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Novel silk fibroin/elastin wound dressings

Contents lists available at Acta Biomaterialia Novel silk fibroin/elastin wound dressings Andreia Vasconcelos Andreia C. Gomes , Artur Cavaco-Paulo , a Universidade do Minho, Departamento de Engenharia Têxtil, Campus de Azurém, 4800-058 Guimarães, Portugalb Centre of Molecular and Environmental Biology (CBMA), Department of Biology, Campus de Gualtar, 4710-057 Braga, Portugal Silk fibroin (SF) and elastin (EL) scaffolds were successfully produced for the first time for the treatment Received 28 October 2011 of burn wounds. The self-assembly properties of SF, together with the excellent chemical and mechanical Received in revised form 12 April 2012 stability and biocompatibility, were combined with elastin protein to produce scaffolds with the ability to Accepted 20 April 2012 mimic the extracellular matrix (ECM). Porous scaffolds were obtained by lyophilization and were further Available online 27 April 2012 crosslinked with genipin (GE). Genipin crosslinking induces the conformational transition from randomcoil to b-sheet of SF chains, yielding scaffolds with smaller pore size and reduced swelling ratios, degradation and release rates. All results indicated that the composition of the scaffolds had a significant effect on their physical properties, and that can easily be tuned to obtain scaffolds suitable for biological applications. Wound healing was assessed through the use of human full-thickness skin equivalents (Epi- dermFT). Standardized burn wounds were induced by a cautery and the best re-epithelialization and the fastest wound closure was obtained in wounds treated with 50SF scaffolds; these contain the highestamount of elastin after 6 days of healing in comparison with other dressings and controls. The cytocom-patibility demonstrated with human skin fibroblasts together with the healing improvement make theseSF/EL scaffolds suitable for wound dressing applications.
Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
parameters in the biomaterials field. Recently, we developed silkfibroin/keratin films incorporating a synthetic inhibitor of elastase, Skin wounds are the disruption of normal skin physiology. From to control the high levels of this enzyme produced in a chronic the moment the wound is created, the healing mechanism is initi- wound environment Silk fibroin/alginate sponges demonstrate ated to re-establish skin continuity. The healing process is complex a higher healing effect than both components acting alone In and involves an integrate response of different cell types and this work, scaffolds based on silk fibroin and soluble elastin were growth factors . Promotion of healing is often accompanied developed and tested.
by the use of biocompatible wound dressings: these should pro- Elastin (EL) is an insoluble extracellular matrix protein that mote a moist environment in the wound and serve as a shield provides elasticity and resilience to the arteries, lungs and skin against external factors like dust and bacteria; enhance water Due to its highly crosslinked nature, elastin is highly and vapor permeation and promote epithelialization by releasing insoluble and difficult to process into new biomaterials. As a con- biological agents to the wounds. Due to its unique properties of sequence, soluble forms of elastin including tropoelastin , high mechanical strength and excellent biocompatibility, silk a-elastin and elastin-like polypeptides are fre- fibroin has been explored for the development of wound dressings.
quently used to develop elastin-based biomaterials. Nevertheless, The degradation rates of electrospun silk materials applied as a crosslinking step is required to obtain an insoluble material.
wound dressings have been evaluated and the incorporation There are several crosslinking methods for elastin including chem- of growth factors into electrospun silk mats has been shown to ical , enzymatic physical and c-irradia- accelerate wound healing . Moreover, silk films have been tion . Among them, chemical crosslinking agents are widely shown to heal full thickness skin wounds in rats faster than tradi- used. Aldehydes and epoxy compounds have been commonly used tional porcine-based wound dressings in biomaterial constructs due to their efficient formation of cross- Blending silk fibroin with other components has been shown to links with amino acid side chains, low antigenicity and sufficient improve the properties of the resulting material. This allows the mechanical strength. Despite these advantages, they exhibit high modulation of biodegradation and release rates, important Genipin (Ge) is a natural covalent crosslink agent isolated from the fruits of Gardenia jasminoides Ellis that offers comparable ⇑ Corresponding author. Tel.: +351 2535 10100; fax: +351 2535 10293.
E-mail address: (A. Cavaco-Paulo).
crosslinking efficacy. It has been reported that genipin binds with 1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 biological tissues and biopolymers , leading to 2.4. Degree of crosslinking matrices with good mechanical properties, reduced swelling extentand significantly reduced cytotoxicity when compared to synthetic The crosslinking degree was determined by the ninhydrin assay crosslink agents like glutaraldehyde and epoxy compounds . Samples (6.0 ± 0.7 mg) were heated with a ninhydrin solution (2% (w/v)) at 100 °C for 20 min. The optical absorbance To our knowledge, elastin has been crosslinked with collagen of the resulting solution was recorded at a wavelength of 570 nm , fibrin and gelatin for the development of using a Hekios c ThermoSpectronic spectrophotometer. The biomaterials, but never with silk fibroin. In this study, we devel- amount of free amino groups in the test sample after heating with oped silk fibroin/elastin (SF/EL) scaffolds crosslinked with genipin.
ninhydrin is proportional to the optical absorbance of the solution.
The resulting materials were characterized by their physical– The concentration of free NH2 groups in the sample was deter- chemical properties and the effect of crosslinking on those proper- mined from a standard curve of glycine concentration vs. absor- ties was evaluated. Moreover, the wound dressing functionality of bance. SF/EL scaffolds prepared without genipin were used as these materials was tested with a real chronic wound exudate and control materials. Triplicate samples were evaluated. The degree the healing ability was assessed through the use of three-dimen- of crosslinking was determined by the following equation: sional (3-D) human skin equivalents.
Degree of crosslinking ð 2. Materials and methods (NH2)nc and (NH2)c are, respectively, the mole fraction of free NH2 in non-crosslinked and crosslinked samples.
Silk cocoons from Bombyx mori were kindly supplied from ‘‘Sezione Specializzata per la Bachicoltura'' (Padova). Elastin solu- 2.5. FTIR spectroscopy ble from bovine neck ligament was purchased from Sigma (Spain).
Genipin is a product of Wako Chemicals (Germany). The BJ5ta cell FTIR spectra of pure SF and SF/EL scaffolds were measured with line (telomerase-immortalized human normal skin fibroblasts) a Perkin-Elmer (Spectrum One FTIR) spectrometer in the spectral was purchased from ATCC through LGC Standards.
region of 4000–650 cm1 with a ZnSe ATR cell. Spectra were ac- Human full-thickness skin equivalents (EpidermFT) were sup- quired for sponges with and without methanol treatment. For EL plied by MatTek Corporation (USA). All other reagents, including samples, FTIR spectra were recorded in KBr pellets using a FTIR- those used in cell culture, were analytical grade and purchased 4100 from Jasco with a resolution of 2 cm1.
from Sigma (Spain).
2.6. Thermal analysis 2.2. Preparation of silk fibroin solution Differential scanning calorimetry (DSC) measurements were Silk was purified from its sericin content as previously de- performed with a DSC-30 instrument (MettlerToledo), from room scribed The cocoons were cut, cleaned from debris and temperature to 120 °C, at a heating rate of 10 °C min1, and kept larvae and autoclaved for 30 min at 120 °C. Fibroin was then thor- at 120 °C for 10 min, to induce dehydration of samples. The tem- oughly washed with distilled water and dried overnight at room perature was lowered to room temperature and increased to temperature. Silk fibroin (SF) solution (2% (w/v)) was prepared by 500 °C at a heating rate of 10 °C min1. Sample weight was dissolving fibroin in 9.6 M LiBr solution at 60 °C for 3 h. The result- 2–3 mg. The open aluminum cell was swept with N2 during the ing solution was filtered, and dialyzed against distilled water until analysis. The analysis was performed in duplicate for scaffolds with salts were completely removed, using cellulose tubing (Sigma, and without methanol treatment.
Spain) (molecular-weight cut-off of 12,000–14,000 Da).
2.7. Scanning electron microscopy (SEM) 2.3. Silk fibroin/elastin blends preparation; crosslinking reaction;scaffold formation Cross-sections were prepared by cutting the SF/EL scaffolds with a razor blade in liquid nitrogen. Before analysis, the scaffolds Elastin (EL) solution was prepared by dissolving the elastin were coated with gold and examined morphologically using a powder in distilled water. SF (2%) and EL (1%) were mixed to pre- NOVA Nano SEM 200 FEI. The morphology was determined before pare blends of 100/0 SF/EL, 80/20 SF/EL and 50/50 SF/EL. Genipin and after methanol treatment.
(GE) powder, 0.1 and 0.5% (w/v) was added to blend solutionsunder constant stirring at room temperature until complete disso-lution of GE powder. The crosslinking reaction was carried out for 2.8. Swelling ratio 3, 6 and 24 h at 37 °C. The resulting solutions were cast on 96-wellplates and frozen at 20 °C for 2 days and freeze dried for 2 days to SF/EL scaffolds, treated with methanol and completely dry remove the solvent completely. SF/EL scaffolds without genipin (60 °C for 24 h) were immersed in phosphate-buffered saline were used as controls and were prepared by the same process de- (PBS; pH 3.0, 7.4 and 11) at 37 °C for 24 h. The excess buffer was scribed above. The control samples were identified as 100SF, 80SF removed and the wet weight of the scaffolds was determined.
and 50SF, which correspond to 0, 20 and 50% of elastin, and cross- The swelling ratio of the film was calculated as follows: linked scaffolds were identified as 100SFyGE, 80SFyGE and50SFyGE, where y is the genipin concentration used. In order to in- Swelling ratio ¼ duce the transition of SF from random coil to b-sheet structure and consequently insolubility, scaffolds were immersed in 90% (v/v)methanol solution for 30 min and then washed in distilled water WS is the mass of the swollen material and Wd is the initial dry and air dried.
A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 curve. Release studies were performed in triplicate samples andfor a period of 7 days. The release behavior of compounds from The porosity of the SF/EL scaffolds with different blending ratios polymeric systems can be determined by fitting the release data was measured by the liquid displacement method . Hexane to the empirical relationship given by the Ritger–Peppas equation was used as the displacement liquid because it is a non-solvent for SF. The scaffolds were immersed in a known volume (V1) of hexane in a graduated cylinder for 30 min. The total volume of hex- ane after impregnation into the scaffold was recorded as V2. The impregnated scaffolds were then removed from the cylinder and Mt/M1 is the fractional drug release at time t; t is the release the residual hexane volume was recorded as V3. For all types of time; k is the kinetic constant that measures the drug release rate, scaffolds, experiments were carried out in triplicate. Data are and n is the diffusion exponent that depends on the release mech- presented as average ± SD. One-way ANOVA analysis of variance anism and the geometry of the matrix. To determine n values, Eq.
with Bonferroni post-tests was performed, with statistically signif- is modified in Eq. and n is determined from the slope of the icant differences when p < 0.001. All calculations were performed plot of log (%released) vs. log t.
using GraphPad software (version 5.03). The porosity of the scaf- log ð%releasedÞ ¼ logðMt=M fold (e) was calculated by the following equation: 2.12. Cytotoxicity The scaffolds were tested for cytotoxicity according to ISO stan- 2.10. In vitro degradation dards (10993-5, 2009). The BJ5ta cell line (normal human skinfibroblasts) was maintained according to ATCC recommendations 2.10.1. Porcine pancreatic elastase (PPE) (four parts Dulbecco's modified Eagle's medium (DMEM) contain- SF/El scaffolds previously treated with methanol, with and ing 4 mM L-glutamine, 4.5 g l1 glucose, 1.5 g l1 sodium bicarbon- without crosslinking, were incubated for 21 days at 37 °C in a solu- ate, and one part of Medium 199, supplemented with 10% (v/v) of tion containing 0.1 mg ml1 of PPE in 100 mM Tris–HCl buffer, fetal bovine serum (FBS), 1% (v/v) of penicillin/streptomycin solu- pH 8.0. The control samples were incubated in PBS buffer solution tion and 10 lg ml1 hygromycin B). The cells were maintained at (pH 7.4) without enzyme and submitted to the same conditions.
37 °C in a humidified atmosphere of 5% CO2. Culture medium The solutions were replaced every 24 h.
was refreshed every 2 to 3 days.
2.10.2. Wound exudate 2.12.1. Test by indirect contact Wound exudate was collected from pressure wounds using a Scaffolds (£=3 mm and 6 mm thickness) were sterilized by vacuum assisted closure system. Wound fluid was diluted ten-fold immersion in ethanol 70% for 30 min, then hydrated and thor- in PBS solution and centrifuged to remove cells and tissue material.
oughly rinsed with PBS. The conditioned media were obtained by SF/EL scaffolds were incubated with exudate in the same condi- incubating the sponges in 1 ml of DMEM in a CO2 incubator at tions described above in a fixed ratio of exudate per mg of scaffold 37 °C for 5 days. The sponges were then removed and the condi- of 6 mg ml1. At designated time points, samples were washed tioned media were obtained. Before use, the conditioned media thoroughly with distilled water, dried in a desiccator and weighted were filtered to remove degraded scaffolds and diluted if necessary to estimate the extent of degradation by the following equation: in complete cell culture medium. Complete cell culture medium subjected to the same conditions but not exposed to the sponges was used as a negative control, whereas a 1% (v/v) solution of Tri- tonÒ X-100 (Sigma) prepared in fresh culture medium was used as m0 and mf are respectively, the initial and final dry mass of the a toxicity positive control. Cells were seeded at a density of 20  103 cells/100 ll/well on 96-well tissue culture polystyrene(TCPS) plates (TPP, Switzerland) the day before experiments and 2.11. In vitro release then incubated with the conditioned media. At each defined timepoint (24, 48 and 72 h), cell viability was assessed using the Alamar The release of a compound from SF/EL scaffolds was examined Blue assay (alamarBlueÒ Cell Viability Reagent, Invitrogen). Resa- by the incorporation of an antibacterial agent, gentamicin zurin, the active ingredient of alamarBlueÒ reagent, is a non-toxic, (2 mg ml1). For control samples, gentamicin was dissolved in cell-permeable compound that is blue in color and reduced to the protein solutions and stirred for 5 min at room temperature.
resorufin, red color compound, by viable cells. The quantity of The resulting solutions were cast in 96-well plates to prepare the resofurin formed is directly proportional to the number of viable SF/EL scaffolds. In the case of crosslinked samples, gentamicin cells. 10 ll of alamarBlueÒ reagent was added to each well contain- was dissolved in the protein solution before crosslinking reaction.
ing 100 ll of culture medium. After 4 h of incubation at 37 °C the Before release studies, control and crosslinked scaffolds were trea- absorbance at 570 nm was measured, using 600 nm as a reference ted with methanol. SF/EL scaffolds were incubated at 37 °C in PBS wavelength, in a microplate reader (Spectramax 340PC). Data are buffer and in a solution containing 0.1 mg ml1 of PPE. Solutions presented as average ± SD of two independent measurements.
were changed every 24 h. At determined time points, aliquots were Two-way ANOVA with Bonferroni post-tests was performed, with taken and gentamicin release was determined using the o-phthal- statistically significant differences when p < 0.001. All calculations dialdehyde method The analysis was carried out by measur- were performed using GraphPad software (version 5.03).
ing the maximum fluorescence of gentamicin-o-phthaldialdehydecomplex using a multiplate reader (Synergy HT W/TRF from Bio- 2.12.2. Cell proliferation Tek) in the fluorescence mode at an emission wavelength of Cell proliferation was determined in terms of DNA content to 456 nm. After each measurement, the samples were added back monitor the effect of the scaffolds on fibroblast. Scaffolds, prepared to the medium to restore the equilibrium conditions. The quantifi- and sterilized as previously described (£=15 mm and 3 mm cation of the release was established by a gentamicin standard thickness), were gently placed in 24-well (TCPS) plates (TPP, A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 Switzerland), then 250 ll of cell suspension (2  105 cells ml1) was loaded onto an upper side of each scaffold and allowed to infil- Degree of crosslinking obtained for SF/EL solutions, for the different reactionconditions, determined by Eq. trate into the scaffold. The scaffolds were then incubated at 37 °Cunder 5% CO Crosslinking treatment Degree of crosslinking (%) 2 conditions for 3 h to allow for initial cell attachment.
After the initial incubation period the wells were then filled with 250 ll of medium and placed into a cell culture incubator and maintained at 37 °C with 5% CO2 for either 3 or 5 days. Culture media were renewed every 2 days. After each indicated time inter- val, cells/scaffold constructs were collected, rinsed with PBS and cell proliferation was determined in terms of DNA content mea- sured with Hoechst 33258 (Invitrogen). Briefly, cells were har-vested from cell-scaffold constructs by incubating with a 0.25%solution of trypsin. Cells were then collected by centrifugationand lysed in a Tris–HCl 15 mM pH 7.4 buffer with consecutivefreeze–thaw cycles. Cell lysates were incubated with equal volume accepted mechanism is similar to that observed for amino-group of 5 lg ml1 Hoechest 33258 solution for 40 min at room temper- containing compounds where the ester groups of genipin ature in the dark. Fluorescence was determined using a FLUOROS- interact with the amino groups of SF and elastin, leading to the for- KAN ASCENT FL plate reader (ThermoScientific) at 350 nm mation of secondary amide linkages. Moreover, the amino groups excitation and 445 nm emission. The relative fluorescence unit initiate nucleophilic attacks which result in the opening of the value obtained from samples was interpolated against a DNA stan- genipin dihydropyran ring. An inherent phenomenon of genipin dard curve constructed using known number of cells, to determine crosslinking is self-polymerization, which occurs by radical reac- the DNA content/number of cells in each sample. Data are tion of two amino-attached open rings Some authors presented as average ± SD of two independent measurements.
reported that genipin preferentially reacts with the amino Two-way ANOVA with Bonferroni post-tests was performed, with acids lysine and arginine. SF and elastin contain respectively, statistically significant differences when p < 0.001. All calculations 0.95% and 1.07% of these amino acids, which is a very low fraction.
were performed using GraphPad software (version 5.03).
The crosslinking sites are thus low in number, which results inlower crosslinking degrees when compared with other genipincrosslinked blend systems The highest crosslinking degree 2.13. Wound healing assay obtained for sponges containing elastin might be related with theslightly higher fraction of lysine and arginine amino acids.
Skin equivalents (EpidermFT) were cultured at the air–liquid The genipin crosslinking of SF/EL scaffolds might induce confor- interface in tissue culture inserts placed in six-well plates accord- mational changes due to the structural rearrangement of chains to ing to manufacturer's instructions. Upon receipt the tissues were form covalent bonds. FTIR spectra of SF and elastin, with and with- placed into new six-well plates containing 2.5 ml of fresh culture out crosslinking, in the range of 600–2000 cm1 are represented on medium, supplied with the skin equivalents, and kept at 37 °C, . SF protein exists in three conformations namely random coil, 5% CO2 overnight. Burn wounds were made by placing a cautery Silk I (a-form) and Silk II (b-sheet conformation). The 100SF spec- on top of the tissue for 10 s. The SF/EL scaffolds were then placed trum show bands at 1640 cm1 for amide I (C@O stretch- over the wounded area. Two burn wounds per tissue were made ing), 1517 cm1 with a shoulder at 1532 cm1 for amide II (N–H to control wound size, and the healing was evaluated in two inde- deformation) and 1238 cm1 for amide III (C–N stretching, C@O pendent assays. Skin equivalents without dressing and treated bending vibration), indicating a random coil/Silk I conformation with a commercial collagen dressing, Suprasorb C (Lohmann & . SF molecules can structurally rearrange due to changes Rauscher, Germany), were used as controls.
in the hydrogen bonding by methanol treatment acquiring a Healing of these wounds was evaluated after 6 days by histolog- b-sheet conformation. Genipin crosslinking is also able to induce ical evaluation. Skin equivalents were fixed in 4% formaldehyde b-sheet conformation of SF molecules. Comparing the SF spectra solution at room temperature. Subsequently, paraffin-embedded obtained after genipin crosslinking, it is clearly the transition from tissues section of 4 lm thickness were obtained and stained with random coil to b-sheet conformation confirmed by the shifting to Haematoxylin and Eosin (H&E). All sections were observed under lower wavenumbers of amide I (1620 cm1) and amide II a light inverted microscope (Olympus IX71).
(1514 cm1) bands The shoulder observed at 1532 cm1for amide II assigned to random coil, progressively disappears with 3. Results and discussion the increase in genipin concentration. Moreover, a characteristicband of genipin at 1105 cm1 (–COH) appeared in the spectra of 3.1. Biochemical and biophysical properties of SF/EL scaffolds 100SF0.1GE and 100SF0.5GE, confirming once again the reactionbetween genipin and SF. The FTIR results evidenced that genipin The formation of covalent bonds on blended systems may pro- crosslinking of SF is followed by protein conformational changes duce stable and ordered materials with beneficial effect on their already shown by other authors b shows the spec- properties. To achieve such effect, genipin was used to crosslink trum for elastin protein that was acquired in powder form using SF/EL scaffolds. Different crosslinking conditions were tested KBr pellets. 100EL spectrum shows characteristic protein bands (). After 3 h of reaction a color change in the solutions is ob- at 1651 (amide I), 1537 (amide II) and 1239 cm1 (amide III), as- served from light yellow to light blue, indicating the reaction be- signed to random coil conformation . It can be seen that tween both SF and elastin with genipin. It is described that genipin induces in elastin structural changes into a more b-sheet genipin reacts with amino acids or proteins to form dark blue conformation. This was confirmed by the shifting to lower wave- pigments associated with the oxygen-radical polymerization of numbers of the amide I band. In addition, the appearance of a genipin . After 6 h of reaction, the solutions became dark new peak at 1104 cm1 and 1113 cm1, characteristic of genipin, blue and the maximum crosslinking degree was reached.
confirms the crosslinking reaction. The intensity of this peak is The exact mechanism behind the interaction of genipin with directly proportional to the amount of genipin used for the cross- both SF and elastin is yet to be fully described. The generally linking. The results obtained after methanol treatment of the

A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 Fig. 1. FTIR absorbance spectra (a) of pure silk fibroin (100SF) and (b) pure elastin(100EL) and crosslinked with genipin (100X0.1GE and 100X0.5GE, where X is SF orEL).
Fig. 2. DSC scans of (a) pure silk fibroin (100SF) and (b) pure elastin (100EL) andcrosslinked with genipin (100X0.1GE and 100X0.5GE, where X is SF or EL).
scaffolds (Data not shown) show no additional changes, for both The thermal behavior of 100SF is typical of an amorphous SF proteins, when compared with genipin crosslinked spectra.
with random coil conformation as previously shown by FTIR re- FTIR spectra of blend systems show slightly changes of the sults. Addition of genipin induces a small decrease in the Tg and wavenumbers and on the areas of the bands due to mixing effects an increase in the decomposition temperature. The increase in of SF with elastin. The areas under the peaks for pure and blend the thermal stability, given by the increase in Td, of 100SF scaffold systems were calculated by integration, and the ratio AN–H (area containing genipin is due to the increase in the extent of covalent of N–H bending, amide II) to AC@O (area of C@O stretching, amide crosslinks. This fact is the confirmation of the crosslinking reaction I) (data not shown). It was shown that the addition of elastin between genipin and SF. Furthermore, the exothermic peak at decrease the ratio of AN–H/AC@O. Moreover, the area of C–O–C, 226 °C shifts to lower temperature (100SF0.1GE) and disappears attributed to genipin, increases along with the ratio AN–H/AC@O for the sample 100SF0.5GE. This result shows once again the due to the carboxyl group from genipin. This fact is evidence of change in the SF conformation from random coil to b-sheet after the crosslinking reaction. The higher decrease in the ratio AN–H/ genipin crosslinking, and how this change is affected by the con- AC@O obtained for the blend systems is the combined effect of addi- centration of crosslinking agent.
tion of elastin and genipin crosslinking.
The DSC curve of elastin b) shows an endothermic shift at The interaction between SF and elastin, crosslinked with geni- 197 °C assigned to the glass transition temperature of soluble pin, was further investigated using thermal analysis (DSC). DSC elastin peptides The thermogram is further characterized by scans for SF and elastin are shown in and b respectively.
a weak and broad endothermic peak at 265 °C, related to the The DSC curve for 100SF shows an endothermic shift at 184 °C that decomposition of small aggregated structures and a more intense corresponds to the glass transition temperature (Tg) of SF. This endothermic peak at 320 °C related to a component decomposition value is in the range of others previously reported for SF with a at high temperature. Addition of genipin caused the decrease in the random coil conformation . The exothermic peak at 226 °C Tg and, although it was not observed, an increase in the decompo- is related to the crystallization of amorphous SF chains caused by sition temperature (320 °C); the weak peak at 265 °C progressively the transition to b-sheet structure The DSC curve of disappears with the addition of genipin. This fact indicates that the SF is also characterized by an intense endothermic peak at 284 °C small aggregates disappeared due to the crosslinking reaction be- (Td) related to the decomposition of SF chains.
tween genipin and elastin. In the blend system (data not shown)

A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 an increase in the decomposition temperature is observed, the interaction between genipin and SF with conformational suggesting once again the crosslinking effect. Nevertheless, the in- changes that are patent of the scaffold morphology already con- crease in Td is not dependent on blend composition because blends firmed by FTIR and DSC results. In the blended system, it can be with higher crosslinking degree will not have higher thermal seen that the loose network obtained upon addition of elastin becomes more closed and compact due to genipin crosslinking.
The 3-D morphology of the SF/EL scaffolds was analyzed by The fibrils observed in 50SF scaffold disappeared after crosslinking, SEM. The images presented are related to control and crosslinked originating thicker walls (f).
Porosity measurement of scaffolds was done by the liquid dis- (a) shows a disordered pore-like structure with a rough sur- placement method, using hexane as a displacement liquid. Hexane face. The pores are interconnected by a number of even smaller was used because it permeates easily through the interconnected pores. Addition of elastin creates a more open and loose structure scaffold pores, causing negligible swelling or shrinkage. Porosity with thinner walls (b and c). In the case of 50SF a fibr- determination is important in tissue engineering as a highly porous ilar structure can be observed. The presence of large pores in the structure provides much surface area that promotes better cell scaffolds facilitates cellular infiltration and growth within the growth through the easer passage of nutrients to the growing cells.
3-D structure However, such a loose network will have All the scaffolds, without genipin crosslinking, showed porosity detrimental effects on mechanical, swelling and release properties.
ranging between 100 and 70%, as shown in B. Addition of To overcome this, genipin crosslinking was performed and the elastin increases the porosity of the scaffolds as can be seen by results clearly evidenced that genipin changes the scaffold mor- SEM analysis A). Crosslinking with genipin significantly phology. 100SF0.5GE scaffold (shows a more ordered pore (p < 0.001) decreases the porosity of the SF/EL scaffolds, and the structure interconnected between sheets, characteristic of a porosity increases accordingly to the elastin content in the scaffold.
b-sheet conformation In addition, the SEM images ob- Water-binding of scaffolds is an important parameter of tained after methanol treatment (data not shown) show the same biomaterials properties. To study the swelling ratio in response morphology observed with genipin crosslinking. This result shows to external pH conditions, SF/EL scaffolds were immersed in PBS Fig. 3. (A) SEM images of SF/EL scaffolds without genipin: (a) 100SF, (b) 80SF and (c) 50SF; after genipin crosslinking: (d) 100SF0.5GE, (e) 80SF0.5GE and (f) 50SF0.5GE. (B)Porosity percentage of SF/EL scaffolds. Each column represents the average ± SD (n = 3) (significant differences between non-crosslinked and crosslinked samples at ⁄⁄p < 0.01and ⁄⁄⁄p < 0.001).

A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 buffer solutions at pH 3, 7.4 and 11 for 24 h at 37 °C and the results correlated with the scaffold compact structures formed after cross- are presented in . The swelling ratio of SF/EL scaffolds was lower in acidic conditions and became progressively higher at neu-tral and alkaline media. The lowest swelling ratio obtained at pH 3 3.2. In vitro and ex vivo biological degradation might be attributed to the formation of hydrogen bonds betweenSF and elastin due to the presence of carboxylic acid groups Degradation rate of matrices plays an essential role in the deter- (–COOH) and hydroxyl groups (–OH). Increasing the pH, the car- mination of the release of entrapped bioactive agents. The in vitro boxylic acid groups became ionized (–COO) and consequently, degradation of SF/EL scaffolds was investigated by incubation in a higher swelling ratios are observed due to a higher swelling force isotonic, physiological pH solution (PBS, pH 7.4) and a protease rich induced by the electrostatic repulsion between the ionized acid medium (PPE and human exudate from chronic wounds) at 37 °C for several days. At determined time points, samples were removed The swelling ratio was found to be dependent on the composi- and washed with distilled water, dried and weighed to determine tion; 50SF scaffolds, with and without genipin, showed maximums the extent of degradation using Eq. The results are presented in swelling ratios ). SEM analysis indicates that 50SF Scaffolds incubated with PBS solution showed almost no samples presented larger pores with a loose network, still observed degradation within 21 days. From the results it can be seen that after crosslinking, which results in a higher hydrodynamic free the degradation is dependent on scaffold composition. Higher volume to accommodate more of the solvent molecules, thus weight loss was obtained for samples containing higher amounts increasing scaffold swelling .
of elastin. After 21 days of incubation, the weight loss obtained Crosslinking with genipin also affects the swelling ratio of the for 100SF, 80SF and 50SF in PPE solution was 26, 36 and 49% scaffolds. Increasing genipin concentration leads to a decrease in respectively. The low weight loss obtained for 100SF is related to the swelling ratios. Generally, the swelling behavior of the scaf- the crystallinity of fibroin due to the presence of b-sheet struc- folds can be controlled by its composition and crosslinking degree.
tures. Therefore, the observed weight loss is probably due to the In the SF/EL scaffolds, genipin crosslinking created stable struc- degradation of the small hydrolytically peptide sequences that tures that hinder the mobility and relaxation of the macromolecu- remain after scaffold crystallization . Nevertheless, this effect lar chains, lowering the swelling ratio due to water restrict is minimized after genipin crosslinking that increases the b-sheet mobility . This effect is more pronounced in 80SF and 50SF content, creating a closed and compact scaffold network d).
scaffolds that attained higher crosslinking degrees when compared This will diminish the diffusion of solution within the scaffold, with 100SF. The decrease in the swelling ratio can also be increasing the resistance to protease degradation. The higher Fig. 4. The pH-dependent swelling ration of 100SF (a) 80SF, (b) and 50SF (c) scaffolds after 24 h of immersion in buffer solutions at 37 °C determined by Eq.

A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 Fig. 5. In vitro degradation of SF/EL scaffolds incubated with 0.1 mg ml1 of PPE andwound exudate (2.4 lg ml1 of total protein content) at 37 °C for 21 days.
Fig. 6. Cumulative release of gentamicin from SF/EL scaffolds incubated with0.1 mg ml1 of PPE at 37 °C for 21 days.
weight loss obtained with scaffolds containing elastin is becauseelastin is a substrate for elastase. In the human body, elastin, one of the major components of connective tissues, is degraded by Model compound release kinetic data obtained from fitting the experimental release human leukocyte elastase (HLE) . In this way, SF/EL scaf- data to Eq.
folds might be used as elastase-specific wound dressings for Kinetic parameters chronic wounds. Moreover, it has already been demonstrated that elastin-based dressings promote a better wound healing either byan improvement of fibroblasts adhesion and proliferation or by the reduction of wound contraction The loose network observed for scaffolds containing elastin (c) is also responsible for the higher weight loss obtained due to the increase in the surface area. As observed before, the gen- ipin crosslinking decreases the weight loss observed. The creationof a more compact structure between SF and elastin hinders scaf-folds degradation. These results show that genipin crosslinking turn cause the release of higher amounts of compounds. Genipin was effective in the control of degradation.
crosslinking induces slower release rates This is attributed The results obtained with wound exudate show the same deg- to the fact that genipin crosslinking enhances the decrease of pore radation pattern but with higher values. The exudate solution used size. In this way, the diffusion of the compounds through the scaffold pores is more difficult and lower release is attained.
(2.4 lg ml1 of total protein content). Nevertheless, the wound To determine the release mechanism present in the SF/EL scaf- exudate is a mixture of several proteases, including HLE, that act folds, the experimental data were fitted to the semi-empirical synergistically, increasing the hydrolysis.
power law model given by the Ritger–Peppas equation(Eq. ). This equation is further modified to determine the diffu- 3.3. In vitro release sional exponent, n (Eq. that depends on the release mechanismand the geometry of the matrix . There are three different The effect of scaffold composition and genipin crosslinking on mechanisms that can be concluded from the n value. Therefore, the release of model compounds was investigated. The release the release, from a cylindrical geometry like the sponges devel- behavior of gentamicin from SF/EL scaffolds in PPE solution is oped, is purely Fickian diffusion when n = 0.45; for 0.45 < n > 0.89 shown in The release of this compound was monitored in anomalous (non-Fickian) transport is present and, for n = 0.89 the PBS solution (data not shown) and the release observed was low.
release is dominated by Case II transport (matrix relaxation or Gentamicin has been used topically in the treatment of superficial infections of the skin since it is effective against many aerobic The results in , for control samples without crosslinking, Gram-negative and some aerobic Gram-positive bacteria. In this showed that the release of gentamicin is dominated by anomalous way, the antibacterial properties of SF/EL scaffolds will also be transport because n values are above and below 0.45. In the blends, exploited. The release profile shown can be divided into three increasing elastin content, the values for release rate, k, became parts: an initial burst release in the initial 24 h, due to the release progressively higher. This indicates that the addition of elastin im- of the compound bound to the surface of the scaffold; a continuous proves the release of drugs from the scaffolds probably due to the phase release from 24 to 72 h; and a stagnant phase release for the increase in swelling ratio and degradation rate, for higher elastin remaining period of time. Furthermore, it was observed that higher content as previously discussed. On the other hand, the decrease release was obtained for scaffolds containing higher amounts of in the values for diffusional exponent, n, closest to 0.45, suggests elastin. The release of a compound from a matrix is governed by that the addition of elastin also improves the diffusion of drugs several factors such as nature and size of the compound, degree from the scaffolds.
and density of crosslinking and pore size among others. From the Addition of genipin gradually changes the mechanism from SEM results discussed previously, it was concluded that higher anomalous transport to Fickian diffusion, especially for the sample elastin content leads to scaffolds with higher pore size which in 80SF0.5GE (n = 0.451). Furthermore, the crosslinking effect on the A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 Fig. 7. Viability of human normal skin fibroblasts after 24 h, 48 h and 72 h ofcontact with conditioned medium (culture medium where scaffolds were incu-bated). Only the positive control (treatment with Triton detergent) revealed Fig. 8. Number of human normal skin fibroblasts cells, determined in terms of DNA diminished cell viability. (⁄⁄⁄ = significantly different from all the other tested content, after 3 and 5 days of direct contact (significantly different from cells conditions, p < 0.001).
control after 3 days of incubation at ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001; signifi-cantly different from cells control after 5 days of incubation at #p < 0.05,##p < 0.01and ###p < 0.001).
scaffold morphology (compact and closed structure with smallerpores) also influences the release rate. It is observed that therelease rate becomes slower (lower k values) for higher amounts production of low levels of the inflammatory mediator TNFa by of elastin, due to the higher crosslinking degree obtained for these these cells after 48 h of incubation when compared to PMA samples as explained before. The release results clearly support the (phorbol 12-myristate-13 acetate), which is known to differentiate notion that the release from SF/EL scaffolds is affected by its com- THP-1 cells into macrophage-like cells and mimic the intrinsic acti- position and that genipin crosslinking can be used to modulate the vation and differentiation signals that macrophages encounter dur- release mechanism and rate of the compounds.
ing the foreign body reaction This additional observationfurther supports the notion that SF/EL scaffolds are non-immuno-genic and represent a safe alternative biomaterial for the treatment 3.4. Cytocompatibility SF/EL scaffolds Biocompatibility of SF/EL scaffolds, with and without genipin crosslinking, was assessed in human skin fibroblasts in in vitro cul- 3.5. Wound healing tures. The results of the indirect contact study after fibroblast incu-bation with material extracts showed no cytotoxicity caused by To determine the effect of SF/EL scaffolds on the wound healing, medium conditioned by the scaffolds regardless of the incubation materials were applied on the top of the wound immediately after time. represents the viability results for cells in contact with causing the burn. Histological evaluation of the healing pro- undiluted conditioned media. In all cases, the metabolic activity of cess after a period of 6 days revealed that SF/EL scaffolds induced cells in contact with the conditioned media was statistically similar fibroblasts and keratinocytes proliferation and migration to the or higher than the one obtained with negative control (complete wound site, especially for wounds treated with scaffolds contain- culture medium). This result constitutes a preliminary study of ing elastin (and b). The healing improvement obtained with the biocompatibility of SF/EL sponges, indicating that these mate- SF/EL scaffolds is similar to the commercial collagen dressing, rials are not cytotoxic.
Suprasorb C, used in several types of wounds including burn Direct contact study was performed by seeding the cells on the scaffolds to evaluate the effect of SF/EL scaffolds on fibroblasts pro- Microscopic observations of the wounds indicated that the con- liferation. The results presented in showed a time-dependent trol sample is characterized by the absence of epithelium increase in the number of cells that may suggest an increase in cell and the dermis is covered with crust from burning. After 6 days of proliferation. Human skin fibroblasts continued to increase in healing, the crust had disappeared from the control sample number over the period examined, indicating that the scaffolds (and from samples treated with different materials. In are able to support fibroblasts proliferation without producing addition, wounds treated with dressings (collagen and SF/EL scaf- toxic effects. It can also be observed that the presence of elastin folds) induced keratinocyte and fibroblast migration from the on the scaffolds favors cell proliferation. Especially after 5 days of margins to the wound ground, which should result in a faster re- incubation. This fact is explained by the increase of hydrophilicity epithelialization and wound closure. Partial-thickness burn introduced by the presence of elastin that enhances cell adhesion wounds heal almost entirely by epithelialization from the skin and subsequent activity. For scaffolds crosslinked with genipin a periphery to the wound core which was also observed in decrease in the number of cells is observed when compared to this study. For this reason, histological examination was done with non-crosslinked scaffolds, which might be related with the de- sections obtained in the center of the wound, so the results pre- crease in the porosity caused by the genipin crosslinking that sented and the differences obtained are related with the healing inhibits cell infiltration.
improvement by SF/EL scaffolds and not by natural artifacts. From Preliminary studies on the immunogenicity of SF/EL scaffolds the histological results obtained it is also visible that wounds trea- were performed in vitro by measuring TNFa production by ted with scaffolds containing higher amounts of elastin (50SF; THP-1 human macrophages exposed to these materials (data not are almost completely closed and covered with new epithe- shown). The results suggested that SF/EL scaffolds induce lium, which was not the case in controls. The results indicated that A. Vasconcelos et al. / Acta Biomaterialia 8 (2012) 3049–3060 Fig. 9. Histological analysis of burn wound tissues stained with H&E: (a) control wound (no dressing) immediately after burning; (b) control wound (no dressing) after 6 daysof healing; (c) wound treated with commercial collagen dressing, Suprasorb C, after 6 days of healing; (d) wound treated with 100SF scaffold after 6 days of healing; (e)wound treated with 80SF scaffold after 6 days of healing; (f) wound treated with 50SF scaffold after 6 days of healing. Bars = 100 lm.
wound size reduction was significantly greater in the order of containing higher amount of elastin accelerates re-epithelializa- 50SF > 80SF > 100SF = Suprasorb C > No dressing.
tion and wound closure. The results presented are important in The characterization results presented earlier indicated that the design and application of tailor-made biomaterials for wound scaffolds containing higher amounts of elastin become more swel- lable, flexible and elastic. These characteristics suggest that theattachment of the cells within the wound to the dressing is im- proved, resulting in a faster re-epithelialization.
Elastin is the major constituent of skin elastic fibers and is ben- We would like to acknowledge FCT – Portuguese Foundation for eficial for dermal regeneration Several studies have explored Science and Technology for the scholarship conceded to Andreia the application of elastin containing materials for wound healing, Vasconcelos; European FP6 project Lidwine, contract no. NMP2- such as scaffolds of collagen and solubilized elastin or dermal CT-2006-026741 and PEst-C/BIA/UI4050/2011.
substitutes coated with elastin Silk fibroin based-biomate-rials have also been used in this field with promising results; nevertheless, the present study exploits for the first time Appendix A. Figures with essential colour discrimination the combination of silk fibroin and elastin for the production ofwound dressing scaffolds.
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4.4 Welche Krankheitsstadien gibt es? Stadium 1: Die Krankheit entwickelt sich aus einem normalen Leistungsniveau. Stadium 2: In der Folge nimmt die/der Betroffene leichte Störungen wahr. Die Merkfähigkeit und das Gedächtnis sind beeinträchtigt. Namen und Termine werden vergessen. Bei manchen Situationen fehlt die Erinnerung und öfters werden Dinge verlegt.

Univ. Sci. 2014, Vol. 19 (1): 11-29 Freely available on line Técnicas analíticas contemporáneas para la identificación de residuos de sulfonamidas, quinolonas y cloranfenicol Y. Verónica Talero-Pérez scar Julio Medina 1, Wilson Rozo-Núñez2 Contemporary analytical techniques to identify residues of sulfonamides,