Specifics of Porous Polymer and Xenogeneic Matrices and of Bone Tissue Regeneration Related to Their Implantation into an Experimental Rabbit Defect.

This paper provides a study of two bone substitutes: a hybrid porous polymer and an osteoplastic matrix based on a bovine-derived xenograft. Both materials are porous, but their pore characteristics are different. The osteoplastic matrix has pores of 300-600 µm and the hybrid polymer has smaller pores, generally of 6-20 µm, but with some pores up to 100 µm across. SEM data confirmed the porometry results and demonstrated the different structures of the materials. Therefore, both materials were characterized by an interconnected porous structure and provided conditions for the adhesion and vital activity of human ASCs in vitro. In an experimental model of rabbit shin bone defect, it was shown that, during the 6-month observation period, neither of the materials caused negative reactions in the experimental animals. By the end of the observation period, restoration of the defects in animals in both groups was completed, and elements of both materials were preserved in the defect areas. Data from morphological examinations and CT data demonstrated that the rate of rabbit bone tissue regeneration with the hybrid polymer was comparable to that with the osteoplastic matrix. Therefore, the hybrid polymer has good potential for use in further research and improvement in biomedical applications.

Features of Changes in the Structure and Properties of a Porous Polymer Material with Antibacterial Activity during Biodegradation in an In Vitro Model.

Hybrid porous polymers based on poly-EGDMA and polylactide containing vancomycin, the concentration of which in the polymer varied by two orders of magnitude, were synthesized. The processes of polymer biodegradation and vancomycin release were studied in the following model media: phosphate-buffered saline (PBS), trypsin-Versene solution, and trypsin-PBS solution. The maximum antibiotic release was recorded during the first 3 h of extraction. The duration of antibiotic escape from the polymer samples in trypsin-containing media varied from 3 to 22 days, depending on the antibiotic content of the polymer. Keeping samples of the hybrid polymer in trypsin-containing model media resulted in acidification of the solutions-after 45 days, up to a pH of 1.84 in the trypsin-Versene solution and up to pH 1.65 in the trypsin-PBS solution. Here, the time dependences of the vancomycin release from the polymer into the medium and the decrease in pH of the medium correlated. These data are also consistent with the results of a study of the dynamics of sample weight loss during extraction in the examined model media. However, while the polymer porosity increased from ~53 to ~60% the pore size changed insignificantly, over only 10 µm. The polymer samples were characterized by their antibacterial activity against Staphylococcus aureus, and this activity persisted for up to 21 days during biodegradation of the material, regardless of the medium type used in model. Surface-dependent human cells (dermal fibroblasts) adhere well, spread out, and maintain high viability on samples of the functionalized hybrid polymer, thus demonstrating its biocompatibility in vitro.

Functionalization of Osteoplastic Material with Human Placental Growth Factor and Assessment of Biocompatibility of the Resulting Material In Vitro.

This article provides the results of a study of the interaction of placental growth factor with adipose-derived stem cells (ASCs) of various origins, as well as the possibility of generating osteoplastic material based on xenogeneic matrix functionalization with human placental growth factor (PLGF). It is demonstrated that the greatest release of this factor from the functionalized material into the medium occurs during the first 3 h of contact with the model medium, but then the levels of the factor being released fall sharply, although release did continue throughout the 7 days of observation. The modified material was not cytotoxic, and its surface provided good cell adhesion. During 3 days of cultivation, the ASCs proliferated and migrated more actively on the surfaces of the modified material than on the surfaces of the control material. This study can serve as the basis for the development of original methods to functionalize such osteoplastic material by increasing PLGF immobilization by creating stronger bonds in order to regulate both factor dosage and the dynamics of the factor release into the environment. Further studies in experimental animals should facilitate assessment of the effectiveness of the functionalized materials. Such studies will be useful in the development of osteoplastic materials with new properties resulting from the inclusion of growth factors and in research on their biological activity.

Specifics of Cryopreservation of Hydrogel Biopolymer Scaffolds with Encapsulated Mesenchymal Stem Cells.

The demand for regenerative medicine products is growing rapidly in clinical practice. Unfortunately, their use has certain limitations. One of these, which significantly constrains the widespread distribution and commercialization of such materials, is their short life span. For products containing suspensions of cells, this issue can be solved by using cryopreservation. However, this approach is rarely used for multicomponent tissue-engineered products due to the complexity of selecting appropriate cryopreservation protocols and the lack of established criteria for assessing the quality of such products once defrosted. Our research is aimed at developing a cryopreservation protocol for an original hydrogel scaffold with encapsulated MSCs and developing a set of criteria for assessing the quality of their functional activity in vitro. The scaffolds were frozen using two alternative types of cryocontainers and stored at either -40 °C or -80 °C. After cryopreservation, the external state of the scaffolds was evaluated in addition to recording the cell viability, visible changes during subsequent cultivation, and any alterations in proliferative and secretory activity. These observations were compared to those of scaffolds cultivated without cryopreservation. It was shown that cryopreservation at -80 °C in an appropriate type of cryocontainer was optimal for the hydrogels/adipose-derived stem cells (ASCs) tested if it provided a smooth temperature decrease during freezing over a period of at least three hours until the target values of the cryopreservation temperature regimen were reached. It was shown that evaluating a set of indicators, including the viability, the morphology, and the proliferative and secretory activity of the cells, enables the characterization of the quality of a tissue-engineered construct after its withdrawal from cryopreservation, as well as indicating the effectiveness of the cryopreservation protocol.

Production of Graft Copolymers of Cod Collagen with Butyl Acrylate and Vinyl Butyl Ether in the Presence of Triethylborane-Prospects for Use in Regenerative Medicine.

Collagen is a suitable material for regenerative medicine because it is characterized by its good biocompatibility. However, due to its fibrillar structure, it cannot organize itself into three-dimensional porous structures without additional modification. The introduction of synthetic monomer elements into the collagen macromolecules is a technique used to form three-dimensional, collagen-based, branched, and crosslinked structures. New types of graft copolymers made from cod collagen with a butyl acrylate and vinyl butyl ether copolymer in aqueous dispersion were obtained in the presence of triethylborane by a radical mechanism. The process of graft copolymer formation proceeded as usual by radical initiation, through radicals formed during triethylborane oxidation by oxygen residues, collagen borination, and reversible inhibition with the participation of a boroxyl radical. The characteristics of the graft copolymers were determined using methods of physical and chemical analysis (GPC, SEM, IR spectroscopy, etc.), while the cytotoxicity was assessed using the MTT assay method. It is shown that the grafting of alternating blocks of butyl acrylate and vinyl butyl ether to the protein macromolecules results in changes in the morphological pattern of the graft co-polymer in comparison with native collagen. This is manifested in the development of consolidations around the collagen fibers of the structural matrices, with the co-polymer cellular structure consisting of interpenetrating pores of unequal size. Additionally, it is important that the graft co-polymer solutions are not toxic at a certain concentration. The above properties confirm the promising nature of the technique's application as the basis for producing new materials for regenerative medicine.

Scaffold Chemical Model Based on Collagen-Methyl Methacrylate Graft Copolymers.

Polymerization of methyl methacrylate (MMA) in aqueous collagen (Col) dispersion was studied in the presence of tributylborane (TBB) and p-quinone: 2,5-di-tert-butyl-p-benzoquinone (2,5-DTBQ), p-benzoquinone (BQ), duroquinone (DQ), and p-naphthoquinone (NQ). It was found that this system leads to the formation of a grafted cross-linked copolymer. The inhibitory effect of p-quinone determines the amount of unreacted monomer, homopolymer, and percentage of grafted poly(methyl methacrylate) (PMMA). The synthesis combines two approaches to form a grafted copolymer with a cross-linked structure-"grafting to" and "grafting from". The resulting products exhibit biodegradation under the action of enzymes, do not have toxicity, and demonstrate a stimulating effect on cell growth. At the same time, the denaturation of collagen occurring at elevated temperatures does not impair the characteristics of copolymers. These results allow us to present the research as a scaffold chemical model. Comparison of the properties of the obtained copolymers helps to determine the optimal method for the synthesis of scaffold precursors-synthesis of a collagen and poly(methyl methacrylate) copolymer at 60 °C in a 1% acetic acid dispersion of fish collagen with a mass ratio of the components collagen:MMA:TBB:2,5-DTBQ equal to 1:1:0.015:0.25.

Application of hydrogel wound dressings in cell therapy-approaches to assessment in vitro.

Cell therapy is actively used to treat skin defects, particularly burn lesions. The effectiveness of its application may depend on the appropriate choice of wound dressings used together with any cellular material. The aim of the study was to investigate the interaction of 4 hydrogel dressings used in clinical practice with human cells in an in vitro model to determine if their use in combination with cell therapy is possible. The effect of the dressings on the growth medium was assessed by considering the changes caused in the medium's acid-base equilibrium (pH) and viscosity. Cytotoxicity was determined by applying an MTT-assay and by direct contact methods. Cell adhesion and viability on the dressing surfaces were analyzed using fluorescence microscopy. Proliferative and secretory cell activity were determined concurrently. Characterized human dermal fibroblast cultures were used as the test cultures. Results: The tested dressings interacted differently with the growth medium and the test cultures. 1-day extracts of all dressings had almost no effect on the acid-base balance, but, after 7 days, the pH of the dressing Type 2 extract had sharply acidified. The viscosity of the media under the influence of dressings of Types 2 and 3 had also markedly increased. MTT-assays showed nontoxicity of all the 1-day-incubated dressing extracts, while incubation for 7-days resulted in extracts with evident cytotoxicity, which was reduced upon dilution. Cell adhesion to the surfaces of the dressings differed, being observed occurring on dressings 2 and 3, and to a limited extent on dressing 4. Cells under dressing 1 showed evident proliferative and secretory activity whereas the other dressings impaired either proliferation or secretion processes. These effects indicate that, in general, comprehensive studies including a variety of methodological approaches at the in vitro stage are needed to allow the selection of appropriate dressings if they are to be used in combination with cell therapy to act as cell carriers. Of those investigated, the Type 1 dressing can be recommended as a protective dressing for use after transplantation of cells into a wound defect area by some other method.

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.

Biological Characteristics of Polyurethane-Based Bone-Replacement Materials.

A study is presented on four polymers of the polyurethane family, obtained using a two-stage process. The first composition is the basic polymer; the others differ from it by the presence of a variety of fillers, introduced to provide radiopacity. The fillers used were 15% bismuth oxide (Composition 2), 15% tantalum pentoxide (Composition 3), or 15% zirconium oxide (Composition 4). Using a test culture of human fibroblasts enabled the level of cytotoxicity of the compositions to be determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, along with variations in the characteristics of the cells resulting from their culture directly on the specimens. The condition of cells on the surfaces of the specimens was assessed using fluorescence microscopy. It was shown that introducing 15% bismuth, tantalum, or zinc compounds as fillers produced a range of effects on the biological characteristics of the compositions. With the different fillers, the levels of toxicity differed and the cells' proliferative activity or adhesion was affected. However, in general, all the studied compositions may be considered cytocompatible in respect of their biological characteristics and are promising for further development as bases for bone-substituting materials. The results obtained also open up prospects for further investigations of polyurethane compounds.

Cryostorage of Mesenchymal Stem Cells and Biomedical Cell-Based Products.

Mesenchymal stem cells (MSCs) manifest vast opportunities for clinical use due both to their ability for self-renewal and for effecting paracrine therapeutic benefits. At the same time, difficulties with non-recurrent generation of large numbers of cells due to the necessity for long-term MSC expansion ex vivo, or the requirement for repeated sampling of biological material from a patient significantly limits the current use of MSCs in clinical practice. One solution to these problems entails the creation of a biobank using cell cryopreservation technology. This review is aimed at analyzing and classifying literature data related to the development of protocols for the cryopreservation of various types of MSCs and tissue-engineered structures. The materials in the review show that the existing techniques and protocols for MSC cryopreservation are very diverse, which significantly complicates standardization of the entire process. Here, the selection of cryoprotectors and of cryoprotective media shows the greatest variability. Currently, it is the cryopreservation of cell suspensions that has been studied most extensively, whereas there are very few studies in the literature on the freezing of intact tissues or of tissue-engineered structures. However, even now it is possible to develop general recommendations to optimize the cryopreservation process, making it less traumatic for cells.

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.

Changes in the Molecular Characteristics of Bovine and Marine Collagen in the Presence of Proteolytic Enzymes as a Stage Used in Scaffold Formation.

Biopolymers, in particular collagen and fibrinogen, are the leading materials for use in tissue engineering. When developing technology for scaffold formation, it is important to understand the properties of the source materials as well as the mechanisms that determine the formation of the scaffold structures. Both factors influence the properties of scaffolds to a great extent. Our present work aimed to identify the features of the molecular characteristics of collagens of different species origin and the changes they undergo during the enzymatic hydrolysis used for the process of scaffold formation. For this study, we used the methods of gel-penetrating chromatography, dynamic light scattering, reading IR spectra, and scanning electron microscopy. It was found that cod collagen (CC) and bovine collagen (BC) have different initial molecular weight parameters, and that, during hydrolysis, the majority of either type of protein is hydrolyzed by the proteolytic enzymes within the first minute. The differently sourced collagen samples were also hydrolyzed with the formation of two low molecular fractions: Mw ~ 10 kDa and ~20 kDa. In the case of CC, the microstructure of the final scaffolds contained denser, closely spaced fibrillar areas, while the BC-sourced scaffolds had narrow, short fibrils composed of unbound fibers of hydrolyzed collagen in their structure.

Porous Polymer Scaffolds based on Cross-Linked Poly-EGDMA and PLA: Manufacture, Antibiotics Encapsulation, and In Vitro Study.

Porous polymer materials derived from poly(ethylene glycol dimethacrylate) (poly-EGDMA) and antibiotic containing polylactide (PLA) are obtained for the first time. Porous poly-EGDMA monoliths with a system of open interconnected pores are synthesized by a visible light-induced radical polymerization of EGDMA in the presence of 70 wt% of porogenic agent, e.g., 1-butanol, 1-hexanol, 1-octanol, or cyclohexanol. The porosity of the obtained polymers is 75-78%. A modal pore size depends on the nature of the porogen and varies from 0.5 µm (cyclohexanol) to 12 µm (1-butanol). The polymer matrix made with 1-butanol features the presence of pores ranging from 1 to 100 µm. The pore surface of poly-EGDMA matrices is inlayered with poly-D,L-lactide (Mn  23 × 103  Da, PDI 1.31). The PLA-modified poly-EGDMA retains a porous structure that is similar to the initial poly-EGDMA but with improved strength characteristics. The presence of antibiotic containing PLA ensures a high and continuous antibacterial activity of the hybrid polymeric material for 7 days. The nontoxicity of all the porous matrices studied makes them promising for clinical tests as osteoplastic materials.

Long-Term Cryostorage of Mesenchymal Stem Cell-Containing Hybrid Hydrogel Scaffolds Based on Fibrin and Collagen.

The most difficult issue when using tissue engineering products is enabling the ability to store them without losing their restorative capacity. The numbers and viability of mesenchymal stem cells encapsulated in a hydrogel scaffold after cryostorage at -80 °C (by using, individually, two kinds of cryoprotectors-Bambanker and 10% DMSO (Dimethyl sulfoxide) solution) for 3, 6, 9, and 12 months were determined, with subsequent assessment of cell proliferation after 96 h. The analysis of the cellular component was performed using fluorescence microscopy and the two fluorochromes-Hoechst 3334 and NucGreenTM Dead 488. The experimental protocol ensured the preservation of cells in the scaffold structure, retaining both high viability and proliferative activity during storage for 3 months. Longer storage of scaffolds led to their significant changes. Therefore, after 6 months, the proliferative activity of cells decreased. Cryostorage of scaffolds for 9 months led to a decrease in cells' viability and proliferative activity. As a result of cryostorage of scaffolds for 12 months, a decrease in viability and proliferative activity of cells was observed, as well as pronounced changes in the structure of the hydrogel. The described scaffold cryostorage protocol could become the basis for the development of storage protocols for such tissue engineering products, and for helping to extend the possibilities of their clinical use while accelerating their commercialization.

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.

Quantitative analysis of cells encapsulated in a scaffold.

One of the urgent problems arising while carrying out research in the field of scaffold technology is achieving an objective, direct, quantitative analysis of cells cultivated within a scaffold; one which allows characterization of the density distribution of the cells, their viability and their proliferative activity when encapsulated within the scaffold. This problem is associated with the peculiarities of cell cultivation in the three-dimensional structure of scaffolds, including limitations imposed on the possibility of direct cell counting using light microscopy. Also, most scaffolds are opaque, so this generally excludes methods of quantitative analysis using light microscopy. There are methods for the quantitative analysis of cells in a scaffold based on the assessment of their metabolic activity (for example: MTT test). However, these methods are indirect and can result in significant errors. This is due to differences in the metabolic activity of the cells, for example, in different phases of mitosis. Methods based on direct counting of the number of cells isolated from the scaffold are also characterized by a high degree of error that is associated with the loss of cells during the destruction of the scaffold. We describe in detail a method that allows the direct quantitation of cells within a scaffold. Modifications of the method make it possible both to analyze the proliferative activity of cells cultivated in a scaffold and to assess their viability and density distribution in the three-dimensional structure.•Direct rather than indirect analysis of the number of cells in the scaffold by counting the number of nuclei.•Carrying out research without destroying the scaffold structure.•Carrying out research without additional preliminary preparation of samples before staining.

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.