[Biocompatibility and osteoinductive properties of collagen and fibronectin hydrogel impregnated with rhBMP-2]. / Biosovmestimost' i osteogennye svoistva kollagen-fibronektinovogo gidrogelya, impregnirovannogo BMP-2.

The study aimed to demonstrate the biocompatibility and osteoinductive properties of a hydrogel based on highly purified collagen and fibronectin impregnated with rhBMP-2. In vitro and in vivo experiments have shown that the minimum effective dosage of rhBMP-2 is 10 µg/ml. The cytocompatibility of the collagen-fibronectin gel was determined using MTT test and staining with PKH-26. There was no inflammation reaction when the material was subcutaneously implanted in rats (n=30) in vivo. The collagen-fibronectin hydrogel containing 10 µg/ml rhBMP-2 showed high osteogenic properties. By the end of 28 days 8±4% of its volume was replaced by newly formed bone tissue in case of subcutaneous implantation, 17±10% in intramuscular implantation and 26±11% in intraosseous implantation in the calvarial critical-size. The optimal combination of biocompatible and osteogenic properties of collagen-fibronectin hydrogel impregnated with BMP-2 allows us to consider it as a promising basis for creating the new generation of osteoplastic materials for dentistry.

In Vitro Modeling of Co-Transplantation of Multipotent Stromal Mesenchymal Cells from Orbital Fat Pad and Lipoaspirate of Human Subcutaneous Adipose Tissue in Organ Culture in Collagen Gel.

The interplay of multipotent stromal cells derived from the orbital fat pads and cells of the lipoaspirate from the subcutaneous adipose tissue was studied using in vitro co-transplantation model in an organ culture in a collagen gel. Microscopy findings and intensity of apoptosis and cell proliferation in cultures of lipoaspirate with and without multipotent stromal cells showed that the cells maintained their viability, proliferation capacity, and cytokine secretion activity. Higher proliferatitive activity of cells in cocultures promotes renewal of fat transplant cells and can help to maintain its stable volume in delayed terms after transplantation.

[The prospects of collagen as a basis for curable and activated osteoplastic materials]. / Perspektivy ispol'zovaniia kollagenovogo gidrogelia v kachestve osnovy dlia otverzhdaemykh i aktivirovannykh kostno-plasticheskikh materialov.

In the review, the structure and biological properties of collagen, variants of its production from natural sources and purification are considered. Methods for modifying the physico-mechanical properties of collagen to create a curable, highly purified collagen hydrogel are described. The advantages of a cured highly purified collagen hydrogel as a basis for osteoplastic material and a means of delivery of growth factors are indicated. The registered osteoplastic materials based on the curable highly purified collagen hydrogel are described, and their comparative analysis is carried out. On the basis of the obtained data, a conclusion was made about the prospects of using collagen as a basis for curable and activated osteoplastic materials.

[Differences in the cytocompatibility of bone-plastic materials from xenogeneic hydroxyapatite with stem cells from human exfoliated deciduous teeth and adipose tissue-derived mesenchymal stem cells]. / Razlichiia tsitosovmestimosti kostno-plasticheskikh materialov iz ksenogennogo gidroksiapatita s mul'tipotentnymi mezenkhimal'nymi stromal'nymi kletkami, poluchennymi iz pul'py vypavshikh molochnykh zubov i podkozhnogo lipoaspirata.

AIM: To compare the cytocompatibility of osteoplastic materials used in dentistry with stem cells from human exfoliated deciduous teeth (SHED) and adipose tissue-derived mesenchymal stem cells (AD-MSC). MATERIAL AND METHODS: Materials of the brands 'Bio-Oss', 'Indost', 'Bioplast', 'Viscoll' and 'Trikafor' were selected for study purposes. Cultures of SHED and AD-MSC were used for testing. The cytotoxic effect of the materials was determined using MTT test and vital staining with trypan blue. Cell adhesion was assessed by the vital staining of PKH-26. RESULTS: Water extracts of bone-plastic materials from xenogeneic hydroxyapatite of the brands 'Bio-Oss', 'Indost' and 'Bioplast' exert a cytotoxic effect on SHED and do not cause the death of AD-MSC. Materials based on collagen and ß-tricalcium phosphate possess high cytocompatibility with all cell cultures under study. CONCLUSION: From the point of cytocompatibility all the examined bone-plastic materials may be considered safe for the restoration of bone defects. It should be noted that SHED transplantation on the surface of materials containing xenogeneic hydroxypatite is unacceptable.

The Technique of Thyroid Cartilage Scaffold Support Formation for Extrusion-Based Bioprinting.

During biofabrication, a tissue scaffold may require temporary support. The aim of this study was to develop an approach of human thyroid cartilage scaffold temporal support formation. The scaffold 3D-model was based on DICOM images. XY plane projections were used to form scaffold supporting part. To verify the technique, collagen hydrogel was chosen as the main scaffold component. Gelatin was applied for the supporting part. To test the applicability of the approach, a model of thyroid cartilage scaffold with the support was printed. The scaffold corresponded to a given model, although some discrepancy in geometry was observed during verification by computed tomography.

[Elimination kinetics of carbonyl-modified low density lipoproteins from bloodstream]. / Kinetika éliminirovaniia karbonil-modifitsirovannykh lipoproteinov nizkoi plotnosti iz krovotoka.

The elimination kinetics of carbonyl-modified low density lipoproteins (LDL) from rabbit bloodstream was studied using isolated LDL of rabbits and humans after preliminary biotinylation or labeling with FITZ. LDL from rabbit or human blood plasma were isolated using differential ultracentrifugation in a density gradient, and then LDL were labeled using biotinylation or FITZ, after which they were modified with various low molecular weight natural dicarbonyls: malondialdehyde (MDA), glyoxal or methylglyoxal. Native and dicarbonyl-modified biotinylated or FITZ-labeled LDL were injected into the ear vein of rabbits and blood samples were taken at certain intervals. To determine the content of biotinylated LDL in blood plasma, an enzyme immunoassay was performed; FITZ-labeled LDL were determined by spectra fluorescence. It is shown that glyoxal- and methylglyoxal-modified LDL in rabbits and humans circulated in the bloodstream for almost the same time as native (unmodified) LDL. At the same time, MDA-modified rabbit and human LDL were extremely quickly eliminated from the rabbit bloodstream. Dicarbonyl-modified LDL from the donors blood plasma were not associated with the red blood cells and endothelial cells. It has been shown that using the kits Oxidized LDL ELISA ("Mercodia", Sweden), it is possible to identify mainly MDA-modified LDL. The level of MDA-modified LDL in the blood plasma of CHD patients sharply decreases during therapy with the hypocholesterolemic drug the PCSK9 inhibitor (evulokumab), which activates LDL reutilization in the liver cells. These results explain the extreme drop in the level of MDA-modified LDL by their increased utilization in hepatocytes. The results obtained indicate a high atherogenicity of glyoxal- and methylglyoxal-modified LDL, long-term circulating in the bloodstream.

Protection of cultured endothelial cells from hydrogen peroxide-induced injury by antibody-conjugated catalase.

The cytoprotective features of catalase-antibody conjugate prepared by covalent conjugation of catalase to rabbit antibody against mouse IgG is described. The bifunctional cross-linking agent m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) was used for conjugation. Functionally active conjugate binds specifically to the plastic-adsorbed mouse IgG and to the surface of live human endothelial cells treated with mouse antiserum against human endothelial cells. Up to 4 units of catalase activity can bind to 1 cm2 of the endothelial monolayer. The targeted catalase protects endothelial cells from cytotoxic action of hydrogen peroxide: the minimal cytotoxic concentration of H2O2 for protected cells is 80-times higher than for intact cells. This effect is attributed partly to local reduction of H2O2 concentration in the cell microenvironment. Targeted catalase was estimated to reduce H2O2 concentration 8-fold near the cell surface with respect to average total concentration.

Binding of human monomeric type I collagen to platelets.

Interaction of platelets with subendothelial collagen is important in primary hemostasis and thrombosis. Although activation of platelets by collagen polymers has been widely investigated, only insufficient data are available concerning the binding of genetically distinct collagen types in their triple helical (monomeric) form to platelets. We report on the binding of 125I-labeled human type I collagen to platelets. The binding assay was performed at 20 degrees C in the presence of arginine in order to prevent polymerization of the collagen monomers. The binding of monomeric 125I-labeled human type I collagen is dose- and time-dependent, saturable and specific, since it is competitively inhibited by unlabeled type I collagen, but not by unlabeled human type V collagen. Scatchard analysis reveals a class of specific high affinity binding sites with a Kd of 2.5 X 10(-8) M. These results suggest that platelets interact with type I collagen through specific binding sites, and that there are various different binding sites on the platelet membrane for the genetically distinct collagen types.

Cytotoxicity of glucose oxidase conjugated with antibodies to target cells: killing efficiency depends on the conjugate internalization.

The cytotoxic action of glucose oxidase conjugated with antibodies against the target cells has been examined in a culture of human endothelial cells. Internalizable (anti-endothelial, MoAb E25) and non-internalizable (anti-fibronectin, MoAb FN) monoclonal antibodies were employed as vectors. Anti-endothelial monoclonal antibody E78 (whether it can be internalized by endothelial cells is unclear) and polyclonal mouse antiserum to the human endothelium were also used. The conjugates were prepared by oxidation of the enzyme carbohydrate moiety with periodate. Free conjugates display similar enzyme activity in glucose solution. In contrast to glucose oxidase, conjugated with no-immune IgG, antibody-conjugated glucose oxidase binds specifically to target cells. The efficiency of targeting was different for various conjugates. Targeting via the anti-fibronectin antibody and anti-endothelial antiserum provided maximal quantitative binding of glucose oxidase to endothelial cells, while the conjugates with MoAb E25 and MoAb E78 monoclonal antibodies provided less effective binding. In the presence of glucose, targeted glucose oxidase generated H2O2. Hydrogen peroxide is relatively stable in buffer, but rapidly decays in the culture medium supplemented with 20% human serum. Though the quantitative binding of MoAb E25-conjugated glucose oxidase was minimal comparing to other conjugates, targeting via MoAb E25 produced the maximal cytotoxic effect as well as targeting via polyclonal antiserum. The killing efficiencies of MoAb FN-conjugated and MoAb E78-conjugated glucose oxidase were about 30-fold lower. The high efficiency of the MoAb E25-conjugated enzyme may be due to its internalization by target cells. Internalization can lead to unaccessibility of generated H2O2 for extracellular scavengers and pH optimization for glucose oxidase activity, which provides valuable advantages for the cytotoxicity of the conjugate. Thus, cytotoxicity of antibody-conjugated glucose oxidase depends not only on the efficiency of specific binding to the target cell, but also on the fate of cell-bound conjugate. Cytotoxicity is extremely effective in case of 'internalizable' conjugate and drastically less effective in case of 'non-internalizable' conjugate.

Fibrillar, fibril-associated and basement membrane collagens of the arterial wall: architecture, elasticity and remodeling under stress.

The ability of a human artery to pass through 150 million liters of blood sustaining 2 billion pulsations of blood pressure with minor deterioration depends on unique construction of the arterial wall. Viscoelastic properties of this construction enable to re-seal the occuring damages apparently without direct immediate participance of the constituent cells. Collagen structures are considered to be the elements that determine the mechanoelastic properties of the wall in parallel with elastin responsible for elasticity and resilience. Collagen scaffold architecture is the function-dependent dynamic arrangement of a dozen different collagen types composing three distinct interacting forms inside the extracellular matrix of the wall. Tightly packed molecules of collagen types I, III, V provide high tensile strength along collagen fibrils but toughness of the collagen scaffold as a whole depends on molecular bonds between distinct fibrils. Apart of other macromolecules in the extracellular matrix (ECM), collagen-specific interlinks involve microfilaments of collagen type VI, meshwork-organized collagen type VIII, and FACIT collagen type XIV. Basement membrane collagen types IV, XV, XVIII and cell-associated collagen XIII enable transmission of mechanical signals between cells and whole artery matrix. Collagen scaffold undergoes continuous remodeling by decomposition promoted with MMPs and reconstitution from newly produced collagen molecules. Pulsatile stress-strain load modulates both collagen synthesis and MMP-dependent collagen degradation. In this way the ECM structure becomes adoptive to mechanical challenges. The mechanoelastic properties of the arterial wall are changed in atherosclerosis concomitantly with collagen turnover both type-specific and dependent on the structure. Improving the feedback could be another approach to restore sufficient blood circulation.

Red blood cell targeting to collagen-coated surfaces.

The interaction of human red blood cells carrying antihuman collagen antibody with collagen-coated surfaces was studied. Avidin was used as bifunctional crosslinking agent for the attachment of antibody to the red blood cell surface. Antibody-carrying red blood cells efficiently and specifically bound to collagen-coated surface covering a significant part of the surface. The components of normal blood had an insignificant effect on red blood cell binding. A model of drug targeting to the injured sites(s) of blood vessel wall is proposed.

Targeting of enzyme immobilized on erythrocyte membrane to collagen-coated surface.

It is suggested to use 'enzyme(s)-erythrocyte-antibody' complex for modulation of the microenvironment in definite compartments of blood circulation. A model system including peroxidase, human erythrocytes and anti-collagen antibodies was chosen to illustrate the principle. Peroxidase was conjugated to the erythrocyte surface via periodate-oxidized enzyme carbohydrate moiety; biotinylated antibodies were linked by avidin to the biotinylated erythrocytes. The properties of the immunocomplexes obtained have been investigated in an artificial system simulating an injured blood vessel wall. The advantages in using erythrocyte-mediated immunoenzyme complexes for enzyme (drug) targeting are discussed.

Red blood cell targeting to smooth muscle cells.

Monoclonal antibody discriminating between endothelial and smooth muscle cells is suggested to be used as a vector for directed transport of drugs to injured (denuded) areas of blood vessel wall. An in vitro model system was used in the studies: vascular smooth muscle or endothelial cells grown on plastic surface were treated with specific mouse monoclonal antibody recognizing an antigen localized on the surface of smooth muscle rather than endothelial cells; then erythrocytes coated with secondary (rabbit antimouse) antibodies were added. The results were analyzed spectrophotometrically or with scanning electron microscopy. Under the experimental conditions, erythrocytes, possible 'containers' for carrying the drugs, were found to bind only to smooth muscle cells. The data show that antibody provides absolute discrimination between endothelial and smooth muscle cells and, thus, may be used as a vector for drug targeting.

Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

Phenotype related changes of intimal smooth muscle cells from human aorta in primary culture.

To study the functional characteristics of smooth muscle cell (SMC) phenotypes, we have investigated myosin expression, cell proliferation, collagen production and low-density lipoprotein (LDL) receptor activity in intimal SMCs of normal human aorta during their growth in primary culture. By staining with rabbit antibodies to smooth muscle myosin (ASMM) 3 cell types could be distinguished in culture: homogeneously stained cells, cells with discontinuous myosin fibrils and myosin-negative cells. The ratio of cell types greatly changed with culture growth: on days 5, 7 and 14 it was 82:1:17%, 70:5:25% and 10:30:60%, respectively. After 5-6 days of culture intimal SMCs began to proliferate and DNA-synthesizing nuclei were seen 1.5-4.3 times more frequently in myosin-negative cells than in cells with homogeneous myosin distribution. At that time the number of cells reacted with monoclonal antibody (MAb) to an epitope shared collagen types I and III started to increase. By double immunofluorescence staining it was shown that the cultured cells containing both ASMM and MAb markers were found 2.0-4.8 times more rarely than MAb-positive staining in myosin-negative cells. During the first 5 days in culture LDL binding and uptake were diminished in intimal cells with intercellular lipid inclusions independently of their myosin staining pattern, but their activity increased with culture growth. Thus, SMCs from human aortic intima change their phenotype on days 6 and 7 in primary culture as manifested by alteration of myosin expression, increased cell proliferation, collagen production and LDL receptor activity. Changes in myosin expression, however, are not an essential prerequisite for cell proliferation and collagen production.

Relative distribution of fibronectin and type I, III, IV, V collagens in normal and atherosclerotic intima of human arteries.

Spatial distribution of fibronectin and type I, III, IV and V collagen has been investigated in normal arterial intima, fatty streaks, and atherosclerotic plaques by indirect immunofluorescence on transverse sections. Two distinct types of extracellular matrix were revealed in atherosclerotic lesions. The fibrous plaques consisted mostly of interstitial collagen types I and III, contained moderate amounts of type V and none of type IV collagen or fibronectin. In the extracellular matrix of the fatty streaks and in some areas of the fibrous plaques containing large amounts of subendothelial cells, some interstitial collagen was revealed, an increased amount of type IV, some type V collagen and a lot of fibronectin. Similarities of the extracellular matrix in atherosclerotic lesions and granulation tissues are discussed.

Rapid blood clearance of biotinylated IgG after infusion of avidin.

The techniques of immunotherapy and radioimmunoimaging suffer from the problem of background: intravenously injected antibodies remain in the circulation much longer than it is necessary for effective binding to the target. Various approaches, including the postinjection of second antibodies, were explored to overcome the problem with some success. The phenomenon of a 100-fold more rapid blood clearance of biotinylated immunoglobulins after postinjection of an equivalent dose of avidin is described. The concentration of 125I-labeled biotinylated IgG in the circulation of rats slowly decreased to 20% of initial in 24 hr. Avidin injection at any interval during this period induced 90-95% reduction of radioactivity in blood in 15 min. Up to 70% of the radioactivity was recovered in the liver. Avidin-induced blood clearance of biotinylated immunoglobulins may find applications in immunotherapy and radio- or nuclear magnetic resonance immunoimaging.

Stromal differentiation and architecture of the human umbilical cord.

In order to assess the characteristics of its stromal cells and the distribution of extracellular matrix proteins, we investigated, immunohistochemically and ultrastructurally, term, first and second trimester human umbilical cords. A differential distribution pattern of the various cytoskeletal proteins of stromal cells and extracellular matrix proteins was observed in different zones of the stroma, the subamniotic stroma, Wharton's jelly, and the vessels' adventitia. All three zones showed immunoreactivities for collagen types I, III and VI and for basement membrane molecules such as collagen type IV, laminin and heparan sulphate proteoglycan. Immunoreactivities for these extracellular matrix molecules were observed around cleft-like territories (stromal clefts) in the Wharton's jelly which were occupied by homogeneous ground substance but void of collagen fibrils and basal lamina molecules. Moreover, between the stromal clefts, slender cells were found which immunohistochemically and ultrastructurally corresponded to various stages of myofibroblastic differentiation. In earlier stages of gestation, stromal cells with a less complex expression pattern prevailed. The stromal clefts and the contractile cells together might serve as a system regulating the turgor of the cord.

Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta.

Confocal and conventional indirect immunofluorescence and immunogold electron microscopic methods were applied to examine the distribution of extracellular matrix constituents (collagens types III and IV) in the villi of immature and term human placentae. The immunofluorescence study revealed that collagen type III is more distinct in the villous stroma of term placenta as compared with that of the first trimester. Collagen type IV was detected mainly in endothelial and epithelial basement membranes and interestingly also to a certain extent in the stroma. Results obtained using immunoelectron microscopy support the proposal that collagen types III and IV are characteristic of stromal and basement membranes, respectively. Stromal collagen type IV is apparently localized in association with the interstitial types of collagen (I and III), in the villous stroma of term placenta.

Collagen-targeted antibodies inhibit platelet-dependent thrombosis in vivo.

Subendothelial collagen is one of the main triggers of platelet-dependent thrombus formation in arteries. The antithrombotic effects of rabbit polyclonal inhibitory antibodies to rat collagen type I-III and of murine non-inhibitory monoclonals to human recombinant single-/two-chain urokinase-type plasminogen activator (rscu-/rtcu-PA), cross-reacting with rat scu-/tcu-PA and their chemically synthesized conjugate, were studied both in vitro and in vivo. Anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombus-like structures induced by rat collagen immobilized on the polystyrene surface in a condition mimicing a high shear rates in the large elastic arteries. Monoclonals to human rscu-/rtcu-PA did not block the collagen-induced platelet activation in vitro. The short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56 +/- 4% (P < 0.05). Bispecific conjugate, directed to collagen and endogenous rat scu/tcu-PA inhibited thrombus formation by the same factor as anticollagen antibodies. The treatment of collagen-adsorbed conjugate by human rtcu-PA did not increase the antithrombotic effect. The present results suggest, that the local administration of the anticollagen antibodies to the site of vascular injury can be an efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries at thrombolysis or percutaneous transluminal coronary angioplasty.