Biocompatibility Study of Hydrogel Biopolymer Scaffold with Encapsulated Mesenchymal Stem Cells.

One of the key and actively developing areas of regenerative medicine is tissue-engineering. There is no doubt that the use of tissue-engineering products can have a significant impact on the efficiency of repair of damaged tissues and organs. However, before being used in clinical practice, tissue-engineering products require thorough preclinical studies to confirm their safety and efficacy, both with in vitro models and in experimental animals. This paper presents preclinical studies of a tissue-engineered construct, based on a hydrogel biopolymer scaffold carrier (consisting of blood plasma cryoprecipitate and collagen) with encapsulated mesenchymal stem cells, to evaluate its biocompatibility in vivo. The results were analyzed using histomorphology and transmission electron microscopy. It was shown that when implanted into animal (rat) tissues, the implants were completely replaced by connective tissue components. We also confirmed that no acute inflammation occurred in response to the scaffold implantation. The observed processes of cell recruitment to the scaffold from the surrounding tissues, the active formation of collagen fibers and the absence of acute inflammation testified that the regeneration process was ongoing in the implantation area. Thus, the presented tissue-engineered construct shows promise for becoming an effective tool for regenerative medicine in the future and may be used, in particular, to repair soft tissues.

Aspects of In Vitro Biodegradation of Hybrid Fibrin-Collagen Scaffolds.

The success of the regenerative process resulting from the implantation of a scaffold or a tissue-engineered structure into damaged tissues depends on a series of factors, including, crucially, the biodegradability of the implanted materials. The selection of a scaffold with appropriate biodegradation characteristics allows for synchronization of the degradation of the construct with the processes involved in new tissue formation. Thus, it is extremely important to characterize the biodegradation properties of potential scaffold materials at the stage of in vitro studies. We have analyzed the biodegradation of hybrid fibrin-collagen scaffolds in both PBS solution and in trypsin solution and this has enabled us to describe the processes of both their passive and enzymatic degradation. It was found that the specific origin of the collagen used to form part of the hybrid scaffolds could have a significant effect on the nature of the biodegradation process. It was also established, during comparative studies of acellular scaffolds and scaffolds containing stem cells, that the cells, too, make a significant contribution to changes in the biodegradation and structural properties of such scaffolds. The study results also provided evidence indicating the dependency between the pre-cultivation period for the cellular scaffolds and the speed and extent of their subsequent biodegradation. Our discussion of results includes an attempt to explain the mechanisms of the changes found. We hope that the said results will make a significant contribution to the understanding of the processes affecting the differences in the biodegradation properties of hybrid, biopolymer, and hydrogel scaffolds.

Biopolymer Hydrogel Scaffold as an Artificial Cell Niche for Mesenchymal Stem Cells.

The activity of stem cell processes is regulated by internal and external signals of the cell "niche". In general, the niche of stem cells can be represented as the microenvironment of the cells, providing a signal complex, determining the properties of the cells. At the same time, the "niche" concept implies feedback. Cells can modify their microenvironment, supporting homeostasis or remodeling the composition and structure of the extracellular matrix. To ensure the regenerative potential of tissue engineering products the "niche" concept should be taken into account. To investigate interactions in an experimental niche, an original hydrogel biopolymer scaffold with encapsulated mesenchymal adipose-derived stem cells (ASCs) was used in this study. The scaffold provides for cell adhesion, active cell growth, and proliferative activity. Cells cultured within a scaffold are distinguished by the presence of a developed cytoskeleton and they form a cellular network. ASCs cultured within a scaffold change their microenvironment by secreting VEGF-A and remodeling the scaffold structure. Scaffold biodegradation processes were evaluated after previous culturing of the ASCs in the scaffolds for periods of either 24 h or six days. The revealed differences confirmed that changes had occurred in the properties of scaffolds remodeled by cells during cultivation. The mechanisms of the identified changes and the possibility of considering the presented scaffold as an appropriate artificial niche for ASCs are discussed.

Hydrogel scaffolds based on blood plasma cryoprecipitate and collagen derived from various sources: Structural, mechanical and biological characteristics.

At present there is a growing need for tissue engineering products, including the products of scaffold-technologies. Biopolymer hydrogel scaffolds have a number of advantages and are increasingly being used to provide means of cell transfer for therapeutic treatments and for inducing tissue regeneration. This work presents original hydrogel biopolymer scaffolds based on a blood plasma cryoprecipitate and collagen and formed under conditions of enzymatic hydrolysis. Two differently originated collagens were used for the scaffold formation. During this work the structural and mechanical characteristics of the scaffold were studied. It was found that, depending on the origin of collagen, scaffolds possess differences in their structural and mechanical characteristics. Both types of hydrogel scaffolds have good biocompatibility and provide conditions that maintain the three-dimensional growth of adipose tissue stem cells. Hence, scaffolds based on such a blood plasma cryoprecipitate and collagen have good prospects as cell carriers and can be widely used in regenerative medicine.

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice.

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.

OCT-guided laser hyperthermia with passively tumor-targeted gold nanoparticles.

The goal of this study is the development of a method of local laser hyperthermia with gold nanoparticles under noninvasive optical monitoring of nanoparticle accumulation in tumor tissue in vivo. Bifunctional plasmon resonant nanoparticles that are optimal for OCT diagnostics and laser heating at the wavelength of 810 nm were used in the study. The OCT examination showed that the accumulation of gold nanoparticles in the tumor invading into skin was maximal 4-5 h after intravenous injection. It was demonstrated that nanoparticle accumulation in tumor allowed more local heating and enhanced thermal sensitivity of tumor tissue. Laser hyperthermia that heated tumor up to 44-45 °C at maximum nanoparticle accumulation induced apoptotic death of tumor cells and inhibited tumor growth by 104% on the 5th day after treatment.