Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

[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.

[Kinetics of methyluracil release from bioresorbable polymeric carriers].

Bioresorbable poly(lactic-co-glycolic) matrix-carriers containing 20 wt. % of 6-methyluracil (MU) have been prepared by supercritical fluid monolithization without organic solvents. Raman spectroscopy was used to analyze both the spatial distribution MU over polymer matrices and the MU release kinetics from the carrier into phosphate buffer solution. It was found that, during the first 24 h, the amount of released MU did not exceed 15-20% of its encapsulated content. After that, the MU release kinetics passed to almost linear regime with simultaneous retarding of the process. On the 40th day of observation, the MU content in solution reached up to 80% of its initial content in the carriers. Thus, using 6-methyluracil as a model, it was shown that the proposed bioresorbable and bioactive composites can be used as matrix-carriers for targeted and long-term drug release.

Design of tissue engineering implants for bone tissue regeneration of the basis of new generation polylactoglycolide scaffolds and multipotent mesenchymal stem cells from human exfoliated deciduous teeth (SHED cells).

Cultures of multipotent mesenchymal stromal cells from the pulp of human deciduous teeth (SHED cells) were characterized. The cells were used for population of 3D biodegradable polylactoglycolide scaffolds; their osteogenic potential was preserved under these conditions. Implantation of the scaffolds to mice induced no negative reactions in the recipients. These results suggest that the use of polylactoglycolide scaffolds populated with SHED cells is a promising approach for creation of implants for bone defect replacement.

Biocompatibility of tissue engineering constructions from porous polylactide carriers obtained by the method of selective laser sintering and bone marrow-derived multipotent stromal cells.

We studied the biocompatibility of porous polylactide carrier matrices obtained by means of surface selective laser sintering. Carrier matrices had no cytotoxic activity, but maintained adhesion and proliferation of cells. Subcutaneous transplantation of tissue engineering constructions from these carriers and bone marrow-derived multipotent stromal cells did not cause the inflammatory response and pathological changes in rats. The conditions for organotypic regeneration were provided at the site of transplantation (high degree of blood supply and considerable amount of immature precursor cells).

Atomic force microscopic study of the surface morphology of apatite films deposited by pulsed laser ablation.

Atomic force microscopy (AFM) has been used to study the surface morphology of apatite films deposited on metallic and polyethylene substrates by laser ablation using KrF and transversely excited atmospheric CO2 lasers. The films are found to consist of a smooth apatite coating with macroparticles scattered on the surface. A wide variety of macroparticles, differing in size, shape and roughness, were found and analysed employing the high spatial resolution of AFM (< 1 nm). We have investigated the correlation between the apatite film morphology and the deposition conditions. Of particular importance are laser fluence, gas pressure, the nature of the target and the substrate temperature. We have explained these dependencies on the basis of a theoretical model which includes evaporation and a cluster-type laser ablation mechanism.

Osteoblast growth on titanium foils coated with hydroxyapatite by pulsed laser ablation.

Pulsed laser ablation is a new method for deposition of thin layers of hydroxyapatite (HA) on to biomaterial surfaces. In this paper, we report activity and morphology of osteoblasts grown on HA surfaces fabricated using different laser conditions. Two sets of films were deposited from dense HA targets, at three different laser fluences: 3, 6 and 9 Jcm(-2). One set of the surfaces was annealed at 575 degrees C to increase the crystallinity of the deposited films. Primary human osteoblasts were seeded onto the material surfaces and cytoskeletal actin organisation was examined using confocal laser scanning microscopy. The annealed surfaces supported greater cell attachment and more defined cytoskeletal actin organisation. Cell activity, measured using the alamar Blue assay, was also found to be significantly higher on the annealed samples. In addition, our results show distinct trends that correlate with the laser fluence used for deposition. The cell activity increases with increasing fluence. This pattern was repeated for alkaline phosphatase production by the cells. Differences in cell spreading were apparent which were correlated with the fluence used to deposit the HA. The optimum surface for initial attachment and spreading of osteoblasts was one of the HA films deposited using 9 J cm(-2) laser fluence and subsequently annealed at 575 degrees C.

Biocompatibility of Laser-deposited Hydroxyapatite Coatings on Titanium and Polymer Implant Materials.

We have investigated the biocompatibility of calcium phosphate coatings deposited by pulsed laser ablation from hydroxyapatite (HA) targets onto polyethylene and Teflon substrates. It was found that the cell density, attachment, and morphology of primary rat calvaria osteoblasts were influenced by both the original polymer and by the nature of the apatite coatings. HA coatings on Teflon were found to have higher biocompatibility in terms of cell adhesion and spreading. In vivo studies of bone response to coatings deposited by KrF excimer and CO2 lasers on commercial Ti6A14V alloy implants show that both deposition techniques suppress fibrous tissue formation and promote osteogenesis. © 1998 Society of Photo-Optical Instrumentation Engineers.

[Enhancing the biocompatibility of dentures made from polymethylmethacrylate by using hydroxyapatite]. / Povyshenie biologicheskoi sovmestimosti zubnykh protezov iz polimetilmetakrilata s pomoshch'iu gidroksiapatita.

Twenty or 50 weight-percent of hydroxyapatite (Polystome) was added in suspensions of AKR-15 copolymer and acid monomers, then the mixtures were molded and subjected to thermal treatment. Admixtures (supercritical CO2) were extracted from the composites in parallel with infrared spectroscopic measurement of the concentration of methylmetacrylate toxic monomers. Hydroxyapatite in 20% concentration decreased the release of toxic substances from the composite, whereas 50% concentration of the agent did not produce this effect. Three patients with intolerance of acrylic plastic were effectively treated using this method.

[The tissue reaction to acrylic plastics modified by supercritical extraction with carbon dioxide]. / Tkanevaia reaktsiia na akrilovye plastmassy, modifitsirovannye sverkhkriticheskoi ékstraktsiei dvuokis'iu ugleroda.

The process of extraction of admixtures from acryl plastic widely used in dentistry by means of supercritical carbon dioxide (sc-CO2) was studied and effects of extraction conditions on biocompatibility and toxicity of resultant materials assessed, sc-CO2 effectively purified the specimens from toxic compounds (monomers and low-molecular oligomers, methylmethacrylate, dichloroethane) and notably improved the biocompatability of polymer implants. Tissue reaction to ethacryl and protacryl depends on the degree of implant polymerization and duration of extraction of toxic substances from polymer.

[Biocompatible coatings for metallic implants obtained by laser spraying]. / Biosovmestimye pokrytiia dlia metallicheskikh implantov, poluchennye lazernym napyleniem.

Use of various lasers and regimens for spraying the coatings and for additional thermal processing makes it possible to purposefully alter the morphology, composition, and crystallinity of the coatings, and hence, the optimal coatings, as regards their biological activity, may be created. Replacement of eximer lasers with CO2 lasers makes the spraying process cheaper and comparable in efficacy with the plasma process, with the quality of coatings and reproducibility of their parameters appreciably improved. Experiments with coatings of three types, sprayed by CO2 lasers, demonstrated that they met the mechanical and physico-chemical requirements of biocompatibility.

[The structural characteristics of the bony tissue at the surface of an implant with hydroxyapatite spray-coated with excimer and CO2 lasers]. / Osobennosti postroeniia kostnoi tkani u poverkhnosti implanta s pokrytiiami iz gidroksiapatita, napylennymi éksimernym i CO2-lazerami.

Rat experiments were performed to study osteogenesis and osseointegration in implanting fragments of dental titanic implants into the spongy bone. The implant spray-coating with hydroxyapatite produced by excimer and CO2 lasers stimulates osteogenesis. Bone tissue integration with implant proceeds more actively in response to CO2 laser radiation. The weakest integration was registered between the metal and bone tissues.

[Biodegradable polymer microparticles with entraped herbal extracts: preparation with supercritical carbon dioxide and use for tissue repair].

Biodegradable microparticles based on poly-D,L-lactide with entrapped mixture of herbal water-soluble extracts of Plantago major and Calendula officinalis were prepared. For preparation of these microparticles the previously developed method based on the usage of supercritical carbon dioxide (SC-CO2) was proposed. Microparticles were obtained by two techniques: 1) by preparing porous polymer monolith containing entrapped mixture of herbal extracts, which was then reduced to fine microparticles (ca. 0.1 mm) by dry ice grinding (called here as "monolithisation technique") and 2) by spraying of this polymer/extracts mixture through a jet (spray technique). In vitro release kinetic profile of herbal extract mixture was found to depend on the microparticle preparation technique, on the microparticle structure as well as on the initial ratio polymer/extracts (w/w). The microparticles were used for gastric ulcer treatment in a rat model. The extracts released from microparticles were found to accelerate tissue repair.

Physical, chemical, and biological characterization of pulsed laser deposited and plasma sputtered hydroxyapatite thin films on titanium alloy.

The physical, chemical, and biological properties of pulsed laser deposited (PLD) and plasma sputtered (PS) hydroxyapatite (HA) coatings were compared. Human osteoblast-like cell responses to these coatings in vitro were assayed for proliferation and phenotypic expression. PS coatings formed smooth and continuous thin films that followed the contours of the substrate surface. PLD coatings consisted of numerous spheroidal micro- and macroparticles. The crystallinity of all coatings was quantified by comparison with the HA target used for both the PS and PLD processes. The XRD and FTIR results indicated that unannealed PLD coatings deposited at room temperature had X-ray spectra consistent with an amorphous structure and were found to dissolve after only a few hours in saline solution. Annealing at 400 degrees C increased the crystallinity (87-98%), which resulted in improved stability and cell activity. The PS coatings showed greater chemical stability than the unannealed PLD coatings and contained an approximate 15% crystalline phase, increasing to 65% postannealing. Cell proliferation and alkaline phosphatase production were significantly higher on unannealed PS specimens than the other coating treatments. There may be benefits in engineering the presence of a minor percentage of a microcrystalline phase in an amorphous or nanometer scale polycrystalline HA structure.

Properties of calcium phosphate coatings deposited and modified with lasers.

Physical, chemical and biological properties of calcium phosphate coatings fabricated by a pulse laser deposition method at room temperature (RT PLD) have been studied. In vitro evaluation of RT PLD coatings on bioresorbable polymers (Poly-epsilon-caprolactone and Poly-L-lactide) have been carried out. It was shown that both polymers support osteoblast growth, with increased cell activity, alkaline phosphatase activity and total protein content on those surfaces that have been coated. The advantages of RT PLD coatings in biomaterials surface optimization are discussed.