CHITOSAN BIOMIMETIC SCAFFOLDS AND METHODS FOR PREPARING THE SAME

The present invention relates to a layered chitosan scaffold wherein said layered scaffold comprises at least two fused layers, wherein at least one of the fused layers comprises a chitosan nanofiber membrane and the other fused layer comprises a porous chitosan support layer. Moreover, the present invention provides a layered chitosan scaffold characterized by (i) a good adhesion between the porous and nanofiber layers, (ii) a tuneable porosity of the nanofiber layer by tuning the distance between the nanofibers, (iii) a stable nanofibers and porous morphology even when immersed in water or other solvents and a process for the preparation of such layered chitosan scaffold. The present invention also provides a process for the preparation of the layered chitosan scaffold.

The following non-limiting examples illustrate the invention:

Electrospinning of Chitosan Solution

Medical grade chitosan with acetylation degree 19.6%, viscosity (1% solution in 1% acetic acid) 21 mPa·s, Mw 77 kDa (L08049), from Kitozyme, Belgium and PEO high molecular weight (HMW, 900000 g/mol) purchased from Aldrich were used as received. Acetic acid, ethanol and sodium hydroxide (Sigma) were analytical grade and used as received.

Solutions of 10.5% chitosan, 4% HMW PEO were prepared by dissolving the appropriate amount of chitosan (in 6.5% acetic acid solution) and PEO (in distilled water), by stirring overnight. The next day, the chitosan and PEO solutions were then mixed to obtain mixtures with weight ratios of chitosan:PEO of 90:10. Two ml of the well homogenized resulting mixture were fed into 5 ml plastic syringes fitted with blunt tipped stainless steel needles (gauge 18 and 21). The solution feed was driven using a syringe pump (Razel Scientific Instruments) and an electrospinning voltage ranging from 15 to 30 kV was applied between the needle and the collector (aluminium foil) by the use of a Spellman SL10 power supply. The positive electrode of a high voltage power supply was connected to a metal capillary by copper wires. The distance needle tip-collector was 15 cm, and the flow rate of the solution was 0.75 ml/h. All electrospinning experiments were performed at room temperature. The nano fibrous nonwoven mats were collected on the surface of aluminum foil.

For improving the water stability of the electrospun material, the as-spun membranes were treated first with ethanol and then with NaOH (1M) solutions. Subsequently, the membranes were extensively rinsed with distilled water and dried under the vacuum.

To analyze the composition of electrospun and stabilized nanofiber mat, infra-red spectra were recorded with a Perkin-Elmer FT-IR 1720× and differential scanning calorimetry (DSC) was carried out with a TA DSC Q100 thermal analyzer calibrated with indium. The electrospun nanofibers were sputter-coated with Pt and their morphology examined with a scanning electron microscope (Jeol JSM 840A).

The as-spun chitosan membranes prepared with the electrospinning method mentioned above are highly positively charged, thus they dissolve in acids and exhibit a low stability in neutral or weak alkaline aqueous media. As far as these membranes are foreseen in tissue engineering, their stability in cell culture and physiological media is required, This has been achieved in the present approach, by reducing the content of PEO necessary for electrospinning below 15%, by neutralizing the chitosan nanofibers after electrospinning and by removing of the PEO by repetitive washings steps. This stabilization process, i.e. without the use of chemical cross-linkers or chlorine-containing organic solvents. results in the long-term stability of the chitosan nanofibers in water and phosphate buffers. Stabilized membranes can be stored in distilled water for months without any apparent change of the morphology. (FIG. 2)

Engineering of the Dressing

As reported above, bandages and dressings for treating wounds have to satisfy various requirements. This explains why the conventional fibrous matrix scaffold obtained so far by electrospinning of chitosan is not used today. Some of these disadvantages are that:a) a fibrous scaffold composed of chitosan nano fibers has poor mechanical properties that prevent easy handling and cause rapid destruction in situ when placed on wounds.b) nano fibrous scaffolds having only 2-dimensional structure are limited in applications since they do not provide protection against outside mechanical influences.c) thin nanofibrous scaffolds are not appropriate to maintain the moisture equilibrium promoting the wound healing process.d) The pores in scaffold made of cross-linked nanofibers are too small to permit cell invasion, which, in certain circumstances, is a disadvantage.

The present invention overcomes all of these problems by providing a wound dressing consisting in multiple sheets or layers. The designation of a “first layer”, “second layer”, and the like, is meant to describe the location of a material relative to the wound bed. For example, the material located adjacent to the wound bed and in contact with it is termed the “first layer”. The material that is placed on top of the first layer (proceeding in a direction away from the wound bed) is termed the “second layer”, and so on. A layer may comprise one material, or two or more materials. In our prototype of dressing, the first layer is made of a non woven chitosan nanofibers mats obtained by electropinning (first layer) on an absorbent neutralized chitosan sponge (Kitozyme) or sponge from Hemcon (ChitoFlex™) being the second layer (FIG. 3). The chitosan sponge used in this invention is porous, and has interconnecting pores having a pore size in the range of about 50-400 microns. When the nano fibers come to the surface of the sponge, they still contain acetic acid that dissolves the surface of the sponge.

Although this process is very limited, it allows adhesion of nano fibers mat on the sponge without using any synthetic adhesive. Interactions between these two layers are strong enough for easy handling of the entire dressing but still loose enough for easy separation of the two layers if required for clinical applications (FIG. 4).

Our dressing retains the flexibility required for optimal adhesion to the wound, although possessing adequate mechanical properties for easy handling. This dressing has also the capacity to absorb large amount of aqueous liquid, for instance exudates in the specific case of wounds.

The electrospinning process is capable of generating fibrous scaffolds from both natural and synthetic polymers. The limitation of scaffolds fabricated using the electrospinning process is the high fiber density and the resultant “fish net effect” (FIG. 2). In other words, fiber density in electrospun mats is often too high to allow cell infiltration. The mean pore radius of electrospun matrices varies with fiber diameter. For example, a 100-nm fiber diameter yields a mean pore radius less than 10 nm at a relative density of 80%. The comparative size of a rounded cell—ranging from 5 to 20 μm—predicts that such small pore sizes would prevent cell infiltration or invasion. A number of methodologies for regulating pore size have been proposed. Recently, cells have been electrosprayed into forming scaffolds. However, issues of layering, sterility, and time to produce thick scaffolds are expected to limit application. A more efficient method for improving cell invasion may be by increasing porosity. Experiments with porous foams and sponges suggested that it exists an optimal pore size for cell infiltration. For fibrous scaffolds, this approach has been addressed by mixing fibers of different diameter. Alternatively, pores have been produced by including sacrificial fibers in a composite scaffold containing heterogeneous fibers that display different solubility in a given solvent: the selective removal of sacrificial fibers increasing porosity and accelerating cell infiltration.

The present invention demonstrates that freeze-drying is a rapid and easy way to provide electrospun membranes containing pores of desired size. While this process has been used to produce porous materials (foam, porous ceramics and the like), it has never been used for the creation of pores in as-spun membranes. After stabilisation treatment, dressing (nanofibers and foam) was washed and frozen at −20° C. during 12 h. During the freezing process growing ice crystals push back and concentrate the fibers into the inter-crystals space. After complete solidification, the frozen dressing is lyophilised (to sublimation of the ice under vacuum), resulting in a fibers mat with pores of the size and shape of the initial ice crystals (FIG. 5). In this condition (−20° C. during 12 h) the pore size obtained in a fibers mat is, ˜50% of 1 to 2 μm pore diameters, ˜30% of 5 to 7 μm pore diameters and ˜10% of 10 to 15 pore diameters. (FIG. 5) However, depending on the freezing conditions the size of the ice crystals can be modulated, regulating accordingly the size of the resulting pores in the nanofiber layer of the mat. Previously, there have been many studies that have investigated the structure of ice crystals formed in various kinds of materials and foods. Thus, it is well-known that rapid freezing rather than slow freezing gives smaller size ice crystals in frozen samples. Also, it has been reported that ice crystals grow in size during storage by recrystallization, depending on the storage time and temperature. This treatment will improve the rapid colonization of the dressing (when desired) and increase its permeability to air, oxygen, water vapour and liquid water.

This structure of dressing (nanofibers with pores onto a sponge) affords rapid absorption of exudates and thereby draws bacteria away from the wound, helping to protect against wound sepsis, as will be discussed below. Dressings of the present invention can provide water absorptive capabilities as high as about 24 grams of water per gram of dressing. It is by the nature of fluidic flow that exudates tend to fill the available volume before progression onto higher levels of the layers. As exudates move into the more voluminous pores of the second layer, the gating or inhibition to flow is reduced and exudates may flow into this new region more rapidly. The effect of this is to provide a cone-like spread to the exudates passage, that is to say exudates are spread over a greater area of the wound dressing upon its passage through the graded density layer 1. The exudate that passes through the first layer is rapidly absorbed by the absorbent layer 2. However, the wound site is still kept moist by the effect of the graduated density of layers. As the exudates are spread by this layer 1, even when the absorbent layer directly above the wound site becomes saturated, the exudates may still pass through and be absorbed at more inclined areas of the layer 2.

Co-Electrospinning of Chitosan with Proteins and Chemical Molecules Having the Ability to Maintain and to Preserve the Biological Activity of Proteins

The β-lactamase BlaP ofBacillus licheniformiswere used as protein models in co-electrospinning experiments. This protein corresponds to a bacterial enzyme with a globular 3D-structure. The recombinant protein was overproduced overproduced inE. coliand purified to homogeneity by using affinity chromatography methods.

For co-electrospinning experiments, the BlaP protein was added in the chitosan/PEO mixture (see Example 1) at a final concentration of 3 mg/ml and electrospun using the same procedure described in Example 1.2.1. In some cases, the mixture was also supplemented with a derivative of β-cyclodextrin.

3.2.2 Detection of Proteins in Co-Electrospun Chitosan Membranes

For the β-lactamase assays performed with BlaP, the co-electrospun polymers were incubated during 4 minutes with 1 ml of 100 μM nitrocefin (a chromogenic β-lactam substrate). Then the absorbance of the hydrolyzed product was measured at 482 nm. This absorbance value is thus directly correlated to the active β-lactamase immobilized on the polymer.

3.3 Results and Discussion:

Chitosan:PEO mixtures were co-electrospun with the class A β-lactamase BlaP, with a derivative of β-cyclodextrin or with these two products together, for investigating the ability of the protein to maintain its post-electrospinning activity, as well as the capability of cyclodextrin to stabilise the protein in the due conditions. The conditions for electrospinning were: 30 kV voltage, 15 cm distance between electrodes, 0.762 ml/h solution flow rate, 30 min electrospinning time. Adding cyclodextrin alone to the chitosan:PEO mixture increased the incidence of spindle-shaped fibers, while adding protein contributed to the production of smooth, bead-free fibers, irrespective of the presence or absence of cyclodextrin in the electrospinning mixture. In addition, we also attempted to measure the β-lactamase activity immobilized on the co-electrospun chitosan polymers obtained in the absence and in the presence of cyclodextrin. The data are presented inFIG. 6. The rate of nitrocefin hydrolysis (ΔA/4 min/mg of polymer) obtained in the presence and in the absence of cyclodextrin were 0.939±0.125 and 0.577±0.111, respectively. These ones indicate that the addition of β-cyclodextrin increases significantly (+39%) the β-lactamase activity recovered in the co-electrospun product. In both cases, we also monitored the immobilized activity during storage of the polymers at room temperature after 6 weeks. No decrease of the immobilized β-lactamase activity was observed neither in absence nor in presence of cyclodextrin.

Conversion of Chitosan Nanofibers into Chitin with Preservation of the Nanofibers Morphology

A medical grade chitosan of fungi origin (kiOmedine-CsUP™, Kitozyme, Belgium) was used. Its degree of acetylation (DA) and molecular mass (MM) were 19% and 68000, respectively. All chemical and biochemical reagents were of analytical grade.

Thirty mg of chitosan were solubilized in 2.5 ml of 0.1 M Sodium acetate (pH3). After an overnight incubation under stirring condition at room temperature, the chitosan solution was diluted 10× with 0.1M phosphate (pH7.4). Next, 150 μl of this chitosan solution were added in each well of an Immobilizer Tm-Amino modules/plates (Exiqon A/S) for 2 h at room temperature. Covalent bonds between the activated groups of the well and the amine groups of chitosan are established. The plates are next washed 3 times with 20 mM Tris (pH8) by using a plate washer Biotrak II (Amersham Biosciences). To block the residual activated groups that have not reacted with chitosan or Tris, two washes of 20 min each are performed with 250 μl/well of 20 mM Tris (pH8).

For chitosan binding assays, the wells were filled with 200 μl of PBS containing 3% BSA and incubated overnight at 4° C. The plates were next washed 3 times with PBS (50 mM phosphate, 150 mM NaCl, pH7.4) by using a plate washer Biotrak II.

4.3 Chemical Conversion of Immobilized Chitosan on Immobilizer™-Amino Modules/Plates

Thirty mg of chitosan were solubilized in 2.5 ml of 0.1 M Sodium acetate (pH3). After an overnight incubation under stirring condition at room temperature, the chitosan solution was diluted 10× with 0.1M phosphate (pH7.4). Next, 150 μl of the new chitosan solution were added in each well of Immobilizer Tm-Amino modules/plates (Exiqon A/S) for 2 h at room temperature. Covalent bonds between the activated groups of the well and the amine groups of chitosan are established. Then, 99% pure acetate anhydride (10.4 M) was diluted in different organic solvents and 60 μl/well of these preparations were added to the wells filled with the chitosan solution. All the acetate anhydride preparations were tested individually for the reacetylation of chitosan. The reacetylation of chitosan was conducted for 1 h at room temperature under stirring condition. The plates were next washed 3 times with PBS (50 mM phosphate, 150 mM NaCl, pH7.4) by using a plate washer Biotrak II.

For chitin binding assays, the wells were filled with 200 μl of PBS additionned with 3% BSA and incubated overnight at 4° C. The plates were next washed 3 times with PBS (50 mM phosphate, 150 mM NaCl, pH7.4) by using a plate washer Biotrak II.

The gene coding for the chitin-binding domain ofBacillus circulansWL-12 chitinase A1 (ChBDA1) was PCR amplified using Pfu polymerase with primers CHBDA1+ and CHBDA1-(5′-GGAACGACAAATCCTGGTGTATCCGCTTGGCAGGTC-3′ (SEQ ID NO:1) and (5′-TCCTTGAAGCTGCCACAATGCTGGAACGTTGGATGG-3′ (SEQ ID NO:2)). The PCR products were successively purified on a GFX™ gel band purification kit (Amersham Biosciences, UK), phosphorylated using T4 polynucleotide kinase and purified again with GFX™ gel band purification kit before to be cloned into the SmaI-digested and dephosphorylated pNY-ESBlap. By using this vector, a full constitutive expression of the β-lactamase BlaP is obtained inE. coli. The resulting genetic construct was called pNYBlapChBDA1. In this construction, the β-lactamase BlaP sequence is preceded by a signal peptide for periplasmic secretion and followed by a (his)6-tag on the C-terminal end to improve its purification by affinity chromatography. The nucleotidic sequence encoding ChBDA1 is cloned into the nucleotidic sequence of BlaP. The chitin binding domain is expressed in a loop of the β-lactamase BlaP which is solvent accessible and diametrically opposed to the active site of the enzyme to avoid steric hindrance (FIG. 7). This technology refers to patent EP1713907 (Hybrid protein of active-site serine β-lactamase).

To achieve production of the hybrid β-lactamase B1apChBDA1,E. coliJM109 transformed respectively with pNYBlapChBDA1, were grown in Terrific Broth supplemented with 75 μg/ml spectinomycin and 10 μg/ml ampicillin at 37° C. Cells from an overnight culture (1 L) were harvested by centrifugation (9000 g for 15 min) and resuspended in 40 ml of TES (20% sucrose, 30 mM Tris-HCl, 5 mM EDTA, pH 8) at 37° C. The bacterial suspension was placed under stirring at 37° C. for 10 min. Cells were harvested by centrifugation (9000 g for 15 min) and the pellet resuspended in 100 ml of 5 mM MgSO4at 4° C. The bacterial suspension was stirred at 4° C. for 10 min. The supernatant containing the periplasmic proteins was harvested by centrifugation (13000 g for 20 min) and diluted with three volumes of 50 mM phosphate (pH 7.4). The periplasmic proteins were loaded on a HisTrap™ Chelating HP column (GE Healthcare) equilibrated in 50 mM phosphate (pH 7.4). The column was successively washed with 2M NaCl and 50 mM phosphate (pH 7.4) supplemented with 10 mM imidazole. The hybrid proteins were eluted by an imidazole linear gradient (10 to 500 mM) in 50 mM phosphate (pH 7.4). Fractions containing the purified hybrid proteins were pooled and dialyzed against PBS (50 mM phosphate, 150 mM NaCl, pH 7.4).

The gene coding for the chitin-binding domain of the human macrophage chitotriosidase (ChBDhmc) was constructed by overlapping PCR and cloned into the pGEM-T-easy vector (Promega) for sequencing. The oligonucleotides used in the overlapping PCR are presented in Table 1 (ChitO1 to ChitO8).

Next, the gene coding for ChBDhmc was PCR amplified using Pfu polymerase with primers CHIT1+ and CHIT1−. The PCR products were successively purified on a GFX™ gel band purification kit (Amersham Biosciences, UK), phosphorylated using T4 polynucleotide kinase and purified again with GFX™ gel band purification kit before to be cloned into the SmaI-digested and dephosphorylated pNY-ESBlap. The resulting genetic construct was called pNYBlapChBDhmc.

To achieve production of the hybrid β-lactamase BlapChBDhmc,E. coliJM109 transformed respectively with pNYBlapChBDhmc were grown in Terrific Broth supplemented with 75 μg/ml spectinomycin and 10 μg/ml ampicillin at 37° C. Cells from an overnight culture (1 L) were harvested by centrifugation (9000 g for 15 min) and resuspended in 40 ml of TES (20% sucrose, 30 mM Tris-HCl, 5 mM EDTA, pH 8) at 37° C. The bacterial suspension was placed under stirring at 37° C. for 10 min. Cells were harvested by centrifugation (9000 g for 15 min) and the pellet resuspended in 100 ml of 5 mM MgSO4at 4° C. The bacterial suspension was stirred at 4° C. for 10 min. The supernatant containing the periplasmic proteins was harvested by centrifugation (13000 g for 20 min) and diluted with three volumes of 50 mM phosphate (pH 7.4). The periplasmic proteins were loaded on a HisTrap™ Chelating HP column (GE Healthcare) equilibrated in 50 mM phosphate (pH 7.4). The column was successively washed with 2M NaCl and 50 mM phosphate (pH 7.4) supplemented with 10 mM imidazole. The hybrid proteins were eluted by an imidazole linear gradient (10 to 500 mM) in 50 mM phosphate (pH 7.4). Fractions containing the purified hybrid proteins were pooled and dialyzed against PBS (50 mM phosphate, 150 mM NaCl, pH 7.4).

4.6 Chitosan and Chitin Binding Assays on Immobilizer™-Amino Modules/Plates

To perform the chitosan and chitin binding assays, we used the plates described in points 2 and 3 of this Example. The binding assays were performed by using the hybrid β-lactamase BlaP ChBDA1 and BlaP ChBDhmc. To attest that the interaction of these hybrid proteins with chitosan is specific, we used the hybrid β-lactamase BlaP Actev as a negative control. The engineered loop of BlaP Actev does not contain chitin-binding domain but two Actev-cleavage sites surrounding the insertion site.

One hundred μl of purified β-lactamase diluted in PBS (5 μg/ml) were added to the coated wells and the plates were incubated for 2 h at room temperature. Next, the plates were washed 3 times with PBS. The direct measurement of the immobilized β-lactamase activity in each well was done by following the hydrolysis of 150 μl of nitrocefin (100 μM) in 50 mM phosphate buffer (pH 7.5) at 482 nm.

Membranes formed by electrospun chitosan nanofibers were prepared as described in Example 1. To improve the water stability of the electrospun material, the as-spun membranes were first soaked successively in pure ethanol and later with 1M NaOH. Next, the membranes were extensively rinsed with distilled water.

4.8 Chemical Conversion of Electrospun Chitosan Nanofibers into Chitin

The electrospun nanofibrous membranes of chitosan were cut into square of 1 cm2for chemical conversion assays. The samples were placed into closed tubes containing the differents acetate anhydride solutions presented in table 2. The polymers were incubated 1 h at room temperature. Next, the polymer were extensively washed in water bath and dried into a dessicator before processing for scanning electron microscopy. In this work, we avoided air drying the polymers because this process caused a dramatic loss of the fibrillary structure of the polymer.

4.9 Scanning Electron Microscopy

The morphology of the electrospun and re-acetylated chitosan nanofibers were sputter-coated with Pd and examined with a scanning electron microscop (Jeol JSM 840A).

4.10 Expression and Purification of the Human Lysozyme

Recombinant human lysozyme was generated using the pPIC9KPichia pastorisexpression vector (Invitrogen). Briefly, the gene encoding the lysozyme (P61626) was subcloned from the pGA HMlyso plasmid into pPIC9K vector (Invitrogen) by using the SnaBI and NotI restriction sites. The pPIC9K plasmid contains the α-factor secretion signal that directs the recombinant protein into the secretory pathway. The constructs were digested with Sal I and used to transformPichia pastorisstrain SMD168 by electroporation. This resulted in insertion of the construct at the AOX1 locus ofPichia pastoris, generating a His+Mut+phenotype. Transformants were selected for the His+phenotype on 2% agar containing regeneration dextrose biotin (1 M sorbitol, 2% dextrose, 1.34% yeast nitrogen base, 4×10−5percent biotin, and 0.005% ofL-glutamic acid,L-methionine,L-lysine,L-leucine, andL-isoleucine) medium and then further selected for high copy number by their ability to grow on 2% agar containing 1% yeast extract, 2% peptone, 2% dextrose medium, and the antibiotic G418 at various concentrations (0.5-4 mg/ml) (Invitrogen). The protein was expressed in a shaker flask and harvested at 72 h after induction by methanol.

The protein was purified by using first a cation-exchange chromatography as follows: the supernatant was dialysed against buffer A (citrate 25 mM, pH5) and applied onto a SP-Sepharose column equilibrated with buffer A. The column was washed with the same buffer. Elution was performed by an increasing linear (0-1M) NaCl gradient in buffer A with ten column volumes. The elution fractions were analysed on SDS-PAGE and the fractions containing the protein of interest were pooled and dialysed against buffer A. The protein was next applied onto a Puros 20HS column equilibrated with buffer A. The column was washed with buffer A. Elution was performed by an increasing linear (0-1M) NaCl gradient in buffer A with ten column volumes. The elution fractions were analysed on SDS-PAGE. The fractions containing the protein of interest were pooled, dialysed against water and lyophilised.

4.11 Expression and Purification of the Human Macrophage Chitotriosidase

Recombinant human chitotriosidase was generated using the pPIC9KPichia pastorisexpression vector (Invitrogen). The gene encoding the chitotriosidase (GenBank, gi:4502808) followed by a His6-tag on the C-terminus end was subcloned from the pGA HMchito plasmid into pPIC9K vector (Invitrogen) by using the SnaBI and NotI restriction sites. The pPIC9K plasmid contains the α-factor secretion signal that directs the recombinant protein into the secretory pathway. The constructs were digested with Sal I and used to transformPichia pastorisstrain SMD168 by electroporation. This resulted in insertion of the construct at the AOX1 locus ofPichia pastoris, generating a His+Mut+phenotype. Transformants were selected for the His+phenotype on 2% agar containing regeneration dextrose biotin (1 M sorbitol, 2% dextrose, 1.34% yeast nitrogen base, 4×10−5percent biotin, and 0.005% of L-glutamic acid,L-methionine,L-lysine,L-leucine, andL-isoleucine) medium and then further selected for high copy number by their ability to grow on 2% agar containing 1% yeast extract, 2% peptone, 2% dextrose medium, and the antibiotic G418 at various concentrations (0.5-4 mg/ml) (Invitrogen). The protein was expressed in a shaker flask and harvested at 72 h after induction by methanol.

The protein was purified by using nickel-nitrilotriacetic acid-agarose (Ni-NTA; Qiagen) as follow: the supernatant was applied onto a Ni-NTA-Sepharose column ((Novagen, USA, 1×10 cm) equilibrated with buffer A. The column was washed with seven column volumes of the same buffer, three column volumes of buffer A+2 M NaCl and three column volumes of buffer A+10 mM imidazole. Elution was performed by an increasing linear (10-500 mM) imidazole gradient in buffer A. Active fractions eluted at 100 mM imidazole and appeared as a single band upon SDS-PAGE analysis.

4.12 In Vitro Degradation of Electrospun Chitosan Nanofibers

The nanofibrous membranes of chitosan and re-acetylated chitosan were cut into square of 1 cm2for in vitro degradation testing. The samples were placed into closed tubes containing human lysozyme, human macrophage chitotriosidase or a mixture of human lysozyme/human macrophage chitotriosidase. The human lysozyme and the human macrophage chitotriosidase were used at a final concentration of 3.5 and 0.35 μM in phosphate buffer saline (pH 7.4), respectively. The degradation assays were monitored during 6 weeks. Every three days, the enzymatic solution was replaced with a fresh solution. At specified time intervals, the electrospun membranes were taken out from the solution, washed with distilled water, dried, and weighted. Electrospun membranes were also incubated in phosphate buffer without enzyme to determine exactly the enzyme-catalysed degradation. The degree of in vitro degradation was expressed as the percentage of the dried sample weight before and after degradation.

The goal of this study consisted to convert electrospun chitosan nanofibers to chitin nanofibers according to a process which permits:To maintain both the fibrillar structure of the nanofibers and the morphology of the biopolymerTo restore the sensitivity to human glycosylhydrolasesTo control the reacetylation rateTo combine chemical, biophysical and biological properties of chitosan and chitin in the same biopolymer

The chemical conversion of chitosan consisted to substitute the C2 amine (—NH2) group by an acetamide group (—NHCOCH3) with acetate anhydride (FIG. 8).

First, we started to study the chemical conversion of chitosan with chitosan covalently immobilized on Immobilizer™-Amino modules/plates. Different acetate anhydride solutions were prepared and tested for the chemical conversion of chitosan (table 2).

TABLE 2Acetate anhydride solutions tested for the reacetylation of chitosan.Acanhydride/OrganicsolventEtha-Meth-Ace-DimethylTetra-(Vol/Vol)nolanoltoneFormamideHeptanehydrofuraneAcetate1/21/21/21/21/21/2anhydride1/41/41/41/41/41/41/81/81/81/81/81/81/161/161/161/161/161/161/321/321/321/321/321/321/641/641/641/641/641/641/1281/1281/1281/1281/1281/1281/2561/2561/2561/2561/2561/256

To check the efficiency of the chemical conversion, chitin-binding assays were performed by using two hybrid β-lactamases harbouring the chitin-binding domains ChBDA1 or ChBDhmc. ChBDA1 and ChBDhmc were isolated from theBacillus circulansWL-12 chitinase A1 and the human macrophage chitotriosidase, respectively. The hybrid β-lactamase BlaP Actev was used as a negative control to attest that the binding to chitin was only due to the presence of the chitin-binding domain into BlaP. The results are presented in table 3 and 4.

TABLE 3Chitin binding assays performed with BlaP ChBDhmc and BlaP Actev. Chitosan was first covalently immobilized on ImmobilizerTm-Amino modules/plates and then treated with various concentrations of acetate anhydride in different solvent. The immobilizedβ-lactamase was monitored by following the hydrolysis of nitrocefin at 482 nm.

TABLE 4Chitin binding assays performed with BlaP ChBDhmc, BlaP ChBDA1 andBlaP Actev. Chitosan was first covalently immobilized on Immobilizer Tm-Amino modules/plates and then treated with various concentrations ofacetate anhydride in different solvent. The immobilized β-lactamase wasmonitored by following the hydrolysis of nitrocefin at 482 nm.All the experiments were performed in triplicate.

In regard to table 3, we demonstrated that the hybrid protein BlaP ChBDhmc detected the presence of chitin when the immobilized chitosan was treated with anhydride acetate diluted in ethanol, methanol, acetone, dimethylformamide or heptane whatever the dilution tested in a range from ½ to 1/256. No specific binding was detected with BlaP ChBDhmc when chitosan was treated with acetate anhydride diluted in tetrahydrofuranne. Globally, the most elevated chitin-binding activities were observed when acetate anhydride was diluted with ethanol and methanol in a large range of dilutions.

In the continuity of this work, we decided to focus on the chemical conversion of chitosan to chitin by using acetate anhydride diluted in ethanol and methanol. The results presented in table 4 were done in triplicate and confirmed the efficiency of acetate anhydride diluted in methanol and ethanol to convert chitosan to chitin.

The next step of this work consisted to check if the treatments described above permit also to convert electrospun chitosan nanofibers to chitin nanofibers without disturbing the fibrillary structure of the nanofibers and the morphology of the biopolymer. The electrospun and re-acetylated chitosan nanofibers were sputter-coated with Pt and examined with a scanning electron microscop. The results are presented inFIGS. 9 and 10. In regard to these figures, we observed that the fibrillary structure of the electrospun re-acetylated nanofibers and the global morphology of the biopolymer were preserved only in some conditions. These conditions were limited to the use of methanol as organic solvent in a confined range of acetate anhydride dilutions from 1/16 to 1/64. All the conditions tested in the presence of ethanol resulted on the complete dislocation of the nanofibers, a drastic decrease of the porosity and the formation of a film.

We also checked if the hybrid β-lactamase BlaP ChBDA1 was also able to bind electrospun chitosan nanofibers when these ones are reacetylated with acetate anhydride diluted in methanol. The graph ofFIG. 11attests that the chemical conversion in the presence of methanol results on the formation of chitin nanofibers because a specific binding of BlaP ChBDA1 is only observed when the chitosan nanofibers are treated. No binding was observed with BlaP Actev confirming that the binding of BlaP ChBDA1 was specific and related to the presence of chitin.

Finally, we compared the biodegradation of electrospun chitosan nanofibers before and after treatment with acetate anhydride diluted in methanol ( 1/32). The weight of the biopolymer was evaluated over a period of 6 weeks. The biopolymers were incubated in the presence of human lysozyme, human macrophage chitotriosidase and a mix of the two enzymes. The protein solutions were changed every two days. The histogram ofFIG. 12Ashows the weak efficiency of human glycosylhydrolases to degrade alone or in combination the electrospun chitosan nanofibers. InFIG. 12B, the histogram indicates that the treatment with acetate anhydride diluted in methanol ( 1/32) increases drastically the susceptibility of the nanofibers to the hydrolytic activities of human lysozyme and human macrophage chitotriosidase.

Characterization of Normal Human Keratinocytes Cultured on Electrospun Chitosan Membranes

Human keratinocytes were isolated by trypsin float technique [11] from normal adult skin samples. In order to study a representative cell population, the keratinocytes from three different donors were mixed in a keratinocyte pool. The pool was stored frozen in liquid nitrogen. The cell cultures were plated at density 30000 cells/cm2and incubated in KGM-2 keratinocyte medium at 37° C. and 5% CO2in a humidified incubator. The medium was renewed every two days.

Scanning electron microscopy (SEM) was used to analyze the adhesion, spreading and morphology, of keratinocytes cultured on electrospun chitosan membranes. The membranes were cut out with a punch (20 mm in diameter) and put into 24-well culture plates, sterilized with 70% ethanol, air-dried in sterile culture hood and equilibrated with culture medium. Keratinocytes were seeded on electrospun chitosan membranes at a high cell density (30 000 cells/cm2) in KGM-2 medium. The cell cultures grown over electrospun chitosan membranes for various periods of time were fixed with 2.5% glutaraldehyde for 20 minutes at room temperature, and then washed for 5 minutes with 0.1 M cacodylate buffer pH 7.4. The membranes were then dehydrated with rising concentrations of ethanol (25%, 50%, 75%, 95% and 100%), and then were subjected to critical point drying. The membranes were sticked to a support for scanning electron microscopy and then covered with a thin gold layer. The samples were examined and photographed in a Phillips XL20 scanning electron microscope.

Histological Analysis and Hematoxilin Staining:

For this analysis, keratinocytes cultured over electrospun chitosan membranes were fixed with 4% paraformaldehyde for 10 minutes, then subjected to dehydration in methanol and toluol, and then embedded vertically into paraffin. Then 6 μm-thick paraffin sections were sliced and mounted onto glass slides. After removal of paraffin, sections were used for histological staining with hematoxilin.

To analyse the phenotype of keratinocytes cultured on electrospun chitosan membranes, we have extracted total RNA after 7, 14 and 21 days in culture with the standard phenol-chloroform procedure for RNA extraction, using the TRI Reagent® (Molecular Research Center, Inc., USA) and chloroform (Merck, Germany). After reverse transcription of RNA with Super Script II RNase H-Reverse transcriptase kit (Invitrogen, Belgium), we have performed real-time quantitative PCR for the analysis of the expression of specific genes (keratins 14 and 10, involucrin). The cDNAs were amplified using Power SYBR Green PCR Master Mix (Applied Biosystems, Belgium) and primer sense and antisense sequences (Sigma-Aldrich, Belgium) in a 7300 real-time PCR machine (Applied Biosystems, Belgium). mRNA levels were normalized to 36B4 (house-keeping gene) mRNA levels determined using the same procedure.

2. Results and Discussion:

The first in vitro experiments were intended to analyse adhesion and proliferation of keratinocytes cultured on electrospun chitosan membranes. Because of the presence of the nanofibers forming the membranes, it was difficult to observe the growth of keratinocytes using phase-contrast microscopy, explaining why most of the observations were performed by scanning electron microscopy (SEM). The SEM images illustrate that normal human keratinocytes were capable to attach to chitosan nanofiber membranes, to proliferate, and reach cell confluence on the top of the membrane (FIG. 13). No penetration of the cells into the nanofiber membrane was observed, probably because of the small spaces left between the fibers forming the membranes. An interesting observation was that freshly seeded keratinocytes formed lamellipodia and filopodia along the fibers (FIG. 14). After few days in culture, the small keratinocyte colonies merged into larger colonies progressively forming nearly complete cell monolayer on the top of chitosan nanofibers. When the cells were observed later during the culture, keratinocytes acquired appearance of postconfluent cultures, i.e. the cells retracted and overlapped and a few cells appear apoptotic. These results indicate that electrospun chitosan membranes are a suitable substrate for keratinocyte growth and differentiation, thus appearing as an excellent candidate for engineering wound dressings. In order to check whether keratinocytes were growing over membranes without penetrating between the fibers, histological staining of vertical sections of paraffin-embedded chitosan membranes cultured with keratinocytes were also performed. These experiments confirm our SEM observations showing that keratinocytes grow over the surface of membranes without penetrating between chitosan fibers (FIG. 15), which mimic the in vivo situation where keratinocytes form a covering layer without penetrating in the underlying dermis.

Keratinocyte cultures are characterized by specific expression of several genes, e.g. those encoding keratinocyte differentiating markers, like keratins and involucrin. Proliferative keratinocyte cultures express keratin 5 and keratin 14, which are markers for the undifferentiated cell phenotype. When the differentiation program of keratinocytes begins, the early markers of epidermal differentiation (suprabasal keratin 10 and involucrin) and the later markers of differentiation (filagrin and loricrin) are then expressed [11]. The results of the real-time PCR analysis showed that the expression of keratin 14, a marker of undifferentiated keratinocytes, slowly decreases with the time in culture. Simultaneously with the increase in cell density, we observed an increase in the expression of keratin 10 and involucrin—two of the markers of epidermal differentiation. These results show that epidermal keratinocytes cultured on nanofiber chitosan membranes are able to undergo differentiation as observed in vivo and in vitro on other physiological substrates (FIG. 16).

Characterization of Normal Human Fibroblasts and Endothelial Cells Cultured on Electrospun Chitosan Membranes

A medical grade chitosan of fungi origin (kiOmedine-CsUP™, Kitozyme, Belgium) was used. Its degree of acetylation (DA) and molecular mass (MM) were 19% and 68000, respectively. All chemical and biochemical reagents were analytical grade.

1.2—Preparation of Chitosan Films and Electrospun Membranes.

Sterile chitosane solutions (1% in 1% acetic acid) were obtained by filtration through a 0.22 μm filter.

Films. For obtaining films, adequate volume of sterile chitosan solution were poured in culture dishes and air-dried for 24 h in a laminar flow culture hood under sterile conditions. After complete drying, films were immersed during 2 h in 1% NaOH in distilled water to neutralise the residual acetic acid. Chitosan films were then rinsed three times in copious amounts of distilled water and dried. Before using to cell culture, the films were equilibrated during 2 h in the culture medium at 37° C.

Electrospun membranes. Membranes formed by electrospun chitosane nanofibers were prepared as described in Example 1. The chitosan membranes were then sterilized in 70% ethanol for 2 hours, washed three times in PBS (30 min each wash) and then equilibrated during 2 h in the culture medium at 37° C.

1.3—Cells and Cell Cultures

Human skin fibroblasts and microvascular endothelial cells (HMEC) were grown culture Petri dishes in Dulbecco's Minimum Essential Medium (DMEM, Lonza) supplemented with 10% fetal bovine serum (FBS, Lonza), antibiotic (100 U/ml penicillin and 100 μg/ml streptomycin, Lonza) and essential amino acid (Lonza) and incubated at 37° C. in humidified atmosphere with 5% CO2. After a confluent cell layer was formed, the cells were detached in PBS containing 0.25% trypsin and 1 mM EDTA and seeded on the various substrates in the same supplemented DMEM medium as described above. The culture medium was changed every 2-3 days.

1.4—Cell Morphology and Proliferation

Cell adhesion, spreading and proliferation were assayed on plastic and on chitosan films and electrospun membranes.

The morphology (adhesion and spreading) of fibroblasts and HMEC was investigated by phase-contrast microscopy or scanning electron microscopy (SEM). Briefly, for the SEM, the chitosan membranes and their adherent cells were harvested and then fixed for 10 min with 2.5% glutaraldehyde solution in Sodium Cacodylate buffer 0.1M pH 7.4, CaCl20.1% at 4° C. After rinsing three times with Sodium Cacodylate buffer 0.1M pH 7.4 with CaCl20.1% for 5 min, the specimens were dehydrated in graded ethanol of 25%, 50%, 75%, 95% and 100%, 5 and then 10 min each. The specimens were coated with a thin layer of gold and then subjected to observation by SEM (ktics).

Both the membrane and the film specimens were used for cell morphology and proliferation tests. For proliferation test, the cells were seeded on chitosan films or membranes in 4 cm2polystyrene disc at a density of 10 000 cells/cm2. They were then incubated for 1, 3, 5 and 7 days at 37° C. in air containing 5% CO2. WST-1 test was carried out to quantify the viability of the cells which adhered on chitosan at each specified seeding times point and completed by evaluating of proliferation rate using the radioactivity incorporation test. The cells attached on chitosan films or membranes were compared to the cell adhesion observed on the plastic material used as a reference.

1.5 Quantification of Viable Cells and Proliferation Rate

For measurement of cells proliferation and viability, a colorimetric method was used. This colorimetric assay is based on the cleavage of the tetrazolium salt WST-1 to a formazan-class dye by mitochondrial succinate-tetrazolium reductase in viable cells. As the cells proliferate, more WST-1 is converted to the formazan product. The quantity of formazan dye is directly related to the number of metabolically active cells, and can be quantified by measuring the absorbance at 420-480 nm (Amax450 nm). Before adding the reagent, the medium was removed and cells culture was washed two times with Dulbecco's. Then, cells were incubated in DMEM with 10% FBS and mixed with WST1 reagent in the ratio 9:1. After 4 h of incubation, 100 μl of the supernatant was carefully transferred to 96-well plates and optical density was measured at 450 nm.

The proliferation rate of cells was also measured by evaluating the incorporation of [3H] thymidine into TCA-precipitable DNA.

2. Results and Discussion:Cell adhesion. The first in vitro experiments were intended to compare the rate of adhesion of human skin fibroblasts and microvascular endothelial cells cultured on plastic, chitosan evaporated films and chitosan electrospun membranes. As compared to evaporated films, electrospun nanofibers induced a better and faster attachment of both cell types (FIG. 17).Cell spreading. Cells on plastic and chitosan films were easily visualized by phase-contrast microscopy (FIG. 18). For cells at the surface of electrospun membranes observation required scanning electron microscopy (SEM) because of a lack of transparency due to the presence of the dense network of nanofibers. Fibroblasts and HMEC in plastic culture dishes are fully spread after one day. As the cultures become confluent, a squeezing of the cells was observed, significant of an excessive proliferation as usually observed in vitro. When seeded on chitosan evaporated films, only few cells were able to attach, confirming our previous data regarding cell adhesion. However, none of them were able to fully spread, as evidenced by comparing the morphology of cells at day 1 on plastic to cells at any time on chitosan films. Moreover, due to the poor adhesion and spreading, these cells tend to form aggregates. These clusters, appearing in our cultures as refringent structures, are an additional evidence of the poor quality of chitosan films as a substrate for cells. In order to confirm that this was not the result of the presence of toxic compounds contaminating the films, floating individual cells and clusters were collected together with the conditioned culture medium and poured in plastic culture dishes (not shown). In these conditions, all the cells (individual or in clusters) attached rapidly and started to proliferate, firmly demonstrating that the poor adhesion and spreading resulted from the inappropriate structure of the chitosan films. On the contrary, cells on electrospun chitosan nanofibers are almost fully spread at day 1 (FIGS. 19-21), indicating favorable interactions between cells and the nanofiber scaffold. Interesting observations were also made at later time points, showing for example that cell filipodia are in close contact with individual nanofibers and even begin to invade the nano fiber structure and that cells are able to proliferate at a reasonable rate.Cell proliferation. Microscopic observations were highly suggestive of cell proliferation on chitosan nano fibers but not on chitosan films. This was further investigated by establishing a proliferative index by measurement of the incorporation of [3H] thymidine into TCA-precipitable DNA. As expected from microscopic studies, HMEC and fibroblasts are able to proliferate on chitosane nanofibers (FIG. 22). At the contrary a reduction of thymidine incorporation was evidenced for cells on films, as a result of cell detachment and/or cell death because of inappropriate interactions between the substrate (a process named anoikis).

Experiments described in this Example 6 have clearly demonstrated that chitosan can be a good substrate for the adhesion, spreading and proliferation of fibroblasts and endothelial cells, but only when manufactured as a scaffold made of nanofibers. Similar data were obtained with keratinocytes (Example 5). These unexpected results may be due to the increased specific surface of the nano fiber biomaterial or to the fact that chitosan nano fibers present structural features that mimic collagen fibers that are the major organic constituent of many tissues in vivo, especially skin, tendons and bones.

These very promising data prompted us to characterize further the biocompatibility and the biological properties of electrospun chitosan in vivo. These experiments are described in Example 7.

Characterization of the Biological Properties of Electrospun Chitosan Scaffold In Vivo

A medical grade chitosan of fungi origin (kiOmedine-CsUP™, Kitozyme, Belgium) was used. Its degree of acetylation (DA) and molecular mass (MM) were 19% and 68000, respectively. All chemical and biochemical reagents were of analytical grade.

Sterile chitosane solutions (1% in 1% acetic acid) were obtained by filtration through a 0.22 μm filter.

Electrospun membranes. Membranes formed by electrospun chitosane nano fibers were prepared as described in Example 1. The chitosan membranes were then sterilized in 70% ethanol for 2 hours, washed three times in PBS (30 min each wash) and then equilibrated during 2 h in the culture medium at 37° C.

Sponges. Due to the absence of three-dimensional structure, evaporated chitosan films were not used in vivo. Instead, chitosan sponges (supplied from Kitozyme) were used. The dried chitosan sponges were then immersed in 70% ethanol solution for sterilization and rinsed many time in large volumes of saline phosphate buffer.

1.3—Biocompatibility of Chitosan Sponges and Membranes

All procedures were performed with the approval of the Animal ethical Committee authorities of University of Liege, Belgium. Male Balb/c mice 8-10 weeks old, weighing about 22 g were kept under specific pathogen free (SPF) conditions and given free access to food and water throughout the experiment. The 18 mice were housed in groups of at least six animals. Before implantation, chitosan membranes and sponges were cut into 8.0-mm diameter pieces, sterilised in 70% ethanol and soaked in sterile saline solution. Before starting the experiment the mice received a subcutaneous injection of Temgesic (0.05 mg/kg) to prevent pain. This treatment was continued twice daily during ten days after the surgery. The hairs on the backs of the mice were shaved. Anaesthesia was performed by successive intramuscular injections of Domitor (500 μg/kg) and Ketamine (60 mg/kg). Chitosan sponges and electrospun nanofiber membranes were subcutaneously implanted through a 1-cm incision and secured to the inner face of skin with nylon sutures. The mice were waked by intramuscular injection of Antisedan (200 μg/kg). Mice were sacrificed at 1, 2, 4, 8 or 12 weeks after implantation. Serums were collected for the potential presence of anti-chitosan antibodies evaluation. Sponges or electrospun membranes were collected, fixed and used (see below) for histological examination or transmission electronic microscope (TEM).

1.4—Preparation of Samples for Immune-Histological Examination and TEM

After sacrifice of mice, the chitosan sponges or electrospun membranes were dissected from mice and divided in several equivalent parts. Some were frozen and stored at −80° C. for protein and RNA analysis or embedded in Tissue Tek for cryostat sectioning. Another part was fixed in 4% neutralized buffered formaldehyde, embedded in paraffin and processed conventionally to produce 5 μm sections. These sections were then stained (Haematoxylin/eosin, Sirius red, yellow Safran/Haematoxylin) or used for immunostaining Finally, some pieces were also processed for transmission electron microscopy (TEM) after fixation overnight at 4° C. in a 2.5% glutaraldehyde solution (in sodium cacodylate buffer 0.1M, pH 7.4, 0.1% CaCl2). After rinsing three times in a sodium cacodylate buffer (0.1M; pH 7.4, with CaCl20.1%) for 5 min, the specimens were further fixed in 1% osmium tetroxide (diluted in 0.1M cacodylate buffer pH 7.4) for 1 h at 4° C. The specimens were dehydrated with graduated concentration of ethanol (25%, 50%, 75%, 95% and 100%) and then in propylene oxide. Afterwards, the specimens were infiltrated and embedded in LX112 resin (LADD, USA), polymerized consecutively at 37° C. for 24 h, 47° C. for 24 h and 60° C. for 48 h. Orientation sections for light microscopy were cut at 2 μm thickness and stained with toluidine blue. Ultra-thin sections (50-70 nm) were cut, stained with 4% uranyl acetate (diluted in 50° ethanol), then contrasted with lead citrate and mounted on uncoated grids. Samples were then observed by TEM (FEI Tecnai 10, USA).

The following primary antibodies were used: (1) monoclonal biotin-conjugated rat anti-mice

CD45 (1:1500; PHARMINGEN) for the labelling of leukocytes; (2) polyclonal Guinea pig antibody to vimentin ((1:20; QUARTETT) for the labelling of mesenchymal cells, especially fibroblasts; (3) a “home-made” rabbit anti-mouse type IV collagen (1:100) which identifies basement membrane, here mainly identifying mature blood vessels and (4) mouse anti-alpha smooth muscle actin (1:400; SIGMA) which identifies smooth muscle cells. Before use, paraffin sections were “deparaffined” and rehydrated. Immunohistochemical staining and visualization using diaminobenzidine (DAB) and counter-staining with haematoxylin/eosin for 30 sec.

96 wells-plates were coated with 180 μg of chitosan solution, incubated for 2 h at room temperature, washed twice with 20 mM Tris buffer (20 min each wash) and then blocked overnight at 4° C. by addition of bovine serum albumin (5% in PBS). Plates were then washed 3 times with PBS containing 0.05% of Tween-20. Serum samples from control or from chitosan-implanted mice (sponges or electrospun membranes) were serially diluted for evaluation. Plates were sequentially (i) incubated overnight at 4° C., (ii) washed with PBS/Tween, (iii) incubated for 2 hours with a horseradish peroxidase-conjugated secondary antibody (at a 1/1000 dilution in PBS) and (iv) washed again in PBS/Tween. Staining was obtained by the addition a solution of 1 mM ABTS containing 0.03% H2O2after incubation for 1 h at 37° C. in the dark. The reaction was stopped by adding a solution of 1% SDS. The OD at 405 nm was measured and used for comparing the various experimental conditions.

2. Results and Discussion

Absence of generation of antibody. Sera were recovered from control mice (that were never in contact with chitosan) or from mice subcutaneously implanted with either electrospun chitosan membranes or chitosan lyophilized sponges. The experimental procedure consisted in an ELISA assay, a standard highly sensitive technique for screening the presence of specific antibodies (FIG. 23). The background values obtained from the serum of control mice were considered as being caused by the low affinity existing between chitosan and proteins found in the serum, including immunoglobulins. Measurements performed on sera collected at increasing time after chitosan implantation were always in the range of values observed for control mice, even for long term implanted mice. These data confirm that chitosan, irrespective of its structure (sponge or nanofibers), do not elicit any specific antibody production, thus confirming by this aspect its biocompatibility in vivo.

Immunohistological examination of implanted chitosan sponges and nanofibers. Mice were subcutaneously implanted with either electrospun chitosan membranes or chitosan lyophilized sponges. After 1 to 12 weeks, implanted material was recovered, fixed, embedded in paraffin, sectioned and stained by hematoxylin/eosin or by the use of specific antibodies. Chitosan electrospun membranes appear as an undulating dense structure, somehow mimicking the extracellular matrix organization seen in skin in vivo (FIGS. 24,25). Cell infiltration and colonization was evidenced and seemed to be time-dependent, starting after less than one week and reaching the center of the biomaterial after 12 to 20 weeks. These cells were shown to be of mesenchymal origin (fibroblasts, myofibroblasts, smooth muscle cells) (FIG. 27) and endothelial cells forming structures resembling functional capillaries (FIG. 28). A limited number of leukocytes were also detected (FIG. 26). As a whole these data are indicative of a physiological progressive remodelling process. By sharp contrast, sponges, that are formed by a multi-lamellae structure containing a large ratio of void space, are not efficiently colonized by living cells and do induce a strong innate immune response (as evidenced by the accumulation of leukocytes and activated mesenchymal cells forming a granuloma at the periphery of the implanted sponges).

Ultrastructural characterization of implanted biomaterial. Electrospun chitosan membranes and chitosan lyophilized sponges recovered at increasing time after implantation were also processed and characterized by transmission electron microscopy (TEM).

Chitosan nanofibers appear as black elongated cylindrical structure in longitudinal section and as black circles in cross sections (FIG. 29). Numerous fibroblasts were identified within the dense chitosan nanofiber network. These fibroblasts are biosynthetically active since they produce collagen accumulating as physiological fibres and fibrils in close contact with chitosan nanofibers. Some macrophages were also identified, possibly participating to a slow chitosan degradation process (FIG. 30). Again, these data strongly suggest a progressive and physiologically ordered remodelling of the nanofiber scaffold.

By contrast, collagen and cell accumulation was found only at the periphery of lyophilized sponges (not shown), confirming immunohistochemical data and illustrating that the nanofibrillar structure of chitosan is crucial for true biocompatibility in vivo.

Three deeply unexpected results were obtained during these in vivo experiments.Although the nano fiber network appeared too dense to allow cell migration (seeFIG. 21), colonization of the entire width (2 mm) of the electrospun membrane was observed within 2 to 3 months. This is due in part to the capacity of cells to change their shape but also to the structure of the scaffold that allows some sliding of any single nanofiber relatively to others because of the absence of reticulation.While chitosan sponges are being recognized by the mouse as “foreign bodies”, and, consequently, encapsulated in a dense capsule (essentially made of collagen, activated mesenchymal cells and leukocytes), electrospun nanofibers are considered as “self” tissue that is progressively filled by host cells and extracellular matrix without any sign of aberrant activation of invading cells. Moreover the limited presence of leukocytes inside the biomaterial is likely to be beneficial for the remodeling process by regulating the properties of other cell types as observed in “in vivo” situation during wound healing.The close association observed between chitosan nanofibers, on one side, and cells and the newly deposited extracellular matrix on the other side, illustrates the fact that the design of the structure of the chitosan nanofiber scaffold (rigidity, fiber orientation, fiber density, . . . ) should strongly influence the properties of the newly formed tissue. This is a crucial importance for any medical application since cell fate and behavior are both strongly regulated by the extracellular environment and since the desired biophysical properties of engineered biomaterials deeply vary according the tissue to be repaired (skin, tendon, bone, etc).As an example, the corrugated structure of the electrospun nanofibers, as they are designed in this study, mimics the organization of collagen fibers found in the dermal compartment of the skin. Therefore such biomimetic structure used as progressively degradable scaffold dressing for skin ulcer should favor the optimal repair of skin in terms of elasticity and functionality.

Effect of Chitosan Nanofibers on Wound Healing in Mice

A medical grade chitosan of fungi origin (KiOmedine-CsUP™, Kitozyme, Belgium) was used. Its degree of acetylation (DA) and molecular mass (MM) were 19% and 68000, respectively. All chemical and biochemical reagents were of analytical grade.

1.2 Preparation of Chitosan Electrospun Membranes

Sterile chitosan solutions (1% in 1% acetic acid) were obtained by filtration through a 0.22 μm filter.

Electrospun membranes. Membranes formed by electrospun chitosan nanofibers were prepared as described in example 1. The chitosan membranes were sterilized in 70% ethanol for 2 hours, washed three times in PBS (30 min each wash) and then air-dry for 24 h in a laminar flow culture hood under sterile conditions.

1.3 Animal Experiments

A total of 30 male Balb/c mice (8-10 weeks) were used, weighing between 20 and 25 g at the time of the experiments. The animal protocols followed in the present study were approved by the Animal Ethical Committee Authorities of University of Liege, Belgium. Before starting the experiment the mice received a subcutaneous injection of Temgesic (0.05 mg/kg) to prevent paint. This treatment was continued twice daily during ten days after the surgery. Mice were individually anesthetized via an intramuscular injection of ketamine (50 mg/kg) and domitor (0.5 mg/kg) for surgery and induction of the excisional wound. The operative skin area was shaved and disinfected using ethanol. Then, the dorsal skin of the animal was excised using a biopsy punch to create an 8-mm wound. The animals were divided into two groups. In group 1, wounds were covered successively with electrospun chitosan membrane, a Tegaderm3Mlayer and a protective elastic bandage (Elastoblast). In group 2, used as control, wounds were only covered with Tegaderm3Mand the elastic bandage (Elastoblast). After surgery, animals were kept in separate cages under specific pathogen free (SPF) conditions and given free access to food and water throughout the experiment. All animals showed good general health conditions throughout the study, as assessed by their weight gain. The animals were sacrificed after 7, 14, and 21 days.

The material from the skin lesions, either controls or covered first by the electrospun chitosan membrane, was formalin fixed and paraffin embedded for routine histological processing. Five μm sections obtained from each paraffin block were stained with hematoxylin and eosin (H&E) and evaluated by using a light microscope, or characterized by immunohistochemistry. For the morphological evaluation of skin lesions after H&E staining, three parameters were considered: 1) nature and duration of the inflammatory reaction, 2) composition and thickness of the granulation tissue layer and 3) thickness and quality of the epithelial layer. Immunohistochemical stainings were also performed using various antibodies that allow to monitor the healing rate and the quality of the repair process. These antibodies can recognize as example collagen, fibronectin, CD45 (for leukocytes), alpha-SMA (for smooth muscles cells), KI-67 (for establishing a proliferative index), cytokeratin and involucrine (for keratinocytes).

2. Results and Discussion

The goal of this study was to assess the effect of electospun chitosan on the rate of the healing process and on the quality of the repaired tissue. Macroscopic findings showed that chitosan nano fibers adhered uniformly to the freshly excised wound surface and also absorbed the exudates from the wound surface. The porosity of electrospun chitosan nanofibers promotes gas exchange, which is fundamental for the wound-healing process. No signs of infections were detected in the skin lesion. Standard Hematoxylin and eosin staining of wounds removed from control mice and chitosan treated-mice at 7, 14 and 21 days postwounding are show inFIG. 31. On days 7 after injury, histological examination of the wounds treated or not with the electrospun chitosan nano fibers showed that epithelialization process had started on the wound, and the wound bed (dermis) was rich in polynuclear and macrophage inflammatory cells that play crucial role for cleasing the wound and initiating the healing process. Wound treated with electrospun chitosan nanofibers showed a largest extend of infiltration of these inflammatory cells. This indicates that chitosan attracted inflammatory cells during the early phase of wound early without the excessive inflammation. At the 14th postoperative day, wounds were epithelialized but the dermis of control wounds still contained an excessive number of inflammatory cells, showing some delay in the healing process as compared to the wound covered by electrospun chitosan. Moreover, the level of myofibroblastic/fibroblastic cells and the number of newly formed capillaries was higher in presence of electrospun chitosan as compare to controls, again demonstrating a faster healing for treated wounds. At the 21st postoperative day, the epithelium of the electrospun chitosan nanofibers treated wound was thin and well organized resembling to a normal epithelium. The deposition of newly synthesized collagen in the dermis was also well organized with fibrils being oriented parallel to the skin surface, showing an optimal repair of the damaged tissue and indicating that the electrospun chitosan nanofibers increases the rate of healing and help to restore tissue architecture. By contrast the repair process was slower for control wounds.

CONCLUSIONS

The electrospun chitosan nanofibers used as a wound dressing is fully biocompatible. It showed excellent oxygen permeability and could inhibit exogenous microorganism invasion due to its inherent antimicrobial property of chitosan. Due to its physical form and its chemical structure, electrospun chitosan nanofibers promote the recruitment of inflammatory cells at the early phase of wound healing. Later it increases the rate of blood vessel formation and the maturation of the entire tissue being repaired, including dermis and epidermis, showing that electropun chitosan has many therapeutic advantages and properties when used as a wound dressing.

LITERATURE REFERENCES