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 aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture.
Replicative senescence and stress-induced premature senescence (SIPS) cells are known to share certain traits. However, whether these cells are different at the protein level is unclear. Thus, this study has utilized proteomics to identify differences in the proteomes of replicative senescence and SIPS cells compared to normal cells. Replicative senescence was induced by serial passage of normal cells in culture. SIPS was established by exposure to H2 O2 at a subcytotoxic concentration of 20 μM for two weeks. Following 2DE, protein profiles were compared and protein spots that changed in abundance were identified by MALDI-TOF MS. Quantitative real-time RT-PCR was then performed to evaluate the transcript expression of selected altered proteins. A total of 24 and 10 proteins were found to have changed in abundance in replicative senescence and SIPS cells, respectively, when compared to young cells. Quantitative RT-PCR revealed that nine genes showed the same direction of change as observed in the proteomics analysis. Very little overlap was observed between proteins that changed in replicative senescence and SIPS cells, suggesting that although both SIPS and replicative senescence cells share hallmarks of cellular senescence, they were different in terms of proteins that changed in abundance.
Tea has been suggested to promote oral health by inhibiting bacterial attachment to the oral cavity. Most studies have focused on prevention of bacterial attachment to hard surfaces such as enamel.
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.
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.
This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.
We have previously reported that 1,25(OH)2D3 inhibits NF-κB activity and thus inhibits growth of OSCC cells in serum-free culture and down-regulates HBp17/FGFBP-1 expression, which is important for cancer cell growth and angiogenesis. Here, we have investigated the effects of ED-71, an analog of vitamin D3 (VD) on OSCC cell lines in serum-free culture. It is known that ED-71 has a stronger inhibitory effect on bone resorption compared to VD and other VD analogs. To the best of our knowledge, there was no report examining the potential of ED-71 as an anti-cancer agent for OSCC. We found that ED-71 is able to inhibit the growth of cancer cell lines at a concentration of hundred times lower than calcitriol. As Cyp24A1 was reportedly induced in cancer cells, we measured the expression of CYP24A1 in OSCC cell lines (NA and UE), A431 epidermoid carcinoma and normal fibroblast cell (gfi) in serum-free culture. As a result, CYP24A1 mRNA and the protein expression in the OSCC cells treated with ED-71 increased in a dose-dependent manner. However, in vivo experiment, in which the A431 cells were implanted in mice, tumor formation was reduced by the ED-71 treatment with no significant difference between Cyp24A1 expression in the tumors of ED-71-treated and control group, as analyzed by western blotting and immunohistochemistry. These results suggest that ED-71 is a potential anti-cancer agent for OSCC.
Zanthoxylum rhetsa is an aromatic tree, known vernacularly as "Indian Prickly Ash". It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin), a berberine alkaloid (columbamine) and a triterpenoid (lupeol) from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF) and mouse melanoma (B16-F10) cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells.
Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.
Both wild-type virulent and mutant strains of pseudorabies virus (PrV) were used in this study. Mutants used were derived from the plaque purified strain PrVmAIP. A total of six drug resistant mutants, three bromodeoxyuridine (BUdR) resistant and three iododeoxyuridine (IUdR) resistant, respectively, were isolated and passaged in chicken embryo fibroblast (CEF) cells. The DNA of these PrVs were compared with the wild-type isolates by means of the restriction fragment pattern (RFP) findings produced with Bam HI, Kpn I, Hind III and Bgl II restriction enzymes (RE). Compared to the wild-type PrVs (PrV-VBA1-parental strain of PrVmAIP; PrV-VBA2; PrV-VBA3), the RFP of PrVmAIP showed the presence of mutations within the RE sites studied. Both PrV-VBA1 and PrV-VBA2 appeared to be closely related but their RFPs differed from PrV-VBA3. Significant differences either in the number, size or migrations of the DNA fragments could also be detected in the BUdR resistant strains. Even though different features of cytopathic effect (GPE) were observed in the IUdR resistant PrVs, the RFP findings remained identical. The PrVs studied showed considerable differences from the reference PrV (PrV-CD).
Bi-directional signaling involved in radiation-induced bystander effect (RIBE) between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to radiation cancer therapy. Using the SPICE-NIRS microbeam, we delivered 500 protons to A549-GFP lung carcinoma cells, stably expressing H2B-GFP, which were co-cultured with normal WI-38 cells. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured up to 24-h post-irradiation in both targeted and bystander cells. As a result, inhibition of gap junction intercellular communication (GJIC) attenuated DSB repair in targeted A549-GFP cells, and suppressed RIBE in bystander WI-38 cells but not in distant A549-GFP cells. This suggests that GJIC plays a two-way role through propagating DNA damage effect between carcinoma to normal cells and reversing the bystander signaling, also called 'rescue effect' from bystander cells to irradiated cells, to enhance the DSB repair in targeted cells.
Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.
Porcupine bezoar (PB) is a calcified undigested material generally found in porcupine's (Hystrix brachyura) gastrointestinal tract. The bezoar is traditionally used in South East Asia and Europe for the treatment of cancer, poisoning, dengue, typhoid, etc. However, limited scientific studies have been performed to verify its anticancer potential to substantiate its traditional claims in the treatment of cancers. Hence, this study was aimed at investigating the in vitro and in vivo anticancer properties of two grassy PB aqueous extract (PB-A and PB-B) using A375 cancer cell line and zebrafish model, respectively. This paper presents the first report on in vitro A375 cell viability assay, apoptosis assay, cell cycle arrest assay, migration assay, invasion assay, qPCR experimental assay and in vivo anti-angiogenesis assay using the grassy PBs. Experimental findings revealed IC50 value are 26.59 ± 1.37 μg/mL and 30.12 ± 3.25 μg/mL for PB-A and PB-B respectively. PBs showed anti-proliferative activity with no significant cytotoxic effect on normal human dermal fibroblast (NHDF). PBs were also found to induce apoptosis via intrinsic pathway and arrest cell cycle at G2/M phase. Additionally, the findings indicated its ability to debilitate migration and invasion of A375 cells. Further evaluation using embryo zebrafish model revealed LC50 = 450.0 ± 2.50 μg/mL and 58.7 ± 5.0 μg/mL for PB-A and PB-B which also exerted anti-angiogenesis effect in zebrafish. Moreover, stearic acid, ursodeoxycholic acid and pregnenolone were identified as possible metabolites that might contribute to the anticancer effect of the both PBs. Overall, this study demonstrated that PB-A and PB-B possess potential in vitro and in vivo anticancer effects which are elicited through selective cytotoxic effect, induction of apoptosis, inhibition of migration and invasion and anti-angiogenesis. This study provides scientific evidence that the porcupine bezoar do possess anti-cancer efficacy and further justifies its traditional utility. However, more experiments with higher vertebrae models are still warranted to validate its traditional claims as an anticancer agent.
Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells has emerged as an invaluable method for generating patient-specific stem cells of any lineage without the use of embryonic materials. Following the first reported generation of iPS cells from murine fibroblasts using retroviral transduction of a defined set of transcription factors, various new strategies have been developed to improve and refine the reprogramming technology. Recent developments provide optimism that the generation of safe iPS cells without any genomic modification could be derived in the near future for the use in clinical settings. This review summarizes current and evolving strategies in the generation of iPS cells, including types of somatic cells for reprogramming, variations of reprogramming genes, reprogramming methods, and how the advancement iPS cells technology can lead to the future success of reproductive medicine.
Polyhydroxyalkanoate (PHA) is a microbial polymer that has been at the forefront of many attempts at tissue engineering. However, the surface of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) is hydrophobic with few recognition sites for cell attachment. Various concentrations of fish-scale collagen peptides (FSCPs) were incorporated into P(3HB-co-4HB) copolymer by aminolysis. Later, FSCPs were introduced onto the aminolyzed P(3HB-co-4HB) scaffolds. Introduction of the FSCP groups was verified using Fourier transform infrared spectroscopy and the ninhydrin method. The effect of the incorporation of FSCPs on hydrophilicity was investigated using the water contact angle. As the concentration of FSCPs increased, the water contact angle decreased. In vitro study demonstrated that P(3HB-co-4HB)/FSCP scaffolds provided better cell attachment and growth of L929 mouse fibroblast cells and better cell proliferation. In vivo study showed that P(3HB-co-4HB)/1.5 wt% FSCPs had a significant effect on wound contractions, with the highest percentage of wound closure (61%) in 7 d.
Copper complexes have been widely studied for the anti-tumour application as cancer cells are reported to take up greater amounts of copper than normal cells. Preliminary study revealed that the newly synthesised copper complex [Cu(SBCM)2] displayed marked anti-proliferative towards triple-negative MDA-MB-231 breast cancer cells. Therefore, Cu(SBCM)2 has great potential to be developed as an agent for the management of breast cancer. The present study was carried out to investigate the mode of cell death induced by Cu(SBCM)2 towards MDA-MB-231 breast cancer cells. The inhibitory and morphological changes of MDA-MB-231 cells treated with Cu(SBCM)2 was determined by using MTT assay and inverted light microscope, respectively. The safety profile of Cu(SBCM)2 was also evaluated towards human dermal fibroblast (HDF) normal cells. Confirmation of apoptosis and cell cycle arrest were determined by flow cytometry analysis. The expression of p53, Bax, Bcl-2 and MMP2 protein were detected with western blot analysis. Cu(SBCM)2 significantly inhibited the growth of MDA-MB-231 cells in a dose-dependent manner with GI50 18.7 ± 3.06 µM. Indeed, Cu(SBCM)2 was less toxic towards HDF normal cells with GI50 31.8 ± 4.0 µM. Morphological study revealed that Cu(SBCM)2-treated MDA-MB-231 cells experienced cellular shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies, suggesting that Cu(SBCM)2 induced apoptosis in the cells, which was confirmed by Annexin-V/PI flow cytometry analysis. It was also found that Cu(SBCM)2 induced G2/M phase cell cycle arrest towards MDA-MB-231 cells. The induction of apoptosis and cell cycle arrest in the present study is possibly due to the down-regulation of the mutant p53 and MMP2 protein. In conclusion, Cu(SBCM)2 can be developed as a targeted therapy for the treatment of triple-negative breast cancer.
The evaluation of the interaction of cells with biomaterials is fundamental to establish the suitability of the biomaterial for a specific application. In this study, the properties of bacterial nanocellulose/acrylic acid (BNC/AA) hydrogels fabricated with varying BNC to AA ratios and electron-beam irradiation doses were determined. The manner these hydrogel properties influence the behavior of human dermal fibroblasts (HDFs) at the cellular and molecular levels was also investigated, relating it to its application both as a cell carrier and wound dressing material. Swelling, hardness, adhesive force (wet), porosity, and hydrophilicity (dry) of the hydrogels were dependent on the degree of cross-linking and the amount of AA incorporated in the hydrogels. However, water vapor transmission rate, pore size, hydrophilicity (semidry), and topography were similar between all formulations, leading to a similar cell attachment and proliferation profile. At the cellular level, the hydrogel demonstrated rapid cell adhesion, maintained HDFs viability and morphology, restricted cellular migration, and facilitated fast transfer of cells. At the molecular level, the hydrogel affected nine wound-healing genes (IL6, IL10, MMP2, CTSK, FGF7, GM-CSF, TGFB1, COX2, and F3). The findings indicate that the BNC/AA hydrogel is a potential biomaterial that can be employed as a wound-dressing material to incorporate HDFs for the acceleration of wound healing.
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.