In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts

The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece, (2) acellular dermal replacement, Alloderm, (3) knitted poly(lactic-co-glycolic acid) (10:90)-poly(-caprolactone) (PLGA-PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [(3)H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece, Alloderm and PLGA-PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece contracted to 10% of the original size while Alloderm supported cell proliferation predominantly on the surface of the material. PLGA-PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.     

In vitro bioactivity and degradation of polycaprolactone composites containing silicate fillers

In spite of numerous publications on the potential use of combinations of polycaprolactone (PCL)/bioactive fillers for bone regeneration, little information exists on the assessment of solid, nonporous composites prepared via solventless routes and consisting of unmodified, slowly degrading homopolymer with relatively low amounts of reactive fillers such as bioglass or calcium silicate (CS). Thus, composites of PCL with commercial CS and a bioactive glass (BG45S5) at 30wt.% were produced by melt mixing in a twin screw extruder. Neat fillers, PCL and their composites were immersed in simulated body fluid (SBF) and phosphate buffer saline and tested for in vitro bioactivity and degradation, respectively, over a 4 month period. Testing methods included scanning electron microscopy with energy dispersive X-ray analysis, X-ray diffraction (XRD), elemental analysis and weight and pH changes before and after immersion. Experiments with neat fillers indicated fast growth of calcium phosphate minerals having different textures; they included clusters and globules of mineral precipitates as well as needle-shaped nanosized crystallites and possibly other calcium phosphate structures with varying Ca/P ratio. The bioactive glass composite initially showed fast growth of the precipitated minerals and partial surface coverage after 1 week, whereas in the CS composite, growth and surface coverage increased as a function of immersion time (over a period of 4 weeks) in the SBF solution. XRD results showed early appearance (1 week) of hydroxyapatite for both types of composites with differences attributed to different dissolution rates and different surface reactions of the fillers. Both fillers appeared to enhance the hydrolytic degradation of the matrix. Overall, the limited observed bioactivity of both composites within the test period may be related to the hydrophobicity of the matrix, insufficient ionic activity since SBF was not replenished and the relatively low content of the low surface areas fillers. Optimization of filler properties, such as surface/volume ratio, surface chemistry and size range, appears as a most important factor that would provide, at the required high filler volume fractions, a balance of melt processability and bioactivity.     

In vitro contraction rate of collagen in sponge-shape matrices

Connective tissue substitute can be made of collagen sponge-shape matrice which is reconstituted by freeze-drying a collagen dispersion. This procedure is then followed by a crosslinking treatment to decrease the in vivo biodegradation rate. In the present study, collagen dispersions made of collagen fibrils with a D-staggered pattern were submitted to the following treatments: (1) cyanamide or glutaraldehyde was introduced during the dispersion step followed by the manufacture of sponges; (2) uncrosslinked sponges were exposed to formaldehyde vapor; or (3) uncrosslinked and crosslinked sponges were severely dehydrated. To characterize the in vitro contraction rate, the surface areas of sponges were sequentially recorded in relation to soaking time. Contraction did not significantly occur when sponges were chemically treated. However, collagen in sponges treated by either severe dehydration or by both cyanamide treatment and severe dehydration contracted. On the other hand, the different treatments of the collagen modified the distribution of the D-staggered pattern within fibrils. After glutaraldehyde treatment, the periodicity of collagen fibrils disappeared and large fibres were observed. These experiments show that the different treatments of the collagen can be useful for designing a contractile as well as a non-contractile biomaterial.     

In vitro mineralization of gelatin-polyacrylic acid complex matrices

Gelatin-polyacrylic acid (gel-PAA) matrices were obtained by slow diffusion of polyacrylic acid into gelatin gels. The matrices were submitted to uniaxial stretching, which induces a preferential orientation of the collagen molecules, and used as biomimetic substrates for the nucleation of hydroxyapatite from simulated body fluid (SBF). The relative amount of hydroxyapatite deposited from 1.5SBF increases as a function of polyelectrolyte content in the matrices, up to about 30 wt%. In the absence of PAA, the inorganic phase is laid down on the surface of the gelatin matrices as hemispherical aggregates. At variance, hydroxyapatite deposition in the gel-PAA composite matrices at relatively low PAA content occurs preferentially in the spaces between the layers on the surface of the matrices and displays a tablet-like morphology. At high polyelectrolyte concentration, an almost uniform layer of hydroxyapatite covers the whole surface of the matrices. The preferential orientation of the (002) hydroxyapatite reflection indicates a close relationship between the inorganic crystals and the collagen molecules.     

In vitro mineralization of gelatin-polyacrylic acid complex matrices

Gelatin-polyacrylic acid (gel-PAA) matrices were obtained by slow diffusion of polyacrylic acid into gelatin gels. The matrices were submitted to uniaxial stretching, which induces a preferential orientation of the collagen molecules, and used as biomimetic substrates for the nucleation of hydroxyapatite from simulated body fluid (SBF). The relative amount of hydroxyapatite deposited from 1.5SBF increases as a function of polyelectrolyte content in the matrices, up to about 30 wt%. In the absence of PAA, the inorganic phase is laid down on the surface of the gelatin matrices as hemispherical aggregates. At variance, hydroxyapatite deposition in the gel-PAA composite matrices at relatively low PAA content occurs preferentially in the spaces between the layers on the surface of the matrices and displays a tablet-like morphology. At high polyelectrolyte concentration, an almost uniform layer of hydroxyapatite covers the whole surface of the matrices. The preferential orientation of the (002) hydroxyapatite reflection indicates a close relationship between the inorganic crystals and the collagen molecules.     

In vitro contraction rate of collagen in sponge-shape matrices.

Connective tissue substitute can be made of collagen sponge-shaped matrice which is reconstituted by freeze-drying a collagen dispersion. This procedure is then followed by a crosslinking treatment to decrease the in vivo biodegradation rate. In the present study, collagen dispersions made of collagen fibrils with a D-staggered pattern were submitted to the following treatments: (1) cyanamide or glutaraldehyde was introduced during the dispersion step followed by the manufacture of sponges; (2) uncrosslinked sponges were exposed to formaldehyde vapor; or (3) uncrosslinked and crosslinked sponges were severely dehydrated. To characterize the in vitro contraction rate, the surface areas of sponges were sequentially recorded in relation to soaking time. Contraction did not significantly occur when sponges were chemically treated. However, collagen in sponges treated by either severe dehydration or by both cyanamide treatment and severe dehydration contracted. On the other hand, the different treatments of the collagen modified the distribution of the D-staggered pattern within fibrils. After glutaraldehyde treatment, the periodicity of collagen fibrils disappeared and large fibres were observed. These experiments show that the different treatments of the collagen can be useful for designing a contractile as well as a non-contractile biomaterial.     

In vitro inflammatory response of nanostructured titania, silicon oxide, and polycaprolactone

Nanostructured materials are ubiquitous in tissue engineering, drug delivery, and biosensing applications. Nonetheless, little is known about the inflammatory response of materials differing in surface nanoarchitecture. Here we report human monocyte viability and morphology, in addition to inflammatory cytokines (IL-1alpha and B, IL-6, IL-10, IFN-alpha and gamma, TNF-alpha, IL-12, MIP-1alpha and beta), and reactive oxygen species production on several nanostructured surfaces, compared to flat surfaces of the same material. The surfaces studied were titiania nanotubes, short and long silicon oxide, and polycaprolactone nanowires. The results indicate that inflammation on titanium, polycaprolactone, and silicon oxide materials can be reduced by restructuring the surface with nanoarchitecture. Nanostructured surfaces display a reduced inflammation response compared to a respective flat control, with significant differences between titanium and nanotubular titanium. Little difference is observed in the inflammatory response between short and long nanowires of PCL and silicon oxide. All surfaces are significantly less inflammatory than the positive control, lipopolysaccharide. Additionally, we show that flat titanium is more inflammatory than silicon oxide and polycaprolactone. This study shows that nanoarchitecture can be used to reduce the inflammatory response of human monocytes in vitro.     

In vitro generated mast cells investigated by monoclonal antibodies

In the present study we have investigated the development of mast cells under in vitro conditions after depletion of mature mast cells of male Wistar rats by intraperitoneal application of distilled water. The studies were carried out both by monoclonal antibodies (MAb) against rat peritoneal mast cells and by cytochemical techniques. During cultivation the amount of MAb-positive cells increased from less than 5% to 64% after 3 weeks, which was accompanied by increased histamine levels and cell size. After 2 weeks the first Alcian blue stained mast cells were detectable, while safranin-positive granules appeared only after 3 weeks. These findings suggest that there are mast cell precursors in the peritoneum which may differentiate into mature mast cells. The antigen on the cell surface recognized by the MAb seems to be expressed during mast cell maturation.     

青霉源抗菌肽类霉肽素的体外抑菌活性研究

目的研究抗菌肽类霉肽素（AF）的体外抑菌活性。方法通过抑菌圈法和二倍稀释法检测AF的抗菌谱和对金黄色葡萄球菌的MIC;通过检测在AF中传代菌的敏感性确定细菌是否容易对AF产生抗药菌;将AF和细菌在不同温度和pH环境作用后检测抑菌活性,明确AF发挥活性的最适温度和pH值。结果AF对大部分供试细菌和抗药菌有效,对金黄色葡萄球菌的MIC为0．8mg／ml,金黄色葡萄球菌在AF中传代200代后敏感性不变。在14℃到30℃之间AF的抑菌活性随温度升高而降低,在pH3到pH10之间AF的活性随pH值升高而降低。结论AF是一种对抗性菌有效的广谱抗菌肽,而且不容易诱导细菌产生抗药性。     

青霉源抗菌肽类霉肽素的体外抑菌活性研究

目的 研究抗菌肽类霉肽素(AF)的体外抑菌活性.方法 通过抑菌圈法和二倍稀释法检测AF的抗菌谱和对金黄色葡萄球菌的MIC;通过检测在AF中传代菌的敏感性确定细菌是否容易对AF产生抗药菌;将AF和细菌在不同温度和pH环境作用后检测抑菌活性,明确AF发挥活性的最适温度和pH值.结果 AF对大部分供试细菌和抗药菌有效,对金黄色葡萄球菌的MIC为0.8 mg/ml,金黄色葡萄球菌在AF中传代200代后敏感性不变.在14℃到30℃之间AF的抑菌活性随温度升高而降低,在pH 3到pH 10之间AF的活性随pH值升高而降低.结论 AF是一种对抗性菌有效的广谱抗菌肽,而且不容易诱导细菌产生抗药性.     

In vitro culture of human thymic epithelial cells in serum-free media

Epithelial cells from human thymus were cultured in vitro at various serum concentrations and under defined serum-free conditions. A total of 238 cultures from 46 thymuses (MG and normal) were analyzed. Cells from fresh thymic tissue were explanted either as fragments or single cells after enzyme treatment. Serum-free as well as fetal calf serum (FCS) containing media based on Dulbecco's minimal essential medium and Ham's F-12 (DMEM/F-12) were found to be superior to MCDB 151 based serum-free media combinations, for the selective growth of thymic epithelial cells. In contrast, cultures based on RPMI 1640 medium supplemented with 1% FCS or more showed less epithelial cell selectivity and also supplement Ultroser G gave less fibroblast contamination. In serum-free media containing less than 0.1 mM ionic Ca, the cells had a smaller surface area and appeared more angular and also contained less keratin as compared to culture media with higher calcium contents. The development of serum-free conditions for in vitro growth of human thymic epithelial cells free of fibroblast contamination will facilitate studies of growth and maturation of the epithelial cells as well as investigations of their possible role in the development of myasthenia gravis.     

In vitro characterization of an artificial dermal scaffold

The treatment of extensive burn injuries has been enhanced by the development of artificial skin substitutes. Integra Artificial Skin, an acellular collagen-glycosaminoglycan (C-GAG) dermal equivalent requires a two-stage grafting procedure. However, preseeding the C-GAG dermal equivalent with cultured fibroblasts and keratinocytes, with the aim of performing a single-stage grafting procedure, may be beneficial in terms of replacing the requirement for traditional split-skin grafts. In this comparative in vitro study, the interactions of cultured human dermal fibroblasts and epidermal keratinocytes in Integra Artificial Skin in comparison to cadaver deepidermalized dermis (DED) was investigated. An increase in cell proliferation and migration in the C-GAG dermal equivalent was observed over time. Cocultures of fibroblasts and keratinocytes on both dermal equivalents showed positive expression of proliferation, differentiation, and extracellular matrix (ECM) protein markers. Organization of keratinocytes in the epidermal layers of DED composites were better compared to the C-GAG composites. Deposition of ECM proteins was enhanced in the presence of keratinocytes in both dermal equivalents. Results demonstrate that in vitro the C-GAG dermal equivalent is biocompatible for cell attachment, migration, proliferation, and differentiation. Preseeding Integra Artificial Skin with cultured autologous fibroblasts and keratinocytes for in vivo application, as a single-stage grafting procedure, warrants testing. A better clinical outcome may be achieved as shown by our in vitro results of the coculture composites.     

In vitro characterization of rat thymic macrophages.

Thymic macrophages have been isolated from Wistar rats and maintained in long-term culture. Day 1-isolated thymic macrophages expressed MHC class I and II antigens, Thy-1, CR3, CD4, ED1, ED2, as well as acid phosphatase and non-specific esterase activities. Whereas cultured macrophages were positive for the activation antigen recognized by the mAb OX48, which was not expressed after isolation, MHC class II and Thy-1 expression were down-regulated during the culture, but could be induced with human rIL-2 or with Con A-SCM, which are also inducers of IL-2R, a cell-surface marker not expressed on Day 1 or on cultured macrophages.     

In vitro characterization of EHV-4 gG-deleted mutant

Equine herpesvirus 4 (EHV-4) is an important pathogen that causes respiratory tract disease in horse populations worldwide. Glycoprotein G (gG) homologs have been identified in several alphaherpesviruses as minor non-essential membrane-anchored glycoproteins. In this study, EHV-4 gG deletion mutant has been generated by using bacterial artificial chromosome technology to investigate the role of gG in viral pathogenesis. Our findings reported here revealed no significant difference between parental EHV-4 and gG-negative strain in their replication cycle in cell culture. Furthermore, virus titers and plaque formation were comparable in both viruses. It is noteworthy that these findings disagree with the previously published study describing gG deletion in another EHV-4 strain.     

In vitro evaluation of random and aligned polycaprolactone/gelatin fibers via electrospinning for bone tissue engineering

Scaffold, as an essential element of tissue engineering, should provide proper chemical and structural cues to direct tissue regeneration. In this study, aligned and random polycaprolactone (PCL)/gelatin fibrous scaffolds with different mass ratio were electrospun. Chemical, structural, and mechanical properties of PCL/gelatin fibrous scaffolds were characterized by FTIR and tensile measurements. The average diameters of different groups were between 334.96?±?41.43?nm and 363.78?±?50.49?nm. Blending PCL with gelatin increased the mechanical properties of the scaffolds. The cell culture results demonstrated that the mass ratio of PCL and gelatin showed no obvious effects on cell behavior, whereas the cell growth behavior was affected by the fibers orientation. Higher elongation ratio, enhanced cell proliferation and elevated alkaline phosphatase activity were observed for cells cultured on aligned fibers. The findings in our research provide insightful information for the design and fabrication of scaffolds for bone tissue engineering.     

Contractile forces generated by articular chondrocytes in collagen-glycosaminoglycan matrices

The objective of the study was to directly measure the force of contraction of adult articular chondrocytes and to examine their contractile behavior in collagen-glycosaminoglycan analogs of extracellular matrix by live cell imaging in vitro. The contractile forces generated by passages 2 and 3 adult canine articular chondrocytes were measured using a cell force monitor. The contractile behavior of the cells was also directly imaged as they were cultured in collagen-glycosaminoglycan scaffolds. Passage 2 cells seeded in a collagen-glycosaminoglycan scaffold were capable of generating a force of 0.3 nN/cell. Chondrocytes subcultured through a third passage generated a force twice that level, paralleling the increase in the alpha-smooth muscle actin (SMA) content of the cells as demonstrated by Western blot analysis. Treatment of passage 3 cells with staurosporine reduced the force of contraction by approximately one-half, reflecting the effects of this agent in reducing the SMA content of the cells and disrupting the microfilaments. These values compare with 1 nN previously reported for lapine dermal fibroblasts of passage 5-7, using the same apparatus. Direct live cell imaging documented the contractile behavior of the articular chondrocytes in the collagen-glycosaminoglycan matrix in the time frame in which the force was directly measured in the cell force monitor. This imaging demonstrated how the cells acted individually and in unison to buckle the collagen struts making up the matrix.Adult articular chondrocytes are capable of generating a SMA-enabled force of contraction sufficient to model extracellular matrix molecules.     

In vitro cultivation of Herpetosoma trypanosomes in insect cell tissue culture media

The cultivation of Herpetosoma trypanosomes in insect tissue culture media supplemented with foetal calf serum is described. Trypanosoma lewisi and T. musculi, which can be grown in blood agar media, were compared with four other species of Herpetosoma trypanosomes, T. microti, T. evotomys, T. grosi and T. nabiasi, in their growth in Schneider's Drosophila medium, Grace's, Mitsuhashi-Maramorosch, RPMI 1640, TCM 199 and nutrient blood agar media. Schneider's Drosophila and Grace's media supplemented with 20% foetal calf serum proved the most suitable media for growth of all parasites except T. nabiasi from rabbits which was not successfully established. Primary cultures were passaged after approximately 3 weeks and were maintained to continuously produce metacyclic trypomastigotes which produced less virulent infections although they maintained their infectivity to their respective hosts. The growth patterns in culture and morphology of the parasites are described.     

In vitro generated mast cells express natural cytotoxicity against tumour cells.

Previously, we found that Klebsiella O3 lipopolysaccharide (KO3 LPS) exhibits very strong adjuvant activity in augmenting the delayed-type hypersensitivity and antibody responses to protein antigens, and also strong ability to enlarge the regional lymph node in mice. By hydrolysis in 1% acetic acid at 100 degrees for 1 hr, KO3 LPS was dissociated into 54-60% polysaccharide fraction and 25-27% lipid A fraction. The lipid A fraction thus prepared retained lethal toxicity for mice, whereas the polysaccharide fraction was essentially non-toxic. However, neither the lipid A fraction nor the polysaccharide fraction could reproduce the strong adjuvanticity of KO3 LPS, and a simple mixture of these two fractions also failed to do so. It is therefore concluded that, for expression of the strong adjuvanticity of KO3 LPS, both the lipid A and polysaccharide moieties are indispensable and must be combined in the form of LPS. By contrast, administration of relatively large amounts of the lipid A fraction alone could elicit enlargement of the regional lymph node to the same degree as that attained by KO3 LPS. The regional lymph node-enlarging activity of the lipid A fraction in lesser amounts was enhanced by addition of the polysaccharide fraction. It is therefore likely that there is no direct correlation between adjuvant activity of the LPS or its derivatives, and their regional lymph node-enlarging activity.     

In vitro and in vivo response to nanotopographically-modified surfaces of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and polycaprolactone

Colloidal lithography and embossing master are new techniques of producing nanotopography, which have been recently applied to improve tissue response to biomaterials by modifying the surface topography on a nano-scale dimension. A natural polyester (Biopol^?), 8% 3-hydroxyvalerate-component (D400G) and a conventional biodegradable polycaprolactone (PCL) were studied, both nanostructured and native forms, in vitro and in vivo. Nanopits (100-nm deep, 120-nm diameter) on the D400G surface were produced by the embossing master technique (Nano-D400G), while nanocylinders (160-nm height, 100-nm diameter) on the PCL surface were made by the colloidal lithography technique (Nano-PCL). L929 fibroblasts were seeded on polyesters, and cell proliferation, cytotoxic effect, synthetic and cytokine production were assessed after 72 h and 7 days. Then, under general anesthesia, 3 Sprague–Dawley rats received dorsal subcutaneous implants of nanostructured and native polyesters. At 1, 4 and 12 weeks the animals were pharmacologically euthanized and implants with surrounding tissue studied histologically and histomorphometrically. In vitro results showed significant differences between D400G and PCL in Interleukin-6 production at 72 h. At 7 days, significant (P < 0.05) differences were found in Interleukin-1 β and tumor necrosis factor-α release for Nano-PCL when compared to Nano-D400G, and for PCL in comparison with D400G. In vivo results indicated that Nano-D400G implants produced a greater extent of inflammatory tissue than Nano-PCL at 4 weeks. The highest vascular densities were observed for Nano-PCL at 4 and 12 weeks. Chemical and topographical factors seem to be responsible for the different behaviour, and from the obtained results a prevalence of chemistry on in vitro data and nanotopography on soft tissue response in vivo are hypothesized, although more detailed investigations are necessary in this field.     

In vitro and in vivo response to nanotopographically-modified surfaces of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and polycaprolactone

Colloidal lithography and embossing master are new techniques of producing nanotopography, which have been recently applied to improve tissue response to biomaterials by modifying the surface topography on a nano-scale dimension. A natural polyester (Biopol), 8% 3-hydroxyvalerate-component (D400G) and a conventional biodegradable polycaprolactone (PCL) were studied, both nanostructured and native forms, in vitro and in vivo. Nanopits (100-nm deep, 120-nm diameter) on the D400G surface were produced by the embossing master technique (Nano-D400G), while nanocylinders (160-nm height, 100-nm diameter) on the PCL surface were made by the colloidal lithography technique (Nano-PCL). L929 fibroblasts were seeded on polyesters, and cell proliferation, cytotoxic effect, synthetic and cytokine production were assessed after 72 h and 7 days. Then, under general anesthesia, 3 Sprague-Dawley rats received dorsal subcutaneous implants of nanostructured and native polyesters. At 1, 4 and 12 weeks the animals were pharmacologically euthanized and implants with surrounding tissue studied histologically and histomorphometrically. In vitro results showed significant differences between D400G and PCL in Interleukin-6 production at 72 h. At 7 days, significant (P < 0.05) differences were found in Interleukin-1beta and tumor necrosis factor-alpha release for Nano-PCL when compared to Nano-D400G, and for PCL in comparison with D400G. In vivo results indicated that Nano-D400G implants produced a greater extent of inflammatory tissue than Nano-PCL at 4 weeks. The highest vascular densities were observed for Nano-PCL at 4 and 12 weeks. Chemical and topographical factors seem to be responsible for the different behaviour, and from the obtained results a prevalence of chemistry on in vitro data and nanotopography on soft tissue response in vivo are hypothesized, although more detailed investigations are necessary in this field.