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.

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.

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.

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.

Contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4.

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.