Physico-Chemical Properties and Biocompatibility of Thermosensitive Chitosan Lactate and Chitosan Chloride Hydrogels Developed for Tissue Engineering Application.

Recently, the modification of the initial structure of biopolymers, mainly chitosan, has been gaining importance with a view to obtain functional forms with increased practicality and specific properties enabling their use in tissue engineering. Therefore, in this article, the properties (structural and biological) of thermosensitive hydrogels obtained from chitosan lactate/chloride and two types of crosslinking agents (ß-glycerol phosphate disodium salt pentahydrate and uridine 5'-monophosphate disodium salt) are discussed. The aim of the research is to identify changes in the structure of the biomaterials during conditioning in water. Structural investigations were carried out by FTIR spectroscopy. The crystallinity of gels was determined by X-ray diffraction analysis. The biocompatibility (evaluation of cytotoxicity and genotoxicity) of chitosan hydrogels was investigated by contact with human colon adenocarcinoma cell line for 48 h. The cytotoxicity was verified based on the colorimetric resazurin assay, and the genotoxicity was checked by the comet assay (percentage of DNA in the comet tail). The conducted research showed that the analyzed types of chitosan hydrogels are non-cytotoxic and non-genotoxic materials. The good biocompatibility of chitosan hydrogels surfaces makes them interesting scaffolds with clinical potential in tissue regeneration engineering.

Influence of chitosan average molecular weight on degradation and stability of electrodeposited conduits.

Tubular chitosan-based hydrogels, obtained in an electrodeposition process, are subject of degradation and stability studies. The implants are prepared from polymer with different average molecular weight. This approach allows fabricating structures that vary in mass and wall thickness. The obtained implants are incubated in phosphate buffered solution (pH 7.4) with or without lysozyme up to 56 days at 37 °C. Subsequently, chemical, physical as well as mechanical properties of implants are evaluated. Although the initial physicomechanical properties are different, they change upon incubation and remain similar over its period. Finally, in vitro biocompatibility of implants is proven after assessing their action towards mHippoE-18 embryonic hippocampal cells and THP1-XBlue™ monocytes. Since dimensions of nerves and the gap length differ across the body and injury, respectively, the possibility to control properties of chitosan applied gives a tool to prepare implants with wall thickness adjusted to the specific peripheral nerve injury case.

Current Progress in Biomedical Applications of Chitosan-Carbon Nanotube Nanocomposites: A Review.

The currently observed development of medical science results from the constant search for innovative solutions to improve the health and quality of life of patients. Particular attention is focused on the design of a new generation of materials with a high degree of biocompatibility and tolerance towards the immune system. In addition, apart from biotolerance, it is important to ensure appropriate mechanical and technological properties of materials intended for intra-body applications. Knowledge of the above parameters becomes the basis for considerations related to the possibilities of choosing the appropriate polymer materials. The researchers' interest, as evidenced by the number of available publications, is attracted by nanobiocomposites based on chitosan and carbon nanotubes, which, due to their properties, enable integration with the tissues of the human body. Nanosystems can be used in many areas of medicine. They constitute an excellent base for use as dressing materials, as they exhibit antimicrobial properties. In addition, they can be carriers of drugs and biological macromolecules and can be used in gene therapy, tissue engineering, and construction of biosensors. For this reason, potential application areas of chitosan-carbon nanotube nanocomposites in medical sciences are presented in this publication, considering the characteristics of the system components.

Epineurium-mimicking chitosan conduits for peripheral nervous tissue engineering.

In this investigation, we report on a fabrication method of epineurium-mimicking tubular conduits based on electrodeposition from chitosan solution. The pre-enrichment of electrodeposition solution with hyaluronic acid and/or collagen components results in structures which structural, morphological, and physicochemical properties can be controlled. In order to determine the optimal composition of the initial chitosan solution resulting in conduits meeting the requirements imposed on peripheral nerve implants, we perform chemical, physical, and biological studies. Both the molecular weight of hyaluronic acid and the concentration of additives are found to be crucial for the final mechanical as well as biological performance of conduits. Because, the obtained structures show biocompatibility when contacting with a mouse hippocampal cell line (mHippoE-18), we further plan to test their application potential on an animal model.

Assessment of degradation and biocompatibility of electrodeposited chitosan and chitosan-carbon nanotube tubular implants.

Designing three-dimensional tubular materials made of chitosan is still a challenging task. Availability of such forms is highly desired by tissue engineering, especially peripheral nerve tissue engineering. Aiming at this problem, we use an electrodeposition phenomenon in order to obtain chitosan and chitosan-carbon nanotube hydrogel tubular implants. The in vitro biocompatibility of the fabricated structures is assessed using a mouse hippocampal cell line (mHippoE-18). As both implants do not induce significant cytotoxicity, they are next subjected to in vitro degradation studies in the environment simulating in vivo conditions for specified periods of time: 7, 14, and 28 days. The mass loss of implants indicates their stability at the tested time period; therefore, the materials are subcutaneously implanted in Sprague Dawley rats. The explants are collected after 7, 14, and 28 days. The assessment of composition and changes in tissues surrounding the implanted materials is made in respect to surrounding tissue thickness as well as the number of blood vessels, macrophages, lymphocytes, and neutrophils. No symptoms of acute inflammation are noticed at any point in time. The observed regular healing process allows concluding that both chitosan and chitosan-carbon hydrogel tubular implants are biocompatible with high application potential in tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2701-2711, 2016.

One week's holiday sun exposure induces expression of photoaging biomarkers.

INTRODUCTION: Skin aging is accompanied by the upregulation of the expression of various matrix metalloproteinases (MMPs). It was shown that exposure to ultraviolet radiation (UVR) may induce skin expression of MMPs and dysregulation of the transforming growth factor beta (TGF-ß)/Smad pathway. The aim of our study was to compare the effects of short holiday UVR exposure and lifetime UVR exposure, on the expression of MMP-8, TGF-ß1, and Smad2 in human skin biopsies. MATERIAL AND METHODS: Skin biopsies were taken from the outer upper arm of 15 elderly people with significant photoaging (mean age 64.1 years) (Group 1) and from 15 healthy young adult volunteers (mean age 24.1 y) who participated in a six-day sun holiday. Biopsies were taken twice: 24 hours before leaving for holiday (Group 2a) and 24 hours after returning (Group 2b). The expression of TGF-ß1, Smad2, and MMP-8 was examined by immunochemistry and measured semiquantitatively by two independent pathologists. RESULTS: The mean expression of TGF-ß1 in dermal fibroblasts and keratinocytes in Group 1 and Group 2b was significantly lower than in Group 2a (0.54% ± 0.44% and 0.48% ± 0.51% vs. 1.48% ± 0.72%, respectively). The percentage of Smad2 (+) cells in Group 1 and Group 2b was lower than in Group 2a (2.13% ± 1.39% and 1.81% ± 1.16% vs. 4.13% ± 1.58%, respectively). The MMP-8 expression in Group 2b was 1.36% ± 0.68% and was significantly higher than in Group 1 (0.34% ± 0.42%) and Group 2a in which the protein was not detected (p < 0.001). CONCLUSIONS: We conclude that the decrease in the expression of TGF-ß1 and Smad2 is a persistent biomarker of skin photoaging, while the increased expression of MMP-8 in keratinocytes can be regarded as a marker of acute sun exposure.

Tubular electrodeposition of chitosan-carbon nanotube implants enriched with calcium ions.

A new approach for obtaining chitosan-carbon nanotube implants enriched with calcium ions in the form of tubular hydrogels is fostered. The intended application of the hydrogels is tissue engineering, especially peripheral nervous tissue regeneration. The fabrication method, based on an electrodeposition phenomenon, shows significant advantages over current solutions as implants can now be obtained rapidly at any required dimensions. Thus, it may open a new avenue to treat patients with peripheral nerve injuries. Either single walled or multiwalled carbon nanotubes enhance the mechanical properties of the tubular hydrogels. The controlled presence of calcium ions, sourced from hydroxyapatite, is also expected to augment the regenerative response. Because in vitro cytotoxic assays on mouse cell lines (L929 fibroblasts and mHippoE-18 hippocampal cells) as well as pro-inflammatory tests on THP-1XBlue™ cells show that the manufactured implants are biocompatible, we next intend to evaluate their immune- and nervous-safety on an animal model.

Chitosan-based hydrogel implants enriched with calcium ions intended for peripheral nervous tissue regeneration.

A new method for fabrication of chitosan-based hydrogel implants intended for peripheral nervous tissue regeneration was developed. The method is based on an electrodeposition phenomenon from a solution of chitosan and organic acid. In order to increase the mechanical strength of the implant, the solution was enriched with hydroxyapatite. Hydroxyapatite served as a source of calcium ions too. The influence of the concentration of the polymer and the additive on chemical, mechanical as well as biological properties of the obtained implant was evaluated. The study showed great dependence of the initial solution composition mainly on the physicochemical properties of the resulting structure. Basic in vitro cytotoxic and pro-inflammatory assays showed biocompatibility of manufactured implants, therefore, animal experimentations may be considered.

Pigment protection factor as a predictor of skin photosensitivity--a Polish study.

Assessment of individual photosensitivity by determining the minimal erythema dose (MED) is commonly accepted. MED objectively describes a single individual response to the irradiation of skin with a particular wavelength (UVB, UVA). Pigment protection factor (PPF) is an objective value to measure skin type. The aim of the project was to analyze PPF values in the population of Lodz and the relationship between PPF, skin phototype, and individual MED. The study was conducted on the group of 270 volunteers: 130 men and 140 women, mean age 28.5 years (OS + 9.66) with either skin phototype II or III, as defined by Fitzpatrick Skin Phototype Classification. Phototesting of each volunteer was undertaken with an increasing dose series (UVB radiation) on six squares (1×1 cm) on the skin of the back. The MED was defined as a perceptible erythema 24 hours later. Starting dose was determined by history, physical examination, and phototype ranged from 0.03-0.07 J/cm2. PPF was measured by a skin reflectance meter UV Optimize 555. The mean MED value was 0.15 J/cm2 and the PPF value was 6.15. A positive correlation between the MED value and PPF (R=0.38; P<0.001), and a positive correlation between phototype and MED and PPF (P< 0.001) were found. Both determination of MED and PPF are objective methods of photosensitivity assessment, but PPF determination is an easy and non-invasive method.

Atopic dermatitis patients carrying G allele in -1082 G/A IL-10 polymorphism are predisposed to higher serum concentration of IL-10.

INTRODUCTION: Atopic dermatitis (AD) is a chronic skin inflammatory disease in which Th2-derived cytokines play an essential role. Aim of the study was to assess interleukin 4, 10 and 13 (IL-4, IL-10 and IL-13) serum concentrations in AD patients and to correlate the values with the occurrence of genotypes of selected polymorphisms in genes encoding these cytokines. MATERIAL AND METHODS: Seventy-six AD patients (mean age 11.4 years) and 60 healthy controls were enrolled in the study. Blood samples were analyzed for IL-4, IL-10 and IL-13 concentrations with ELISA assay and genotyping for -590C/T IL-4, -1082A/G IL-10 and -1055C/T IL-13 polymorphisms with PCR-RFLP. RESULTS: The obtained results revealed statistically higher serum concentration of IL-10 and IL-13 in AD patients when compared to healthy controls (10.30 pg/ml vs. 8.51 pg/ml for IL-10 and 5.67 pg/ml vs. 4.98 pg/ml for IL-13). There were no significant differences between AD patients and controls in regard to IL-4 serum level (5.10 pg/ml vs. 7.1 pg/ml). Analyzing the association between level of the examined cytokines and genotype polymorphisms -590 C/T for the IL-4 gene, -1082 A/G for the IL-10 gene and -1055 C/T for the IL-13 gene, we found a statistically higher IL-10 serum level among carriers of the G allele in the -1082 G/A IL-10 polymorphism both in AD and control groups. We did not find any significant differences between serum level of IL-4 and IL-13 in regard to genotype occurrence in examined polymorphisms: -590 C/T for the IL-4 gene and -1055 C/T for the IL-13 gene. CONCLUSIONS: The obtained results confirm the genetic background of IL-10 synthesis in the Polish population.

Can biologic treatment induce cutaneous focal mucinosis?

Skin mucinosis is a rare skin disease which clinically manifests as firm papules and waxy nodules. We report a case of a 66-year-old female psoriatic patient who developed skin mucinosis during biological therapy. Because of a previous lack of response to the local and conventional systemic treatment of psoriasis, the patient received biological therapy (infliximab from June 2008 to May 2009 - initial clinical improvement and loss of treatment effectiveness in the 36(th) week of the therapy; adalimumab from June 2009 to January 2010 - lack effectiveness; ustekinumab from March 2012 to the present). Throughout 2 months we observed a manifestation of the skin mucinosis as well-demarcated, yellow and brown, papulo-nodular lesions of 5-10 mm in diameter, localized on the back. Histopathological examination with alcian blue staining demonstrated mucin deposits in the dermis. On the basis of clinical and histopathological findings, the diagnosis of cutaneous focal mucinosis was established. We present the case because of the extremely rare occurrence of the disease. Scarce literature and data suggest that there is an association between focal mucinosis and thyroid dysfunction, as well as possible adverse effects of biological therapy with TNF-α antagonists.

Sonic hedgehog pathway dysregulation in skin basal-cell carcinoma of a Polish population.

Sonic hedgehog (Shh) pathway impairment plays a key role in the pathogenesis of basal-cell carcinomas (BCC), the most frequent skin tumor among Caucasians. Shh, Smo, and Gli2 family proteins are necessary for adequate and controlled cell proliferation. The aim of this study was to evaluate Shh, Smo, and Smo expression in BCC skin biopsies taken from sun-exposed areas. 41 BCC skin biopsies and 22 healthy skin specimens (the control group) taken from the same areas served as material for the study. All specimens were immunohistochemically stained with monoclonal antibodies directed against the chosen proteins. Shh and Smo expression (cytoplasmic pattern) were recorded semiquantitatively using a four-grade score (0-3). Gli2 expression (nuclear pattern) was determined using an image analysis system (semiautomatic function). The immunoexpression of the Shh and Smo proteins significantly increased in the BCC group, as compared with the normal controls (for Shh, the mean intensity was 1.67 in BCC vs. 1.17 in the control group, p < 0.001; for Smo, the mean intensity was 1.46 in BCC vs. 0.99 in the control group, p < 0.001). The staining for Gli2 in the BCC group was completely negative, but indicated the presence of Gli2 in the control patients (1.15 Gli2+ cells/100 cells). Sonic hedgehog pathway dysregulation may play an important role in skin cancerogenesis leading to BCC development.

Effective treatment of nodular amyloidosis with carbon dioxide laser.

BACKGROUND: Nodular amyloidosis is a rare form of localized cutaneous amyloidosis that is characterized by nodules located on the extremities, trunk, genitalia, or face. In treatment regimens, many approaches have been described, including carbon dioxide (CO2) laser therapy. OBJECTIVE: We present a case of a 60-year-old white male with a 20-year history of disseminated waxy, purpuric, yellowish, and bullous skin lesions on the trunk and extremities. The skin changes were accompanied by pain during palpation and were temporarily pruritic. METHOD: Based on histologic and direct immunofluorescence test findings, the diagnosis of cutaneous nodular amyloidosis was established. Skin lesions were treated with a CO2 laser. During surgery, treated tissue was found to be slightly friable, and there was a little problem with hemostasis that correlated with amyloid infiltration of the dermis and blood vessels. However, after 8 weeks, we observed clinical improvement of all treated areas with the presence of atrophic scars. In the regions of laser therapy, no recurrence of the disease was observed during a 12-month follow-up. CONCLUSION: Based on these results, we conclude that CO2 laser has a beneficial effect in the treatment of nodular amyloidosis; however, surgery procedures may be associated sometimes with tissue friability and poor hemostasis.

An enhanced risk of basal cell carcinoma is associated with particular polymorphisms in the VDR and MTHFR genes.

BACKGROUND: Vitamin D and folate are influenced by ultraviolet radiation (UVR), and both are implicated in skin carcinogenesis. Polymorphisms in the genes involved in the metabolism of these two compounds may alter the risk of basal cell carcinoma (BCC). OBJECTIVE: To assess the frequency of four polymorphisms in the gene encoding the vitamin D receptor (VDR) (FokI, BsmI, TaqI and ApaI) and two in the gene encoding methylenetetrahydrofolate reductase (MTHFR) (677C/T and 1286A/C) in 142 patients of Polish origin with BCC and the same number of controls. The expression of VDR and MTHFR proteins in the skin, and the vitamin D status of a subset of patients and controls were also measured. PATIENTS/METHODS: The polymorphisms were assayed by PCR-RFLP, the VDR and MTHFR proteins by immunoblotting and vitamin D status as 25-hydroxyvitamin D (25(OH)D) level in the serum by RIA. RESULTS: The presence of the TT genotype in the FokI VDR polymorphism resulted in a >10-fold higher risk of BCC development. The CT genotype in 677C/T MTHFR polymorphism and CC genotype in 1286A/C MTHFR polymorphism also significantly increased the risk of BCC development. The expression of the VDR and MTHFR proteins was significantly higher in BCCs of the patients than in the healthy skin of the controls. The median serum level of 25(OH)D was significantly higher in the control group compared with the patients with BCC. CONCLUSIONS: Certain VDR and MTHFR gene polymorphisms increase the risk of BCC development in individuals of Polish origin.

Risk factors in Central Poland for the development of superficial and nodular basal cell carcinomas.

INTRODUCTION: In the last decades the number of skin carcinomas has dramatically increased, which is mainly connected with changes in lifestyle, especially with common use of artificial light sources such as sunbeds. Basal cell carcinoma (BCC) is the most common form of skin cancer in white populations. Basal cell carcinomas are divided into subtypes, depending on their clinical picture and histology. The main groups are nodular (nBCC) and superficial (sBCC) ones. The major recognized risk factors for basal cell carcinoma (BCC) are exposure to chronic and intermittent burning doses of sunlight. Other risk factors leading to the development of the nBCC and sBCC subtypes of BCC are not well established. MATERIAL AND METHODS: An analysis of 123 patients with either nBCC or sBCC, living in Lodz, Poland, regarding various intrinsic and environmental parameters was undertaken following the histological diagnosis of BCC. RESULTS: No statistical differences were observed between the BCC subtype and sex, age, hair colour, eye colour, smoking, family history of skin cancer, occupation, or past episodes of sunburn. While sBCCs tended to occur on unexposed body sites in phototype I/II subjects who mainly avoided direct sunlight, nBCCs tended to occur on sun-exposed body sites in phototype III subjects who were frequently in direct sunlight. CONCLUSIONS: Thus the development of particular BCC subtypes is partially dependent on phototype and personal sun behaviour.