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.
Gelatin possesses biological properties that resemble native skin and can potentially be fabricated as a skin substitute for full-thickness wound treatment. The native property of gelatin, whereby it is easily melted and degraded at body temperature, could prevent its biofunctionality for various applications. This study aimed to fabricate and characterise buffalo gelatin (Infanca halal certified) crosslinked with chemical type crosslinker (genipin and genipin fortified with EDC) and physicaly crosslink using the dihydrothermal (DHT) method. A porous gelatin sponge (GS) was fabricated by a freeze-drying process followed by a complete crosslinking via chemical-natural and synthetic-or physical intervention using genipin (GNP), 1-ethyl-3-(3-dimethylaminopropyl) (EDC) and dihydrothermal (DHT) methods, respectively. The physicochemical, biomechanical, cellular biocompatibility and cell-biomaterial interaction of GS towards human epidermal keratinocytes (HEK) and dermal fibroblasts (HDF) were evaluated. Results showed that GS had a uniform porous structure with pore size ranging between 60 and 200 µm with high porosity (>78.6 ± 4.1%), high wettability (<72.2 ± 7.0°), high tensile strain (>13.65 ± 1.10%) and 14 h of degradation rate. An increase in the concentration and double-crosslinking approach demonstrated an increment in the crosslinking degree, enzymatic hydrolysis resistance, thermal stability, porosity, wettability and mechanical strength. The GS can be tuned differently from the control by approaching the GS via a different crosslinking strategy. However, a decreasing trend was observed in the pore size, water retention and water absorption ability. Crosslinking with DHT resulted in large pore sizes (85-300 µm) and low water retention (236.9 ± 18.7 g/m2·day) and a comparable swelling ratio with the control (89.6 ± 7.1%). Moreover no changes in the chemical content and amorphous phase identification were observed. The HEK and HDF revealed slight toxicity with double crosslinking. HEK and HDF attachment and proliferation remain similar to each crosslinking approach. Immunogenicity was observed to be higher in the double-crosslinking compared to the single-crosslinking intervention. The fabricated GS demonstrated a dynamic potential to be tailored according to wound types by manipulating the crosslinking intervention.
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.
Curcuminoids have been used for the management of burns and wound healing in traditional Chinese medicine practices but the wide application of curcuminoids as a healing agent for wounds has always been a known problem due to their poor solubility, bioavailability, colour staining properties, as well as due to their intense photosensitivity and the need for further formulation approaches to maximise their various properties in order for them to considerably contribute towards the wound healing process. In the present study, a complex coacervation microencapsulation was used to encapsulate curcuminoids using gelatin B and chitosan. This study also focused on studying and confirming the potential of curcuminoids in a microencapsulated form as a wound healing agent. The potential of curcuminoids for wound management was evaluated using an in vitro human keratinocyte cell (HaCaT) model and the in vivo heater-inflicted burn wound model, providing evidence that the antioxidant activities of both forms of curcuminoids, encapsulated or not, are higher than those of butylated hydroxyanisole and butylated hydroxytoluene in trolox equivalent antioxidant capacity (TEAC) and (2,2-diphenyl-1-picryl-hydrazyl-hydrate) (DPPH) studies. However, curcuminoids did not have much impact towards cell migration and proliferation in comparison with the negative control in the in vitro HaCaT study. The micoencapsulation formulation was shown to significantly influence wound healing in terms of increasing the wound contraction rate, hydroxyproline synthesis, and greater epithelialisation, which in turn provides strong justification for the incorporation of the microencapsulated formulation of curcuminoids as a topical treatment for burns and wound healing management as it has the potential to act as a crucial wound healing agent in healthcare settings.
In the field of tissue engineering and reconstruction, the development of efficient biomaterial is in high demand to achieve uncomplicated wound healing. Chronic wounds and excessive scarring are the major complications of tissue repair and, as this inadequate healing continues to increase, novel therapies and treatments for dysfunctional skin repair and reconstruction are important. This paper reviews the various aspects of the complications related to wound healing and focuses on chitosan because of its unique function in accelerating wound healing. The proliferation of keratinocytes is essential for wound closure, and adipose-derived stem cells play a significant role in wound healing. Thus, chitosan in combination with keratinocytes and adipose-derived stem cells may act as a vehicle for delivering cells, which would increase the proliferation of keratinocytes and help complete recovery from injuries.
Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion.
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.
Recently, transdermal monitoring and drug delivery have gained much interest, owing to the introduction of the minimally invasive microneedle (MN) device. The advancement of electroactive MNs electrically assisted in the capture of biomarkers or the triggering of drug release. Recent works have combined conducting polymers (CPs) onto MNs owing to the soft nature of the polymers and their tunable ionic and electronic conductivity. Though CPs are reported to work safely in the body, their biocompatibility in the skin has been insufficiently investigated. Furthermore, during electrical biasing of CPs, they undergo reduction or oxidation, which in practical terms leads to release/exchange of ions, which could pose biological risks. This work investigates the viability and proliferation of skin cells upon exposure to an electrochemically biased MN pair comprising two differently doped poly(3,4-ethylenedioxy-thiophene) (PEDOT) polymers that have been designed for skin sampling use. The impact of biasing on human keratinocytes and dermal fibroblasts was determined at different initial cell seeding densities and incubation periods. Indirect testing was employed, whereby the culture media was first exposed to PEDOTs prior to the addition of this extract to cells. In all conditions, both unbiased and biased PEDOT extracts showed no cytotoxicity, but the viability and proliferation of cells cultured at a low cell seeding density were lower than those of the control after 48 h of incubation.
Naegleria fowleri and Balamuthia mandrillaris are protist pathogens that infect the central nervous system, causing primary amoebic meningoencephalitis and granulomatous amoebic encephalitis with mortality rates of over 95%. Quinazolinones and their derivatives possess a wide spectrum of biological properties, but their antiamoebic effects against brain-eating amoebae have never been tested before. In this study, we synthesized a variety of 34 novel arylquinazolinones derivatives (Q1-Q34) by altering both quinazolinone core and aryl substituents. To study the antiamoebic activity of these synthetic arylquinazolinones, amoebicidal and amoebistatic assays were performed against N. fowleri and B. mandrillaris. Moreover, amoebae-mediated host cells cytotopathogenicity and cytotoxicity assays were performed against human keratinocytes cells in vitro. The results revealed that selected arylquinazolinones derivatives decreased the viability of B. mandrillaris and N. fowleri significantly (P < 0.05) and reduced cytopathogenicity of both parasites. Furthermore, these compounds were also found to be least cytotoxic against HaCat cells. Considering that nanoparticle-based materials possess potent in vitro activity against brain-eating amoebae, we conjugated quinazolinones derivatives with silver nanoparticles and showed that activities of the drugs were enhanced successfully after conjugation. The current study suggests that quinazolinones alone as well as conjugated with silver nanoparticles may serve as potent therapeutics against brain-eating amoebae.
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.
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.
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.
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.
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.
Previous studies suggested telomerase activity as a determinant of cell replicative capacity by delaying cell senescence. This study aimed to evaluate the feasibility of adopting telomerase activity as a selection criterion for in vitro expanded skin cells before autologous transplantation. Fibroblasts and keratinoctyes were derived from the same consenting patients aged 9-69 years, and cultured separately in serum-supplemented and serum-free media, respectively. Telomerase activity of fresh and cultured cells were measured and correlated with cell growth rate, donor age and passage number. The results showed that telomerase activity and cell growth were independent of donor age for both cell types. Telomerase was expressed in freshly digested epidermis and dermis and continued expressing in vitro. Keratinocytes consistently showed 3-12 folds greater telomerase activity than fibroblast both in vivo and in vitro. Conversely, growth rate for fibroblast exceeded that of keratinocyte. Telomerase activity decreased markedly at Passage 6 for keratinocytes and ceased by Passage 3 for fibroblasts. The decrease or cessation of telomerase activity coincided with senescence for keratinocyte but not for fibroblast, implying a telomerase-regulated cell senescence for the former and hence a predictor of replicative capacity for this cell type. Relative telomerase activity for fibroblasts from the younger age group was significantly higher than that from the older age group; 69.7% higher for fresh isolates and 31.1% higher at P0 (p<0.05). No detectable telomerase activity was to be found at later subcultures for both age groups. Similarly for keratinocytes, telomerase activity in the younger age group was significantly higher (p<0.05) compared to that in the older age group; 507.7% at P0, 36.8% at P3 and the difference was no longer significant at P6. In conclusion, the study provided evidence that telomerase sustained the proliferation of keratinocytes but not fibroblasts. Telomerase activity is an important criterion for continued survival and replication of keratinocytes, hence its positive detection before transplantation is desirable. Inferring from our results, the use of keratinocytes from Passage 3 or lesser for construction of skin substitute or cell-based therapy is recommended owing to their sustained telomerase expression.
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.
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.
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.
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.
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.