Development of a Two-Layer Porous Scaffold Based on Porcine Nasal Septal Cartilage for Orthopedics.

The aim of the study was to design a construct based on a nasal septal cartilage plate providing required cell differentiation in different layers to replace a deep osteochondral defect and develop an algorithm of chemical and physical effect sequence to create non-immunogenic two-layer porous structure with requisite elasto-mechanical properties. MATERIALS AND METHODS: The plates derived from porcine nasal septal hyaline cartilage covered by perichondrium were multi-stage treated including freezing, equilibrating in a hypotonic saline solution (type I specimens); trypsinization, point IR-laser effect, re-trypsinization (type II specimens); a stabilizing effect of crosslinking agents - glyceraldehyde/ribose in an acidic medium - washing (type III specimens).For all type specimens:there were established stability parameters (collagen denaturation temperature using a thermal analysis; and Young's modulus using a mechanical analysis);there were determined morphological characteristics using light and polarization microscopy with classical staining and nonlinear optical microscopy in second-harmonic generation mode. RESULTS: Thermal, mechanical, and morphological properties in type I specimens slightly differed from those of the initial nasoseptal system. A considerable part of cells had destroyed membranes.In type II specimens, thermal stability of collagen frame was significantly lower; Young's modulus decreased more than fourfold compared to type I specimens. Collagen structure of hyaline cartilage appeared to be disarranged, although the morphological differences of the hyaline part and perichondrium preserved. The construct matrix was almost completely decellularized. Successive exposure to laser radiation and trypsin resulted in the formation of partial holes in the matrix, ~100 µm in diameter.In type III specimens, both the thermal stability of the collagen frame and Young's modulus (E) increased. Glyceraldehyde was more effective than ribose, E having reached the value typical for intact hyaline cartilage. Collagen fibers in type III specimens were thicker than in type I and II specimens. The morphological differences of the hyaline part and perichondrium and partial holes were preserved. CONCLUSION: Due to sequential treatment by salts, trypsin, IR-laser radiation, and nontoxic crosslinking agents, nasal septal cartilage plate forms porous acellular construction consisting of two layers formed by type I (from perichondrium) and type II (from hyaline part) collagen fibers. In the present construction, stability, mechanical properties, and size of the partial holes can be assigned for cell colonization. It enables to use the construction to replace articular cartilage defects.

Broad-spectrum antibacterial and pro-regenerative effects of photoactivated Photodithazine-Pluronic F127-Chitosan polymer system: In vivo study.

Emerging global danger of multidrug resistant microbes makes it essential to explore new approaches to treat infections. We studied antibacterial and pro-regenerative effects of photodynamic therapy (PDT) performed with water solutions of photodithazine and its complexes with Pluronic F127 and chitosan in rat model of full thickness wound (n = 24) infected by an associated Gram-negative and Gram-positive bacteria culture. Laboratory rats were exposed to PDT 24 and 72 h after the injury. Exudate samples were collected before and after PDT for a microbiological study. Autopsy tissues were excised and fixed in formalin on day 4 of the experiment. Fixed tissues were processed and poured into paraffin. Paraffin sections were stained with hematoxylin and eosin and studied by an experienced pathologist. Microbiological analysis revealed that the photoactivation of photodithazine and its complexes suppressed the associated microflora in vivo and inhibited suppurative inflammation in the wounds. The triple Photodithazine-Pluronic F127-Chitosan system possessed the highest antibacterial activity. The morphological study revealed that PDT with photodithazine polymer complexes accelerated wound healing, promoted restoration of microcirculation, facilitated proliferation of fibroblast and vessels and stimulated collagen synthesis. The Photodithazine-Pluronic F127-Chitosan complex may be successfully applied for PDT to prevent and treat suppurative inflammatory diseases of the skin and soft tissues.

[Experimental evidence for regenerative treatment of chronic tympanic membrane perforation]. / Eksperimental'noe obosnovanie metoda tkanevoi inzhenerii dlya zakrytiya stoikikh perforatsii barabannoi pereponki.

OBJECTIVE: The aim of our study was an experimental evaluation of tissue engineering approach to chronic tympanic membrane perforation closure. MATERIALS AND METHODS: Chronic tympanic membrane perforation models were created both sides in 12 chinchillas. Right sided perforations were divided into two equal groups (A and B) according to treatment; left sided perforations were used as a control group. Group A perforations were treated with collagen scaffold and fibroblast growth factor, group B perforations were treated with collagen scaffold only. During follow-up, we provided otovideoendoscopy for closure rates assessement every 2 weeks. In case of perforation closure, a morphological investigation of the regenerate was performed. RESULTS: Group A perforations were totally closed 2 weeks after treatment in all animals. In group B, complete closure of perforation was achieved after the third treatment procedure in one case. There were no spontaneous perforation closure in the control group. According to morphological investigation, the restoration of trilaminar structure was observed only in tissue engineering group tympanic membranes.

[Costal cartilage changes in children with pectus excavatum and pectus carinatum]. / Izmeneniia rebernogo khriashcha pri voronkovidnoi i kilevidnoi deformatsii grudnoi kletki u detei.

Pectus excavatum (PE) and pectus carinatum (PC) in children are the most common congenital deformities that cause complications in the thoracic organs; however, the role of chondrocytes and cartilage canals in the pathogenesis of these conditions remains unexplored. OBJECTIVE: To investigate qualitative and quantitative changes of cartilage lacunae and canals in the costal cartilages in children with PE and PC compared to those with normal chests. SUBJECT AND METHODS: Costal cartilages were investigated in 10 children with normal chests (a control group), in 12 children with PE, and in 12 children with PC. Tissue fragments were fixed in 10% neutral formalin and embedded in compacted paraffin. Sections were stained with hematoxylin and eosin. Slides were examined by light microscopy. Cartilage lacunae, hyper- and hypolacunar zones, and cartilage canals were morphometrically examined, followed by statistical data analysis. RESULTS: There was a significant decrease in the number of cartilage lacunae and in the frequency of hyperlacunar zones and an increase in that of hypolacunar zones in the PE and PC groups. There were no significant differences in these parameters between the PE and PC groups; however, there was a tendency to the smallest number of cartilage lacunae and canals in the PC group and that to the preponderance of empty lacunae in the PE group. Only the PC group showed also negative correlations between the proportions of empty lacunae and the age of children. CONCLUSION: The pathogenesis of PE and PC in children is related to the impaired trophism of costal cartilages due to the smaller number of cartilage channels containing vessels and lacunae with chondrocytes. The development of PE and PC is associated with specific costal cartilage morphological changes that suggest that PE and PC are different manifestations of the same disease, namely connective tissue dysplasia.

[Costal cartilage structural and functional changes in children with a funnel or keeled chest]. / Strukturnye i funktsional'nye izmeneniia rebernykh khriashchei pri voronkovidnoi i kilevidnoi deformatsii grudnoi kletki u detei.

Congenital chest wall deformities (CCWDs) in children are severe diseases leading to cosmetic defects and diseases of the respiratory and cardiovascular systems. The most common of these deformities are funnel-shaped (pectus excavatum, FD) and keeled (pectus carinatum, KD) ones. The pathogenesis of CCWDs and the role of costal cartilage structural and functional changes in their pathogenesis have now been not well studied, which makes it difficult to elaborate pathogenetic approaches to correcting these diseases. Analysis of the literature has shown that structural and functional changes occur in the matrix and chondrocytes from the costal cartilage in FD. Similar costal cartilage changes are observed in KD. It is still unknown exactly which pathological processes are present in the costal cartilage and how they result in the development of one or other type of CCWDs. The role of amianthoid transformation (AT) of costal cartilages in these processes is also unknown. It is not improbable that it is AT drastically changing the native cartilage matrix, which is one of the key mechanisms leading to changes in its properties and to the subsequent development of FD or KD.

Reconstruction of Ligament and Tendon Defects Using Cell Technologies.

We studied the possibility of restoring the integrity of the Achilles tendon in rabbits using autologous multipotent stromal cells. Collagen or gelatin sponges populated with cells were placed in a resorbable Vicryl mesh tube and this tissue-engineered construct was introduced into a defect of the middle part of the Achilles tendon. In 4 months, histological analysis showed complete regeneration of the tendon with the formation of parallel collagen fibers, spindle-shaped tenocytes, and newly formed vessels.

Bioreactor-Based Tumor Tissue Engineering.

This review focuses on modeling of cancer tumors using tissue engineering technology. Tumor tissue engineering (TTE) is a new method of three-dimensional (3D) simulation of malignant neoplasms. Design and development of complex tissue engineering constructs (TECs) that include cancer cells, cell-bearing scaffolds acting as the extracellular matrix, and other components of the tumor microenvironment is at the core of this approach. Although TECs can be transplanted into laboratory animals, the specific aim of TTE is the most realistic reproduction and long-term maintenance of the simulated tumor properties in vitro for cancer biology research and for the development of new methods of diagnosis and treatment of malignant neoplasms. Successful implementation of this challenging idea depends on bioreactor technology, which will enable optimization of culture conditions and control of tumor TECs development. In this review, we analyze the most popular bioreactor types in TTE and the emerging applications.

[The morphological and morphometric study of amianthoid transformation of the costal cartilage in health and in keeled chest deformity in children]. / Morfologicheskoe i morfometricheskoe issledovanie amiantoidnoi transformatsii rebernykh khryashchei v norme i pri kilevidnoi deformatsii grudnoi kletki u detei.

Amianthoid transformation (AT) is the accumulations of abnormal collagen structures (amianthoid fibers) in the hyaline cartilages, tumors, and tendons. Neither functional value of costal cartilage matrix AT, nor its role in the pathogenesis of congenital chest deformities is known now. AIM: to examine the morphological features of AT in the costal cartilage of children with the normal and keeled chest. SUBJECTS AND METHODS: Costal cartilages were studied in 6 children with the normal chest (autopsy material) and in 5 ones with keeled chest (surgical material). Tissue fragments were fixed in 10% neutral formalin and embedded in compacted paraffin. The sections were stained with hematoxylin and eosin, picrofuchsin by van Gieson, with picrosirius, toluidine blue and by the Malaurie method modified by Gallego. The specimens were examined by light, phase-contrast, dark-field, fluorescence, and polarization microscopy. The frequency of AT sites and their area were morphometrically studied and the findings were then statistically processed. RESULTS: Various types of AT in the costal cartilages were described as both the normal and keeled chest. According to their morphological features, classic, fine-fiber, twisted, and intralacunar types were identified. There were statistically significant increases in the incidence of all types (except the intralacunar one) and in the area of the fine-fiber AT type in keeled chest deformity as compared to health. There were positive correlations between the area of classic, intralacunar, and twisted types in both groups and between the area of a classic type and age in the controls. CONCLUSION: A classification of AT areas varying in structures in health and disease has been given for the first time; their relation to each other and to the presence of keeled deformity shown, which, in our opinion, suggests that AT is implicated in the pathogenesis of the disease.

[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.

Submicron polyacrolein particles in situ embedded with upconversion nanoparticles for bioassay.

We report a new surface modification approach of upconversion nanoparticles (UCNPs) structured as inorganic hosts NaYF4 codoped with Yb(3+) and Er(3+) based on their encapsulation in a two-stage process of precipitation polymerization of acrolein under alkaline conditions in the presence of UCNPs. The use of tetramethylammonium hydroxide both as an initiator of acrolein polymerization and as an agent for UCNP hydrophilization made it possible to increase the polyacrolein yield up to 90%. This approach enabled the facile, lossless embedment of UCNPs into the polymer particles suitable for bioassay. These particles are readily dispersible in aqueous and physiological buffers, exhibiting excellent photoluminescence properties, chemical stability, and also allow the control of particle diameters. The feasibility of the as-produced photoluminescent polymer particles mean-sized 260 nm for in vivo optical whole-animal imaging was also demonstrated using a home-built epi-luminescence imaging system.

[Oxidative stress in uveitis and its correction with superoxide dismutase antioxidative enzyme (experimental study)].

OBJECTIVE: to study the influence of experimental uveitis on those biochemical parameters of aqueous humor that reflect inflammation acuity as well as local antioxidant and local antiproteolytic activity; to study the effect of topical superoxide dismutase (SOD) on the clinical course of uveitis and ocular metabolism. MATERIAL AND METHODS: Acute uveitis was induced in rabbits by a double injection (subcutaneous and intravitreal) of normal horse serum. The following parameters of aqueous humor were measured: protein concentration, antioxidant activity, SOD activity, alpha2-macroglobulin level, total nitrates and nitrites, and leukocyte number. Clinical assessment and histopathological study were performed. RESULTS: It was found that uveitis is associated with a statistically significant increase in protein concentration, leukocyte number, SOD activity, and alpha2-macroglobulin level in aqueous humor as well as a decrease in anti-hydroxyl radical activity. SOD instillations contributed to the reduction of the listed parameters and improvement of the antioxidant activity. Clinical presentations of uveitis also became less pronounced. CONCLUSION: SOD instillations for oxidative stress correction help reduce clinical presentations of uveitis, which is confirmed by biochemical examination.

[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.

[Retromolar mandibular anesthesia. Radiological and topographical study of an additional method of lower teeth anesthesia].

The aim of the study was to estimate the clinical efficacy of mandibular third molars anesthesia with various combinations of local anesthesia methods. Anatomical, radiological and morphological methods were used in the study. The topographic features of retromolar triangle were thoroughly examined. The neurovascular bundle revealed its relationship with retromolar area structures. According to the results of the clinical study the most effective combinations of mandibular third molars analgesia were selected.

[The wound healing stimulation].

The aim of the study is to work out the method of stimulation of postoperative wound healing after emergency abdominal surgery. Morphologic and morphometric studies showed that serotonin and/or NO-therapy use in the treatment of surgical laparotomic wounds furthers its healing by decreasing distrophic and inflammatory processes and favouring fibroblast proliferation, neoagiogenesis, collagen production etc. Results of 763 patients with laparotomic wounds treated with serotonin, mexidole and NO postoperatively demonstrate faster wound healing and absence of infectious suppurative complications.

[The morphologic characteristics of scar tissues and the new clinicomorphologic classification of human skin scars].

The morphological study of 200 intraoperative biopsy specimens of skin scars of various etiology identified criteria for the differential diagnosis and mechanisms responsible for formation of 4 major types of scar tissues and their structural and functional varieties. In addition to the earlier known normotrophic, hypertrophic, and keloid scar tissues; the special type of scar tissue - the fibrously altered derma) was defined. Most skin scars were found to comprise a combination of a few of scar tissues and a relationship was established between the clinical type of a scar and its histological structure. The principles and mechanisms responsible for changes occurring in the structural composition of scars as the latter mature are described. The new scar clinicomorphological classification provides the basis for the pathogenetically substantiated choice of methods for the prevention and treatment of scars and for the prediction of recurrent scarring.

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.

[Dinitrosyl-iron complexes with cysteine or glutathione accelerate skin wound healing in animals].

The beneficial effect of NO-donors, dinitrosyl-iron complexes with cysteine or glutathione on the healing of skin wound in rats was demonstrated by hystological and hystochemical methods: dinitrosyl-iron complexes accelerated efficiently repair processes in wound tissue after a twofold injection of an aqueous solution of a dinitrosyl-iron complex into wound tissue at a total dose of 5 mmol on days 1 and 2 after skin wounding, and the granulocyte volume increased 3-4 times on the fourth day after wounding compared with the control. Higher doses of dinitrosyl-iron complex provoked an inflammation process in the wound. Similar experiments with of another NO donor S-nitrosoglutathione affected adversely the wound. S-Nitrosoglutathione was added to the wound at a total dose of 10 mmol, which ensured the administration of NO to the wound tissue in the amount equal to that introduced upon the injection of dinitrosyl-iron complex. The addition of dinitrosyl-iron complex with glutathione at a dose of 2.5 mmol was accompanied by the formation of protein-bound dinitrosyl-iron complex in wound tissue. The formation of dinitrosyl-iron complex was also observed after the injection of S-nitrosoglutathione. However, the amount of complexes was more than 25 times less than that after the administration of dinitrosyl-iron complex. The beneficial effect of dinitrosyl-iron complex on the wound was suggested to be due to the formation of a self-regulated chemical system in wound tissue, which is characterized by the mutual transformation of low-molecular dinitrosyl-iron complex and S-nitrosoglutathione. This system ensures a regulated delivery of NO to its intracellular targets without the formation of high amounts of peroxynitrite which could adversely affect the intracellular processes. It was assumed that the self-regulated system of dinitrosyl-iron complex and S-nitrosoglutathione is not formed after the addition of S-nitrosoglutathione to the wound, probably due to a low amount of intracellular iron which could provide the formation of dinitrosyl-iron complex. The rapid decomposition of S-nitrosoglutathione results in the appearance of high amounts of NO and hence peroxynitrite, which adversely affects the wound.

[Hypoxic training -- effective method of raising skin vitality in cases of local plastic operations].

In spite of the complications after expander use the advantages of local plastics predetermine constant return to the method. Systemic use of hypoxic training produces stimulating effect on the skin microcirculation and can be used widely in clinical surgical practice in cases of local plastic operations to prevent possible complications reducing consequences of blood supply disturbance and tissue oxygen provision. The method of hypoxic adaptation let us receive as non-medicamentous mean to prepare for shorter period of time more vital flap.