Widefield surface plasmon resonance (WSPR) microscopy provides high resolution imaging of interfacial interactions. We report the application of the WSPR imaging system in the study of the interaction between keratinocytes and liquid crystals (LC). Imaging of fixed keratinocytes cultured on gold coated surface plasmon substrates functionalized with a thin film of liquid crystals was performed in air using a 1.45NA objective based system. Focal adhesion of the cells adhered to glass and LC were further studied using immunofluorescence staining of the vinculin. The imaging system was also simulated with 2×2 scattering matrix to investigate the optical reflection of the resonant plasmonic wave via the glass/gold/cell and glass/gold/LC/cell layers. WSPR imaging indicated that keratinocytes are less spread and formed distinct topography of cell-liquid crystal couplings when cultured on liquid crystal coated substrates. The simulation indicates that glass/LC shifted the surface plasmon excitation angle to 75.39° as compared to glass/air interface at 44°. The WSPR microcopy reveals that the cells remodelled their topography of adhesion at different interfaces.
The unique potential of mesenchymal stromal cells (MSC) has generated much research interest recently, particularly in exploring the regenerative nature of these cells. Previously, MSC were thought to be found only in the BM. However, further studies have shown that MSC can also be isolated from umbilical cord blood, adipose tissue and amniotic fluid. In this study, we explored the possibility of MSC residing in the cornea.
Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration.
Skin plays an important role in defense against infection and other harmful biological agents. Due to its fragile structure, skin can be easily damaged by heat, chemicals, traumatic injuries and diseases. An autologous bilayered human skin equivalent, MyDerm™, was engineered to provide a living skin substitute to treat critical skin loss. However, one of the disadvantages of living skin substitute is its short shelf-life, hence limiting its distribution worldwide. The aim of this study was to evaluate the shelf-life of MyDerm™ through assessment of cell morphology, cell viability, population doubling time and functional gene expression levels before transplantation. Skin samples were digested with 0.6% Collagenase Type I followed by epithelial cells dissociation with TrypLE Select. Dermal fibroblasts and keratinocytes were culture-expanded to obtain sufficient cells for MyDerm™ construction. MyDerm™ was constructed with plasma-fibrin as temporary biomaterial and evaluated at 0, 24, 48 and 72 hours after storage at 4°C for its shelf-life determination. The morphology of skin cells derived from MyDerm™ remained unchanged across storage times. Cells harvested from MyDerm™ after storage appeared in good viability (90.5%±2.7% to 94.9%±1.6%) and had short population doubling time (58.4±8.7 to 76.9±19 hours). The modest drop in cell viability and increased in population doubling time at longer storage duration did not demonstrate a significant difference. Gene expression for CK10, CK14 and COL III were also comparable between different storage times. In conclusion, MyDerm™ can be stored in basal medium at 4°C for at least 72 hours before transplantation without compromising its functionality.
Keratinocyte traction forces play a crucial role in wound healing. The aim of this study was to develop a novel cell traction force (CTF) transducer system based on cholesteryl ester liquid crystals (LC). Keratinocytes cultured on LC induced linear and isolated deformation lines in the LC surface. As suggested by the fluorescence staining, the deformation lines appeared to correlate with the forces generated by the contraction of circumferential actin filaments which were transmitted to the LC surface via the focal adhesions. Due to the linear viscoelastic behavior of the LC, Hooke's equation was used to quantify the CTFs by associating Young's modulus of LC to the cell induced stresses and biaxial strain in forming the LC deformation. Young's modulus of the LC was profiled by using spherical indentation and determined at approximately 87.1±17.2kPa. A new technique involving cytochalasin-B treatment was used to disrupt the intracellular force generating actin fibers, and consequently the biaxial strain in the LC induced by the cells was determined. Due to the improved sensitivity and spatial resolution (∼1μm) of the LC based CTF transducer, a wide range of CTFs was determined (10-120nN). These were found to be linearly proportional to the length of the deformations. The linear relationship of CTF-deformations was then applied in a bespoke CTF mapping software to estimate CTFs and to map CTF fields. The generated CTF map highlighted distinct distributions and different magnitude of CTFs were revealed for polarized and non-polarized keratinocytes.
Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.
The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.
Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.
Collagen is the most abundant extracellular matrix (ECM) protein in the human body, thus widely used in tissue engineering and subsequent clinical applications. This study aimed to extract collagen from ovine (Ovis aries) Achilles tendon (OTC), and to evaluate its physicochemical properties and its potential to fabricate thin film with collagen fibrils in a random or aligned orientation. Acid-solubilized protein was extracted from ovine Achilles tendon using 0.35M acetic acid, and 80% of extracted protein was measured as collagen. SDS-PAGE and mass spectrometry analysis revealed the presence of alpha 1 and alpha 2 chain of collagen type I (col I). Further analysis with Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDS) confirms the presence of triple helix structure of col I, similar to commercially available rat tail col I. Drying the OTC solution at 37°C resulted in formation of a thin film with randomly orientated collagen fibrils (random collagen film; RCF). Introduction of unidirectional mechanical intervention using a platform rocker prior to drying facilitated the fabrication of a film with aligned orientation of collagen fibril (aligned collagen film; ACF). It was shown that both RCF and ACF significantly enhanced human dermal fibroblast (HDF) attachment and proliferation than that on plastic surface. Moreover, cells were distributed randomly on RCF, but aligned with the direction of mechanical intervention on ACF. In conclusion, ovine tendon could be an alternative source of col I to fabricate scaffold for tissue engineering applications.
Fibrin has excellent biocompatibility and biological properties to support tissue regeneration and promote wound healing. However, the role of diluted fibrin in wound healing has yet to be elucidated as it is commonly used in high concentration. This study was aimed to examine the effects of diluted plasma-derived fibrin (PDF) on keratinocyte and fibroblast wound healing in term of cell proliferation, migration, extracellular matrix (ECM) production and soluble factor secretion. Two PDF concentrations, 10 and 20% (v/v) were tested on keratinocytes and fibroblasts indirectly co-cultured in the transwell system. The control group was cultured with 5% FBS. Results showed that PDF reduced the keratinocyte growth rate and fibroblast migration, and increased the fibroblast ECM gene expression whereby significant differences were found between the 20% PDF group and the 5% FBS group. Similar trend was seen for the 10% PDF group but the differences were not significant. Comparison of the soluble factors between the PDF groups demonstrated that the level of growth-related oncogene alpha, interleukin-8 and epithelial neutrophil-activating peptide-78 were significantly higher in the 10% PDF group, whilst interleukin-1 alpha and granulocyte-macrophage colony stimulating factor were significantly more concentrated in the 20% PDF group. Our results suggested that PDF selectively elevated the expression of collagen type 1 and collagen type 3 in fibroblasts but slowed down the migration in concentration-dependent manner. These novel findings provide new insight into the role of PDF in wound healing and may have important implications for the use of fibrin in skin tissue engineering.
Bacterial cellulose (BC)/acrylic acid (AA) hydrogel has successfully been investigated as a wound dressing for partial-thickness burn wound. It is also a promising biomaterial cell carrier because it bears some resemblance to the natural soft tissue. This study assessed its ability to deliver human epidermal keratinocytes (EK) and dermal fibroblasts (DF) for the treatment of full-thickness skin lesions. In vitro studies demonstrated that BC/AA hydrogel had excellent cell attachment, maintained cell viability with limited migration, and allowed cell transfer. In vivo wound closure, histological, immunohistochemistry, and transmission electron microscopy evaluation revealed that hydrogel alone (HA) and hydrogel with cells (HC) accelerated wound healing compared to the untreated controls. Gross appearance and Masson's trichrome staining indicated that HC was better than HA. This study suggests the potential application of BC/AA hydrogel with dual functions, as a cell carrier and wound dressing, to promote full-thickness wound healing.
In the present study in vitro expansion of human keratinocytes by supplementing dermal fibroblasts conditioned medium (DFCM) has been reported. Effect of two different DFCM acquired by culturing fibroblasts in keratinocyte-specific medium (defined keratinocytes serum free medium, DFCM-DKSFM) and fibroblast-specific serum free medium (F12: DMEM nutrient mix, DFCM-FD) have been compared. Growth kinetics of keratinocytes in terms of efficiency of cell attachment, expansion index, apparent specific growth rate and growth potential at the end of culture was evaluated in culture supplemented with DFCM-DKSFM and DFCM-FD in comparison with control i.e. DKSFM only. Results indicated that supplementation of DFCM caused significant increase in keratinocyte attachment. Efficiency of keratinocyte attachment in culture supplemented with bFCM-DKSFM was significantly higher compared to those cultured in DFCM-FD and DKSFM. In addition, the expansion index of keratinocytes in cultures supplemented with DFCM-DKSFM and DFCM-FD were 3.7 and 2.2 times higher than that of control condition even though the apparent growth rate and proliferative potential was found significantly lower. These results suggested that supplementation of DFCM enhanced expansion of keratinocyte by increasing efficiency of cell attachment, and DFCM-DKSFM provided suitable condition for in vitro expansion of keratinocytes compared to DFCM-FD and control condition.
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.
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.
Phenytoin-loaded alkyd nanoemulsions were prepared spontaneously using the phase inversion method from a mixture of novel biosourced alkyds and Tween 80 surfactant. Exposure of human adult keratinocytes (HaCaT cells) for 48 h to alkyd nanoemulsions producing phenytoin concentrations of 3.125-200 μg/mL resulted in relative cell viability readings using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide of 100% confirming nontoxicity and suggesting cell proliferation activity. Phenytoin-loaded alkyd nanoemulsions generally resulted in higher mean cell viability compared with equivalent concentration of phenytoin solutions, suggesting that the nanoemulsions provided a controlled-release property that maintained the optimum phenytoin level for keratinocyte growth. HaCaT cell proliferation, measured by 5-bromo-2-deoxyuridine uptake, was found to increase following exposure to increasing phenytoin concentration from 25 to 50 μg/mL in solution or encapsulated in nanoemulsions but declined at a drug concentration of 100 μg/mL. An in vitro cell monolayer wound scratch assay revealed that phenytoin solution or nanoemulsions producing 50 μg/mL phenytoin concentration resulted in 75%-82% "scratch closure" after 36 h, similar to medium containing 10% fetal bovine serum as a cell growth promoter. These findings indicate that phenytoin-loaded alkyd nanoemulsions show potential for promoting topical wound healing through enhanced proliferation of epidermal cells.
Hertwig's epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.
In dental implant treatment, the long-term prognosis is dependent on the biologic seal formed by the soft tissue around the implant. The in vitro investigation of the implant-soft tissue interface is usually carried out using a monolayer cell-culture model that lacks a polarized-cell phenotype. This study developed a tissue-engineered three-dimensional oral mucosal model (3D OMM) to investigate the implant-soft tissue interface.
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.
Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.
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.