Carrageenan films as promising mucoadhesive ocular drug delivery systems.

Polymer mucoadhesive films being developed for use in ophthalmology represent a new tool for drug delivery and are considered an alternative to traditional dosage forms. Due to their mucoadhesive properties, carrageenans (CRGs) are widely used in various forms for drug delivery. In this study, films based on CRGs of various structural types (κ/ß, κ, x, and λ) for use in ophthalmology were studied. The films were loaded with the active substance echinochrome (ECH), a sea urchin pigment used in ophthalmology. Spectral data showed that ECH remained stable after its incorporation into the CRG films and did not oxidize for at least six months. Hydrophilic CRG films with a thickness of 10-12 µm were characterized in terms of their swelling and mucoadhesive properties. The rheological properties of solutions formed after film dissolution in artificial tears were also assessed. κ- and κ/ß-CRG films with ECH exhibited pseudoplastic behavior after rehydrating films with an artificial tear solution. The CRG-loaded films had different swelling characteristics depending on the structure of the CRG used. The films based on highly sulfated CRGs dissolved in artificial tears, while the films of low-sulfated κ/ß-CRG exhibited limited swelling. All studied ECH-loaded films exhibited mucoadhesive properties, which were evaluated by a texture analyzer using mucous tissue of the small intestine of the pig as a model. There was a slight prolongation of ECH release from CRG films in artificial tears. The effect of CRG/ECH on the epithelial cell lines of the outer shell of the human eye was investigated. At low concentrations, ECH in the composition of the CRG/ECH complex had no cytotoxic effect on corneal epithelial and conjunctival human cells. The use of ECH-containing films can prevent the drug from being immediately washed away by tears and help to retain it by increasing viscosity and having mucoadhesive properties.

Corneal Reconstruction with EGFP-Labelled Limbal Mesenchymal Stem Cells in a Rabbit Model of Limbal Stem Cell Deficiency.

Ocular surface reconstruction is essential for treating corneal epithelial defects and vision recovery. Stem cell-based therapy demonstrates promising results but requires further research to elucidate stem cell survival, growth, and differentiation after transplantation in vivo. This study examined the corneal reconstruction promoted by EGFP-labeled limbal mesenchymal stem cells (L-MSCs-EGFP) and their fate after transplantation. EGFP labeling allowed us to evaluate the migration and survival rates of the transferred cells. L-MSCs-EGFP seeded onto decellularized human amniotic membrane (dHAM) were transplanted into rabbits with a modeled limbal stem cell deficiency. The localization and viability of the transplanted cells in animal tissue were analyzed using histology, immunohistochemistry, and confocal microscopy up to 3 months after transplantation. EGFP-labeled cells remained viable for the first 14 days after transplantation. By the 90th day, epithelialization of the rabbit corneas reached 90%, but the presence of viable labeled cells was not observed within the newly formed epithelium. Although labeled cells demonstrated low survivability in host tissue, the squamous corneal-like epithelium was partially restored by the 30th day after transplantation of the tissue-engineered graft. Overall, this study paves the way for further optimization of transplantation conditions and studying the mechanisms of corneal tissue restoration.

Labial Mucosa Stem Cells: Isolation, Characterization, and Their Potential for Corneal Epithelial Reconstruction.

Purpose: The purpose of this study was to characterize labial mucosa stem cells (LMSCs) and to investigate their potential for corneal epithelial reconstruction in a rabbit model of total limbal stem cell deficiency (LSCD). Methods: Rabbit LMSCs (rLMSCs) and human (hLMSCs) LMSCs were derived from labial mucosa and characterized in terms of their proliferation activity by the evaluation of proliferation index (PI) and colony forming efficiency (CFE), cell senescence, and differentiation abilities. The expression of various limbus-specific, stem cell-specific, and epithelial markers was assessed via immunocytochemistry. Flow cytometry was used to evaluate mesenchymal and hematopoietic cell surface markers expression. Chromosomal stability of the derived cells was examined using the conventional GTG-banding technique. To assess the impact of LMSCs on corneal epithelial reconstruction, rLMSCs were seeded onto a decellularized human amniotic membrane (dHAM), thereafter their regeneration potential was examined in the rabbit model of total LSCD. Results: Both rLMSCs and hLMSCs showed high proliferation and differentiation abilities, entered senescence at later passages, and expressed different stem cell-specific (ABCB5, ALDH3A1, ABCG2, and p63α), mesenchymal (vimentin), and epithelial (CK3/12, CK15) markers. Cell surface antigen expression was similar to other described mesenchymal stem cells. No clonal structural chromosome abnormalities (CSCAs) and the low percentage of non-clonal structural chromosome abnormalities (NSCAs) were observed. Transplantation of rLMSCs promoted corneal epithelial reconstruction and enhanced corneal transparency. Conclusions: LMSCs have significant proliferation and differentiation abilities, display no detrimental chromosome aberrations, and demonstrate considerable potential for corneal repair.

Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology.

The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.

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.

Experimental bladder regeneration using a poly-l-lactide/silk fibroin scaffold seeded with nanoparticle-labeled allogenic bone marrow stromal cells.

In the present study, a poly-l-lactide/silk fibroin (PL-SF) bilayer scaffold seeded with allogenic bone marrow stromal cells (BMSCs) was investigated as a potential approach for bladder tissue engineering in a model of partial bladder wall cystectomy in rabbits. The inner porous layer of the scaffold produced from silk fibroin was designed to promote cell proliferation and the outer layer produced from poly-l-lactic acid to serve as a waterproof barrier. To compare the feasibility and efficacy of BMSC application in the reconstruction of bladder defects, 12 adult male rabbits were divided into experimental and control groups (six animals each) that received a scaffold seeded with BMSCs or an acellular one, respectively. For BMSC tracking in the graft in in vivo studies using magnetic resonance imaging, cells were labeled with superparamagnetic iron oxide nanoparticles. In vitro studies demonstrated high intracellular incorporation of nanoparticles and the absence of a toxic influence on BMSC viability and proliferation. Following implantation of the graft with BMSCs into the bladder, we observed integration of the scaffold with surrounding bladder tissues (as detected by magnetic resonance imaging). During the follow-up period of 12 weeks, labeled BMSCs resided in the implanted scaffold. The functional activity of the reconstructed bladder was confirmed by electromyography. Subsequent histological assay demonstrated enhanced biointegrative properties of the PL-SF scaffold with cells in comparison to the control graft, as related to complete regeneration of the smooth muscle and urothelium tissues in the implant. Confocal microscopy studies confirmed the presence of the superparamagnetic iron oxide nanoparticle-labeled BMSCs in newly formed bladder layers, thus indicating the role of stem cells in bladder regeneration. The results of this study demonstrate that application of a PL-SF scaffold seeded with allogenic BMSCs can enhance biointegration of the graft in vivo and support bladder tissue regeneration and function.

Two-stage implantation of the skin- and bone-integrated pylon seeded with autologous fibroblasts induced into osteoblast differentiation for direct skeletal attachment of limb prostheses.

Angio- and osteogenesis following the two-stage (TS) implantation of the skin- and bone-integrated pylon seeded with autologous fibroblasts was evaluated. Two consecutive animal substudies were undertaken: intramedullary subcutaneous implantation (15 rabbits) and a TS transcutaneous implantation (12 rabbits). We observed enhanced osseointegrative properties of the intramedullary porous component seeded with fibroblasts induced into osteoblast differentiation, as compared to the untreated porous titanium pylon. The three-phase scintigraphy and subsequent histological analysis showed that the level of osteogenesis was 1.5-fold higher than in the control group, and significantly so (p < 0.05). The biocompatibility was further proved by the absence of inflammatory response or encapsulation and sequestration on the histology assay. Treatment of the transcutaneous component with autologous fibroblasts was associated with nearly a 2-fold decrease in the period required for the ingrowth of dermal and subdermal soft tissues into the implant surface, as compared to the untreated porous titanium component. Direct dermal attachment to the transcutaneous implant prevented superficial and deep periprosthetic infections in rabbits in vivo.

Porous composite prosthetic pylon for integration with skin and bone.

This article presents results of the further development and testing of the "skin and bone integrated pylon" (SBIP-1) for percutaneous (through skin) connection of the residual bone with an external limb prosthesis. We investigated a composite structure (called the SBIP-2) made of titanium particles and fine wires using mathematical modeling and mechanical testing. Results showed that the strength of the pylon was comparable with that of anatomical bone. In vitro and in vivo animal studies on 30 rats showed that the reinforcement of the composite pylon did not compromise its previously shown capacity for inviting skin and bone cell ingrowth through the device. These findings provide evidence for the safe and reliable long-term percutaneous transfer of vital and therapeutic substances, signals, and necessary forces and moments from a prosthetic device to the body.

Enhancement of the blood growth promoting activity after exposure of volunteers to visible and infrared polarized light. Part I: stimulation of human keratinocyte proliferation in vitro.

The systemic mechanisms of the wound healing effect of low intensity lasers remain largely uninvestigated. The goal of this randomized, placebo controlled, double blind study is to prove that irradiation of a small area of the human body with visible and infrared polarized (VIP) light (400-3400 nm, 95% polarization, 40 mW cm(2), 12 J cm(2)) leads to an increase of the growth promoting (GP) activity of the entire circulating blood for primary cultures of human keratinocytes (KCs). Thirty minutes after the VIP-irradiation of a sacral area of volunteers, the GP activity of circulating blood was seen to increase through the elevation of the number of KCs cultured with the isolated plasma by 20 +/- 3%, p < 0.001. A similar increase in GP activity was seen in plasma derived from the in vitro irradiated blood of each volunteer, and from the mixture of irradiated and non-irradiated autologous blood 1:10. Enhanced GP activity was also recorded at 24 h after the 1st and 4-9th daily phototherapeutic sessions. Hence, exposure of volunteers to VIP light leads to a fast increase in the GP activity of the entire circulating blood for human KCs in vitro, which is a consequence of the transcutaneous photomodification of blood and its effect on the rest of the circulating blood volume.