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Skin regeneration requires a three-dimensional (3D) scaffold for cell adhesion, growth and proliferation. A type of the scaffold offering a 3D structure is a nonwoven material produced via a melt-blown technique. Process parameters of this technique can be adapted to improve the cellular response. Polylactic acid (PLA) was used to produce a nonwoven scaffold by a melt-blown technique. The key process parameters, i.e., the head and air temperature, were changed in the range from 180–270 °C to obtain eight different materials (MB1–MB8). The relationships between the process parameters, morphology, porosity, thermal properties and the cellular response were explored in this study. The mean fiber diameters ranged from 3 to 120 µm. The average material roughness values were between 47 and 160 µm, whereas the pore diameters ranged from 5 to 400 µm. The calorimetry thermograms revealed a correlation between the temperature parameters and crystallization. The response of keratinocytes and macrophages exhibited a higher cell viability on thicker fibers. The cell-scaffold interaction was observed via SEM after 7 days. This result proved that the features of melt-blown nonwoven scaffolds depended on the processing parameters, such as head temperature and air temperature. Thanks to examinations, the most suitable scaffolds for skin tissue regeneration were selected.

Standard cell culturing on plastic plates (two dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. The three dimensional (3D) systems, composed of extracellular matrix and/or pure amino acids which form a scaffold for cells, are more accurate in this respect. 3D cultures were primarily developed for cancer cells but there is also a need for their application in studies on inflammatory leukocytes. Herein we describe our approach to study neutrophil-like cells in the 3D system. We describe measures taken to establish a neutrophil-like cell line (nHL-60) and selection of 3D scaffolds (PuraMatrix alone or enriched with collagen type I) for their culturing. We focus on challenges in measurement of neutrophil viability in 3D cultures and based on our data we suggest application of resazurin, rather than tetrazolium-based dyes or trypan blue exclusion, for evaluation of neutrophil viability.

Here, we report on modification of one of the model biomedical polymers, poly L-lactide-co-glycolide (PLGA; 85:15), by reactive ion etching (RIE) oxygen plasma treatment. PLGA's major disadvantage is high hydrophobicity which restrains binding of cell-adhesive proteins and host cells. In the current approach, we aimed to answer two questions: (1) will only short (10 s) and moderate (20-200 mTorr, 45-90 W) RIE oxygen plasma treatment, leading to decrease of water contact angle by only up to 10°, sufficiently improve PLGA adherence to cells, and (2) how will this affect osteoblasts and activation of the immune system? All obtained modified PLGAs had improved hydrophilicity but unaltered roughness (as revealed by water contact angle measurements, X-ray photoelectron spectroscopy, and atomic force microscopy) resulting in significantly improved adhesion of osteoblasts (MG-63) and their low activation. Importantly, macrophages (RAW 264.7), one of the key cells initiating inflammation and bone resorption, responded significantly less vigorously to the modified polymers, expressing/releasing lower amounts of nitric oxide, matrix metalloproteinases (MMP-9), and pro-inflammatory cytokines (TNF-α, IL-6, IL-12p70, IFN-γ, IL-10). We conclude that already slight RIE oxygen plasma modification of PLGA is sufficient to improve its surface properties, and enhance cytocompatibility. Most importantly, this type of modification prevents excessive immune response.

Biodegradable aliphatic polyesters have numerous biomedical applications and their capacity to degrade in biological fluids provides the significant advantage of their removal. Three glycolide-containing aliphatic polyesters: a copolymer of glycolide and L-lactide (PGLA), a terpolymer of glycolide, L-lactide and e-caprolactone (PGLCap) and a copolymer of glycolide, and e-caprolactone (PGCap) were tested to evaluate their biocompatibility towards osteoblasts and fibroblasts. Each of the polymer units was previously reported to have acceptable biological properties and good biodegradability, and PGLA is already used for biomedical applications. Here we report that both PGLCap and PGCap affected cell adherence, and compromised cell viability as estimated by flow cytometric analyses of apoptotic and necrotic cells. The two polymers enhanced also production of numerous inflammation-related factors: nitric oxide, matrix metalloproteinases (MMP-2 and MMP-9), and cytokines, including pro-inflammatory TNF-α, IL-1β, IL-6, and chemokines (IL-8 or MCP-1) attracting leukocytes. The effects of PGLCap and PGCap were similar despite the fact that they possess different characteristics: amorphous/smooth surface and semicristalline/rough surface, respectively. However, their common feature, distinctive from PGLA, is a presence of e-caprolactone units in their structure. This compound is considered to be acceptably biocompatible but our data suggest that its copolymerization with glycolide and L-lactide does not provide satisfactory biocompatibility towards osteoblasts and fibroblasts. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

There is a constant search for biodegradable polymers with biocompatible characteristics. However, the reported materials are rarely tested for their immunostimulatory properties, which is an important issue as immune cells activated by the polymers might cause their rejection and lead to further injury to the host tissues. Therefore, the aim of the present study was to determine if biodegradable polymers are able to activate RAW 264.7 macrophages. Aliphatic polyesters, poly(L-lactide) (PLLA), poly(L-lactide-co-trimethylene carbonate) (PLTMC), poly(glycolide-co-L-lactide) (PGLA), poly(glycolide-co-L-lactide-co-ε-caprolactone) (PGLCap) and poly(glycolide-co-ε-caprolactone) (PGCap), processed into foils by slip-casting, were characterized in terms of their structure ((1)H-NMR, GPC, DSC) and surface properties (chemical composition, water contact angle, surface free energy, topography and roughness). RAW 264.7 cells were cultured on the materials for 3 or 5 days and their adherence, numbers of apoptotic/necrotic cells, as well as production of several cytokines/chemokines and other inflammation-related molecules (matrix metalloproteinases, nitric oxide) was evaluated. The study demonstrated that PLLA and PGLA did not influence macrophage activation and survival. In contrast, PLTMC, PGLCap and PGCap significantly decreased macrophage adherence, increased ratio of apoptosis and up-regulated synthesis/release of numerous inflammatory mediators. Thus, the latter materials might initiate an undesired inflammatory reaction. The above effects of the polymers were attributed to their high hydrophobicity and low polarity due to the presence of ε-caproyl blocks (PGLCap and PGCap), and/or high flexibility and susceptibility to mechanical deformation due to low glasstransition temperature (PLTMC, PGLCap and PGCap). In conclusion, while PLLA and PGLA do not affect macrophage functioning, the other materials (PLTMC, PGLCap, PGCap) up-regulate macrophage activity.

The aim of the study was to examine potential differences in the morphological, rheological and biochemical blood parameters of winter swimmers who remained physically active during the period between the end of one winter swimming season and the beginning of another. The study included a group of healthy winter swimmers (n = 17, all between 30 and 60 years of age). Six months following the end of winter season, the levels of mean corpuscular hemoglobin concentration and mean corpuscular hemoglobin turned out to be significantly higher, while erythrocyte count and hematocrit level significantly lower than at the baseline. Moreover, the break in winter swimming was reflected by a significant increase in median erythrocyte elongation index at all shear stress levels ≥ 1.13 Pa. The only significant changes in biochemical parameters of the blood pertained to an increase in the concentration of transferrin and to a decrease in the total protein, albumin and beta-1 globulin concentrations. Seasonal effort of winter swimmers between the end of one winter swimming season and the beginning of another has a positive influence on morphological, rheological and biochemical blood parameters.

Here, we report on modification of one of the model biomedical polymers, poly L-lactide-co-glycolide (PLGA; 85:15), by reactive ion etching (RIE) oxygen plasma treatment. PLGA's major disadvantage is high hydrophobicity which restrains binding of cell-adhesive proteins and host cells. In the current approach, we aimed to answer two questions: (1) will only short (10 s) and moderate (20-200 mTorr, 45-90 W) RIE oxygen plasma treatment, leading to decrease of water contact angle by only up to 10°, sufficiently improve PLGA adherence to cells, and (2) how will this affect osteoblasts and activation of the immune system? All obtained modified PLGAs had improved hydrophilicity but unaltered roughness (as revealed by water contact angle measurements, X-ray photoelectron spectroscopy, and atomic force microscopy) resulting in significantly improved adhesion of osteoblasts (MG-63) and their low activation. Importantly, macrophages (RAW 264.7), one of the key cells initiating inflammation and bone resorption, responded significantly less vigorously to the modified polymers, expressing/releasing lower amounts of nitric oxide, matrix metalloproteinases (MMP-9), and pro-inflammatory cytokines (TNF-α, IL-6, IL-12p70, IFN-γ, IL-10). We conclude that already slight RIE oxygen plasma modification of PLGA is sufficient to improve its surface properties, and enhance cytocompatibility. Most importantly, this type of modification prevents excessive immune response.

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.

Morphine coinjection with zymosan inhibits pain and leukocyte accumulation during peritonitis in several strains of mice, and affects systems of endogenous opioids. Present investigations focus on Met-enkephalin (Met-ENK) in the inflamed peritoneal cavity and brain centers of Swiss mice. Males of Swiss mice were IP injected with zymosan or zymosan supplemented with morphine. At the selected time points the peritoneal leukocytes were counted and the Met-ENK level was measured in exudatory fluid and leukocytes, striatum, hypothalamus, and pituitary gland. The Met-ENK content in peritoneal fluid rised sharply after zymosan injection, which corresponded with its decline in exudatory leukocytes, hypothalamus, and striatum. Morphine coinjection enhanced intraperitoneal accumulation of Met-ENK and its release from exudatory leukocytes, but inhibited its early fluctuations in hypothalamus and striatum. Effects of morphine-modulated inflammation on the Met-ENK system lasted longer than 7 days.

Bone tissue loss due to injury or disease often requires application ofautologous tissue grafts or artificial biomaterials to fill the fracture. Synthetic biomaterials provide temporary structural support for bone tissue and can be subsequently colonized by host tissue-specific cells. One of the most investigated groups of biomaterials are degradable polymers that naturally decompose in tissues with time. In particular aliphatic polyesters such as polylactides were reported to fulfill biocompatibility requirements as they induce a minor or lack an immune response and integrate with the surrounding tissue. Here we report on the biological effects of two polymers: poly(L-lactide) (PLLA) and a copolymer of L-lactide and trimethylene carbonate (PLTMC) on osteoblasts (MG-63) and fibroblasts (L-929). Osteoblasts are bone forming cells that are in the closest contact with the potential implant while fibroblasts produce the stroma forming the extracellular matrix (ECM) and along with macrophages initiate inflammation. We detected that both types of cells adhered better to PLLA than to PLTMC which might be related to the more rough surface of the former. However, both polymers, but especially PLTMC, increased apoptotic death of both cell types. Moreover, in contrast to PLLA, PLTMC modulated the production of some immune-related mediators by fibroblasts: it increased nitric oxide production and synthesis of numerous pro-inflammatory factors, cytokines (TNF-a and IL-6) activating leukocytes, and ECM-degrading MMP-9 which facilitates leukocyte migration. Thus, overall, our data suggest that PLTMC is less cytocompatible than PLLA.

Porous titanium is one of the most widely used implant materials because of its mechanical properties, however, it is also characterised by low bioactivity. To improve the above parameter we prepared three modifications of the porous (30 wt%) titanium (Ti) surface by covering it with bioactive hydroxyapatite (HA), bioglass (BG) and calcium silicate (CS). Subsequently we tested the impact of the modifications on macrophages directing the inflammatory response that might compromise the implant bioactivity. In the study we investigated the in vitro effects of the materials on murine cell line RAW 264.7 macrophage adherence, morphology and activation (production/release of metalloproteinase MMP-9 and pro- and anti-inflammatory cytokines). CS Ti decreased the macrophage adherence and up-regulated the release of several pro-inflammatory mediators, including TNF-α, IL-6, IL-12. Also HA Ti reduced the cell adherence but other parameters were generally not increased, except of TNF-α. In contrast, BG Ti improved macrophage adherence and either decreased production of multiple mediators (MMP-9, TNF-α, IFN-γ, MCP-1) or did not change it in comparison to the porous titanium. We can conclude that analyzing the effects on the inflammatory response initiated by macrophages in vitro, calcium silicate did not improve the biological properties of the porous titanium. The improved bioactivity of titanium was, however, achieved by the application of the hydroxyapatite and bioglass layers. The present in vitro results suggest that these materials, HA Ti and especially BG Ti, may be suitable for in vivo application and thus justify their further investigation.

Increased vascular permeability lead- ing to vascular leakage is a central feature of all inflammatory reactions and is critical for the for- mation of an inflammatory exudate. The leakage occurs because of gap formation between endothe- lial cells and breakdown of the basement mem- brane barriers. The present study aimed to inves- tigate the role of gelatinase B (matrix metallopro- teinase 9 (MMP-9)), known to be involved in neutrophil exudation, in changes of vascular per- meability at the early stages of acute zymosan peri- tonitis. We show that although MMP-9 is being released already within the first minutes of perito- nitis, its lack, induced pharmacologically or genet- ically, does not decrease but rather increases va- sopermeability. In mice treated with an inhibitor of gelatinases (A and B), a tendency to increased va- sopermeability existed, and in MMP-9/ mice (knockout (KO)), the difference was statistically significant in comparison with their controls. More- over, in intact KO mice, significantly augmented production of prostaglandin E2 (PGE2) of cycloox- ygenase 1 (COX-1) origin was detected, and deple- tion of peritoneal macrophages, but not mast cells, decreased vasopermeability in KO mice. Thus, the increase of vasopermeability observed on KO mice is a result of the increased production of COX-1- derived PGE2 by peritoneal macrophages. We con- clude that genetic deficiency in gelatinase B might lead to the development of a compensatory mech- anism involving the COX pathway. J. Leukoc. Biol. 80: 125-132; 2006.

Murine zymosan-induced peritonitis represents a well-defined model of acute inflammation. However, the molecular mechanisms by which leukocytes degrade basement membranes during extravasation into the peritoneum are not clear. Gelatinase B (MMP-9) is thought to participate in cellular migration, yet its role in leukocyte transmigration through endothelia during inflammation remains controversial. The aim of the present study was to evaluate the role of MMP-9 in the cell influx during zymosan-induced experimental peritonitis. In zymosan-treated Balb/c mice MMP-9 and its natural inhibitor (tissue inhibitor of metalloproteinase 1 - TIMP-1) were present in the peritoneal fluid and plasma at the time of peritoneal neutrophil (polymorphonuclear leukocyte - PMN) infiltration and persisted there until the time of monocytes/macrophages influx. To probe the function of gelatinases, gelatinase B-deficient mice (MMP-9(-/-)) were used as well as Balb/c mice treated with cyclic CTTHWGFTLC (INH), a specific peptide inhibitor of gelatinases. The studies revealed that in either group of mice deprived of MMP-9 activity, PMN infiltration was impaired at the time of their maximal extravasation (6h) while tumor necrosis factor alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant (KC) and interleukin 10 (IL-10) levels were not changed. At later stages (24 h post-zymosan) a significant increase in PMNs was observed in MMP-9(-/-) mice, but not in the inhibitor-treated mice, in comparison to their respective controls. Moreover, intraperitoneal (i.p.) injection of recombinant mouse pro-MMP-9 induced leukocyte accumulation in peritoneum. Collectively, the findings indicate that gelatinase B participates in leukocyte transmigration; however, its function can be compensated by other mechanisms.

Adult male mice were kept for one week either one or four animals per cage. Some were maintained under the same social conditions for an additional 9 days (controls); their counterparts were either grouped (4 per cage) or isolated (1 per cage). Changes in housing conditions caused a significant increase of plasma corticosterone measured 30 minutes after separation or grouping of SWISS, C57C3H, and BALB/c but not of C57BL/6 mice. Peritoneal inflammation was induced by i.p. zymosan injection on day 9 after changes in housing conditions when corticosterone was again at its initial level in each group. Peritonitis-connected pain symptoms, exudatory PMN numbers, and cytokine (IL-1beta and MPC-1) and corticosterone levels were compared between animals living in stable social conditions with those shifted 9 days earlier from separation to the group or vice versa. These factors were unaffected by social stress in C57BL/6 mice and in SWISS animals transferred from the group to isolation. In all other instances at least two parameters were significantly different in the post-stressed and control animals, being either enhanced or inhibited. In conclusion, social stress had long-term consequences on the course of inflammation in three out of four investigated strains of mice.

The local increase in Met-enkephalin level during zymosan-induced peritonitis may be related to its local production by exudatory leukocytes (as evidenced by our previous experiments) and influx from distal centers, as its level is significantly decreased in blood, inguinal lymph nodes, and some brain areas (striatum, hypothalamus, and pituitary gland).

Zymosan- or thioglycollate-induced experimental peritoneal inflammation in mice may serve as a convenient model for investigations of involvement of opioid peptides derived from exudatory leukocytes in the inflammatory processes. During peritonitis, the influx of neutrophils and monocytes/macrophages correlated with a sequential appearance of proinflammatory cytokines (IL-1beta and TNFalpha). After both kinds of stimulation, the expression of PENK mRNA was much higher in exudatory peritoneal leukocytes than its basal level in steady state.

Acute peritonitis induced in the goldfish by intraperitoneal injection of a sterile Thioglycollate solution shows a typical pattern with intraperitoneal exudation of serum proteins followed by influx of leucocytes (mainly heterophils/macrophages) correlated with elevated levels of chemotactic factors in peritoneal fluid and blood plasma. Supplementation of Thioglycollate with morphine (20 mg kg(-1) b.w.) does not affect the leakage of serum proteins into peritoneum. In contrast, it reduces the number of exudate peritoneal leucocytes (among them heterophils/macrophages) to the control level and decreases the level of peritoneal fluid/plasma chemoattractants, both effects being reversed by naltrexone pretreatment. Morphine itself acts as a chemokinetic factor for fish leucocytes as it increases their random movements. Therefore inhibitory effects of morphine on accumulation of exudate cells might be explained by inhibition of the production/release of chemotactic factors and/or reduced sensitivity of leucocytes to chemotactic signals. The effects of morphine on the goldfish peritonitis are in concordance with those described recently in Atlantic salmon and CB6 mice.

SWISS mice, edible frogs and goldfish i.p. injected with zymosan (Z groups) develop peritoneal inflammation connected with a massive intraperitoneal accumulation of leukocytes, which is significantly diminished in mice and fish (but not frogs) by supplementation of zymosan with morphine (ZM groups). In order to check the putative role of resident peritoneal macrophages in morphine-modulated zymosan-induced peritonitis, some animals were depleted of resident macrophages by repeated i.p. injections of clodronate-liposomes (CL) followed by Z or ZM injection. In SWISS mice such CL-induced removal ofMac-3-positive cells (macrophages) resulted in an enhanced influx and prolonged accumulation of polymorphonuclear leukocytes (PMNs) in CL-Z and CL-ZM groups in comparison with their counterparts with intact macrophages. Nevertheless, supplementation of zymosan with morphine inhibited the early stages of peritonitis in CL-treated animals as it did in untreated mice. This indicates that intact peritoneal macrophages of SWISS mice are important for limiting PMN accumulation, perhaps mainly through the release of IL-10, but are not critical for the induction of anti-inflammatory effects of morphine during the early stages of peritonitis. Unexpectedly, macrophage depletion in CL-treated frogs and fish resulted in a lack of a typical peritonitis in both Z and ZM groups of these ectothermic animals.

This paper describes F-based dry etching and resulting surface properties of biocompatible silicone elastomer. The etch rate of polysiloxane and surface morphology was found to be highly temperature dependent. An increase in temperature results in a significantly higher etch rate and a lower surface roughness. Possible mechanisms of the etching process and the roughness formation on an elastomer surface are discussed. The polysiloxane surface was proved to have hydrophobic characteristics both prior to and after plasma exposure. The results of the preliminary cytotoxicity study are very promising: cell viability on a raw and plasma treated polysiloxane was found to be very high and comparable to control. Due to the acceptable etch rate and absence of toxic contaminations, a F-containing plasma is considered an excellent method for microprocessing of silicone elastomers intended for biomedical applications.