MEMBRANE TO RE-ESTABLISH CONTINUITY OF INJURED BIOLOGICAL TISSUES

Abstract

A membrane for re-establishing the continuity of injured biological tissues includes a non-woven fabric of fibres of a polyhydroxyalkanoate (PHA) containing 3-hydroxybutyrate monomer units, wherein the fibers have an average diameter between 800 nm to 3500 nm. Preferably, the PHA is a poly-3-hydroxybutyrate homopolymer and at least one active ingredient is associated with the fibers of the non-woven fabric. The membrane has the advantages of promoting the healing of injured biological tissues relatively quickly, without inducing excessive inflammatory or rejection reactions, thus demonstrating a high biocompatibility, thereby providing a valid support in the healing process of biological tissue injuries and preventing the formation of dehiscences and incisional hernias.

Claims

1. A membrane for re-establishing the continuity of injured biological tissues, comprises a non-woven fabric of fibres of a polyhydroxyalkanoate (PHA) containing 3-hydroxybutyrate monomer units, wherein said fibers have an average diameter comprised from 800 nm to 3500 nm.

2. The membrane according to claim 1, wherein the PHA is a poly-3-hydroxybutyrate homopolymer (P3HB) or a copolymer containing at least 20 mol % of 3-hydroxybutyrate monomer units, the remainder being hydroxyalkanoates monomer units other than 3-hydroxybutyrate.

3. The membrane according to claim 2, wherein the hydroxyalkanoate monomer units other than 3-hydroxybutyrate derive from: 4-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundec-10-enoate, 4-hydroxyvalerate.

4. The membrane according to claim 2, wherein the PHA is a poly-3-hydroxybutyrate homopolymer (P3HB).

5. The membrane according to claim 1, wherein the PHA has a weight average molecular weight (M.sub.W) comprised in the range from 5,000 to 1,500,000 Da.

6. The membrane according to claim 1, wherein the PHA fibers of the non-woven fabric are produced by electrospinning.

7. The membrane according to claim 1, wherein the non-woven fabric has dimensions comprised from 1 cm.sup.2 to 50 cm.sup.2, a rectangular, rhomboidal, square, oval, round or irregular shape and a thickness comprised from 10 μm to 700 μm.

8. The membrane according to claim 1, wherein at least one active ingredient is associated with the fibers of the non-woven fabric.

9. The membrane according to claim 8, wherein the at least one active ingredient is selected from the group consisting of: pentoxifylline, doxycycline, ibuprofen, cefazolin, rifampicin, paclitaxel, itraconazole, mefoxin, tetracycline hydrochloride, and mixtures thereof.

10. The membrane according to claim 9, wherein the active ingredient is doxycycline.

11. The membrane according to claim 10, wherein the doxycycline has a concentration comprised from 0.1% w/w to 4% w/w, with respect to the weight of the PHA.

12. The method for producing a membrane comprising a non-woven fabric according to claim 1, the method including the following steps: preparing a spinning solution by solubilization of the polyhydroxyalkanoate (PHA) containing 3-hydroxybutyrate monomeric units in an organic solvent, and subjecting the spinning solution to an electrospinning process by means of a spinneret and a rotating support placed substantially perpendicular to the spinning direction, using a configuration called horizontal arrangement, so as to obtain the non-woven fabric.

13. The method according to claim 12, wherein the organic solvent usable for the preparation of the spinning solution is selected from the group consisting of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), chloroform, N,N-dimethylformamide (DMF), and mixtures thereof.

14. The method according to claim 12, wherein in the spinning solution, the PHA containing 3-hydroxybutyrate monomer units has a concentration comprised from 1% to 20% w/v.

15. The method according to claim 12, wherein the spinning solution further comprises at least one active ingredient.

Description

DETAILED DESCRIPTION OF THE DISCLOSURE

[0060] The following embodiment examples are provided purely for illustrative purposes of the present disclosure and should not be interpreted as limiting the scope of protection defined by the accompanying claims.

Example 1: Production of the P3HB Non-Woven Fabric

[0061] The purified P3HB was dissolved in chloroform so as to obtain a spinning solution with a concentration of 6% w/v. The solution was stirred on a heating plate at 800 rpm at a temperature of 80° C. for about 2 hours in order to facilitate the complete dissolution of the polymer.

[0062] After complete dissolution, N,N-dimethylformamide (DMF) was added to the solution until a chloroform/DMF ratio of 90:10 v/v was achieved.

[0063] The solution was stirred at 800 rpm at a temperature of 25° C. for about 2 hours.

[0064] Thereafter, the solution was cooled, poured into a 10 mL plastic syringe and placed inside the electrospinning system to be processed. The P3HB filament deposition was made by means of a spinneret and a rotating cylindrical support placed substantially perpendicular to the spinning direction, using a configuration called “horizontal arrangement”. Once the deposition on the rotating support had been carried out, the deposition was opened and laid in order to obtain the non-woven fabric in the form of a flat membrane.

[0065] The deposition of the P3HB filament on the rotating cylindrical support therefore allowed obtaining the non-woven fabric.

[0066] The following parameters were used for the electrospinning process

[0067] Spinning solution: purified P3HB dissolved in chloroform/DMF (90:10 v/v) at 6% w/v;

[0068] solution volume processed: 120 mL;

[0069] solution rate: 8 mL/hour;

[0070] translational speed of the spinneret: 8 mm/s;

[0071] distance between the tip of the spinneret and the cylindrical support (gap): 15 cm;

[0072] voltage applied to the electrodes: 20 kV;

[0073] rotation speed of the cylindrical support: 150 rpm;

[0074] diameter of the spinneret outlet hole: 2.15 mm;

[0075] FIG. 1 shows the images of the non-woven fabric of Example 1 obtained by scanning electronic microscopy (SEM).

Example 2: Production of the P3HB and Doxycycline Non-Woven Fabric

[0076] The purified P3HB was dissolved in chloroform so as to obtain a spinning solution with a concentration of 6% w/v. The solution was stirred on a heating plate at 800 rpm at a temperature of 80° C. for about 2 hours in order to facilitate the complete dissolution of the polymer.

[0077] At the same time, a stock solution of doxycycline was prepared by dissolving the active ingredient in N,N-dimethylformamide (DMF). The resulting solution was stirred on a plate at 800 rpm at a temperature of 25° C. for about 1 hours.

[0078] The stock solution of doxycycline in DMF was diluted three times in order to obtain three solutions having different concentrations of doxycycline: [0079] solution 1: P3HB (6% w/v) and doxycycline (0.1% w/v) in chloroform/DMF (90/10 v/v); [0080] solution 2: P3HB (6% w/v) and doxycycline (0.25% w/v) in chloroform/DMF (90/10 v/v); [0081] solution 3: P3HB (6% w/v) and doxycycline (0.5% w/v) in chloroform/DMF (90/10 v/v).

[0082] The three solutions were stirred at 800 rpm at a temperature of 25° C. for about 2 hours.

[0083] Thereafter, the three solutions were made to cool, were respectively poured into three 10 mL plastic syringes and one at a time placed inside the electrospinning system to be processed in succession. The P3HB+doxycycline filament deposition was made by means of a spinneret and a rotating cylindrical support placed substantially perpendicular to the spinning direction, using a configuration called “horizontal arrangement”. Once deposition on the rotating cylindrical support had been carried out, the deposition was opened and laid in order to obtain the non-woven fabric admixed with doxycycline in the form of a membrane. The deposition of the P3HB+doxycycline filament on the rotating cylindrical support therefore allowed obtaining the non-woven fabric added of the active ingredient.

[0084] The following parameters were used for the electrospinning process of solution 1:

[0085] spinning solution 1: purified P3HB (6% w/v) and doxycycline (0.1% w/v) dissolved in chloroform/DMF (90:10 v/v);

[0086] solution 1 volume processed: 50 mL;

[0087] solution 1 rate: 7 mL/hour;

[0088] translational speed of the spinneret: 6 mm/s;

[0089] distance between the tip of the spinneret and the cylindrical support (gap): 20 cm;

[0090] voltage applied to the electrodes: 20 kV;

[0091] movement speed of the flat support: 200 rpm;

[0092] diameter of the spinneret outlet hole: 2.15 mm;

[0093] FIG. 2 shows the images of the non-woven fabric added of doxycycline (0.1% w/v) obtained by scanning electronic microscopy (SEM).

[0094] The following parameters were used solution 2 for the electrospinning process of solution 2 spinning solution 2: purified P3HB (6% w/v) and doxycycline (0.25% w/v) dissolved in chloroform/DMF (90:10 v/v);

[0095] solution 2 volume processed: 50 mL;

[0096] solution 2 rate: 7 mL/hour;

[0097] translational speed of the spinneret: 6 mm/s;

[0098] distance between the tip of the spinneret and the cylindrical support (gap): 20 cm;

[0099] voltage applied to the electrodes: 20 kV;

[0100] rotation speed of the cylindrical support: 200 rpm;

[0101] diameter of the spinneret outlet hole: 2.15 mm;

[0102] FIG. 3 shows the images of the non-woven fabric admixed with doxycycline (0.25% w/v) obtained by scanning electronic microscopy (SEM).

[0103] The following parameters were used solution 3 for the electrospinning process of solution 3 spinning solution 3: purified P3HB (6% w/v) and doxycycline (0.5% w/v) dissolved in chloroform/DMF (90:10 v/v);

[0104] solution 3 volume processed: 50 mL;

[0105] solution 3 rate: 7 mL/hour;

[0106] translational speed of the spinneret: 6 mm/s;

[0107] distance between the tip of the spinneret and the cylindrical support (gap): 20 cm;

[0108] voltage applied to the electrodes: 20 kV;

[0109] movement speed of the cylindrical support: 200 rpm;

[0110] diameter of the spinneret outlet hole: 2.15 mm;

[0111] FIG. 4 shows the images of the non-woven fabric added of doxycycline (0.5% w/v) obtained by scanning electronic microscopy (SEM).

[0112] The concentration of doxycycline trapped within the three non-woven fabrics was then determined by the following method.

[0113] A rectangular portion of about 150 mg of weight was cut from each non-woven fabric. Each portion was in turn cut and reduced into small pieces, in order to promote the release of trapped doxycycline. The pieces of each non-woven fabric were immersed, within a 50 mL falcon, in 10 mL 0.1 M NaOH. The three suspensions obtained were stirred overnight on a rotating plate and subsequently put in centrifuge at 6000 rpm for 15 minutes. The supernatant solution of each falcon was diluted and analyzed by UV-Vis (results reported in Table 1).

TABLE-US-00001 TABLE 1 concentration (% w/w) of doxycycline in the three non-woven fabrics Theoretical Actual doxycycline doxycycline compared to the compared to the weight of P3HB weight of P3HB Efficiency Sample (% w/w) (% w/w) (%) P3HBDOX1 1.2% 0.5% .sup. 25% P3HBDOX2 4.2% .sup. 1% 21.5% P3HBDOX3 8.3% 3.6% 43.6%

Example 3: Preclinical Study—Intraperitoneal Implant of Membranes Comprising Non-Woven Fabrics of Examples 1 and 2

[0114] a) Materials and Methods

[0115] The study was conducted according to the good practices described in the following documents:

[0116] “ISO 10993-2:2006 Biological evaluation of medical devices Part 2: Animal welfare requirements”;

[0117] “ISO 10993-6:2016 Biological evaluation of medical devices—Part 6: Tests for local effects after implantation”;

[0118] “F763-04 (2016) Standard Practice for Short-Term Screening of Implant Materials”;

[0119] “F1904-14 Standard Practice for Testing the Biological Responses to Particles in vivo”;

[0120] “F1983-14 Standard Practice for Assessment of Selected Tissue Effects of Absorbable Biomaterials for Implant Applications”.

[0121] Adult Sprague-Dawley male rats weighing 225-250 g each were used.

[0122] 42 animals (of the total 120 used) were randomly allocated to the groups: [0123] P3HB group (membrane comprising the non-woven fabric of Example 1); [0124] P3HB+Doxy group (membrane comprising the non-woven fabric of Example 2 in a 1:1000 ratio of doxycycline).

[0125] b) Biocompatibility

[0126] The animals were subjected to an intraperitoneal implant of a portion of 1 cm.sup.2 of membrane comprising non-woven fabric to be tested. After laparotomy on the midline, the membrane was implanted in the abdomen adhering to the parietal peritoneum about 1 cm to the left of the white line.

[0127] An incision of 2 cm was performed on the median plane comprising skin, subcutis and the white line.

[0128] The membrane was then implanted as described by “Azab AK, Doviner V, Orkin B, Kleinstern J, Srebnik M, Nissan A, Rubinstein A: Biocompatibility evaluation of crosslinked chitosan hydrogels after subcutaneous and intraperitoneal implantation in the rat; J Biomed Mater Res A 2007, November; 83(2):414-422”. Briefly, a single 1 cm.sup.2 portion was inserted through the surgical incision and fixated at about 1 cm from the white line to the left abdominal wall with a single simple suture point. The abdominal wound was then closed on two planes with a simple continuous suture with glycomer 631 USP 3-0 for the band and USP 3-0 nylon for the skin.

[0129] In the negative control group, the procedure was performed in the same way but without insertion of the membrane.

[0130] The surgical procedures lasted about 20 minutes each, while the anaesthesia time of each rat was about 1 hour.

[0131] During and after the procedures, the rats were kept on a heated plate and under an infrared lamp to avoid hypothermia and 5 ml of isotonic physiological solution (0.9%) were administered subcutaneously before putting them in their respective cages.

[0132] In the post-operative period, the administration of analgesics (Carprofen 5-10 mg/kg subcutaneously for 2-3 days), antibiotics (penicillin+dihydrostreptomycin 0.2-0.3 mg/kg subcutaneously every 2 days 2 times a day) was assured.

[0133] The subjects were kept in single cages and then subjected to euthanasia on day 14, and then for each rat the left abdominal wall, in the portion comprising the membrane, was removed and fixated in formalin. Thereafter, the tissue was dissected, stained with hematoxylin and eosin and examined by a pathologist not aware of the sample assignment group.

[0134] The following parameters were considered to histologically define the tissue response: [0135] neovascularization; [0136] fibrosis; [0137] adipose infiltration.

[0138] For each parameter, a score was assigned on a scale from 0 (absent) to 4 (very abundant).

[0139] FIG. 5 shows an example of a histological preparation of Example 3.

[0140] Furthermore, the expression of COX-2 (cyclooxygenase 2) was evaluated to define the degree of inflammation of the tissue.

[0141] All the data obtained were analyzed to verify the normality thereof by the Shapiro-Wilk test.

[0142] Non-parametric tests (Kruskal-Wallis) were then used to determine their significance.

[0143] c) Results

[0144] Table 2 reports the results concerning neovascularization.

TABLE-US-00002 TABLE 2 (neovascularization) P3HB P3HB + Doxy Median (range) 3(2-4)* 2(1-2)* *p = 0.0163 IQ range 2.25-3.08 1.67-2.12

[0145] These results indicate that the two membranes comprising non-woven fabrics are comparable with respect to neovascularization.

[0146] Table 3 reports the results related to fibrosis.

TABLE-US-00003 TABLE 3 (fibrosis) P3HB P3HB + Doxy Median (range) 3(2-4) 4(3-4)* *p = 0.0001 IQ range 2.8-3.53 3.49-4.1

[0147] These results indicate that P3HB+DOXY induces a more pronounced fibrosis compared to P3HB. This is attributable to the fact that doxycycline has an inhibiting effect on the metalloproteases responsible for the inhibition of collagen remodeling.

[0148] Table 4 shows the results concerning adipose infiltration.

TABLE-US-00004 TABLE 4 (adipose infiltration) P3HB P3HB + Doxy Median (range) 2.5(1-4).sup.b 1(1-2).sup.a, b .sup.ap = 0.0143 IQ range 1.86-3.13 0.9-1.64 .sup.bp = 0.0156

[0149] These results indicate that P3HB+DOXY induces a lower adipose infiltration compared to P3HB. This result can also be explained by the inhibition of metalloproteases by doxycycline which, potentially, causes a substitution of adipose tissue with fibrous tissue.

[0150] COX-2 expression was not significantly different between the two groups (data not shown). This indicates that the degree of inflammation induced by the presence of the two membranes comprising the non-woven fabrics is comparable.

[0151] d) Conclusions

[0152] The membrane comprising the P3HB+DOXY non-woven fabric was found to induce a superior tissue response compared to the membrane comprising the P3HB non-woven fabric, causing greater fibrous tissue apposition.

[0153] Not having shown any signs of toxicity and not having caused reactions different from those expected for this type of implant, the two membranes comprising the tested non-woven fabrics have proven to be largely biocompatible, capable of promoting healing of the injured biological tissue relatively quickly, without inducing inflammatory or rejection reactions. The membranes comprising the tested non-woven fabrics have therefore proved to be a valid support in the healing process of biological tissue injuries, capable of preventing the formation of dehiscences and incisional hernias.