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The sterilization of porous collagen sponges remains a challenging procedure. Gamma irradiation denatures collagen, resulting in dramatic changes to its structure. Ethylene oxide leaves toxic residues requiring weeks to evaporate. This study investigated the impact on cell behavior of gas plasma treatment when combined with vacuum freeze-drying. The goal of this procedure is to eliminate the molecules of hydrogen peroxide remaining after the sterilization process, together with their decomposition products, from the scaffolds. These molecules hinder the immediate use of the porous designs. Collagen and EDC/NHS-heparinized collagen scaffolds were sterilized with gas plasma. H2O2 released by the collagen specimens was measured by peroxidase test both immediately and also 1 week after the plasma treatment. Further measurements were done 24, 36, 48 and 72 h after vacuum freeze-drying. The activity of these scaffolds was further evaluated in relation to the proliferation, migration and differentiation of human umbilical vein endothelial cells (HUVECs). Both immediately after exposure to gas plasma and also 1 week later, the collagen designs contained significantly higher concentrations of H2O2 than scaffolds having also undergone vacuum freeze-drying. This procedure achieved faster decontamination of the remaining H2O2. Following vacuum freeze-drying, sponges already allowed HUVEC proliferation after 48 h, but in non-lyophilized specimens after gas plasma treatment alone, cell death occurred as early as only 1 week later. These data highlight the advantages of carrying out vacuum freeze-drying following gas plasma sterilization. The results show the substantial impact of sterilization of porous materials made for tissue engineering. [ABSTRACT FROM AUTHOR]

Apple (Malus domestica Borkh.) trees were propagated by budding from selected fully grown hybrids that ranged in height from 1.5 to 8 m. The growth and development of the selected budded trees after 7 years in the orchard was similar to that of the parent trees. Additional grafting studies showed that the dwarfism was not associated with the roots. Differences in photosynthetic activity and associated processes were not related to the size difference between tissue culture-propagated orchard-grown standard cv. Golden Delicious and dwarf hybrid trees. Applications of GA3 did not stimulate elongation of shoots of dwarf trees. Shoots of both standard and dwarf trees started to develop in mid-April when they contained nearly the same amounts of GA1, GA3 and GA8, but standard shoots contained higher concentrations of GA19, GA20 and GA29. On 2 June standard shoots were almost three times the length of dwarf shoots, but the number of leaves and area per leaf were nearly the same. The relative amounts of GAs on 12 May and 2 June for both plant types were similar to those on 20 April, except that GA19, GA20, GA1 and GA29 levels had declined. Gibberellin levels in standard shoots declined further between 2 and 22 June, after which there was no further shoot elongation or production of new leaves. Between 2 June and the end of the growing season, when summer temperatures were high, dwarf shoots continued to elongate slowly and to develop new leaves, which expanded little. During this time, the GA19 content of dwarf shoots nearly doubled, whereas the amounts of GA20, GA1, GA29 and GA8 declined. By the end of the season, standard shoots were 40 cm in length with 20 leaves and dwarf shoots were 28 cm in length, but with 36 leaves. High summer temperatures appear to induce loss of GA-responsiveness in orchard-grown dwarf trees and to cause a reduced rate of conversion of GA19 to GA20 in these genotypes. [ABSTRACT FROM AUTHOR]

Orchard-grown dwarf apple (Malus domestica Borkh.) trees selected from a hybrid population were propagated by tissue culture but had a growth pattern similar to standard cv. Golden Delicious plants when grown at constant 27°C instead of the expected dwarf pattern of growth. Shoot elongation was markedly reduced, with or without gibberellin A1 (GA1) or GA4 treatment, when trees were grown in an environment where day temperature was maintained at 35°C for 2 h in a ramped regime (night 20°C, day ramped to 35°C, held for 2 h and ramped down to 20°C night over a 14-h photoperiod). Application of GA1 or GA4 partially overcame growth retardation resulting from prior paclobutrazol treatment of both standard and dwarf trees grown at constant 27°C and of standard trees grown in the ramped environment. However, these GAs had no effect on paclobutrazol-treated or untreated dwarfs grown in the ramped regime. Gas chromatography-mass spectrometry with labelled internal standards was used to quantify GA1, GA3, GA8, GA19, GA20 and GA29 in extracts from standard and dwarf plants grown either at a constant 27°C or in a 20–30–20°C ramped temperature regime. Standard plants, which elongate quite rapidly in either environment, had similar levels of these GAs in both temperature regimes. The slowly growing dwarfs in the ramped temperature environment contained three times more GA19 than the rapidly elongating dwarfs grown at 27°C. The concentrations of the other GAs were reduced to ca 40% or less in plants grown in the ramped temperature regime compared with those grown at 27°C. These data suggest that shoot elongation of dwarf plants is sensitive to elevated temperatures both as a result of reduced responsiveness to GAs and because of a reduction in the concentration of GA1, apparently as a result of a lower rate of conversion of GA19 to GA20. It is possible that the altered GA metabolism may be a consequence of the change in GA sensitivity. [ABSTRACT FROM AUTHOR]

Tissueculturepropagated own-rooted cv. Spartan apple trees (Malus domestica Borkh.) planted in 1979 were treated in 1983 and 1985 via a soilline trunk drench with the plant growth retardant paclobutrazol [(2RS, 3RS)1(4chlorophenyl)4,4dimethyl-2-(1,2, 4-triazol1yl) pentan-3-ol]. Seeds of immature fruits from untreated and treated trees were sampled in 1989 ca 75 days after full bloom. After seeds were freezedried, gibberellins (GAs) were extracted, purified and fractionated via C18 reversedphase highperformance liquid chromatography (HPLC). Gibberellins A1, A3, A4, A7, A8, A9, A15, A17, A19, A20, A24, A34, A35, A44, A51, A53, A54, A61, A62, A63 and A68 were identified by using C18 HPLC, gas chromatography-selected ion monitoring and Kovats retention indices. Eight of the Gas identified were also quantified by using deuterated internal standards. The paclobutrazol applications caused a 55% reduction of vegetative shoot elongation in 1989, but both treated and untreated trees had developed a biennial bearing pattern by that time (heavy bloom or "on year" in 1989). Levels of early 13hydroxylation pathway GAs, viz. GA53, GA19, GA20, GA1 and also GA3, were not altered by treatment. However, GA4, GA7 and GA9 were increased 13.4, 6.5 and 3.8 times, respectively, in seeds of fruit from treated compared to untreated trees [ABSTRACT FROM AUTHOR]

High temperature has been implicated as the major factor responsible for dwarfing of selected apple (Malus domestica Borkh.) trees of a hybrid population of cv. Goldspur Delicious × cv. Redspur Delicious. Dwarf plants grew only 2.2 cm in 63 days under a ramped temperature regime (night 15°C, day ramped up to 38°C, held for 2 h and ramped down to 15°C - 14 h daylength), whereas semi-dwarf plants grew 26.3 cm. At a constant 27°C (14 h daylength), both dwarf and semi-dwarf plants grew nearly 50 cm. The gibberellin biosynthesis inhibitor, paclobutrazol, retarded growth of semi-dwarf plants in both ramped and constant environments and dwarf plants in the constant 27°C environment. It did not further reduce the size of dwarf plants growing under the ramped regime. Gibberellin (GA3) treatment reversed the inhibition of growth caused by paclobutrazol for all plants except it did not restore growth of dwarf plants in the ramped environment. These data suggest that neither pacobutrazol-treated nor untreated dwarf plants growing in the ramped environment (or in the orchard during hot summer months) are able to respond to GA3. In constrast, GA3 was utilized by the paclobutrazol-inhibited dwarf plants growing at constant 27°C, enabling shoot elongation to take place. It appears that high temperature may have caused alterations in GA target tissues in dwarf plants so that they no longer had the capacity to respond to GA. [ABSTRACT FROM AUTHOR]

The role of environment on the dwarfing (short internode) phenomenon of apple (Malus domestica Borkh.) was investigated and defined in controlled environmental chambers. Orchard-grown very dwarf, dwarf and semi-dwarf trees obtained by natural sibcrossing of spur-type cv. Golden Delicious and cv. Delicious, as well as standard cv. Golden Delicious, were propagated via in vitro techniques. Growth was rapid and none of the 4 types exhibited dwarf-like characteristics when grown at constant 27°C with 12, 14 or 16 h daylengths. Standard and very dwarf plants grew at nearly the same rate at constant 30°C, whereas growth nearly ceased on both types at constant 35°C after 7 days. Dwarf and very dwarf plants responded differently from standard and semi-dwarf plants when grown under alternating (ramped) night/day temperatures (15 or 20°C night ramped up to a daytime maximum over 8 h of 23, 28, 33 or 38°C. held for 2 h and then ramped down over 5 h to the night temperature). As the night/maximum day temperature differential increased from 0 to 23° under the ramping environments, growth of dwarf plants decreased markedly as compared to standard plants. When the same night/maximum day temperature differential occurred, the effect on decreasing shoot length was greater at the higher (20°C) night temperature. Increasing maximum day temperatures under the ramped environment also reduced leaf area plant-1 but did not markedly affect leaf number, resulting in short internodes. When a period of constant temperature was followed by ramped temperatures or vice versa, the sequence of constant vs ramped environments made little difference in the final growth of the 4 plant types. The data point to high temperature as the major factor for causing dwarfing of the sensitive plant types. Increasing the differential between night and maximum day temperature resulted in short internode, dwarf plants with small leaves similar to orchard-grown dwarf trees. [ABSTRACT FROM AUTHOR]

'York Imperial' apple seedlings (Malus domestica Borkh.) were continuously supplied via the roots with paclobutrazol [(2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl- 2-(1,2,4-triiazol-1-yI)pentan-3-oI)], a triazole GA biosynthesis inhibitor, at 0.68 μM in a nutrient solution. In comparison to controls, seedlings treated with paclobutrazol for 66 days showed a 91% reduction in shoot length, a 66% reduction in leaf area but only a 17% reduction in leaf number. This effect could be reversed by GA, applied to the foliage at 71.4 μM 0, 19 or 35 days after paclobutrazol was initially supplied and leaf area values for paclobutrazol-treated seedlings given both treatments did not differ significantly from controls. Plots of growth data indicate linearity of shoot longitudinal growth of GA3-treated seedlings. Leaf area increase was non-linear alter GA3 treatment up to approximately 30 days, when the rate dropped. On a per shoot basis, leaf weight closely followed leaf area but on a per unit area basis, paclobutrazol-treated leaves were heavier than controls; GA3 applications temporarily reversed this trend. [ABSTRACT FROM AUTHOR]

High temperature has been implicated as the major factor responsible for dwarfing of selected apple (Malus domestica Borkh.) trees of a hybrid population of cv. Goldspur Delicious × cv. Redspur Delicious. Dwarf plants grew only 2.2 cm in 63 days under a ramped temperature regime (night 15°C, day ramped up to 38°C, held for 2 h and ramped down to 15°C - 14 h daylength), whereas semi-dwarf plants grew 26.3 cm. At a constant 27°C (14 h daylength), both dwarf and semi-dwarf plants grew nearly 50 cm. The gibberellin biosynthesis inhibitor, paclobutrazol, retarded growth of semi-dwarf plants in both ramped and constant environments and dwarf plants in the constant 27°C environment. It did not further reduce the size of dwarf plants growing under the ramped regime. Gibberellin (GA3) treatment reversed the inhibition of growth caused by paclobutrazol for all plants except it did not restore growth of dwarf plants in the ramped environment. These data suggest that neither pacobutrazol-treated nor untreated dwarf plants growing in the ramped environment (or in the orchard during hot summer months) are able to respond to GA3. In constrast, GA3 was utilized by the paclobutrazol-inhibited dwarf plants growing at constant 27°C, enabling shoot elongation to take place. It appears that high temperature may have caused alterations in GA target tissues in dwarf plants so that they no longer had the capacity to respond to GA. [ABSTRACT FROM AUTHOR]

Changes of metabolic activities during dormancy and breaking of dormancy in the cherry flower buds of early blooming (EB) cultivar (Prunus avium L. cv. Coeur de Pigeon) and late blooming (LB) cultivar (Prunus serrulata Lindl. cv. Kwanzan) were determined. The LB buds had higher polyamines, protein and 1‐(malonylamino) cyclopropane‐1‐carboxylic acid (MACC) content than the EB buds. During the dormant state, the DNA, RNA, protein and polyamines in the EB buds were low but increased slowly and steadily, whereas those in the LB buds remained at a consistently higher level. The transition from dormancy to the active state in both cultivars was characterized by a sharp increase in DNA, RNA, protein, polyamines, S‐adenosyl‐methionine (SAM), 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and MACC. After initial swelling and development of flowers, the levels of all these components decreased. Polyamine and ethylene biosyntheses did not seem to be competing for their common substrate, SAM, during flower bud development. [ABSTRACT FROM AUTHOR]

Some physiological and biochemical changes in apple seedlings (Malus domestica Borkh cv. `York imperial') caused by an inhibitor of gibberellin biosynthesis, paclobutrazol [(2RS. 3RS)-1-(4-chlorophenyl)-4.4-dimethyl-2-(1 ,2,4-triazol-1-yl) pentan- 3-ol], were determined. Paclobutrazol shifted assimilate partitioning from leaves to roots, increased carbohydrates in all parts of apple seedlings, increased chlorophyll content on a leaf area basis, increased soluble protein in leaves, increased mineral element concentration in leaf tissue and increased root respiration. Foliar application of gibbercllic acid (GA3) counteracted the effects induced by paclobutrazol. [ABSTRACT FROM AUTHOR]

The homologous series of n‐alkyl esters (C1–C10) of 3,4‐dichlorobenzoic acid was synthesized and their effects in inhibiting the growth of Nicotiana meristems were studied. The inhibition of growth was considered in terms of penetration of chemicals into the plant tissue and subsequent cell membrane disruption. Penetration was investigated by applying the emulsified ester to the meristem and then measuring the compound recovered with the isolated surface lipids. Decreasing amounts of 3,4‐dichlorobenzoate esters penetrated into the plant as the alkyl chain length of the ester moeity was increased. Essentially no penetration occurred with the n‐C7 through C10 esters tested. The effect of the ester homologues on cell membranes was studied by measuring the efflux of betacyanin from beet root cells. Decreasing amounts of pigments were released as the alkyl chain length of the ester was increased. Minimal cell membrane disruption was found for the C7–C10 esters. Inhibition of the plant meristem may result from the more rapid penetration of the short chain ester homologues into the plant. [ABSTRACT FROM AUTHOR]

Serum macrophage migration inhibitory factor (MIF) and procalcitonin (PCT) concentrations as well as leucocyte numbers were evaluated in a retrospective study with 23 patients with severe burn injuries. The MIF and PCT concentrations as well as the number of leucocytes (LEU) were monitored over a period of 5 days. The total body surface area (TBSA) and sepsis-related organ failure assessment (SOFA) scores were also evaluated. The MIF, PCT concentrations and leucocyte counts were profoundly increased in all patients with severe burn wounds. At the time of admission into the intensive care unit, no significant differences were observed for the MIF and PCT levels between patients with a TBSA<60% (Group 1) and patients with a TBSA>60% (Group 2). After 48 h, however, the MIF and PCT levels reached very high levels in a subgroup of the patients, whereas these levels became normal again in other subgroups. The group of patients with a TBSA>60% was, therefore, subdivided in three groups (subgroups 2a-c). The MIF and PCT data pairs in these subgroups appeared to correlate in an inhomogeneous manner. These levels in the subgroup 2a (i.e., lethal within 5 days) were strongly elevated over those observed in Group 1 (TBSA<60%) and highly increased concentrations of both MIF and PCT correlated with lethal outcome. The combined determination of MIF and PCT might, therefore, be useful to discriminate between post-burn inflammation and systemic inflammatory response syndrome (SIRS) or sepsis with lethal outcome. [ABSTRACT FROM AUTHOR]

This study aimed to analyze the role of endothelial progenitor cell (EPC)-derived angiogenic factors and chemokines in the multistep process driving angiogenesis with a focus on the recently discovered macrophage migration inhibitory factor (MIF)/chemokine receptor axis. Primary murine and murine embryonic EPCs (eEPCs) were analyzed for the expression of angiogenic/chemokines and components of the MIF/CXC chemokine receptor axis, focusing on the influence of hypoxic versus normoxic stimulation. Hypoxia induced an upregulation of CXCR2 and CXCR4 but not CD74 on EPCs and triggered the secretion of CXCL12, CXCL1, MIF, and vascular endothelial growth factor (VEGF). These factors stimulated the transmigration activity and adhesive capacity of EPCs, with MIF and VEGF exhibiting the strongest effects under hypoxia. MIF-, VEGF-, CXCL12-, and CXCL1-stimulated EPCs enhanced tube formation, with MIF and VEGF exhibiting again the strongest effect following hypoxia. Tube formation following in vivo implantation utilizing angiogenic factor-loaded Matrigel plugs was only promoted by VEGF. Coloading of plugs with eEPCs led to enhanced tube formation only by CXCL12, whereas MIF was the only factor which induced differentiation towards an endothelial and smooth muscle cell (SMC) phenotype, indicating an angiogenic and differentiation capacity in vivo. Surprisingly, CXCL12, a chemoattractant for smooth muscle progenitor cells, inhibited SMC differentiation. We have identified a role for EPC-derived proangiogenic MIF, VEGF and MIF receptors in EPC recruitment following hypoxia, EPC differentiation and subsequent tube and vessel formation, whereas CXCL12, a mediator of early EPC recruitment, does not contribute to the remodeling process. By discerning the contributions of key angiogenic chemokines and EPCs, these findings offer valuable mechanistic insight into mouse models of angiogenesis and help to define the intricate interplay between EPC-derived angiogenic cargo factors, EPCs, and the angiogenic target tissue.

Macrophage migration inhibitory factor (MIF), a structurally and functionally unique pleiotropic mediator in inflammation and immune processes, was identified decades ago. There is now strong evidence that MIF promotes revascularization and is involved in wound healing processes. However, its exact role in wound healing is still a matter of debate. A cohort of 33 patients was recruited, including 14 patients with acute and 19 patients with chronic wounds. Both serum and wound fluid samples were collected from each patient, and MIF and cortisol concentrations were determined. To functionally underscore MIF's potential role in wound revascularization, a chemotaxis assay was adapted to test whether and to what extent serum samples and wound fluids of each group promote the chemotactic migration of endothelial progenitor cells (EPCs). MIF serum levels were significantly higher in chronic wound patients than in acute wound patients. Wound exudates of chronic wounds, however, contained a significantly lower concentration of MIF. In chronic wound patients, EPC migration might be delayed, as suggested by in vitro chemotaxis experiments. Despite the overall descriptive nature of this study, we conclude that MIF is correlated with occurrence of chronic wound. The increased MIF levels in the serum of chronic wound patients might be due to MIF's systemic effect of its proinflammatory activities, while its locally decreased levels in chronic wound exudates might be responsible for impaired recruitment of EPCs. Additional prospective data and detailed in vivo models are needed for a more comprehensive understanding of the role of MIF in chronic wound healing., (© 2012 by the Wound Healing Society.)

Background: Endothelial progenitor cells (EPCs) promote angiogenesis and play an important role in tissue revascularization and wound healing. Yet, the exact stimuli and mechanisms for the mobilization remain understood poorly. Macrophage migration inhibitory factor (MIF), which is a structurally unique pleiotropic cytokine, has been suggested to play a role in EPC recruitment and thus was a target of this study., Methods: This study included 20 patients who underwent flap operation. Subjects were divided into 3 groups according to the pattern of flap applied. The number of circulating EPCs and serum levels of MIF or CXCL12 were determined at different time intervals. In vitro chemotaxis experiments using Transwell devices were carried out to test whether MIF promotes the chemotactic migration of EPCs. To underscore functionally the chemotactic potential of MIF toward EPCs in flap patients, the chemotactic effects of serum samples from all groups were also examined in the presence and absence of monoclonal anti-macrophage migration inhibitory factor and anti-CXCL12 antibodies on EPC recruitment using in vitro migration chambers., Results: In flap patients, the number of circulating EPCs and serum levels of MIF but not CXCL12 serum levels were increased markedly compared with preoperative levels at day 1 after operation, especially in the group of free microvascular flaps. Serum levels of CXCL12 in the flap patients were increased only significantly compared with the healthy control group. An analysis between EPCs and MIF revealed a significant correlation, whereas no correlation was observed for CXCL12. MIF exerted a dose-dependent, prochemotactic effect on isolated human EPCs, and serum samples from all flap patients promoted EPC migration. Importantly, this effect was blocked partially by anti-macrophage migration inhibitory factor and to a weaker extent by anti-CXCL12 antibodies., Conclusion: We conclude that MIF plays an important role in the mobilization of EPCs, which is dependent on the degree of ischemia. Enhancement by MIF of chemotactic EPCs migration in vitro underpins its proposed in vivo function., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non-cognate ligand of the CXC-family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine-like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC-derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia-induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non-classical mechanism and originated from pre-formed MIF stores. Early hypoxia-triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia-inducible factor (HIF)-1α-induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF-1α-induced de novo synthesis of MIF. To functionally investigate the role of hypoxia-inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL(+) KDR(+) CD31(+) EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose-dependent bell-shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia-conditioned HUVECs, an effect that was completely abrogated by anti-MIF- or anti-CXCR4-antibodies. Thus, hypoxia-induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia-induced myocardial damage., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Fibrocytes were first described in 1994 as fibroblast-like, peripheral blood cells. These bone marrow-derived mesenchymal progenitor cells migrate into regions of tissue injury. They are unique in their expression of hematopoietic and monocyte lineage markers and extracellular matrix proteins. Several studies have focused on the specific role of fibrocytes in the process of wound repair and tissue regeneration. We discuss herein the biology and mechanistic action of fibrocytes in wound healing, scar formation, and maintenance of tissue integrity. Fibrocytes synthesize and secrete different cytokines, chemokines, and growth factors, providing a wound milieu that supports tissue repair. They further promote angiogenesis and contribute to wound closure via pathways involving specific cytokines, leukocyte-specific protein-1, serum amyloid P, and adenosine A(2A) receptors. Fibrocytes are involved in inflammatory fibrotic processes in such diseases as systemic fibrosis, atherosclerosis, asthma, hypertrophic scarring, and keloid formation. Accumulating literature has emphasized the important role of fibrocytes in wound healing and fibrosis. Detailed mechanisms nevertheless remain to be investigated to elucidate the full therapeutic potential of fibrocytes in the treatment of fibrosing disorders and the enhancement of tissue repair., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The high degree of degradation and the low angiogenic capabilities of temporary tissue substitutes still represent a major challenge in the field of tissue engineering. In an attempt to meet some of these challenges we covalently incorporated Astragalus polysaccharides, a plant extract with angiogenic properties, into collagen matrices. This contribution aims at developing a three-dimensional scaffold for temporarily covering tissue defects in tissue engineering and wound healing e.g. third degree burn wounds. Collagen matrices were modified by incorporating Astragalus polysaccharides (Ap) by means of covalent cross-linking with the watersoluble carbodiimide EDC. Matrices with different Ap/EDC ratios were prepared. After intensively washing of collagen matrices, the Ap modified and non-modified collagen matrices were exposed to the chorioallantoic membrane or implanted into subcutaneous pockets of rats. The number of capillaries in the chorioallantoic membrane in the vicinity of the samples, the hemoglobin contents within the explants and the hydroxyproline contents in the tissues attached to the explants were enhanced. Immunohistochemical evaluation of the explants revealed an increase in the recruitment of CD34+-cells in the modified matrices, indicative of improved angiogenic capabilities. To explore the underlying mechanisms, human umbilical vascular endothelial cells (HUVECs) were exposed to varying concentrations of Ap, collagen I and combinations thereof. The proliferative and chemotactic activities of HUVECs, as well as the protein expression of integrin αV, were strongly enhanced. The modification of collagen matrices with Astragalus polysaccharides of Ap with the cross-linking agent EDC leads to matrices with an increased angiogenic potential. The angiogenic capabilities of the modified collagen matrices appeared to depend on the Ap to EDC ratio. The presented results demonstrate that the incorporation of Astragalus polysaccharides into collagen matrices is an interesting and promising alternative for making wound dressings more angiogenic and improving their capabilities for covering tissue defects.

The porous TiO(2)/glass composite Ecopore has potential applications in hard tissue replacement. We describe the modification of Ecopore with the growth factor bone morphogenetic protein-2 (BMP-2) to add osteoinductive properties. Ecopore covalently coated with BMP-2 caused a weak induction of alkaline phosphatase in murine embryonal fibroblasts. In a rabbit bone defect model, BMP-2-coated Ecopore had moderately higher bone apposition rates and ingrown bone quantities at 6 weeks after implantation. To overcome loss of function due to chemical surface coupling, we filled the pore system of Ecopore with heparinized collagen sponge and loaded this secondary matrix with BMP-2. Heparinization of collagen filling increased the BMP-2 loading capacity of the matrix approximately 1.28-fold. Within 96 h, 17.0+/-0.1 and 10.1+/-0.2% of the used BMP-2 was released from non-modified and heparinized Ecopore/collagen, respectively, indicating that the heparin modification retarded BMP-2 release. Revealed by energy-dispersive X-ray spectroscopy analysis of implant cross-sectional areas, BMP-2-loaded Ecopore/collagen had significantly higher bony ingrowth quantities in rabbits, with the heparinized modification yielding the highest value (16.09+/-3.51%, p<0.005) compared with the non-heparinized matrix (10.72+/-4.07%, p<0.05) and the BMP-2-free controls (5.60+/-1.47%). This suggested a beneficial effect of the biomimetic modification of Ecopore with heparinized collagen for bone healing and integration.

The poor vascularization rate of matrices following cell invasion is considered to be one of the main shortcomings of scaffolds used in tissue engineering. In the past decade much effort has been directed towards enhancing the angiogenic potential of biomaterials. A great many studies have appeared reporting about enhancement of vascularization by immobilizing angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-2 (FGF-2). We have also tried to achieve this goal by modifying collagen matrices by covalent incorporation of heparin into the matrices and loading them with VEGF. We and others have observed that loading angiogenic factors to heparinized materials markedly increases angiogenic capacity. In the present paper we also investigated the angiogenic properties of collagen matrices which were only cross-linked, i.e. in the absence of heparin. The angiogenic capacity of the modified matrices was evaluated using the chorioallantoic membrane assay. Differences in angiogenic potential were deduced from macroscopic and microscopic analyses of the chorioallantoic membrane, as well as from dry weight changes. Cross-linked only matrices and matrices both cross-linked and heparinized appeared to show a significantly larger angiogenic potential than unmodified matrices. As previously observed, loading VEGF to these matrices further stepped up angiogenic potential. Quite surprisingly, cross-linking had a substantial impact on angiogenic potential. In terms of magnitude, this effect was similar to the effect of loading VEGF to heparinized matrices. Both modification procedures resulted in an increase of average pore size within the collagen matrices, and this observation may explain the more rapid invasion of mouse fibroblasts into cross-linked and heparinized matrices. Form changes of the implants were also monitored during the in vivo contacts: cross-linked and heparinized matrices showed far better resistance against contraction, as compared to unmodified matrices. Results from the chorioallantoic membrane assay experiments were compared with data obtained from rat model experiments, which confirmed the results from the chorioallantoic membrane assay. This relatively simple assay was again shown to be extremely helpful in evaluating and predicting the angiogenic capabilities of biomaterials for use in tissue engineering and wound healing.

MIF was recently redefined as an inflammatory cytokine, which functions as a critical mediator of diseases such as septic shock, rheumatoid arthritis, atherosclerosis, and cancer. MIF also regulates wound healing processes. Given that fibroblast migration is a central event in wound healing and that MIF was recently demonstrated to promote leukocyte migration through an interaction with G-protein-coupled receptors, we investigated the effect of MIF on fibroblast migration in wounded monolayers in vitro. Transient but not permanent exposure of primary mouse or human fibroblasts with MIF significantly promoted wound closure, a response that encompassed both a proliferative and a pro-migratory component. Importantly, MIF-induced fibroblast activation was accompanied by an induction of calcium signalling, whereas chronic exposure with MIF down-regulated the calcium transient, suggesting receptor desensitization as the underlying mechanism.