Technical field

[0001] The present disclosure relates to a matrix or a mesh composition comprising collagen nanofibres and agglomerates of calcium phosphate for the repair and/or regeneration of tissues and in treatment of bone diseases, osteoarthritis or skin diseases.

Background

[0002] Several strategies in the field of bioengineering of skin have been investigated in order to overcome the limitations of conventional approaches, such as full-thickness skin grafts or tissue transplant. In particular, in the treatment of burned skin there is a need to prevent infection while promoting the reconstruction of several layers of tissue, such as the dermis and hypodermis near the bone tissue. Numerous types of cells, molecules and ions are recruited to the site of injury because they have a leading role in this process. For example, mesenchymal cells can differentiate themselves into bone or skin cells according to the constitution of the surrounding media in the reconstruction of various tissues. They are also important in the differentiation of macrophages in order to decrease inflammation levels. It is described in the literature that calcium ions play an important role in the regeneration and repair of skin. In addition, in a more advanced stage of the skin regeneration process, these ions regulate the activation of metalloproteinases that play a role of matrix degradation so that there is subsequent recruitment and adhesion of epidermal cells, namely keratinocytes. They are also relevant in the growth and differentiation of keratinocytes. All these factors led to consider the use of the above described composite for the regeneration and repair of skin.

[0003] The electrospinning technique allows the production of structures constituted by fibres whose diameters range from a few microns to less than 10 nm, much lower than those reported by other conventional methodologies. [0004] Until now, all collagen-hydroxyapatite composites obtained by electrospinning were prepared from a mixture (or dope) of collagen and hydroxyapatite (HA). As a consequence, the subsequently formed composite's surface was covered with collagen or HA. This fact prevents direct cell/protein contact with both organic and inorganic components as it occurs in the natural bone environment (Hild, N.; Schneider, O. D.; Mohn, D.; Luechinger, N. A.; Koehler, F. M.; Hofmann, S.; Vetsch, J. R.; Thimm, B. W.; Muller, R.; Stark, W. J. Nonoscole 2011, 3, 401-409 and Teng, S. H.; Lee, E. J.; Wang, P.; Kim, H. E. Mater Lett 2008, 62, 3055-3058). There is also the need to apply non-cytotoxic reagents during the manufacturing processes used in the development of materials for biomedical and biological scientific research application. Since natural polymer solutions, including collagen, are very difficult to solubilize and to originate electrospun fibres, most published works report the use of toxic organic solvents, such as 1,1,1,3,3,3- hexafluoro- 2-propanol, which besides being highly toxic, partially denatures the native structure of collagen. It is also common practice to add synthetic polymers such as polycaprolactone to the collagen solution. However, the chemicals or monomers released by the degradation of such polymer may induce local and systemic host reactions that may cause clinical problems.

[0005] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

General description

[0006] The increased life expectancy in contemporary societies raises growing social and economic concerns in the treatment of diseases associated with aging, such as skin repair and renewal, osteoporotic bone loss, cancer related bone metastasis, as well as degeneration of the cartilage tissue. Accordingly, it is crucial to produce biomaterials similar to extracellular matrices of the various tissues that can be chemically modified by the immobilization of molecules, growth factors or proteins to promote cell response for the regeneration and reconstruction of skin, cartilage or bone. Calcium phosphate/collagen composites are ideal biomaterials for tissue engineering applications and for regenerative medicine since they mimic bone extracellular matrix components, such as the inorganic part (calcium phosphate) and organic part (collagen nanofibres). The application of these composites also covers skin repair and regeneration.

[0007] The mesh composition/biocomposite material of the invention surprisingly disclosed component combination and structure renders a more effective mesh composition suitable for mimic the bone tissue and it is also absorbable by the organism after the reconstitution of the tissue, this new mesh composition allows a better and effective tissue (bone, skin, cartilage) regeneration.

[0008] The present solution describes, in particular, a three-dimensional composite structure or mesh (network) made of collagen and calcium phosphate in particular, with a thickness exceeding 0.2 mm, capable of promoting regeneration and reconstruction of bone tissue, cartilage and skin. It may also serve to support active agents in therapy of bone diseases, skin or cartilage. Some examples of applications include bone and skin infections, dermatitis, burns, osteoarthritis, as well as skin aging. It can also be used as a model substrate for cell culture experiments and tests in scientific research area.

[0009] In view of the drawbacks to the prior art, the disclosure subject matter describes a mesh (network) composition to improve the treatment, reparation and/or regeneration of tissues and in the treatment of bone diseases, osteoarthritis or skin diseases.

[0010] The disclosure subject matter is related a mesh composition for repairing or the regeneration of tissues comprising collagen fibres obtainable by electrospinning; calcium phosphate agglomerates obtainable by electrospraying; the collagen fibres having a diameter less than or equal to 100 nm and the calcium phosphate agglomerates having a size less than or equal to 1.5 μιη.

[0011] The innovative mesh composition/biocomposite material of the present subject-matter forms a network of collagen nanofibers whose average diameters are 30 nm and nanocrystalline agglomerates of calcium phosphates, of dimensions less than 100 nm, processed using non-toxic solvents.

[0012] In an embodiment, the collagen fibres of the mesh composition of the present invention may have a diameter inferior to 50 nm, preferably inferior to 30 nm.

[0013] In an embodiment, the calcium phosphate agglomerates of the mesh may comprise calcium phosphate crystal with a size between 8 - 100 nm.

[0014] In an embodiment, the calcium phosphate agglomerates of the mesh composition of the present invention may have a size between 50 - 1500 nm.

[0015] In an embodiment, the ratio of collagen and calcium phosphate may be 20:1 - 1:1, preferably 10:1 - 2:1, more preferably 5:1 - 2:1.

[0016] In an embodiment, the collagen fibres of the mesh composition of the present invention may be type I collagen, type II collagen, type III collagen, type IV collagen or their mixtures.

[0017] In an embodiment, the mesh composition of the present disclosure may be woven or nonwoven.

[0018] In an embodiment, said collagen fibres can be specifically oriented, in particular with a parallel alignment.

[0019] In an embodiment, the mesh composition of the present invention is interwoven mesh or oriented type I collagen or mixtures of type I collagen and type III collagen.

[0020] In an embodiment, the calcium phosphate agglomerates can be nanophased hydroxyapatite, carbonated hydroxyapatite, fluoroapatite, beta-tricalcium phosphate, tetracalcium phosphate or octacalcium phosphate or their mixtures; hydroxyapatite partially replaced with Se ^2~ , Ba ²⁺ , Sr ²⁺ , Zn ²⁺ , Ag ⁺ , Sb ^3~ , Si ⁴⁺ , Mg ²⁺ or F ^" ions or their mixtures; calcium phosphate agglomerates are mixtures of hydroxyapatite with bioactive glasses or T1O2 or ZnO or their mixtures.

[0021] In an embodiment, the mesh composition of the present invention may further comprises an active agent that may be selected from the following list of growth factors, amino acids, proteins, protein fragments, peptides, nucleic acids.

[0022] In an embodiment, the proteins comprised in the mesh may be selected from the following list: fibronectin, vitronectin, and osteopontin, sialoprotein, osteonectin, chondronectin, glucosoamines, cartilage morphogenetic proteins, keratin, elastin or their mixtures.

[0023] In an embodiment, the mesh composition of the present invention further may comprise hyaluronic acid, urea or their mixtures.

[0024] In an embodiment, the mesh composition of the present invention may further comprise a crosslinking agent selected from the following list: N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide, N-hydroxysuccinimide, transglutaminase enzymes tyrosinase enzymes and lysyl oxidase enzymes or their mixtures.

[0025] In an embodiment, the mesh composition of the present invention may have a two-dimensional or three-dimensional shape.

[0026] In an embodiment, the mesh composition of the present invention may have a thickness between 100 - 5000 μιη.

[0027] In an embodiment, the mesh composition of the present invention may be for use in veterinary or human medicine or cosmetic, in particular in the treatment of bone diseases, bone defects, bone regeneration, bone reconstruction, bone infections, cartilage diseases, cartilage regeneration, cartilage reconstruction, oncologic diseases, skin lesions, skin infections, skin rejuvenation, dermatitis, burns, osteoarthritis, skin aging. [0028] Another aspect of the present subject-matter is related to a mesh, a strip, net, fibres bundle or scaffolds comprising the mesh composition of the disclosure subject matter.

[0029] Another aspect of the present subject-matter is related to a medical prosthesis that may comprise the mesh composition wherein said mesh composition is a prosthesis coating. Furthermore, the medical prosthesis may be a dental implant or a bone implant.

[0030] Another aspect of the present subject-matter relates to a method for obtaining the mesh, previously described, and comprising the following steps:

preparing a collagen solution with a concentration of 1% - 50% (w/v);

preparing a calcium phosphate suspension with a concentration of 0.5% - 80%

(v/v);

electrospinning of the collagen solution;

electrospraying of the calcium phosphate suspension with a concentration of 0.5% - 80% (v/v),

additionally adding a crosslink agent.

[0031] In an embodiment, the electrospinning and electrospraying steps are performed simultaneously.

[0032] In an embodiment, the collagen solution used has a concentration of 1% - 50% (w/v), preferably 5 - 30 % (w/v), more preferably 10% - 15% (w/v) and the collagen is dissolved in non-denaturing conditions and using non-toxic solvents selected from the following list: acetic acid, water, ethyl acetate, or mixtures thereof, in particular the collagen is dissolved in a solution of acetic acid, water, ethyl acetate with a ratio of 40:30:30, respectively.

[0033] In an embodiment, the suspension of calcium phosphate nanocrystals has a concentration of 0.5% - 80% (v/v), preferably 1% - 20%, more preferably 2.5% - 5% (v/v). [0034] The fibres diameter or the granulates/agglomerates size (scanning electron microscopy), can be measured with a custom code image analysis implemented in the program ImageJ as described in (Ribeiro N., Sousa S.R., van Blitterswijk C.A., Moroni L, Monteiro F.J. (2014) A biocomposite of collagen nanofibres and nanohydroxyapatite for bone regeneration. Biofabrication 6 035015).

[0035] In an embodiment the mesh composition/biocomposite product of the present invention mimics the extracellular matrix of a tissue, in particular a trabecular bone structure, and comprises an interwoven mesh or oriented collagen type I or mixtures of collagen type I and type III with an average diameter of 30 nm, significantly lower than those reported in the literature, typically above 200 nm, incorporating agglomerates of calcium phosphate nanocrystals. Examples of calcium phosphate agglomerated structures can be nanophased HA or carbonated HA or fluoroapatite, or beta-tricalcium phosphate or tetracalcium phosphate and octacalcium phosphate, or HA partially replaced with Se ^2~ ions or Ba ²⁺ , or Sr ²⁺ , or Zn ²⁺ or Ag ⁺ , or Sb ^3~ , or Si ⁴⁺ , or Mg ²⁺ , or F ^~ , or mixtures of HA with bioactive glasses or titanium oxide or zinc oxide.

[0036] In an embodiment, the results obtained indicate that, in an unexpected way, the use of an aqueous-based solvent allows to obtain nanoscale fibres with diameters of 30 nm. As mentioned above, the structure of the biocomposite material now disclosed and its chemical composition mimic extra-celular bone matrix, facilitating the access of cells, proteins or peptides or other macromolecules such as cell growth factors to both organic and inorganic components of the mesh/biocomposite. This mesh/biocomposite is obtained by simultaneous use of the techniques: electrospinning of collagen and electrospraying of nanostructured calcium phosphates, operating with non-denaturing and non-toxic solvents. The crosslinking of collagen nanofibres is performed using also non-toxic substances (such as NHS/EDC) or enzymes able to catalyze the formation of covalent intra- or inter-chain of the polymer. This product can be re-sized to the desired scale in the form of two-dimensional structures, like membranes or films or coatings, or presented as three-dimensional structures (scaffolds). The unique network architecture consisting of an interwoven or oriented mesh of collagen nanofibres incorporating agglomerates of calcium phosphate nanocrystals allows access to both organic and inorganic components, as occurs in natural bone environment. This innovative mesh/biocomposite is important for regenerating various connective tissues and in the treatment of bone diseases, osteoarthritis or skin diseases. This material is cytocompatible and suitable for adhesion and growth of bone cells (osteoblasts) as well as undifferentiated stem cells, skin cells (fibroblasts, keratinocytes), endothelial cells and their progenitors, tumour cells derivated from cancer that metastasize preferentially to bone.

[0037] This material/mesh allows binding to proteins, peptides, protein fragments, growth factors and other molecules that interfere with bone regeneration mechanisms. For example, the immobilization of adhesive proteins such as fibronectin, vitronectin, osteopontin, bone sialoprotein and fibrinogen, facilitating the adhesion and proliferation of osteoblasts or differentiation of stem cells to osteoblasts. It also facilitates the simultaneous adhesion of endothelial cells and their progenitors that causes vascularization of regenerated tissues, allowing the supply of oxygen and nutrients that are essential for the survival of the new tissue cells as well as the removal of their metabolites. In addition, it allows the immobilization of extracellular matrix counter- adhesive proteins such as osteonectin, which influences and regulates the formation of new bone via mechanisms of collagen fibrils binding to calcium phosphate crystals, promoting the mineralization of new matrix. This material also supports the interaction between the osteonectin and tumour cells that metastasize to the bone, for countering this phenomenon.

[0038] Its application may cover the regeneration of cartilage tissue, adapting to the collagen type required (type II), following a procedure similar to that described above, but involving chondrocytes adhesion and proteins such as chondronectin. This material can be applied to skin regeneration, reconstruction or its rejuvenation through tailoring the collagen type, and engineering the surface with proteins or other macromolecules involved in these processes, such as keratin, elastin, hyaluronic acid, nucleic acids and urea, and supporting relevant cells for these processes like fibroblasts, keratinocytes and epithelial cells.

[0039] In an embodiment, the material/mesh composition can be used as a model/cell supporting substrate in order to mimic the physiological environment of bone matrix, cartilage or dermis, and replicate disease states such as cancers that metastasize preferentially to bone, skin contraction, diabetic wounds, pigmentation, burns, wound healing or osteoarthritis surprisingly improving the tissue regeneration. Moreover, this material can be used in orthopaedics, dentistry, maxillofacial surgery, plastic surgery in the treatment of burns, dermatology, repair and skin rejuvenation (cosmetics), in oncology for treating bone metastases or even serve as a model in the area scientific research for the prevention/treatment of bone diseases, osteoarthritis or skin diseases.

[0040] The present solution describes a mesh composition/structured biocomposite material comprising collagen nanofibres and calcium phosphate nanocrystalline agglomerates to be use as a supporting substrate for cell tissue regeneration and controlled drug release, while permitting the immobilization of active agents.

[0041] The present disclosure also describes the use of the mesh/biocomposite material for regeneration of skin in dermatologic applications, in particular in regeneration and reconstruction of burned skin; for the regeneration of bone tissue and cartilaginous tissue; for the treatment of bone diseases and/or for applications in orthopaedics, maxillofacial surgery.

[0042] This mesh composition/biocomposite material is produced by simultaneously combining two methodologies, namely electrospinning of a collagen solution to generate nanofibres, and electrospraying of a calcium phosphate nanocrystals suspension. The collagen solution used for electrospinning has a concentration of 1% - 50% (w/v), preferably 10% - 15% (w/v). The calcium phosphate suspension for electrospraying has a concentration of 0.5% - 80% (v/v), preferably 1% - 20%, more preferably between 2.5% and 5% (v/v). [0043] In an embodiment the mesh composition/biocomposite material may comprise collagen nanofibres that may be either randomly distributed, or oriented, preferably parallelly and it further comprises randomly distributed calcium phosphate agglomerates that may be hydroxyapatite nanocrystals.

[0044] The mesh/biocomposite material also comprises collagen fibrils with a diameter less than 100 nm, preferably less than 50 nm, and more preferably less than 30 nm, while the calcium phosphate nanocrystals used have a size between 8 to 100 nm.

[0045] The mesh/biocomposite material according to any preceding claim, wherein the size of agglomerates of calcium phosphate nanocrystals is less than 1.5 μιη.

[0046] The mesh/biocomposite material comprises a ratio of collagen and calcium phosphate between 20:1 - 1:1, preferably between 10:1 to 2:1, more preferably between 5:1 to 2:1, respectively

[0047] The solvent used to prepare the solution of collagen and calcium phosphate suspension is an aqueous-based solvent. The collagen solution is dissolved under non- denaturing conditions and using non-toxic solvents, selected from the following list including acetic acid, water, ethyl acetate, or mixtures thereof. In the particular case of using a mixture of acetic acid, water, ethyl acetate the ratio should preferably be of 40:30:30, respectively.

[0048] In an embodiment, the type I collagen, comprised in the mesh/biocomposite material, is cross linked between fibrils through the use of non-toxic cross linkers, including N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide and N-hydroxysuccinimide (EDC NHS), or enzymes capable of catalysing the formation of covalent bonds, including transglutaminases, tyrosinases and lysyl oxidase.

[0049] In an embodiment, the calcium phosphate comprising the mesh/biocomposite material may be selected from hydroxyapatite, carbonated hydroxyapatite, fluoroapatite, tricalcium phosphate or tetracalcium beta, octacalcium, or any combination of these phosphates, as well as non-stoichiometric hydroxyapatite, where Ca ²⁺ ions or hydroxyl ions are partially replaced by Ba ²⁺ , Sr ²⁺ , Zn ²⁺ , Ag ⁺ , Si ⁴⁺ , Mg ²⁺ , Se ^2~ ,Sb ^3~ , or F ^" ions, or mixtures thereof, or mixtures of calcium phosphates and metal oxides such as titanium dioxide or zinc oxide.

[0050] In an embodiment, the collagen fibres comprising the mesh/biocomposite material may be collagen type I, collagen type II, collagen IV, and may include mixtures of collagen type I and III.

[0051] In an embodiment, the mesh composition/biocomposite material is functionalized with an active agent selected from growth factors, amino acids, proteins, protein fragments, or peptides.

[0052] In an embodiment, the mesh composition/biocomposite material is cytocompatible according to the preceding claims for adhesion, growth, proliferation and differentiation of cells selected from osteoblasts, fibroblasts, endothelial cells, epithelial cells, chondrocytes, keratinocytes and tumor cells.

[0053] In an embodiment, the mesh composition/biocomposite material is functionalized with proteins selected among chondronectin, glucosoamines and cartilage morphogenetic proteins, with adhesive proteins selected among fibronectin, vitronectin, and osteopontin, sialoprotein, with active agents and skin proteins selected among keratin, elastin, hyaluronic acid, nucleic acids and urea. Furthermore, the mesh composition/biocomposite may also be functionalized with counter-adhesion proteins, preferably with osteonectin.

[0054] Furthermore, the present disclosure may be used for bone regeneration and reconstruction-based method for treatment of bone diseases and oncological applications, to combat bone metastases from primary tumors that metastasize preferentially to the bone, as prostate cancer and breast cancer. [0055] The present disclosure may also be used as a method of skin and/or cartilage tissue regeneration and reconstruction using the mesh composition/biocomposite material for the treatment of skin lesions, skin rejuvenation treatments, cartilage tissue diseases and/or lesions.

[0056] The use of the mesh composition/biocomposite material structures may also be used for promoting regeneration through the transport to the host of undifferentiated stem cells or progenitor cells of tissues or co-cultures of these cells from the host.

[0057] In an embodiment the structured mesh/biocomposite material may be use as a model matrix for the research of regeneration and reconstruction of tissues and cell adhesion, growth, proliferation and differentiation.

[0058] In an embodiment, in order to obtain the collagen fibres with calcium phosphate in better conditions, the fibres may be produced by

electrospinning of collagen with a relative humidity between 20 - 60%, preferably between 30 - 45%; the distance from the rotating cylinder and the needle tip of electrospinning and

calcium phosphate are obtaining by electrospraying, wherein the electrospraying devices may be between 80 - 200 mm, preferably 120 mm; the flow rate of electrospraying may be between 0.5 - 3 ml h ^"1 , preferably 2 ml h ^"1 ;

the flow rate of electrospinning may be between 0.05 - 0.5 ml hf ¹ , preferably 0.1 ml h ^"1 ; the samples obtained after electrospinning and electrospraying may be subjected to chemical cross-linking.

Brief description of the drawings

[0059] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of solution. [0060] Figure 1: A schematic diagram of the laboratory set-up used for the simultaneous electrospinning (1) and electrospraying (2) techniques and wherein (3) represents the collector.

[0061] Figure 2: FT-IR spectra of electrospun collagen (4) and collagen-nanoHA (5) composites obtained using the simultaneous electrospinning and electrospraying techniques.

[0062] Figure 3: SEM images of electrosprayed nanoHA (i), electrospun collagen nanofibres (ii) and collagen-nanoHA composites obtained using the simultaneous electrospinning and electrospraying techniques (iii).

[0063] Figure 4: A histogram of the electrospun collagen fibre diameter distribution obtained by electrospinning.

[0064] Figure 5: Surface topography of the electrospun collagen nanofibres (A) and the collagen-nanoHA composite (B): height (i), phase (ii) and 3D (iii) images. All AFM images were obtained under Tapping mode ^® (image scale 3 x 3 μιη ² ). Arrows indicate nanoHA agglomerates.

[0065] Figure 6: Metabolic activity (A) and morphology and cytoskeletal organization (B) of MC3T3-E1 cells cultured on the electrospun collagen nanofibres and the collagen- nanoHA composites obtained using simultaneous electrospinning and electrospraying techniques versus time. In (A) the results are expressed in terms of relative fluorescence units (RFU); in (B) F-actin is indicated in red while the cells' nuclei were counterstained in blue with DAPI dye. MC3T3-E1 cells cultured on coverglasses coated with PDL were used as the control. Values are the average ± SD of six cultures. * p 0.05.

[0066] Figure 7: Metabolic activity (A) and morphology and cytoskeletal organization (B) of fibroblasts cultured on the collagen-nanoHA composites obtained using simultaneous electrospinning and electrospraying techniques versus time. In (A) the results are expressed in terms of relative fluorescence units (RFU), (B) SEM images. Fibroblasts cultured on coverglasses coated with PDL were used as the control. Values are the average ± SD of six cultures. * p<0.05

[0067] Figure 8: SEM images of the electrospun 1.5% collagen in 10% acetic acid at a rate of 0.6-4 mL/mL with an applied voltage of 15-18 kV.

[0068] Figure 9: SEM images of the electrospun 8% collagen in 30:20:50 (acetic acid ethyl acetate: water) at a rate of 0.6 mL/mL with an applied voltage of 20 kV.

Detailed description

[0069] The present solution describes a mesh composition/structured biocomposite material comprising collagen nanofibres and calcium phosphate nanocrystalline agglomerates for use as a support (substrate) for cell tissue regeneration and controlled release while allowing the immobilization of active agents.

[0070] The present disclosure also describes the use of the mesh composition/biocomposite material for regeneration of skin in dermatologic applications, in particular in regeneration and reconstruction of burned skin; for the regeneration of bone tissue and cartilaginous tissue; for the treatment of bone diseases and/or for applications in orthopaedics, maxillofacial surgery.

[0071] In an embodiment, it is described in detail and described herein a form of presentation of the biocomposite materials and their influence on the behaviour of osteoblastic cell line MC3T3-E1 cells and fibroblasts (Human Neonatal Dermal fibroblasts) with reference to the examples of Figures 1 to 7 described below. However, it is important to note that in vitro assays were performed with other cell types, including endothelial cells (Human Pulmonary microvasculature Endothelial Cells) and tumor prostate cancer cells (PC3) and microbiology assays. [0072] In an embodiment, the material is constituted by a synthetic 3D scaffold matrix that mimics the extracellular matrix of bone. Accordingly, a new approach has been implemented based on two simultaneously applied techniques, namely electrospinning of type I collagen and electrospraying of nanophased hydroxyapatite (nanoHA), operating under non-denaturing conditions and using non-toxic solvents. While for electrospinning of collagen an aqueous solution containing acetic acid and ethyl acetate was used, in the crosslinking method the reagents N-ethyl-N'- [3-dimethylaminopropyl] carbodiimide (EDC) and N-hydroxy succinimide (NHS) were used. The results obtained using scanning electron microscopy (SEM) or atomic force microcopy (AFM), revealed a 3D mesh composed by nanoHA agglomerates and collagen nanofibres whose diameters lie within nanometric scale (30.25 ± 23.25 nm) and are significantly lower than those reported in the literature, that typically exceed 200 nm. The collagen integrity as well as the presence of nanoHA in the composites formed by collagen and nanoHA were confirmed by Fourier transformed infrared spectroscopy (FT-IR). Finally the network architecture allows cells access to both collagen nanofibres and nanoHA crystals, as it occurs in the natural bone environment (extra-cellular matrix). The inclusion of nanoHA agglomerates by electrospraying into the type I collagen mesh induced high adhesion and metabolic activity both of the MC3T3-E1 osteoblasts and fibroblastic cells, whose morphology presented an elongated shape. The present disclosure indicates that this new collagen nanofibre-nanoHA composite has great potential for the regeneration of skin and bone, as well as for biomedical application in the treatment of bone and skin diseases.

[0073] Considerable attempts have been made to produce adequate matrices or scaffolds that mimic bone extracellular matrix (ECM) for applications in tissue engineering and regenerative medicine. These biomaterials should be specifically designed to be biocompatible, biodegradable and osteoconductive. Nanohydroxyapatite/collagen nanocomposites are ideal biomaterials for bone regeneration and target molecule delivery systems for the treatment of bone diseases. These types of biomaterials are suitable for bone contact and substitution, particularly novel natural polymer-based composites reinforced with bioactive components, such as nanophased hydroxyapatite (nanoHA). They represent respectively the major inorganic and organic component assembly as in natural bone, where the HA crystals of the mineral part are bound to collagen fibres, that correspond to 90-95% of the bone organic matrix. The mineral phase is responsible for providing adequate mechanical compressive strength, while collagen provides tensile properties and flexibility.

[0074] Electrospinning has recently attracted great interest in generating nanoscale fibres of biomaterials ranging from polymers and ceramics to their composite fibrous scaffolds for tissue engineering applications, with fibres diameters range from a few microns to less than 100 nm. This type of nanofibrous structure is regarded as a promising architecture in the sense that natural bone ECM exhibits collagen fibrils with diameters ranging from 20 nm to 40 μιη, that are far smaller than those that can be achieved with conventional processing methods. The processing of natural polymers such as collagen, is highly complex, namely the production of collagen fibres by electrospinning. For that reason, synthetic polymers such polyglycolic acid (PGA), polyL-lactic acid (PLLA), polylacticcoglycolic acid (PLGA) or polycaprolactone (PCL) are often added to the collagen solution. However, the chemicals (additives, traces of catalysts, inhibitors) or monomers (glycolic acid, lactic acid) released from polymer degradation may induce local and systemic host reactions that may cause clinical problems. Another way to overcome this problem is the use of organic toxic reagents, mainly highly volatile fluoroalcohols such as 1,1,1,3,3,3- hexafluoro-2-propanol (HFP) and 2,2,2-trifluoroethanol (TFE). Natural polymers, including collagen, are very difficult to electrospin due to their high viscosity and low solubility in general organic solvents, as reported in most published works concerning the production of collagen fibrillar meshes (Hild, N.; Schneider, O. D.; Mohn, D.; Luechinger, N. A.; Koehler, F. M.; Hofmann, S.; Vetsch, J. R.; Thimm, B. W.; Muller, R.; Stark, W. J. Nanoscale 2011, 3, 401-409). However, these solvents are highly toxic and partially denature the native structure of collagen through the disruption of its characteristic triple-helical structure, decreasing its denaturation temperature and resulting in significant amounts of collagen lost during electrospinning (Zeugolis, D. I.; Khew, S. T.; Yew, E. S. Y.; Ekaputra, A. K.; Tong, Y. W.; Yung, L. Y. L; Hutmacher, D. W.; Sheppard, C; Raghunath, M. Biomaterials 2008, 29, 2293-2305). Increasing efforts towards applying non-toxic aqueous systems, such as PBS/ethanol or acetic acid, for medical applications have started to emerge. In addition, post-fabrication cross-linking confers mechanical strength through the binding of carboxylic groups between collagen fibrils, which is fundamental for in vitro assays and for the translation of these collagenous meshes in preclinical and clinical settings. In the present solution, EDC/NHS was used as a non-toxic cross-linking system, in contrast with most of the studies in the literature, that apply toxic reagents such as glutaraldehyde. Here, an innovative approach is reported based on two simultaneous methods, type I collagen electrospinning and nanophased HA electrospraying, using non-toxic reagents. Simultaneous electrospinning and electrospraying techniques have been applied to gelatin in only very few studies (Francis, L; Venugopal, J.; Prabhakaran, M. P.; Thavasi, V.; Marsano, E.; Ramakrishna, S. Acta Biomoter 2010, 6, 4100-4109 and Gupta, D.; Venugopal, J.; Mitra, S.; Dev, V. R. G.; Ramakrishna, S. Biomaterials 2009, 30, 2085-2094. The physicochemical properties of this mesh/biocomposite were investigated as well as its influence on MC3T3-E1 osteoblast and fibroblast cell performance in terms of morphology, adhesion and metabolic activity. This construct has revealed to have a non-cytotoxic effect and the ability to support osteoblasts and fibroblasts adhesion and viability.

[0075] Figure 1 illustrates the schematic diagram of the laboratory set-up used for the simultaneous electrospinning of collagen and electrospraying of nanoHA techniques. Type I collagen was suspended in acetic acid:ethyl acetate:water, in particular in a acetic acid:ethyl acetate:water solution with a ratio of40:30:30, respectively, to obtain a collagen solution suitable for electrospinning technique. The addition of ethyl acetate improved the spinnability of the nanofibres and reduced the acidity of the solvent (acetic acid). Since it was desired to preserve the nanometric scale of the HA agglomerates resulting from the electrospraying technique, a nanoHA gel (Fluidinova, nanoXIM»HApl02) was used instead of nanoHA powder. Also, the nanoHA solution was subjected to a set of ultrasonic cycles before the electrospraying process. The sizes of the HA agglomerates were assessed by Zetasizer Nano ZS. There was a steady decrease in size with increasing number of ultrasonic pulses. Comparing the HA agglomerates' size before and after ultrasonic pulse cycles, a reduction in size from 277.6 ± 29.9 nm to 126.3 ± 1.8 nm was observed. The collagenous integrity as well as the presence of nanoHA in the nanostructured collagen-nanoHA composite was confirmed by Fourier transformed infrared spectroscopy (FT-IR).

[0076] The spectrum of electrospun collagen nanofibres in Figure 2 depicts characteristic absorption bands at 1657, 1536 and 124 cm ^"1 , corresponding to amides I, II and III, respectively. The amide I absorption arises predominantly from protein amide C=0 stretching vibrations, amide II is made up of amide N-H bending vibrations and C-N stretching vibrations while amide III arises predominantly from C-N stretching and N-H in-plane bending from amide linkages. The integrity of collagen's triple helix can be evaluated by the ratio between the absorbance at 1235 and 1450 cm ^"1 . Ratio values for denatured collagen are around 0.5 and those for intact structures are around 1. For the analyzed samples, the value obtained was 1.07, indicating that the addition of nanoHA and the applied conditions did not de-stabilize the collagen's triple helix. There was no band at 1706 cm ^"1 , suggesting that there was no free acetic acid in the sample. Furthermore, the FT-IR spectrum of the collagen-nanoHA composites obtained using the simultaneous electrospinning and electrospraying techniques, in addition to the collagen characteristic bands above referred , revealed characteristic bands of nanophased HA, OH- vibrational (633 cm ^"1 ) bands and P0 ₄ ^3" (u3~1093 and 1032 cm ^"1 ; ul~ 962 cm ^"1 , u4 601 and 564 cm ^"1 ) bands. The characteristic bands of the carbonate group can also be observed, namely those corresponding to the u3 vibration of C-0 (1452 cm ^"1 ) and the u2 vibrations (875 cm ^"1 ). In addition to the Zeta sizer results, the nanometric scale of the nanoHA agglomerates was confirmed using scanning electron microscopy (SEM) image analysis, (Figure 3 (a)). The SEM images of the electrospun collagen revealed a random mesh of collagen nanofibres. The diameter measurements of twenty collagen fibres randomly chosen from six different SEM images, with a custom code image analysis implemented in the program ImageJ, allowed the calculation of average and median values, 37.2 ± 23.2 nm and 30.2 ± 23.2 nm, respectively (Figure 4). These diameter values are within the nanometer range (<100 nm) and are significantly lower than those reported in the literature, that typically exceed 200 nm. Figure 3 (c) shows a representative SEM image of the collagen-nanoHA composites obtained using simultaneous electrospinning and electrospraying techniques. A random arrangement of collagen nanofibres and irregular structures of nanoHA incorporated between them can be observed. In this nano-network both collagen and nanoHA are accessible, resembling the bone tissue extracellular matrix organization. Until now, all collagen-HA composites obtained by electrospinning were prepared from solutions containing mixtures of collagen and hydroxyapatite. As a consequence, and opposed to the system hereby presented, the composite surface typically was showing either only collagen , more commonly, or only by HA, thus preventing direct cell/protein contact with both organic and inorganic components (Hild, N.; Schneider, O. D.; Mohn, D.; Luechinger, N. A.; Koehler, F. M.; Hofmann, S.; Vetsch, J. R.; Thimm, B. W.; Muller, R.; Stark, W. J. Nanoscale 2011, 3, 401-409 and Teng, S. H.; Lee, E. J.; Wang, P.; Kim, H. E. Mater Lett 2008, 62, BOSS- BOSS). The cross-linking procedure did not affect the morphological arrangement of the electrospun meshes. Atomic force microcopy (AFM) studies confirmed the nanoscale dimensions of the collagen fibres, confirming the unprecedented resolution achieved with respect to the methodologies used so far. Moreover, the AFM images presented in Figure 5 A and B reveal a three-dimensional arrangement of collagen nanofibres. The capability of phase imaging AFM to distinguish samples with different surface viscoelastic properties enabled the visualization of the nanoHA agglomerates between the collagen nanofibres (Figura 5 B (ii)).

[0077] The diameters of twenty fibres randomly chosen from six different SEM (scanning electron microscopy) images, each one corresponding to a distinct sample, were measured with a custom code image analysis implemented in the program ImageJ as described in (Ribeiro N., Sousa S.R., van Blitterswijk C.A., Moroni L, Monteiro F.J. (2014) A biocomposite of collagen nanofibres and nanohydroxyapatite for bone regeneration. Biofabrication 6 035015).

[0078] The mesh/biocomposite new disclosed was also tested for in vitro cell studies. The network architecture of the mesh/biocomposite allows cells access to both collagen nanofibres and HA crystals, as it occurs in the natural bone environment. MC3T3-E1 cell performance in terms of cell metabolic activity, cell distribution and morphology was investigated for the 3D scaffolds produced herein and compared with a control comprised of coverglasses coated with PDL (Poly-D-Lysine hydrobromide).

[0079] The pattern of metabolic activity found in collagen/nanoHA composites was similar to control samples and higher when compared to the collagen fibres after 96 hours of cell culture (Figura 6 (A)). The cell distribution and morphology of MC3T3-E1 osteoblasts on the materials were followed by inverted epi-fluorescence microscopy after 4, 24 e 96 hours of cell culture. According to metabolic activity data, both collagen and biocomposite constructs presented a non-cytotoxic effect and had the ability to support osteoblast cell adhesion, as observed in Figure 6(B). At 24 hours of cell culture, MC3T3-E1 cells were attached and were spread out across the surface, presenting a characteristic elongated shape with fusiform fibroblastic appearance. The inclusion of nanoHA agglomerates by electrospraying within type I collagen mesh induced the appearence of these elongated shapes and improved the adhesion of osteoblasts after 96 hours of cell culture.

[0080] Regarding the fibroblasts cell study, the pattern of cell metabolic activity increased with time of culture in both substrates. This fact indicates that the biocomposite presented a non-cytotoxic effect and had the ability to support fibroblast cell adhesion and growth. At 7 days of cell culture, the metabolic activity values found in scaffolds were lower than those observed in the control samples. But after 14 days, a recovery of metabolic activity by the fibroblasts cultured on scaffolds was observed, exceeding the values found in control samples after 21 days of incubation Figure 7 (A). The SEM imaging analysis corroborated the metabolic activity data Figure 7 (B). The biocomposite influenced the morphology and distribution of the fibroblasts. In fact, the fibroblasts cultured on control surfaces acquired an elongated shape and cell guidance in their distribution. The same did not happen on mesh composition/biocomposite scaffold surface where fibroblasts appeared more spread and randomly distributed. After 14 days, the mesh composition/biocomposite somehow retrieved the oriented distribution found in other materials. According to SEM images it is possible to observe fibroblasts closely binding to the material and reaching a fusion state, making it difficult to distinguish, between parts of the cell and the substrate constituents.

[0081] Type I collagen was suspended in acetic acid:ethyl acetate:water (40:30:30) by stirring overnight at 4 °C to obtain a 12% (w/v) collagen suspension. The solution was loaded into a syringe (5 ml) with a 21 G needle and electrospun at 0.1 ml/h, under a high electrostatic field (20 kV) onto coverglasses attached on aluminium foil wrapped on a rotating cylinder collector. Simultaneously, electrospraying of nanoHA was carried out. NanoHA gel suspended in methanol was subjected to a set of ultrasonic cycles in order to decrease nanoparticle agglomeration. The solution was loaded into a syringe (10 mL) with a 21 G needle under a high electrostatic field (20 kV) onto the collagen fibres. The simultaneous electrospinning and electrospraying process was continuously performed over 1 h at room temperature (22 °C). The samples obtained were subjected to chemical cross-linking in ethanol 90% (v/v) containing 20 mM EDC and 10 mM NHS at 4 °C over 4 hours in the case of the electrospun collagen fibres and 24 hours in the case of the electrospun collagen fibres plus electrosprayed HA agglomerates. The cross-linked scaffolds were washed three times with ethanol 90% (v/v) and twice with water and dried overnight at room temperature in a desiccator before chemical and morphological characterization and cell culture studies.

[0082] The substrate characterization was performed as follows: The size of the HA agglomerates was determined using a Zetasizer Nano ZS (Malvern Instruments, U.K.), equipped with a 4mW HeNe laser beam with a wavelength of 633 nm and a scattering angle of 173°. The size measurements were performed, at 25 °C in a polystyrene cell (ZEN0040), using the 'General Mode' analysis model, which is suitable for the analysis of the majority of samples and dispersions. Size results were automatically calculated by the software, DTS Nano v.6.30, using the Stokes-Einstein equation. Chemical characterization of the developed structures was performed using Fourier transformed infrared spectroscopy (FT-IR), with a Perkin-Elmer 2000 FT-IR spectrometer. For this purpose, 0.2 g of sample material (electrospun collagen fibres or composites of collagen and nanoHA obtained by simultaneous electrospinning and electrospraying) was ground and analyzed as KBr pellets at a spectral resolution of 4 cm ^"1 . One hundred scans were accumulated per sample. The surface characterization of substrates was examined using scanning electron microscopy (SEM). SEM analyses were performed using a FEI Quanta 400FEG/EDAX GenesisX4M scanning electron microscope under high vacuum conditions. The samples were sputtercoated with a thin palladium-gold film, using a sputter coater (SPI-Module) in an argon atmosphere before observation. The diameters of twenty fibres randomly chosen from six different SEM images were measured with a custom code image analysis implemented in the program ImageJ. The results referred to as diameter measurements correspond to the average and median ± standard deviation (SD). Atomic force microscopy (AFM) studies were carried out using a Veeco Multimode NanoScope IVa scanning probe microscope. Each sample was imaged with a 16x16 μιη ² piezo-scanner. Imaging analyses were performed at room temperature, in Tapping mode ^® , using a silicon cantilever with a spring constant of 25-75 N/m (tip radius <10 nm).

[0083] In vitro cell culture studies were also carried out. MC3T3-E1 cells, established as an osteoblastic cell line from normal mouse calvaria, were grown in an alpha minimum essential medium (a-MEM, Gibco) supplemented with 10% (v/v) foetal bovine serum (FBS) (Invitrogen) and 1% penicillin-streptomycin (Gibco). Cells were cultured in 75 cm ² plastic culture flasks, and incubated in a humidified incubator (37 °C and 5% C0 ₂ ). Freshly confluent MC3T3-E1 cells were rinsed with PBS, followed by incubation in trypsin/EDTA (0.25% trypsin, 1 mM EDTA; Sigma) for 10 min at 37 °C and then re-suspended in supplemented medium. Fibroblasts were cultured in dermal fibroblast culture medium (DMEM, Gibco) supplemented with 15% (v/v) foetal bovine serum and 1% penicillin- streptomycin. Cells were cultured in 75 cm ² plastic culture flasks, and incubated in a humidified incubator (37 °C and 5% C0 ₂ ). Confluent cells were rinsed with PBS, followed by incubation in trypsin/EDTA (0.25% trypsin, 2.21 mM EDTA; Sigma) for 5 min at 37 °C and then re-suspended in supplemented medium. The substrates were sterilized by immersion in a series of dilute ethanol solutions of 90, 70 and 50% (v/v) over 10 min, and incubated with a- MEM for 30 min. After rinsing three times in PBS, the MC3T3-E1 cells were seeded on both substrates (electrospun collagen fibres and collagen-nanoHA composites obtained by co-electrospinning/electrospraying) at a cell seeding density of 4 x 10 ⁴ cells/well. While fibroblasts were seeded on collagen-nanoHA composites obtained by co-electrospinning/ electrospraying at a cell seeding density of 2500 cells /well. Coverglasses coated with Poly-D-lysine hydrobromide (PDL) were used as a control. MC3T3-E1 cells were cultured for periods of 4 h and 1 and 4 days. Fibroblasts were cultured for periods of 1, 7, 14 e 21 days. For each material and culture period, six samples without cells were incubated with complete medium in the same way and used as blanks. The cell metabolic activity of both cell types cultured on substrates after the culture period established was evaluated using a resazurin-based assay. Thus, 50 μΙ of resazurin (Sigma) at a concentration of 0.1 mg/mL were added to each well. After 3 h of reaction time, 100 μΙ of supernatant were transferred to the wells of a black-walled 96- well plate. Fluorescence was read using Xex = 530 nm and Xem = 590 nm in a microplate reader (Biotek, Synergy MX). The fluorescence value corresponding to the unseeded substrates was subtracted. The results correspond to the mean ± standard deviation of six cultured samples.

[0084] The MC3T3-E1 cell distribution and morphology on the 3D scaffolds was assessed using inverted epi-fluorescence microscopy. For immunostaining of the F-actin cytoskeleton and nuclei, the cell-seeded surfaces were rinsed twice with PBS and fixed with 4% para-formaldehyde for 15 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 for 5 min and incubated in 1% BSA for 30 min at room temperature. Cell cytoskeleton filamentous actin was visualized by treating the cells with Alexa Fluor ^® 594 Phalloidin (1:80 in BSA 1%, Molecular Probes ^® ) for 20 min in the dark. Finally the cells were washed with PBS and the cell nuclei were counterstained with 4', 6- diamidino-2-phenylindole (Vectashield/DAPI) dye. The images were acquired on an inverted epi-fluorescence microscope (Axiovert 200M, Zeiss) using a Axion Vision Rel. 4.8 software.

[0085] The fibroblast cell distribution and morphology cultured on substrates was assessed using scanning electron microscopy (SEM). Cell-seeded samples were fixed with 1.5% glutaraldehyde, dehydrated with an increasing ethanol-water gradient and dried using hexamethyldisilazane. SEM analyses were performed using the same scanning electron microscope equipment previously described. Samples were collected at days 1, 4, 7, 14 and 21 of fibroblasts culture on the substrates. Statistical analysis was assessed using t-Student test with a significance level of p 0.05.

[0086] In the present disclosure, a novel composite structure based on collagen nanofibres and nanoHA agglomerates was successfully obtained using simultaneous electrospinning/electrospraying. Its influence on MC3T3-E1 osteoblast and fibroblast cell growth was investigated. The collagen integrity as well as the nanoscale dimensions of both the biocomposite components (collagen and nanoHA) were preserved as confirmed by FT-IR spectra, and SEM and AFM image analysis. In the development of the construct, water-based solvents (ethyl acetate, acetic acid and water) and non-collagen denaturing conditions were applied. The diameters of the electrospun collagen nanofibres, estimated from the SEM images (30.25 ± 23.25 nm), are far below those stated in the literature, thus offering a roadmap to obtain a further level of biomimicry in matrix design strategies. This novel construct allows cells access to both collagen nanofibres and HA crystals, as it happens in the natural bone micro-and nano-environments. Regarding interactions with cells, these structures were cytocompatible and able to withstand adhesion and growth of MC3T3-E1 osteoblasts and fibroblasts. The results indicate that this new collagen nanofibre-nanoHA composite is adequate to promote skin and bone tissue regeneration, and for the treatment of several bone and skin diseases.

[0087] The present solution is not, obviously, in any way restricted to the herein described embodiments and a person with average knowledge in the area can predict many possibilities of modification of the same solution and substitutions of technical characteristics by others equivalent, depending on the requirements of each situation, as defined in the appended claims.

[0088] The embodiments described above can be combined with each other. The following claims further define the preferred embodiments of the present solution.