Experimental Study of the Possibility of Culturing of Mesenchymal Stromal Cell and Induction of Osteogenic Differentiation on Collagen-Based Scaffolds of Various Modifications.

We analyzed the suitability of various collagen-based scaffolds for culturing and osteogenic differentiation of mesenchymal stromal cells (MSC). Decellularized and lyophilized swine intestinal submucosa (SIS) and porous collagen sponge made from reconstructed bovine derma (PCS) were the most effective in promoting MSC adhesion, survival, and growth. MSC from rat and mouse bone marrow and rat adipose tissue successfully adhered to the scaffold surface and penetrated into its deep layers. These scaffolds were also the most effective in inducing osteogenesis. These results indicate that microarchitectonics of PCS and SIS is optimal for support of MSC growth and osteogenic differentiation.

2D/3D buccal epithelial cell self-assembling as a tool for cell phenotype maintenance and fabrication of multilayered epithelial linings in vitro.

Maintaining the epithelial status of cells in vitro and fabrication of a multilayered epithelial lining is one of the key problems in the therapy using cell technologies. When cultured in a monolayer, epithelial cells change their phenotype from epithelial to epithelial-mesenchymal or mesenchymal that makes it difficult to obtain a sufficient number of cells in a 2D culture and to use them in tissue engineering. Here, using buccal epithelial cells from the oral mucosa, we developed a novel approach to recover and maintain the stable cell phenotype and form a multilayered epithelial lining in vitro via the 2D/3D cell self-assembling. Transitioning the cells from the monolayer to non-adhesive 3D culture conditions led to formation of self-assembling spheroids, with restoration of their epithelial characteristics after epithelial-mesenchymal transition. In 7 days, the cells within spheroids restored the apical-basal polarity, and the formation of both tight (ZO1) and adherent (E-cadherin) intercellular junctions was shown. Thus, culturing buccal epithelial cells in a 3D system allowed us to recover and durably maintain the morphological and functional characteristics of epithelial cells. The multilayered epithelial lining formation was achieved after placing spheroids for 7 days onto a hybrid matrix, which consisted of collagen layers and reinforcing poly (lactide-co-glycolide) fibers and was proven promising for replacement of the urothelium. Thus, we offer an effective technique of forming multilayered epithelial linings on carrier-matrices using cell spheroids that was not previously described elsewhere and can find a wide range of applications in tissue engineering, replacement surgery, and regenerative medicine.

Reinforced Hybrid Collagen Sponges for Tissue Engineering.

We created an anisotropic material based on collagen sponge and reactive polylactide structured by laser photopolymerization. The combination of collagen with reactive polylactide improves the resistance of the formed matrices to biodegradation in comparison with collagen sponge, while the existence of sites with different mechanical characteristics and cell affinity on the matrix provides directed cell growth during their culturing. It was shown that reinforcement of the collagen sponges 7-fold increased the mean Young's modulus for the hybrid matrix without affecting its cytotoxicity. The developed matrix provides cell adhesion and proliferation along reinforcement lines and can be used for fabrication of tissue engineering constructs.

[TISSUE-ENGINEERED SUBSTITUTION URETHROPLASTY BASED ON DECELLULARIZED VASCULAR MATRIX AND AUTOLOGOUS CELLS OF THE BUCCAL MUCOSA: THE FIRST EXPERIENCE].

Urethral strictures and anomalies remain a challenging urological problem. Reconstructive plastic surgery has been shown to be the most effective way to treat them. There are two main types of urethroplasty: anastomosis (anastomotic urethroplasty) and expansion of the urethral lumen using of flaps and grafts (substitution urethroplasty). Currently the ideal material for substitution urethroplasty does not exist. Tissue engineering of the urethra seems to be one of the most promising approaches to address this problem. Various tissues-engineering techniques were proposed for substitution urethroplasty. In this study, tissue-engineering design was based on the decellularized cadaveric arterial wall. The study results demonstrated the feasibility of creating stable tissue-engineered structures with autologous cultured epithelial cells of the buccal mucosa and decellularized matrix from human cadaveric arterial wall (DMCAW). There was a complete engraftment of tissue-engineering design based on DMCAW and buccal mucosa cells, used for substitution urethroplasty in a patient with the bulbar urethral stricture. Postoperatively (within 4 months after surgery) no complications and/or adverse events were observed. However, in the late postoperative period (12 months) there was recurrence of urethral stricture in the middle of the tissue-engineering design and the native urethra that warranted another surgery. Tissue-engineering design based on DMCAW and autologous buccal mucosa is safe as a material for substitution urethroplasty. Further research is required to ascertain the effectiveness of the method.

[PLGA mesh-collagen hybrid scaffold and tissue-engineered product in substitution urethroplasty: experimental validation].

Urethral strictures are a pressing issue in modern medicine. Substitution urethroplasty is considered one of the most effective treatment methods. However, despite the surgery showing good results, many problems remain unresolved, one being substitute material deficiency in extensive or recurrent strictures, as well as in cases requiring multistage surgeries, including those used to treat hypospadias. Graft removal also leaves the donor area prone to diseases and increases the length of surgery leading to a higher risk of intra- and postoperative complications. Tissue engineering (namely tissue-engineered products comprised of scaffolds and cells) may be a useful tool in dealing with these issues. The authors assessed the characteristics of a novel hybrid scaffold created from "reconstructed" collagen and a poly(lactic-co-glycolic acid) mesh. The resulting composite product showed good mechanical properties and functional performance. The hybrid scaffold was non-cytotoxic and provided an adequate base for cell adhesion and proliferation. Biodegradation resulted in the scaffold being replaced by urothelium and urethral mucosa. The newly formed tissues possessed adequate structural and functional properties. Only one rabbit out of 12 developed urethral stricture at the site of scaffold implantation. The above-mentioned facts suggest that the novel hybrid scaffold is a promising tissue-engineered product with potential implication in substitution urethroplasty.

[Morphology of collagen matrices for tissue engineering (biocompatibility, biodegradation, tissue response)].

OBJECTIVE: to perform a comparative morphological study of biocompatibility, biodegradation, and tissue response to implantation of collagen matrices (scaffolds) for tissue engineering in urology and other areas of medicine. MATERIAL AND METHODS: Nine matrix types, such as porous materials reconstructed from collagen solution; a collagen sponge-vicryl mesh composite; decellularized and freeze-dried bovine, equine, and fish dermis; small intestinal submucosa, decellularized bovine dura mater; and decellularized human femoral artery, were implanted subcutaneously in 225 rats. The tissues at the implantation site were investigated for a period of 5 to 90 days. Classical histology and nonlinear optical microscopy (NLOM) were applied. RESULTS: The investigations showed no rejection of all the collagen materials. The period of matrix bioresorption varied from 10 days for collagen sponges to 2 months for decellularized and freeze-dried vessels and vicryl meshes. Collagen was prone to macrophage resorption and enzymatic lysis, being replaced by granulation tissue and then fibrous tissue, followed by its involution. NLOM allowed the investigators to study the number, density, interposition, and spatial organization of collagen structures in the matrices and adjacent tissues, and their change over time during implantation. CONCLUSION: The performed investigation could recommend three matrices: hybrid collagen/vicryl composite; decellularized bovine dermis; and decellularized porcine small intestinal submucosa, which are most adequate for tissue engineering in urology. These and other collagen matrices may be used in different areas of regenerative medicine.

[Experimental validation of the developing a matrix based on decellularized vascular wall for subsequent substitution urethroplasty].

Urethral strictures are urgent urological problem. Anastomotic and substitution urethroplasty are the most effective treatments. For substitution urethroplasty, buccal mucosa is most often used. There are the following difficulties associated with the substitution urethroplasty: complications in the donor area, the lack of tissue for substitution, an additional incision, and increased timing of surgery due to the need to obtain a flap or graft. Tissue engineering can be useful in solving the above problems. Tissue engineering involves the use a matrix without cells and matrix with one or more types of cells (tissue-engineering designs). In our study we have evaluated the ability to create a matrix for the substitution urethroplasty in animal experiments. The decellularized cadaveric arterial wall was used as a matrix. Decellularization was performed using enzymatic method. At the first stage, we transplanted matrix fragments in interscapular region in rats. An extremely weak bioactivity dof decellularized matrix of cadaveric arterial wall (DMCAW) due to the low immunogenicity of the material was revealed. Thus resorption of DMCAW was quite slow (60-90 days). At the second stage, in an experiment on rabbits, substitution urethroplasty using tubular DMCAW was successfully performed. Intraoperative urethral defect up to 1.8 cm was created, which was replaced by a tubular DMCAW. The use of this type of matrix has showed good structural and functional results: urethral strictures did not arise, the rejection of the matrix was not observed. A slow degradation of the matrix and progressive epithelialization of onnective tissue capsule were revealed. Decellularized matrix based on cadaveric arterial wall can be considered as a material for substitution urethroplasty.

[Treatment of IIIa grade burns with the biologic band].

The biologic band is created on the base of type I collagen and platelet derived growth factor. It was applied for extensive burns of IIIa grade at 1-2nd day after a trauma, which led to epidermis reconstruction at 5-7th day. Authors assume that the effect of the band is determined by the time of its use--1-2nd day, when stimulating exudation qualities combine with healing properties of the bandage. The effectiveness of the collagen band showed to be equal to those of fibroblast transplantation.

[Effect of short-term application of a lactate-containing probiotic on the pharyngeal microflora in healthy subjects].

Daily dynamics of pharyngeal microflora was evaluated in the absence of preventive treatment and using a local lactate-producing probiotic preparation during 24 hour-long simulated microgravity. The study showed that the short-term application of the local lactate-producing probiotic (four times daily) significantly reduced titers of opportunistic pathogenic microflora and increased the amount of symbionts in the pharynx.

Stimulation of regeneration of hyaline cartilage in experimental osteochondral injury.

A model of osteochondral intra-articular defect in rats is presented. During spontaneous healing, the stage of formation of granulation tissue is followed by its replacement with bone and fibrous tissue. Chondroinductive properties of collagen 1 sponge used for defect filling manifested in the formation of fibrous cartilage with fields of hyaline cartilage. Filling of the defect with collagen 1 sponge containing bone powder stimulated regeneration of the bone tissue and hyaline cartilage.

The use of bioactive wound dressing, stimulating epithelial regeneration of IIIa-degree burn wounds.

Clinical and morphological comparison of wound healing after transplantation of living cultured allofibroblast on days 1-2 after the injury, collagen-1-based dressing with PDGF-BB, and traditional dressing with levomecol ointment showed that bioactive dressing accelerated wound epithelialization (5-7 days vs. 20-22 days with gauze dressing); the incidence of suppurative complications decreased, no crust formed, and epithelialization was not associated with the formation of a hypertrophic cicatrix. Biological dressing based on living cultured allofibroblasts and collagen-1 with PDGF-BB exhibited equal stimulatory effects on burn wound healing.

[Investigation of dysbiosis developing during "dry" immersion and methods of correction].

Dysbiotic shifts in intestinal and integumentary microflora were studied in 10 test-subjectes during 7-day "dry" immersion. Essentially every test-subject reduced significantly fecal lactoflora and developed dysbiotic shifts in the microbial landscape of various types of integument. Pharyngeal microflora was analyzed in 22 normal volunteers for 7-day "dry" immersion. Two probiotic preparations were administered to prevent pharyngeal dysbiosis: peroral dry lactobacterin and topical collagen-immobilized lactobacterin. The peroral probiotic stimulated growth of pharyngeal opportunistic pathogens preventing translocations of the intestinal microflora. The topical probiotic, on the opposite, reduced the content of opportunistic pathogens in the throat; however, it provoked gastrointestinal dysbiosis. It appears that the most effective prophylaxis of pharyngeal dysbiosis can be reached by prescription of both topical and peroral probiotics.

[Experimental grounds for administration of probiotic drugs in conditions of hydroweightlessness].

We studied pharyngeal microflora in 22 healthy volunteers after 7-day dry immersion. For prophylactic pharyngeal dysbiosis, we used two probiotic drugs: oral -- "lactobacterin dry" and local --"lactobacterin immobilized on collagen". Administration of oral probiotic was accompanied with growth of pharyngeal opportunistic microflora preventing translocation of intestinal microflora. Local probiotic, on the contrary, decreased opportunistic microflora in the pharynx, but was associated with gastrointestinal dysbiosis. It is evident that combined use of topic and oral probiotic provides maximal effective prophylaxis of pharyngeal dysbiosis.

[Development and use of periodontal dressing of collagen and Lactobacillus casei 37 cell suspension in combined treatment of periodontal disease of inflammatory origin (a microbiological study)]. / Razrabotka i primenenie parodontal'noi poviazki iz kollagena i suspenzii kletok Lactobacillus casei 37 v kompleksnom lechenii vospalitel'nykh zabolevanii parodonta (rezul'taty mikrobiologicheskikh issledovanii).

Periodontal dressing consisting of collagen and Lactobacillus casei 37 cell suspension (cell concentration 108 cells/ml) was created and used in combined treatment of patients with chronic generalized parodontitis. Efficacy of the developed isolation was explained by a considerable decrease of the number and frequency of isolation of aggressive microbial representatives (pigment synthesizing Bacteroids, Actinomyces and Str. intermedius) in periodontal pockets and also Fungus (Candida albicans). This periodontal dressing provided remission up to 10-12 months.

[Application hemostatic drug in capillary-parenchymatous bleeding]. / Applikatsionnoe sredstvo gemostaza pri kapilliarno-parenkhimatoznom krovotechenii.

Specific activity of a new hemostatic drug thrombocol was studied in experiments. Laboratory rabbits were used for management of experimental capillary-parenchymatous hemorrhage. The action of thrombocol was compared with hemostatic effect of other drugs: carbocol, combutek-2, super-4 (USA), collastipt (Germany), collagen sponge (Germany). It was found that thrombocol was highly hygroscopic, showed good adhesion to wounded surface, high plasticity and better hemostatic effect compared with other collagen-containing drugs.

[Method of protecting of sutures in the stomach and intestines]. / Sposob zashchity shvov na zheludke i kishechnike.

The authors have developed and described the method for intraoperative prophylaxis of complications caused by incompetence of the sutures on the stomach and bowels. It consists in closure of the sutures along the line of contiguity by two-layers of compact-porous collagenous explants, which possess antibacterial properties, the period of resorption in abdominal cavity being 15-20 days. The characteristics of the explants are presented and their advantages compared to commonly used methods covering by pellicle or glue film peritonization by sero-muscular sutures. Application of collagenous explants from "Sanguicol" preparation in surgical treatment of 155 patients with external intestinal fistulas, compared to peritonization by sero-muscular sutures (66 patients), has demonstrated a high effectiveness of proposed explants in prevention of postoperative intraabdominal complications.

[The dynamics of bone defect healing after the implantation of collagen and hydroxyapatite complexes (an experimental morphological study)]. / Dinamika zazhivleniia kostnykh defektov pri implantatsii v nikh kompleksov kollagena i gidroksiapatita (éksperimental'no-morfologicheskoe issledovanie).

Healing of standard bone defects in the distal third of the femoral bone was studied in experiments on 48 rats. In order to repair the defect, colapol or a new composition with structurized collagen and hydroxyapatite powder and granulate KP-2 were used. Histological study showed that healing was optimal after repair with KP-2 composition: callus formed rapidly, as did the typical osseogranular complexes. Newly formed bone structures mature much sooner than after implantation of colapol. The authors come to a conclusion that the use of KP-2 composition holds good promise as material for maxillofacial surgery in plastic repair of bone defects.

[Gapkol--a new osteoplastic material]. / Gapkol--novyi osteoplasticheskii material.

Hapcol, a new osteoplastic material, representing a biological composition on the basis of a natural biopolymer collagen with biologically compatible osteotropic mineral hydroxyapatite has been developed and allowed for clinical use. It is intended for use in dentistry in surgical treatment of periodontal diseases, deformations of the abutment tissues of the face, filling of bone defects after cystectomy, surgery for bone plasty, etc. Hapcol is characterized by antiinflammatory action, stimulates repair osteogenesis and collagenogenesis. Joint stock company Polistom starts commercial manufacture of the new material.

[Experimental premises and 1st experiences in the clinical use of antibacterial vascular prostheses under circumstances of increased risk of suppuration]. / Eksperimental'nye predposylki i pervyi opyt klinicheskogo primeneniia antibakterial'nykh sosudistykh protezov v usloviiakh povyshennogo riska nagnoeniia.

It was shown on the basis of laboratory and experimental data that created antibacterial vascular prostheses are marked, on the main, by local bacteriostatic and anticoagulative effect. It was found that heparin concentration of 3 to 500 U per 1 kg collagen causes a hyperadhesive effect on blood platelets and facilitates the formation of the primary thrombocytic thrombus. The free surface energy grows in this case from 34 +/- 0.8 to 66.3 +/- erg/cm2. The optimal thromboresistant effect occurs in heparin content of 1 U per 1 mg collagen. That this choice is correct is confirmed on examination of the inner surface of the prostheses with an electron microscope after being in the blood flow for 45 minutes. Bifurcation and linear antibacterial prostheses were used in 9 patients with a high risk of suppuration verified by cultures of discharge from ulcers and lymph nodes, and the presence of lymphangitis and lymphadenitis. The effect was positive in 8 patients, 2 of them had superficial suppuration of the operative wounds.