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The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

Made available in DSpace on 2018-12-11T17:24:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-09-10 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Skin graft successful depends on reduction of local inflammation evoked by the surgical lesion and efficient neovascularization to nutrition the graft. It has been shown that N-terminal portion of the Annexin A1 protein (AnxA1) with its anti-inflammatory properties induces epithelial mucosa repair and presents potential therapeutic approaches. The role of AnxA1 on wound healing has not been explored and we investigated in this study the effect of the peptide Ac2-26 (N-terminal AnxA1 peptide Ac2-26; AnxA12-26) on heterologous skin scaffolds transplantation in BALB/c mice, focusing on inflammation and angiogenesis. Treatment with AnxA12-26, once a day, from day 3-60 after scaffold implantation improved the take of the implant, induced vessels formation, enhanced gene and protein levels of the vascular growth factor-A (VEGF-A) and fibroblast influx into allograft tissue. It also decreased pro- while increasing anti-inflammatory cytokines. The pro-angiogenic activity of AnxA12-26 was corroborated by topical application of AnxA12-26 on the subcutaneous tissue of mice. Moreover, treatment of human umbilical endothelial cells (HUVECs) with AnxA12-26 improved proliferation, shortened cycle, increased migration and actin polymerization similarly to those evoked by VEGF-A. The peptide treatment instead only potentiated the tube formation induced by VEGF-A. Collectively, our data showed that AnxA12-26 treatment favors the tissue regeneration after skin grafting by avoiding exacerbated inflammation and improving the angiogenesis process. São Paulo State University (Unesp) Institute of Biosciences Humanities and Exact Sciences (Ibilce) Department of Clinical and Toxicological Analysis Faculty of Pharmaceutical Sciences University of São Paulo From the Post-Graduation in Structural and Functional Biology Federal University of São Paulo Department of Surgical Research Northwick Park Institute for Medical Research University College London São Paulo State University (Unesp) Institute of Biosciences Humanities and Exact Sciences (Ibilce)

Patients with laryngeal disorders may have severe morbidity relating to swallowing, vocalization, and respiratory function, for which conventional therapies are suboptimal. A tissue-engineered approach would aim to restore the vocal folds and maintain respiratory function while limiting the extent of scarring in the regenerated tissue. Under Good Laboratory Practice conditions, we decellularized porcine larynges, using detergents and enzymes under negative pressure to produce an acellular scaffold comprising cartilage, muscle, and mucosa. To assess safety and functionality before clinical trials, a decellularized hemilarynx seeded with human bone marrow-derived mesenchymal stem cells and a tissue-engineered oral mucosal sheet was implanted orthotopically into six pigs. The seeded grafts were left in situ for 6 months and assessed using computed tomography imaging, bronchoscopy, and mucosal brushings, together with vocal recording and histological analysis on explantation. The graft caused no adverse respiratory function, nor did it impact swallowing or vocalization. Rudimentary vocal folds covered by contiguous epithelium were easily identifiable. In conclusion, the proposed tissue-engineered approach represents a viable alternative treatment for laryngeal defects.

Made available in DSpace on 2018-12-11T17:06:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-10-11 Biocompatibility of two newly developed porcine skin scaffolds was assessed after 3, 14, 21 and 90 days of implantation in rats. Both scaffolds showed absence of cells, preservation of ECM and mechanical properties comparable to non-decellularised skin before implantation. Host cell infiltration was much prominent on both scaffolds when compared to Permacol (surgical control). At day 3, the grafts were surrounded by polymorphonuclear cells, which were replaced by a notable number of IL-6-positive cells at day 14. Simultaneously, the number of pro-inflammatory M1-macrophage was enhanced. Interestingly, a predominant pro-remodeling M2 response, with newly formed vessels, myofibroblasts activation and a shift on the type of collagen expression was sequentially delayed (around 21 days). The gene expression of some trophic factors involved in tissue remodeling was congruent with the cellular events. Our findings suggested that the responsiveness of macrophages after non-crosslinked skin scaffolds implantation seemed to intimately affect various cell responses and molecular events; and this range of mutually reinforcing actions was predictive of a positive tissue remodeling that was essential for the long-standing success of the implants. Furthermore, our study indicates that non-crosslinked biologic scaffold implantation is biocompatible to the host tissue and somehow underlying molecular events involved in tissue repair. Post-Graduation in Structural and Functional Biology Federal University of São Paulo (UNIFESP) Department of Biology Instituto de Biociências Letras e Ciências Exatas São Paulo State University (UNESP) Department of Surgical Research Northwick Park Institute for Medical Research University College London (UCL) Department of Biology Instituto de Biociências Letras e Ciências Exatas São Paulo State University (UNESP)

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

This study aimed at evaluating the potential effect of gallium-incorporated phosphate-based glasses towards periodontitis-associated bacteria, Porphyromonas gingivalis, and matrix metalloproteinase-13. Periodontitis describes a group of inflammatory diseases of the gingiva and supporting structures of the periodontium. They are initiated by the accumulation of plaque bacteria, such as the putative periodontal pathogen Porphyromonas gingivalis, but the host immune response such as elevated matrix metalloproteinases are the major contributing factor for destruction of periodontal tissues. Antibacterial assays of gallium-incorporated phosphate-based glasses were conducted on Porphyromonas gingivalis ATCC 33277 using disc diffusion assay on fastidious anaerobe agar and liquid broth assay in a modified tryptic soy broth. In vitro study investigated the effect of gallium on purified recombinant human matrix metalloproteinase-13 activity using matrix metalloproteinase assay kit. In vivo biocompatibility of gallium-incorporated phosphate-based glass was evaluated in rats as subcutaneous implants. Antibacterial assay of gallium displayed activity against Porphyromonas gingivalis (inhibition zone of 22 ± 0.5 mm compared with 0 mm for control glass, c-PBG). Gallium in the glass contributed to growth inhibitory effect on Porphyromonas gingivalis (up to 1.30 reductions in log 10 values of the viable counts compared with control) in a modified tryptic soy broth. In vitro study showed gallium-incorporated phosphate-based glasses inhibited matrix metalloproteinase activity significantly ( p ≤ 0.01) compared with c-PBG. Evaluation of in vivo biocompatibility of gallium-incorporated phosphate-based glasses in rats showed a non-toxic and foreign body response after 2 weeks of implantation. The results indicate that gallium ions might act on multiple targets of biological mechanisms underlying periodontal disease. Moreover, gallium-incorporated phosphate-based glasses are biocompatible in a rat model. The findings warrant further investigation and will have important clinical implications in the future treatment and management of periodontitis.

Off-the-shelf availability of tissue-engineered skin constructs, tailored by different combinations of reagents to produce a highly preserved biological matrix is often the only means to help patients suffering skin damage. This study assessed the effect of five different decellularisation methods on porcine dermal scaffolds with regard to matrix composition, biomechanical strength, and cytotoxicity using an in vitro biocompatibility assay. Results demonstrated that four out of the five tested decellularisation protocols were efficient in producing acellular scaffolds. Nevertheless, decellularisation method using osmotic shock without enzymatic digestion showed to be efficient not only in removing cellular material and debris from dermal scaffolds but was also beneficial in the preservation of extracellular matrix components (glycosaminoglycans and collagen). Histological assessment revealed that the dermal architecture of coarse collagen bundles was preserved. Examinations by scanning electron microscopy and transmission electron microscopy showed that the arrangement and ultrastructure of collagen fibrils in the scaffolds were retained following non-enzymatic method of decellularisation and also after collagen crosslinking using genipin. Moreover, this decellularised scaffold was not only shown to be biologically compatible when co-cultured with bone marrow-derived mesenchymal stem cells and fibroblasts, but also stimulated the cells to release trophic factors essential for tissue regeneration.

Skin is a multilayer composite and exhibits highly non-linear, viscoelastic, anisotropic material properties. In many consumer product and medical applications (e.g. during shaving, needle insertion, patient re-positioning), large tissue displacements and deformations are involved; consequently large local strains in the skin tissue can occur. Here, we present a novel imaging-based method to study skin deformations and the mechanics of interacting skin layers of full-thickness skin. Shear experiments and real-time video recording were combined with digital image correlation and strain field analysis to visualise and quantify skin layer deformations during dynamic mechanical testing. A global shear strain of 10% was applied to airbrush-patterned porcine skin (thickness: 1.2–1.6 mm) using a rotational rheometer. The recordings were analysed with ARAMIS image correlation software, and local skin displacement, strain and stiffness profiles through the skin layers determined. The results of this pilot study revealed inhomogeneous skin deformation, characterised by a gradual transition from a low (2.0–5.0%; epidermis) to high (10–22%; dermis) shear strain regime. Shear moduli ranged from 20 to 130 kPa. The herein presented method will be used for more extended studies on viable human skin, and is considered a valuable foundation for further development of constitutive models which can be used in advanced finite element analyses of skin