caco-2 cell line revealed that the Complex has higher reduction in cell migration (78.3%) compared to everolimus (EV) alone (58.4%) which is possibly due to the synergistic effect of these molecules when used as a combined treatment. Moreover, the Complex significantly decreased the cell motility in cell scratch assay, less than 10% recovery compared to the control which has ~ 45% recovery. The Complex inhibited colony formation by ~ 75% compared to the control. Moreover, the Complex has the ability to inhibit Gal-3 with minimum inhibitory concentration of 33.46 and 41 for β-CD and EV, respectively. Additionally, β-CD and β-CD:EV were able to bind to FGF-7 and decreased the level of FGF-7 more than 80% in cell supernatant. This confirms Swiss Target Prediction result that predicted β-CD could target FGF. These findings advance the understanding of the biological effects of the Complex which reduced cell migration, cell motility and colony formation and it is possibly due to inhibiting circulating proteins such as; Gal-3 and FGF-7.
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.
Skin injury is quite common, and the wound healing is a complex process involving many types of cells, the extracellular matrix, and soluble mediators. Cell differentiation, migration, and proliferation are essential in restoring the integrity of the injured tissue. Despite the advances in science and technology, we have yet to find the ideal dressing that can support the healing of cutaneous wounds effectively, particularly for difficult-to-heal chronic wounds such as diabetic foot ulcers, bed sores, and venous ulcers. Hence, there is a need to identify and incorporate new ideas and methods to design a more effective dressing that not only can expedite wound healing but also can reduce scarring. Calcium has been identified to influence the wound healing process. This review explores the functions and roles of calcium in skin regeneration and reconstruction during would healing. Furthermore, this review also investigates the possibility of incorporating calcium into scaffolds and examines how it modulates cutaneous wound healing. In summary, the preliminary findings are promising. However, some challenges remain to be addressed before calcium can be used for cutaneous wound healing in clinical settings.
The treatment of major burn injuries are a formidable challenge to the burn surgeon. Early aggressive surgery for deep to full thickness burn injuries is vital in the prevention of infection. The ultimate goal in major burn injuries is to prevent the onset of multi-resistant organisms and achieve early wound cover. The field of tissue engineering can help to expedite the healing of these burn wounds. The development of keratinocyte culture delivery system can be used clinically to fasten the healing process and save many lives.
A major factor limiting survival following extensive thermal injury is insufficient availability of donor sites to provide enough skin for the required grafting procedures. Limitation of autologous grafting promotes the usage of allograft skin substitutes to promote wound healing. Here, we investigated the wound healing potential of allograft single layered tissue engineered skin which comprises of either keratinocytes (SLTES-K) or fibroblast (SLTES-F) with fibrin as the delivery system. Results from gross and microscopic evaluation showed our single layered tissue engineered skin constructed with keratinocytes or fibroblast after gamma radiation with the dosage of 2Gy could serve as allograft for the treatment of skin loss.
The interrelationship between malignant epithelium and the underlying stroma is of fundamental importance in tumour development and progression. In the present study, we used cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), tumours that are characterized by the loss of genes such as TP53 and p16INK4A and with extensive loss of heterozygosity, together with CAFs from their more genetically stable (GS) counterparts that have wild-type TP53 and p16INK4A and minimal loss of heterozygosity (GS-OSCC). Using a systems biology approach to interpret the genome-wide transcriptional profile of the CAFs, we show that transforming growth factor-β (TGF-β) family members not only had biological relevance in silico but also distinguished GU-OSCC-derived CAFs from GS-OSCC CAFs and fibroblasts from normal oral mucosa. In view of the close association between TGF-β family members, we examined the expression of TGF-β1 and TGF-β2 in the different fibroblast subtypes and showed increased levels of active TGF-β1 and TGF-β2 in CAFs from GU-OSCC. CAFs from GU-OSCC, but not GS-OSCC or normal fibroblasts, induced epithelial-mesenchymal transition and down-regulated a broad spectrum of cell adhesion molecules resulting in epithelial dis-cohesion and invasion of target keratinocytes in vitro in a TGF-β-dependent manner. The results demonstrate that the TGF-β family of cytokines secreted by CAFs derived from genotype-specific oral cancer (GU-OSCC) promote, at least in part, the malignant phenotype by weakening intercellular epithelial adhesion.
To study ROCK2 activation in carcinogenesis, mice expressing 4-hydroxytamoxifen (4HT)-activated ROCK2 (K14.ROCK(er)) were crossed with mice expressing epidermal-activated ras(Ha) (HK1.ras(1205)). At 8 weeks, 4HT-treated K14.ROCK(er)/HK1.ras(1205) cohorts exhibited papillomas similar to HK1.ras(1205) controls; however, K14.ROCK(er)/HK1.ras(1205) histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma (wdSCC), exhibiting p53 loss, increased proliferation and novel NF-κB expression. By 12 weeks, K14.ROCK(er)/HK1.ras(1205) wdSCCs exhibited increased NF-κB and novel tenascin C, indicative of elevated rigidity; yet despite continued ROCK2 activities/p-Mypt1 inactivation, progression to SCC required loss of compensatory p21 expression. K14.ROCK(er)/HK1.ras(1205) papillomatogenesis also required a wound promotion stimulus, confirmed by breeding K14.ROCK(er) into promotion-insensitive HK1.ras(1276) mice, suggesting a permissive K14.ROCK(er)/HK1.ras(1205) papilloma context (wound-promoted/NF-κB(+)/p53(-)/p21(+)) preceded K14.ROCK(er)-mediated (p-Mypt1/tenascin C/rigidity) malignant conversion. Malignancy depended on ROCK(er)/p-Mypt1 expression, as cessation of 4HT treatment induced disorganized tissue architecture and p21-associated differentiation in wdSCCs; yet tenascin C retention in connective tissue extracellular matrix suggests the rigidity laid down for conversion persists. Novel papilloma outgrowths appeared expressing intense, basal layer p21 that confined endogenous ROCK2/p-Mypt1/NF-κB to supra-basal layers, and was paralleled by restored basal layer p53. In later SCCs, 4HT cessation became irrelevant as endogenous ROCK2 expression increased, driving progression via p21 loss, elevated NF-κB expression and tenascin C-associated rigidity, with p-Mypt1 inactivation/actinomyosin-mediated contractility to facilitate invasion. However, p21-associated inhibition of early-stage malignant progression and the intense expression in papilloma outgrowths, identifies a novel, significant antagonism between p21 and ras(Ha)/ROCK2/NF-κB signalling in skin carcinogenesis. Collectively, these data show that ROCK2 activation induces malignancy in ras(Ha)-initiated/promoted papillomas in the context of p53 loss and novel NF-κB expression, whereas increased tissue rigidity and cell motility/contractility help mediate tumour progression.
The effects of locally produced chitosan (CPSRT-NC-bicarbonate) in the intervention of keloid pathogenesis were investigated in vitro. A human keratinocyte-fibroblast co-culture model was established to investigate the protein levels of human collagen type-I, III and V in a western blotting analysis, the secreted transforming growth factor-β1 (TGF-β1) in an enzyme-linked immunosorbent assay (ELISA) and the mRNA levels of TGF-β1's intracellular signaling molecules (SMAD2, 3, 4 and 7) in a real-time PCR analysis. Keratinocyte-fibroblast co-cultures were maintained in DKSFM:DMEM:F12 (2:2:1) medium. Collagen type-I was found to be the dominant form in primary normal human dermal fibroblast (pNHDF) co-cultures, whereas collagen type-III was more abundant in primary keloid-derived human dermal fibroblast (pKHDF) co-cultures. Collagen type-V was present as a minor component in the skin. TGF-β1, SMAD2 and SMAD4 were expressed more in the pKHDF than the pNHDF co-cultures. Co-cultures with normal keratinocytes suppressed collagen type-III, SMAD2, SMAD4 and TGF-β1 expressions and CPSRT-NC-bicarbonate enhanced this effect. In conclusion, the CPSRT-NC-bicarbonate in association with normal-derived keratinocytes demonstrated an ability to reduce TGF-β1, SMAD2 and SMAD4 expressions in keloid-derived fibroblast cultures, which may be useful in keloid intervention.
This study aimed to explore a potential use of fish scale-derived gelatin nanofibrous scaffolds (GNS) in tissue engineering due to their biological and economical merits. Extraction of gelatin was achieved via decalcification, sonication and lyophilization of mixed fish scales. To fabricate nano-scale architecture of scaffolds analogous to natural extracellular matrix, gelatin was rendered into nanofibrous matrices through 6-h electrospinning, resulting in the average diameter of 48 ± 12 nm. In order to improve the water-resistant ability while retaining their biocompatibility, GNS were physically crosslinked with ultraviolet (UV) irradiation for 5 min (UGN5), 10 min (UGN10) and 20 min (UGN20). On average, the diameter of nanofibers increased by 3 folds after crosslinking, however, Fourier transform infrared spectroscopy analysis confirmed that no major alterations occurred in the functional groups of gelatin. A degradation assay showed that UGN5 and UGN10 scaffolds remained in minimum essential medium for 14 days, while UGN20 scaffolds degraded completely after 10 days. All UGN scaffolds promoted adhesion and proliferation of human keratinocytes, HaCaT, without causing an apparent cytotoxicity. UGN5 scaffolds were shown to stimulate a better growth of HaCaT cells compared to other scaffolds upon 1 day of incubation, whereas UGN20 had a long-term effect on cells exhibiting 25% higher cell proliferation than positive control after 7 days. In the wound scratch assay, UGN5 scaffolds induced a rapid cell migration closing up to 79% of an artificial wound within 24 h. The current findings provide a new insight of UGN scaffolds to serve as wound dressings in the future. In the wound scratch assay, UGN5 induced a rapid cell migration closing up to 79% of an artificial wound within 24 h.
Split-skin grafting (SSG) is the gold standard treatment for full-thickness skin defects. For certain patients, however, an extensive skin lesion resulted in inadequacies of the donor site. Tissue engineering offers an alternative approach by using a very small portion of an individual's skin to harvest cells for propagation and biomaterials to support the cells for implantation. The objective of this study was to determine the effectiveness of autologous bilayered tissue-engineered skin (BTES) and single-layer tissue-engineered skin composed of only keratinocytes (SLTES-K) or fibroblasts (SLTES-F) as alternatives for full-thickness wound healing in a sheep model. Full-thickness skin biopsies were harvested from adult sheep. Isolated fibroblasts were cultured using medium Ham's F12: Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, whereas the keratinocytes were cultured using Define Keratinocytes Serum Free Medium. The BTES, SLTES-K, and SLTES-F were constructed using autologous fibrin as a biomaterial. Eight full-thickness wounds were created on the dorsum of the body of the sheep. On 4 wounds, polyvinyl chloride rings were used as chambers to prevent cell migration at the edge. The wounds were observed at days 7, 14, and 21. After 3 weeks of implantation, the sheep were euthanized and the skins were harvested. The excised tissues were fixed in formalin for histological examination via hematoxylin-eosin, Masson trichrome, and elastin van Gieson staining. The results showed that BTES, SLTES-K, and SLTES-F promote wound healing in nonchambered and chambered wounds, and BTES demonstrated the best healing potential. In conclusion, BTES proved to be an effective tissue-engineered construct that can promote the healing of full-thickness skin lesions. With the support of further clinical trials, this procedure could be an alternative to SSG for patients with partial- and full-thickness burns.
The angiogenic potential of native skin (NS), keratinocytes single skin equivalent (SSE-K), fibroblasts single skin equivalent (SSE-F) and bilayered skin equivalent secreting angiogenic growth factors such as transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) in the in vitro systems at 24, 48, 72 hours and 7 days was compared using Enzyme-Linked Immunosorbent Assay (ELISA). Bilayered skin equivalent exhibit highest release of growth factors within 24 hours to 7 days of culture compared to NS, SSE-K and SSE-F. This proved the potential of bilayered skin equivalent in producing and sustaining growth factors release to enhance angiogenesis, fibroblasts proliferation, matrix deposition, migration and growth of keratinocytes.
Biopolymer chitosan (beta-1,4-d-glucosamine) comprises the copolymer mixture of N-acetylglucosamine and glucosamine. The natural biocompatibility and biodegradability of chitosan have recently highlighted its potential use for applications in wound management. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability, but it is unknown as to what degree. Hence, the biocompatibility of the chitosan porous skin regenerating templates (PSRT 82, 87 and 108) was determined using an in vitro toxicology model at the cellular and molecular level on primary normal human epidermal keratinocytes (pNHEK). Cytocompatibility was accessed by using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay from 24 to 72h. To assess the genotoxicity of the PSRTs, DNA damage to the pNHEK was evaluated by using the Comet assay following direct contact with the various PSRTs. Furthermore, the skin pro-inflammatory cytokines TNF-alpha and IL-8 were examined to evaluate the tendency of the PSRTs to provoke inflammatory responses. All PSRTs were found to be cytocompatible, but only PSRT 108 was capable of stimulating cell proliferation. While all of the PSRTs showed some DNA damage, PSRT 108 showed the least DNA damage followed by PSRT 87 and 82. PSRT 87 and 82 induced a higher secretion of TNF-alpha and IL-8 in the pNHEK cultures than did PSRT 108. Hence, based on our experiments, PSRT 108 is the most biocompatible wound dressing of the three tested.
Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra.
This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.