Effect of Functionalization of the Polycaprolactone Film Surface on the Mechanical and Biological Properties of the Film Itself.

The lack of suitable functional groups for cell adhesion on the surface of Polycaprolactone (PCL) is one of the main limitations in order to use PCL for biomedical applications. The aim of this research is to modify the PCL film surface using arginine, via an aminolysis reaction. In this regard, after PCL films formation by casting method, they were immersed in arginine solutions of various concentration at room temperature or then heated to 40 °C and in the presence of isopropanol or without it. To assess the structure of the modified surface, its wettability, and mechanical properties, methods of measuring the contact angle and the strip tensile test were used, and to compare the degree of attachment and the rate of cell proliferation, the method of fluorescent staining of cultured cells was used. The change in protein synthesis by cells on the modified surface was assessed using Western blotting. The results obtained show that the treatment of PCL films with an aqueous solution of arginine at room temperature for 1 day increases the hydrophilicity of the surface. Wherein surface modification led to a two-fold decrease of mechanical strength and flow stress, but elongation increase by about 30% for PCL films after modification in 0.5 M aqueous arginine solution at room temperature. Moreover, cell attachment and proliferation, as well as collagen synthesis, were significantly enhanced after arginine modification. The proposed simple and effective method for modifying PCL films with arginine significantly expands the possibilities for developing biocompatible scaffolds for tissue engineering.

Protecting the skin-implant interface with transcutaneous silver-coated skin-and-bone-integrated pylon in pig and rabbit dorsum models.

Implant-associated soft tissue infections at the skin-implant interface represent the most frequent complications in reconstructive surgery and lead to implant failures and revisions. Titanium implants with deep porosity, called skin-and-bone-integrated-pylons (SBIP), allow for skin ingrowth in the morphologically natural direction, thus restoring a reliable dermal barrier and reducing the risk of infection. Silver coating of the SBIP implant surface using physical vapor deposition technique offers the possibility of preventing biofilm formation and exerting a direct antimicrobial effect during the wound healing phase. In vivo studies employing pig and rabbit dorsum models for assessment of skin ingrowth into the pores of the pylon demonstrated the safety of transcutaneous implantation of the SBIP system. No postoperative complications were reported at the end of the follow-up period of 6 months. Histological analysis proved skin ingrowth in the minipig model without signs of silver toxicity. Analysis of silver release (using energy dispersive X-ray spectroscopy) in the model of intramedullary-inserted silver-coated SBIP in New Zealand rabbits demonstrated trace amounts of silver after 3 months of in-bone implantation. In conclusion, selected temporary silver coating of the SBIP implant surface is powerful at preventing the periprosthetic infections without imparing skin ingrowth and can be considered for clinical application.

Evaluation of the temporary effect of physical vapor deposition silver coating on resistance to infection in transdermal skin and bone integrated pylon with deep porosity.

Periprosthetic infection via skin-implant interface is a leading cause of failures and revisions in direct skeletal attachment of limb prostheses. Implants with deep porosity fabricated with skin and bone integrated pylons (SBIP) technology allow for skin ingrowth through the implant's structure creating natural barrier against infection. However, until the skin cells remodel in all pores of the implant, additional care is required to prevent from entering bacteria to the still nonoccupied pores. Temporary silver coating was evaluated in this work as a means to provide protection from infection immediately after implantation followed by dissolution of silver layer in few weeks. A sputtering coating with 1 µm thickness was selected to be sufficient for fighting infection until the deep ingrowth of skin in the porous structure of the pylon is completed. In vitro study showed less bacterial (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) growth on silver coated tablets compared to the control group. Analysis of cellular density of MG-63 cells, fibroblasts, and mesenchymal stem cells (MSCs) showed that silver coating did not inhibit the cell growth on the implants and did not affect cellular functional activity. The in vivo study did not show any postoperative complications during the 6-month observation period in the model of above-knee amputation in rabbits when SBIP implants, either silver-coated or untreated were inserted into the bone residuum. Three-phase scintigraphy demonstrated angiogenesis in the pores of the pylons. The findings suggest that a silver coating with well-chosen specifications can increase the safety of porous implants for direct skeletal attachment. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 169-177, 2019.

Aortic Graft at Coronary Artery Bypass Surgery as a Source of Human Aortic Smooth Muscle Cells.

One of the serious obstacles of the aortopathies research is a considerable shortage of human aortic smooth muscle cells (SMCs), which can be used to model the disease. SMC in most cases come from the whole aorta of transplant donors, which are rather difficult to access. In the course of coronary artery bypass graft (CABG) surgery, a fragment of aortic tissue is excised to make a bypass root. In this study, we show a possibility to use CABG leftover fragments of thoracic aorta as a source of human SMC for in vitro research. We isolated SMC from the fragments of aortic tissues obtained during CABG procedure and compared these cells to the cells that were isolated from aortic tissue of transplant donors. The content of key SMC contractile markers (SMA, SM22α, and vimentin) as well as proliferation and migration rates, metalloproteases MMP-2 and MMP-9 activities were similar in CABG-derived SMC and in transplant donor-derived SMC. In conclusion, leftovers of ascending thoracic aorta obtained during CABG can be used as a source of human aortic SMCs for in vitro research.

Knock-down of Hdj2/DNAJA1 co-chaperone results in an unexpected burst of tumorigenicity of C6 glioblastoma cells.

The chaperone system based on Hsp70 and proteins of the DnaJ family is known to protect tumor cells from a variety of cytotoxic factors, including anti-tumor therapy. To analyze whether this also functions in a highly malignant brain tumor, we knocked down the expression of Hsp70 (HSPA1A) and its two most abundant co-chaperones, Hdj1 (DNAJB1) and Hdj2 (DNAJA1) in a C6 rat glioblastoma cell line. As expected, tumor depletion of Hsp70 caused a substantial reduction in its growth rate and increased the survival of tumor-bearing animals, whereas the reduction of Hdj1 expression had no effect. Unexpectedly, a reduction in the expression of Hdj2 led to the enhanced aggressiveness of the C6 tumor, demonstrated by its rapid growth, metastasis formation and a 1.5-fold reduction in the lifespan of tumor-bearing animals. The in vitro reduction of Hdj2 expression reduced spheroid density and simultaneously enhanced the migration and invasion of C6 cells. At the molecular level, a knock-down of Hdj2 led to the relocation of N-cadherin and the enhanced activity of metalloproteinases 1, 2, 8 and 9, which are markers of highly malignant cancer cells. The changes in the actin cytoskeleton in Hdj2-depleted cells indicate that the protein is also important for prevention of the amoeboid-like transition of tumor cells. The results of this study uncover a completely new role for the Hdj2 co-chaperone in tumorigenicity and suggest that the protein is a potential drug target.

Phenotypic and Functional Changes of Endothelial and Smooth Muscle Cells in Thoracic Aortic Aneurysms.

Thoracic aortic aneurysm develops as a result of complex series of events that alter the cellular structure and the composition of the extracellular matrix of the aortic wall. The purpose of the present work was to study the cellular functions of endothelial and smooth muscle cells from the patients with aneurysms of the thoracic aorta. We studied endothelial and smooth muscle cells from aneurysms in patients with bicuspid aortic valve and with tricuspid aortic valve. The expression of key markers of endothelial (CD31, vWF, and VE-cadherin) and smooth muscle (SMA, SM22α, calponin, and vimentin) cells as well extracellular matrix and MMP activity was studied as well as and apoptosis and cell proliferation. Expression of functional markers of endothelial and smooth muscle cells was reduced in patient cells. Cellular proliferation, migration, and synthesis of extracellular matrix proteins are attenuated in the cells of the patients. We show for the first time that aortic endothelial cell phenotype is changed in the thoracic aortic aneurysms compared to normal aortic wall. In conclusion both endothelial and smooth muscle cells from aneurysms of the ascending aorta have downregulated specific cellular markers and altered functional properties, such as growth rate, apoptosis induction, and extracellular matrix synthesis.

Glu-Trp-ONa or its acylated analogue (R-Glu-Trp-ONa) administration enhances the wound healing in the model of chronic skin wounds in rabbits.

The management of chronic skin wounds represents a major therapeutic challenge. The synthesized dipeptide (Glu-Trp-ONa) and its acylated analogue (R-Glu-Trp-ONa) were assessed in the model of nonhealing dermal wounds in rabbits in relation to their healing properties in wound closure. Following wound modeling, the rabbits received a course of intraperitoneal injections of Glu-Trp-ONa or R-Glu-Trp-ONa. Phosphate-buffered saline and Solcoseryl® were applied as negative and positive control agents, respectively. An injection of Glu-Trp-ONa and R-Glu-Trp-ONa decreased the period of wound healing in animals in comparison to the control and Solcoseryl-treated groups. Acylation of Glu-Trp-ONa proved to be beneficial as related to the healing properties of the dipeptide. Subsequent zymography analyses showed that the applied peptides decreased the proteolytic activity of matrix metalloproteinases MMP-9, MMP-8, and MMP-2 in the early inflammatory phase and reversely increased the activity of MMP-9, MMP-8, and MMP-1 in the remodeling phase. Histological analyses of the wound sections (hematoxylin-eosin, Mallory's staining) confirmed the enhanced formation of granulation tissue and re-epithelialization in the experimental groups. By administering the peptides, wound closures increased significantly through the modulation of the MMPs' activity, indicating their role in wound healing.

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L).

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens', was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.