In this paper, we have shown for the fi rst time the soft tissue response of novel silver/ poly(vinyl alcohol)/graphene (Ag/PVA/Gr) and silver/poly(vinyl alcohol)/chitosan/ graphene (Ag/PVA/CHI/Gr) nanocomposite hydrogels aimed for medical applications. These novel hydrogels were produced by in situ electrochemical synthesis of silver nanoparticles in the polymer matrices as described in our previously published works. Both Ag/PVA/Gr and Ag/PVA/CHI/Gr, as well as controls Ag/PVA, Ag/PVA/CHI and commercial Suprasorb©hydrogel discs, were implanted in the subcutaneous tissue of rats. Implants with the surrounding tissue were dissected after post-implantation on days 7, 15, 30 and 60, and then processed for histological examination. The tissue irritation index (TIrI) score, according to ISO 10993-6, 2007, as well as the number of leukocytes in the peri-implant zone and connective tissue capsule thickness were examined. The results show that each TIrI score, the leukocyte number around the implanted materials and capsule thickness gradually decreased during the observation period. At the endpoint of follow-up, the Ag/PVA/CHI/Gr implant was surrounded with a thinner capsule, while both the TIrI score and the number of leukocytes of the peri-implant zone were greater compared to the Ag/PVA/Gr implant. Despite the observed differences, we can conclude that our in vivo experiment suggested that both novel hydrogels were biocompatible and suitable for medical use.
A newly produced biomaterial is necessarily subject of standards, which are performed in vivo on animal models. For the evaluation of soft tissue regenerative possibilities after subcutaneous implantation of biomaterials – silver/poly(vinyl alcohol) (Ag/PVA) and novel silver/poly(vinyl alcohol)/graphene (Ag/PVA/Gr) provided for clinical use, sixteen rats were used, according to the instructions of international standards, ISO 10993-6, 2007. Histological sections were observed 7, 15, 30 and 60 days after grafting. These hydrogels were produced by in situ electrochemical synthesis of silver nanoparticles in the polymer matrices, which enabled obtaining completely safe and biocompatible materials, free from any additional toxic chemical reducing agents. Surgical implantation of hydrogels was done according to the permission of the Ethical Committee of the Faculty of Veterinary Medicine, University of Belgrade. Immunohistochemical (IHC) studies included the assessment of smooth muscle expression actin in blood vessels (α-SMA), the expression of laminin and type I and type III collagen in the skin structures, and, the determination of cell proliferation marker expression (Ki-67) keratinocytes. The results were assessed in a semiquantitative manner. The data were analyzed in the statistical software package IBM SPSS 20. The conclusions indicated that Ag/PVA/Gr might be used as wound dressings to enhance the tissue healing potential and established faster integration and shorter retention in the tissue.
Biocompatibility of materials is one of the most important conditions for their successful application in tissue regeneration and repair. Cell-surface interactions stimulate adhesion and activation of macrophages whose acquaintance can assist in designing novel biomaterials that promote favorable macrophage–biomaterial surface interactions for clinical application. This study is designed to determine the distribution and number of macrophages as a means of biocompatibility evaluation of two newly synthesized materials [silver/poly(vinyl alcohol) (Ag/PVA) and silver/poly(vinyl alcohol)/graphene (Ag/PVA/Gr) nanocomposite hydrogels] in vivo, with approval of the Ethics Committee of the Faculty of Veterinary Medicine, University of Belgrade. Macrophages and giant cells were analyzed in tissue sections stained by routine H&E and immunohistochemical methods (CD68+). Statistical relevance was determined in the statistical software package SPSS 20 (IBM corp). The results of the study in terms of the number of giant cells localized around the implant showed that their number was highest on the seventh postoperative day (p.o.d.) in the group implanted with Ag/PVA hydrogels, and on the 30th p.o.d. in the group implanted with Ag/PVA/Gr. Interestingly, the number of macrophages measured in the capsular and pericapsular space was highest in the group implanted with the commercial Suprasorb© material. The increased macrophage number, registered around the Ag/PVA/Gr implant on 60th p.o.d. indicates that the addition of graphene can, in a specific way, modulate different biological responses of tissues in the process of wound healing, regeneration, and integration.
Maternal hypothyroidism in its overt form affects skeletal development of the offspring, but these data are not available for the subclinical form which is becoming very frequent among pregnant women. We hypothesized that the subclinical form of hypothyroidism in rat dams, infl uences the process of offspring endochondral ossifi cation affecting proliferation and differentiation of chondrocytes, osteoclasts and osteoblasts in pups. Seven-day-old male pups (n=18) derived from control dams and dams treated with a low dose (1.5 mg/L) or high dose (150 mg/L) of propylthiouracil in drinking water during pregnancy and lactation were used. Histomorphometric analysis of pups’ tibia proximal growth plate, expression of mRNA, immunohistochemical and histochemical visualization of extracellular matrix components was performed. The length of the tibia was reduced in hypothyroid pups. Secretion of type 2 and 10 collagens in the subclinical and overt form were lower while the amount of glycosaminoglycans was higher when compared with controls. Down-regulated tartrate resistant acid phosphatase mRNA indicated altered osteoclasts function while lower expression of dentin matrix acid protein-1 mRNA and reduced synthesis of type 1 collagen accentuated a compromised bone formation in the overt form of hypothyroidism. The subclinical form of maternal hypothyroidism had a negative effect on the differentiation of hypertrophic chondrocytes and calcifi ed cartilage removal in 7-day-old pups. In addition, overt hypothyroidism had a negative effect on the proliferation of chondrocytes and deposition of osteoid. Both forms of hypothyroidism resulted in a decrease of tibia length due to changes in growth plate formation.
Epidermis stem cells have a crucial role through the processes of proliferation and differentiation, to replace cells that are constantly lost during tissue turnover or following injury. On the other hand, thyroid hormones regulate the proliferation and differentiation of epidermal cells and thus significantly influence the homeostasis of the skin. It is well known that maternal hypothyroidism during pregnancy leads to impaired development of many organ systems in their offspring. However, there is a lack of data about the influence of maternal subclinical hypothyroidism during pregnancy and lactation on the development of the skin and its derivatives in the litter. The aim of this study was to investigate the effects of maternal thyroid dysfunction on the development of the skin and its derivatives in their offspring in the early postnatal period. Antithyroid substance 6-n-propyl-2-thiouracil was added into the drinking water to female Albino Oxfords rats from the beginning of pregnancy and during lactation, with the aim to induce subclinical and overt form of hypothyroidism. Skin samples were taken from male pups within twenty-four hours and seven days after birth. The main findings of this investigation were that both forms of maternal hypothyroidism lead to serious damage of the epidermis in pups in terms of pronounced hyperkeratosis and reduction of the germinal layer along with a reduced number of hair follicles and their delayed morphogenesis. Epidermal impairments were more pronounced in pups with the overt form of hypothyroidism while offspring with the subclinical form had impairments that were less pronounced and delayed in occurrence.
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