WO2021205307A1

WO2021205307A1 - Medical kit for treating skin lesions

Info

Publication number: WO2021205307A1
Application number: PCT/IB2021/052777
Authority: WO
Inventors: Francesca BOCCAFOSCHI; Luca Fusaro
Original assignee: Tissuegraft S.R.L.; Passalacqua Escavazioni S.R.L.
Priority date: 2020-04-09
Filing date: 2021-04-02
Publication date: 2021-10-14
Also published as: IT202000007564A1

Abstract

Process for obtaining an electrospun layer, said process providing that said layer is obtained by inserting a mixture of two solutions in an electrospinning instrument: a first solution being composed of extracellular matrix obtained by decellularizing bovine pericardium, a second solution being obtained by dissolving gelatin derived from pig skin, which can be found on the market, in a range variable between 10% and 40% by weight/volume in a solution variable between 90% and 100% acetic acid and between 0 and 10% water, said two solutions being mixed in a ratio variable between 2/1 and 1/3 matrix/gelatin, a crosslinking agent then being added to these mixtures, such agent selected in the group constituted by: 3-glycidoxypropyltrimethoxysilane (GPTMS), genepin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC); said process finally providing for the obtainment of a material having fibers comprised between 0.3 and 10 m, with a porosity variable between 15% and 50% with respect to the surface of the material.

Description

“Medical kit for treating skin lesions”

Description Field of the art

The present invention refers to the medical field. More in detail, the present invention refers to a particular process for obtaining an electrospun layer for use in treating skin lesions and especially in treating ulcers, typically but not exclusively those from diabetic foot. Prior art

An ulcer is a lesion on the skin or on a mucous membrane, accompanied by the disintegration of the tissue.

Ulcers can cause the complete loss of the epidermis and often portions of the dermis and even of the subcutaneous fat. Skin ulcers are more common on the skin of the lower limbs. An nicer which appears on the skin is often visible as an inflamed tissue with an area of reddened skin. A cutaneous ulcer is often visible in the case of hot or cold exposure, irritation or problems of blood circulation. In particular, the pressure sore (or decubitus ulcer) is a tissue lesion, with necrotic evolution, that affects the epidermis, the dermis and the subcutaneous layers, up to reaching, in the most serious cases, the muscles and bones. Rather commonly it is also termed “sore”. A decubitus lesion is the direct consequence of a high or prolonged compression, or of shear (or stretching) forces, causing a mechanical stress at the tissues and the constriction of blood vessels. This is due to the persistent pressure which, exceeding 32 mm Hg, causes a construction of the blood vessels, with consequent tissue necrosis. An ulcer develops in 4 stages: inflammation and destruction of the epidermis; destruction of the dermis; destruction of the subcutaneous and of the panniculus adiposus; necrosis of the muscles, of the periosteum and possibly of the bone.

By diabetic foot it is intended a chronic complication of diabetes mellitus, which causes anatomical-functional alterations of the foot and angle, determined by the neuropathy and/or by the peripheral occlusive arteriopathy. Considered today as a decidedly incapacitating syndrome, diabetic foot can involve two pathological situations that are quite distinct, based on the damage mechanism that the diabetes causes the foot: 1. neuropathic diabetic foot

2. neuroischemic foot

Diabetic foot is neuropathic when the damage is caused by the altered levels of glucose in the blood on the nerve. We speak therefore of neurological damage, i.e. the diabetes damages the sensitive, motor and vegetative nerve fibers of the limb. In this case, the neuropathy is on one hand manifested with progressive loss of thermal, tactile and pain sensitivity, and on the other hand with an anatomical alteration of the foot (irregular plantar support) which involves hyper-load areas that tend to be ulcerate. In other words the patient hit by diabetic neuropathy tends to walk badly, wounding himself, but since his/her sensitivity is compromised he/she is also unable to detect the ulcerative lesions of the skin in a timely manner, or the bloody sores and infections which, if neglected, can spread to the adjacent tissues and evolve into tissue necrosis (gangrene).

Neuroischemic foot is instead caused by the second damage mechanism, which is due to the arterial vascular deficit, i.e. the patient is subjected to reduction of the blood flow to the foot caused by the alteration of the blood vessels of the lower limbs, which in this manner become ischemic and more subject to the development of necrosis and gangrene.

The ulcerative lesions of the skin due to circulatory problems (vasculopathy) or nerve problems (neuropathy) can be manifested in the foot and ankle, causing the risk of dangerous infections that can progress and, if not suitably treated, they can represent a high risk factor for amputation of the limb or of septicemia that threatens the life of the patient. From research it is inferred that the value of the market relative to skin ulcer treatments, hence also comprising decubitus sores, reached a total of 7 billion dollars in 2017, with an increase expected of 11% by 2026, due to the improvement of living conditions worldwide, which is associated with an increase of the average age and increase of cases of obesity and diabetes.

Generally, different devices are present for treating ulcers, including decompression devices, anti-infection products, compressing medications, products for cleaning and debriding (removal of the lacerated tissue), moist medications, such as alginates, hydrocolloids, hydrogels, films and foams, products of biological origin such as artificial skin, collagen and growth factors. (Bhattacharya et al., 2015).

All these products are either of synthetic origin or they are obtained by isolating a single active principle, such as in the case of products based on collagen or hyaluronic acid. These products are therefore made of inert materials with respect to the tissue that they are to protect, and even if showing good biocompatibility, in many cases they have an effectiveness that can vary from case to case, and in various reports do not show significant improvements in the tissue regeneration with respect to the simple gauze soaked with saline solution, if not for the fact that they are easier to use than the latter (Westby et al., 2017).

The present invention has set the objective of overcoming the aforesaid critical issues by proposing a new type of tissues with applications analogous to those of aforesaid prior art, but which are consistently effective during their use. The latter is also extended to the adaptation to the platelet-rich plasma (PRP) derived from peripheral blood (autologous).

PRP, by now widely used in maxillofacial surgery, in reconstructive plastic surgery, in odonto-stomatological surgery and in aesthetic medicine, and antiaging, in eye surgery and in sport traumatology, is a source of growth factors that sustain the growth of the bone and of the soft tissues (skin, subcutaneous, myofascial tissues), improving the response to biological damages and facilitating the healing of wounds. It is obtained by concentrating the autologous platelets (i.e. of the patient himself/herself) and deriving therefrom the platelet growth factors (PDGF : Platelet Derived Growth Factors) which, when added to the surgical wounds or to grafts, sustain and improve and accelerate the healing process.

Regarding such matter according to the present invention, described in detail hereinbelow, a new type of electrospun layers will be defined, i.e. bandages or films, which are particularly effective in acting as a support for the application of the PRP on the zones to be treated, as well as in allowing a prolonged release of the growth factors, which is essential for the optimization of the tissue regeneration process.

Descriotion of the invention The present description refers to a particular layer of electrospun material and more specifically to a kit for use as medication in treating skin lesions and typically, but not exclusively, in treating diabetic foot. More in detail the present description refers to a kit comprising at least a layer of electrospun material.

Preferably said kit comprises at least a layer of electrospun material and at least a phial for a syringe containing a suitable hydrogel.

The layer of electrospun material has dimensions of 30 - 70 mm x 30 -70 mm, preferably of 50 mm x 50 mm, while the thickness can vary depending on the application between 0.1 and 2.5 mm, preferably between 0.1 and 1.0 mm.

The electrospun material is obtained by inserting, in the electrospinning instrument, a mixture of two solutions.

The first solution is composed of extracellular matrix (ECM) obtained by decellularizing pericardium of bovine origin.

The decellularization process consists of three steps, the first of which provides for the immersion of the pericardium in a solution of 8 mM CHAPS, 25 mM EOT A, 1 M NaCl, for 1 h at 37 °C under stirring; the second step instead provides for the immersion in a solution of 18 mM SDS, 25 mM EDTA, 1 M NaCl to be kept for 1 h at 37 °C under stirring, finally the pericardium is immersed in a solution of 0.02 mg/ml DNAse, 0.1 M MgCh, 1 M NaCl for 16 h at room temperature. The decellularized matrix is subsequently lyophilized for 16 h at -50 °C temperature and about 0 Pa pressure. The lyophilized tissue is then pulverized by means of suitable grinding machinery, obtaining particles of size variable between 10 and 2000 pm. The pulverized matrix is suspended at a concentration of 10 mg/ml, in a solution of 0.01 M hydrochloric acid and 1 mg/ml of pepsin, so as to proteolyse the ECM particles into water-soluble peptides. All this is incubated under stirring for 65 hours.

At the end, in order to deactivate the pepsin, the pH of the solution is brought to 7 by adding 1 M NaOH and PBS (Phosphate-buffered saline, 1.37 M NaCl, 27 mM KC1, 100 mM Na₂HP0₄, 18 mM KH2PO4).

The obtained solution is once again lyophilized for 16 h at -50 °C temperature and about 0 Pa pressure, and the obtained matrix is diluted in a range between 10% and 20% weight/volume in a solution 90% acetic acid and 10% water.

The second solution is obtained by dissolving gelatin derived from pig skin, which can be found on the market, in a range variable between 10% and 40% by weight/volume in a solution variable between 90% and 100% acetic acid and between 0 and 10% water.

The two solutions are mixed in a ratio variable between 2/1 and 1/3 matrix/gelatin, and a crosslinking agent is added to these mixtures, such agent varying between 3- glycidoxypropyltrimethoxysilane (GPTMS), genepm, 1 -Ethyl-3 -(3- dimethylaminopropyl)carbodiimide (EDC). The mixture thus obtained is placed under stirring for a time variable between 40 minutes and 2 hours depending on the crosslinking agent.

The electrospinner is composed of a syringe with metallic needle, with gauge comprised between 0.2 and 1.5 mm, connected to a pump from which the mixture is injected, and of a manifold constituted by two rotating cylinders on which a sheet of material capable of conducting electricity is placed.

Between the syringe and the manifold, an electrical voltage comprised between 10 and 50 kV is placed. The liquid flow made to pass through the needle is comprised between 0.5 and 3 ml/h, while the distance between the needle and the manifold varies between 5 and 25 cm. At the end of the process, a material will be obtained that is composed of fibers comprised between 0.3 and 10 pm, with a porosity variable between 15% and 50% with respect to the surface of the material.

In order to ensure the best possible performances of the material, the electrospun layer must fall within specific physical parameters. In particular, the Young’s modulus must be comprised between 15 and 70 MPa, the tensile strength between 100 and 900 kPa. A further important parameter is the permeability, which for water is on the order of magnitude of 10- 14 m², for cell culture terrain is higher and on the order of magnitude of 10-12 m². This represents an advantage, since the culture terrain is constituted by a mixture of sugars and proteins, and hence for some surgical applications, where the layer is for example soaked with platelet-rich plasma (PRP, with chemical composition very close to the culture terrain) in order to facilitate the tissue regeneration, the electrospun patch can ensure greater absorption of PRP, and in particular of the growth factors present in the PRP which contribute to speeding up the regenerative process of the damaged tissue.

The layer can be divided into pieces with 10 mm x 10 mm minimum size, by means of strap, using pre-pi eced sections obtained by means of molding during production phase.

The kit can be used both in outpatient context and autonomously by the final user as self- medication. One of the main uses of the kit regards the treatment of ulcers, which can be treated by differently combining the elements of the kit depending on the size and on the depth of the ulcer itself. In the case of surface ulcers, which only regard the epidermis, the layer of electrospun material can be used on its own, while in the case of deep ulcers the wound is first filled with the gel (of autologous derivation or hydrogel for tissue regeneration of animal or synthetic origin) and subsequently covered with the electrospun layer. The layer can also be associated with a cover for the use of the therapy with negative pressure, which is commonly used in treating wounds, both of traumatic and chronic nature, such as the pressure ulcers or in the case of ulcers from diabetic foot.

Other possible applications fall within the scope of aesthetic surgery, and it can also be used in the cosmetics industry.

Description of the figures

The invention will be described in detail hereinbelow also with reference to the enclosed figures, in which:

FIGURE 1 shows an image of the electrospun material obtained from a solution of 15% by weight of ECM and 30% by weight of gelatin in 1:1 ratio, with the addition of GPTMS at 6%, by means of the following parameters: Flux: 2.20ml/h; Voltage: 50 kV. Distance between manifold and needle: 20 cm. Needle diameter: 0.72 mm.

FIGURE 2 shows the SEM images of the electrospun material and distribution of the fiber dimensions.

FIGURE 3 shows the SEM images of the electrospun material after various incubation times. FIGURE 4 shows the graph for measuring the fibers of the electrospun material after the different incubation times.

FIGURE 5 shows the cellular vitality by means of MTS assay. FIGURE 6 shows the microscopic analysis of the cellular morphology. Detailed description of the invention

In one of the preferred embodiments according to the present invention, the electrospun layer, obtainable starting from bovine pericardium and gelatin, was subjected to characterization tests, such as the measurement of the fibers and of the porosity by means of scanning electron microscope (SEM). The fibers were measured and show an average diameter of 0.9 pm. The diameter is nevertheless quite heterogeneous and it is possible to show fibers with diameter less than 0.5 pm, just like fibers having a diameter of about 2-2.5 pm, while with regard to the porosity of the images one obtained a porosity of about 25% with respect to the surface of the material (Fig. 2).

Mechanical tests have also been carried out, in order to measure the elasticity and the strength of the material subjected to uniaxial traction.

Specifically: Elasticity Property (Young’s modulus, MPa); Tensile strength (kPa) Elongation at break and tenacity (kJ*m^‘3): Measurement 44.07 ± 13.41 637.46 ± 271.29 0.17 ± 0.006

5.65 ± 3.48. Table 1. Mechanical properties of the electrospun material

Subsequently, the permeability of the material was evaluated so to be able to evaluate the capacity of the electrospun layer to be soaked with a fluid: (permeability of cell culture terrain = (1.49 ± 0.30)* 10^'14 for water and (2.47 ± 0.27)* 1 O^'12 for the cell culture terrain).

Table 2. Values of permeability of the electrospun material

From these results, it is inferred that the permeability in the cell culture terrain is higher with respect to water, an aspect due to the chemical composition of the culture terrain, rich in proteins and carbohydrates, which therefore has a greater affinity with the material composed of proteins.

The degree of degradation of the fibers was also evaluated, following the subjection of the material at physiological conditions. The material was immersed in culture terrain, incubated at 37 °C and with a controlled atmosphere (5% C02) for a time up to 2 months, and the degradation of the fibers was evaluated both morphologically and by means of measuring the thickness of the fibers.

With regard to quality, it is possible to appreciate the great difference that is observed at the different times due above all to a decrease in the density of the fibers and to a progressive loss of integrity of the same.

After two months, the fibers appear highly segmented and rough, a sign of loss of the initial compactness of the material that is quite visible at time 0 (Fig. 3). The measurement well illustrates the loss of initial heterogeneity of the fibers and the progressive decrease of the average diameter of the same. Such decrease is quite pronounced between time 0 and time 1 month, while between 1 month and 2 months the diameter does not decrease in such an evident manner but shows a further loss of heterogeneity of the fibers (Fig. 4). It is likely that the degradation times can decrease when in the presence of biological fluids and processes which intervene during the tissue regeneration, due to the presence of proteolytic enzymes absent in our experimental test.

In order to verify the biocompatibility of the material, the vitality of keratinocytes - cells which are found in the epidermis - was measured, diffused on the electrospun material and on a control in plastic optimized for cell adhesion and cell growth, used for comparison purposes.

The results show that there are no significant differences in terms of vitality between the material and the control, for any of the considered time points. Hence the electrospun material has a capacity for cell adhesion and cell proliferation suitable for the type of cells present in the tissue, which must facilitate the regeneration (Fig. 5).

Finally, the cellular morphology was studied by means of fluorescence microscope (Fig. 6). The images confirm that with regard to numbers, there are no significant differences between the cells adhered on the electrospun material and on the control on plastic; with regard to the morphology - even if the form of the single cells does not change between the control and the electrospun material - on the electrospun material the cells are more grouped, a characteristic which can represent an advantage in the in vivo application for the formation of a cellular single-layer which induces the regeneration of the tissue. Bibliography

Bhattacharya S, Mishra RK. Pressure ulcers: Current understanding and newer modalities of treatment. Indian JPlast Surg. 20J5;48(1):4-16.

Westby MJ, Dumville JC, Soares MO, Stubbs N, Norman G. Dressings and topical agents for treating pressure ulcers. Cochrane Database of Systematic Reviews 2017, Issue 6. Art.

No.: CD011947.

Claims

1. Process for obtaining an electrospun layer, said process providing that said layer is obtained by inserting, in an electrospinning instrument, a mixture of two solutions: a first solution being composed of an extracellular matrix obtained by decellularizing bovine pericardium, in which the decellularization process provides for:

- the immersion of the pericardium in a solution of 8 mM CHAPS, 25 mM EOT A, 1 M NaCl, for 1 h at 37 °C under stirring; the subsequent immersion in a solution of 18 mM SDS, 25 mM EDTA, 1 M NaCl to be kept for 1 h at 37 °C under stirring;

- the immersion in a solution of 0.02 mg/ml DNAse, 0.1 M MgCh, 1M NaCl for 16 h at room temperature; said decellularized matrix being subsequently lyophilized for 16 h at -50 °C temperature and about 0 Pa pressure up to obtaining a lyophilized tissue, the latter then being pulverized by means of suitable grinding machinery, obtaining particles of size variable between 10 and 2000 pm, said pulverized matrix being suspended at a concentration of 10 mg/ml, in a solution of 0.01 M hydrochloric acid and 1 mg/ml pepsin, so as to proteolyse the ECM particles into water-soluble peptides, all being incubated under stirring for 65 hours; said process then providing for the deactivation of the pepsin, bringing the pH of the solution to 7 by adding 1 M NaOH and PBS, the latter being phosphate-buffered saline,

1.37 M NaCl, 27 mM KC1, 100 mM Na2HP0₄, 18 mM KH2PO4; the solution thus obtained being newly lyophilized for 16 h at -50 °C temperature and about 0 Pa pressure up to obtaining a matrix that is then diluted in a range between 10% and 20% weight/volume in a solution of 90% acetic acid and 10% water; said process also providing that:

- the second solution is obtained by dissolving gelatin derived from pig skin, which can be found on the market, in a range variable between 10% and 40% by weight/volume in a solution variable between 90% and 100% acetic acid and between 0 and 10% water, said two solutions being mixed in a ratio variable between 2/1 and 1/3 matrix/gelatin, a crosslinking agent then being added to these mixtures that is selected in the group constituted by: 3 -glycidoxypropyltrimethoxy silane (GPTMS), genipin, 1 -Ethyl-3 -(3- dimethylaminopropyl)carbodiimide (EDC); said mixture thus obtained being placed under stirring for a time variable between 40 minutes and 2 hours depending on the crosslinking agent; said process also providing that the electrospinner be composed of a syringe with metallic needle, with gauge comprised between 0.2 and 1.5 mm, connected to a pump from which the mixture is injected, and of a manifold constituted by two rotating cylinders on which a sheet of material is placed that is capable of conducting electricity; said process also providing that between the syringe and the manifold, an electrical voltage is placed comprised between 10 and 50 kV and that the liquid flow made to pass through the needle is comprised between 0.5 and 3 ml/h, while the distance between the needle and the manifold varies between 5 and 25 cm; said process finally providing for the obtainment of a material having fibers comprised between 0.3 and 10 pm, with a porosity variable between 15% and 50% with respect to the surface of the material.

2. Electrospun layer obtainable starting from an extracellular matrix from decellularized bovine pericardium and gelatin, said electrospun layer having fibers comprised between 0.3 and 10 pm with a porosity variable between 15% and 50% with respect to the surface of the material, said electrospun layer having a Young’s modulus comprised between 15 and 70 MPa, the tensile strength between 100 and 900kPa and a permeability per culture terrain of 10-12 m².

3. Electrospun layer according to the preceding claim having a Young’s modulus of 44.07± 13.41 MPa, a tensile strength of 637.46 ± 271.29 KPa and a tenacity of 5.65 ±

3.48Kj*m^-3.

4. Electrospun layer according to the preceding claim that can be divided into pieces of 10 mm x 10 mm minimum size, said electrospun layer having pre-pi erced straps.

5. Kit comprising at least one electrospun layer as defined according to any one of claims

2 - 4.

6. Kit according to the preceding claim further comprising a gel, the latter being a hydrogel for tissue regeneration of animal or synthetic origin or of autologous derivation.

7. Kit according to the preceding claim wherein the gel is contained in a syringe.

8. Electrospun layer according to any one of claims 2 - 4 for use as medication.

9. Electrospun layer for use according to the preceding claim in treating skin ulcers.

10. Electrospun layer for use according to the preceding claim in a method for treating diabetic foot.

11. Electrospun layer for use according to claim 8 in an esthetic surgery treatment method.

12. Kit according to one of claims 5 - 7 for use as medication.

13. Kit for use according to the preceding claim in treating skin ulcers.

14. Kit for use according to the preceding claim in a method for treating diabetic foot.

15. Kit for use according to claim 12 for use in an aesthetic suigeiy treatment method.