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WO2020224960A1 - Hydrophilic nonwoven nanofibers membrane for promoting bone regeneration - Google Patents

Hydrophilic nonwoven nanofibers membrane for promoting bone regeneration Download PDF

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Publication number
WO2020224960A1
WO2020224960A1 PCT/EP2020/061184 EP2020061184W WO2020224960A1 WO 2020224960 A1 WO2020224960 A1 WO 2020224960A1 EP 2020061184 W EP2020061184 W EP 2020061184W WO 2020224960 A1 WO2020224960 A1 WO 2020224960A1
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WIPO (PCT)
Prior art keywords
membrane
hydrophilic nonwoven
copolymer
bone
hea
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PCT/EP2020/061184
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English (en)
French (fr)
Inventor
Manuel TOLEDANO PÉREZ
Raquel OSORIO RUIZ
Antonio Luis MEDINA CASTILLO
Original Assignee
Nanomateriales Y Polimeros, S.L.
Universidad De Granada
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Application filed by Nanomateriales Y Polimeros, S.L., Universidad De Granada filed Critical Nanomateriales Y Polimeros, S.L.
Publication of WO2020224960A1 publication Critical patent/WO2020224960A1/en

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    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/40—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties
    • D04H1/42—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties characterised by the use of certain kinds of fibres insofar as this use has no preponderant influence on the consolidation of the fleece
    • D04H1/4326—Condensation or reaction polymers
    • D04H1/435—Polyesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0003—Not used, see subgroups
    • A61C8/0004—Consolidating natural teeth
    • A61C8/0006—Periodontal tissue or bone regeneration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0012—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools characterised by the material or composition, e.g. ceramics, surface layer, metal alloy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A61K31/78—Polymers containing oxygen of acrylic acid or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K6/00—Preparations for dentistry
    • A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
    • A61K6/884—Preparations for artificial teeth, for filling teeth or for capping teeth comprising natural or synthetic resins
    • A61K6/887—Compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L31/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an acyloxy radical of a saturated carboxylic acid, of carbonic acid or of a haloformic acid; Compositions of derivatives of such polymers
    • C08L31/02—Homopolymers or copolymers of esters of monocarboxylic acids
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/40—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties
    • D04H1/42—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties characterised by the use of certain kinds of fibres insofar as this use has no preponderant influence on the consolidation of the fleece
    • D04H1/4382—Stretched reticular film fibres; Composite fibres; Mixed fibres; Ultrafine fibres; Fibres for artificial leather
    • D04H1/43838—Ultrafine fibres, e.g. microfibres
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
    • D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
    • D04H1/724—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged forming webs during fibre formation, e.g. flash-spinning
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the invention relates to a hydrophilic nonwoven nanofiber membrane based on acrylate and methacrylate copolymers and its process of preparation. Furthermore, the invention refers to its hydrolysed form further functionalised with a divalent cation selected from Zn +2 , Ca +2 , Mg +2 and Sr +2 , an antibacterial agent and any of the combinations thereof.
  • the invention refers to a non-resorbable membrane for promoting bone regeneration and a non-resorbable periodontal membrane comprising said hydrophilic nonwoven nanofiber membrane, its hydrolysed form or its hydrolysed form further functionalised with a divalent cation selected from Zn +2 , Ca +2 , Mg +2 and Sr +2 , an antibacterial agent and any of the combinations thereof.
  • GBR Guided Bone Regeneration
  • resorbable tissue-engineered matrices to induce bone formation, when additional support is needed, is not always successful.
  • a major limitation of resorbable materials is the inability to exert spatiotemporal control over the wound-healing process.
  • Most of the employed resorbable membranes e.g. collagen, polylactide-co- glycolide, polycaprolactone
  • bone graft substitutes e.g. hydroxyapatite -HAp- and other calcium phosphates
  • employed resorbable materials may be disadvantageous, as dissolution behaviors are not as long-lasting as required.
  • some degradation products from these resorbable materials have low pH, they may not be cytocompatible and could also alter the remineralization processes [Ivanovski S, Vaquette C, Gronthos S, Hutraum DW, Bartold PM (2014) Multiphasic Scaffolds for Periodontal Tissue Engineering. J Dent Res 93(12):1212-1221 ] [Shimauchi H, Nemoto E, Ishihata H, Shimomura M (2013) Possible functional scaffolds for periodontal regeneration. Japan Dent Sci Rev 49:1 18- 130].
  • non-resorbable synthetic membranes of polytetrafluoroethylene still represent the gold-standard for clinicians, due to the higher predictability of their effects when compared to resorbable membranes.
  • PTFE possess important disadvantages: I) low adhesiveness for cells, II) total absence of the capability of connecting to the bone tissue and providing osseointegration, without formation of a connective tissue interlayer; then a second surgery is required to remove the non- integrated membrane, and finally III) lack of antibacterial properties, being infections frequently observed [Sam G, Pillai BRM (2014) Evolution of Barrier Membranes in Periodontal Regeneration-Are the third Generation Membranes J of Clin Diagn Res 8: 14-17] Therefore, a successful membrane for GBR should resemble the morphology of natural bone.
  • Natural bone is a hybrid of inorganic-organic tissue composed of hydroxyapatite nanocrystals and collagen fibers (with diameters ranging from 50 to 500nm) assembled into a porous mesh, with interconnected pores. Bone is nanostructured, so nanosized materials should be the best choice for bone substitutes.
  • a first aspect of the present invention relates to a hydrophilic nonwoven nanofibers membrane (herein“the membrane of the invention”) characterised in that it comprises a blend of
  • hydrophilic nonwoven nanofibers membrane refers to a membrane formed by long fibers having a diameter of between 150nm and 400nm. Said membranes are nonwoven, this means that they are like a felt, which are neither woven nor knitted: they are made from long fibers (continuous long), bonded together by chemical, mechanical, heat or solvent treatment, and have a hydrophilic character.
  • each needle produces a single fiber that is wound on the drum from the beginning to the end of the electrospinning process (kilometric).
  • the reality is that fibers are cut intermittently along the electrospinning process.
  • copolymer with statistical topology refers to statistical copolymers, that is to say, a copolymer in which the distribution of the monomers in the chain is random since all the monomers present in the solution have the same affinity/probability to react both with monomers of the same chemical nature (with themselves) and with monomers of a different chemical nature.
  • Said membrane exhibits abrasion resistance, flexibility, elasticity, stress resistance, and thus it can be easily manipulated: can be cut, bend and twist.
  • One of the most important parameter in the electrospinning process is the molecular weight of the polymer. Higher molecular weight is generally preferred as there will be greater chains entanglement which facilitates the formation of fibers during spinning. In contrast, lower molecular weight may break up into droplets forming beads or beads combined with short fibers, resulting in heterogeneous materials with unwanted physical properties: irregular surface, low specific surface, low resistance to abrasion, and stress, loss of elasticity.
  • the first copolymer of (MA) 3 -CO-(HEA) 2 has molecular weight between 50000 Da and 3-10 6 Da, preferably above 80000 Da. More preferably, the first copolymer of (MA) 3 -co-(HEA) 2 has molecular weight between 1 10 6 Da and 3- 10 6 Da.
  • the second copolymer of (MMA)i-co-(HEMA)i has molecular weight between 50000 Da and 1 10 6 Da.
  • the membrane further comprises Si0 2 nanoparticles (NPs-Si0 2 ), and said Si0 2 nanoparticles are
  • Silicon dioxide (Si0 2 ) is able to improve not only bioactivity of materials but also cell adhesion and proliferation on artificial tissues, facilitating osteogenic cells differentiation.
  • Si0 2 is considered to be osteoinductive and a catalyst for bone formation. Therefore, in order to improve the bioactivity of membranes they were doped with Si0 2 nanoparticles.
  • the Si0 2 nanoparticles can be introduced in the membranes by two ways: 1 ) suspending them in the electrospinning solution, and then carry out the electrospinning process. In this case the NPs-Si0 2 are trapped homogeneously into the whole fiber volume, forming a solid solution (composite); 2) by physical adsorption in the surface of the fibers once the membrane is made: the membrane is soaked with a suspension of NPS-S1O2, and then the water is evaporated.
  • the option (1 ) is preferred because NPs-SiC>2 are retained in the fibers more efficiently and its leaching is minimized; by (1 ) the NPs- S1O2 can remain in the membrane for longer times than by (2).
  • a second aspect of the present invention relates to a process of preparation of the membrane of the present invention (herein“the process of the invention”) that includes the preparation of the copolymers which are electrospun to produce said membrane.
  • the copolymers of the present invention can be prepared by Conventional Free- Radical Polymerization or by Metal-Catalysed Living Radical Polymerization (MC-LRP) such as normal Atom Transfer Radical Polymerization (Normal ATRP), reverse Atom Transfer Radical Polymerization (reverse ATRP) and activator generated by electron transfer atom transfer radical polymerization (AGET ATRP).
  • the process is characterised in that it comprises the following steps:
  • step (c) preparation of a nanofibers membrane comprising a blend, said blend comprising the first copolymer obtained in step (a) and the second copolymer obtained in step (b) by electrospinning, and
  • step (c) heat treatment of the nanofibers membrane obtained in step (c), wherein the heat treatment is applied in the form of hot water at a temperature range between 30 e C and 80 e C, for instance for at least 4 hours, and wherein the nanofibers membrane obtained in step (c) is kept tensioned by means of a frame.
  • Step (a) of the process of the invention refers herein to the synthesis of the first copolymer of (MA) 3 -co-(HEA) 2 by Metal-Catalysed Living Radical Polymerization using a metal catalytic system and step (b) refers to the synthesis of the second copolymer of (MMA)i-co-(HEMA)i by Reverse Atom Transfer Radical Polymerization using a metal catalytic system.
  • Method “Metal-catalysed living radical polymerization” refers to polymerization methods based on establishing a rapid dynamic equilibration between a minute amount of growing free radicals and a large majority of the dormant species, in which a low oxidation state metal complex acts as the catalyst.
  • Reverse Atom Transfer Radical Polymerization refers to the polymerization methods based on establishing a rapid dynamic equilibration between a minute amount of growing free radicals and a large majority of the dormant species, in which a low oxidation state metal complex acts as the catalyst, the dormant species are alkyl halides, and the reaction is initiated by a conventional radical initiator and a Cu" complex.
  • the term“metal catalytic system” refers to the catalyst used in the Metal-Catalysed Living Radical Polymerization of step (a) and to the catalyst used in the reverse Atom Transfer Radical Polymerization of step (b).
  • Said metal catalytic system comprises a metal, a ligand and an initiator and uses a particular solvent.
  • the metal catalytic system of step (a) and step (b) is a copper amine complex.
  • the metal of the metal catalytic system of step (a) and/or step (b) comprises a transition metal or a mixture of transition metals in different oxidation states. More preferably, the metal of the metal catalytic system of step (a) and step (b) is independently selected from the list consisting of Cu, Fe, Co, Ni, Ru, PI, Rh, Re, Cr and Mo.
  • said metal of the metal catalytic system of step (a) and/or step (b) is in a weight percentage comprised between 0.00001 % and 0.1 %.
  • the ligand of the metal catalytic system of step (a) and/or step (b) is a multidentate aliphatic amine which can be linear or branched. More preferably, the ligand of the metal catalytic system of step (a) and step (b) is independently selected from the list consisting of N,N,N',N",N"-Pentamethyldiethylenetriamine (PMDETA) Tris(2-pyridylmethyl)amine, Tris[2-(dimethylamino)ethyl]amine, 2,2'-Bipyridyl, N,N,N',N'- Tetrakis(2-pyridylmethyl)ethylenediamine and 1 ,1 ,4,7,10,10- Hexamethyltriethylenetetramine.
  • said ligand is in a weight percentage between 0.0001 % and 0.2%.
  • step (a) and step (b) are preferably different.
  • the initiator of the metal catalytic system of step (a) is independently selected from the list consisting of Dodecyl 2-bromoisobutyrate, Ethyl a- bromoisobutyrate, Ethyl a-bromoisobutyrate, Octadecyl 2-bromoisobutyrate, Methyl a- bromoisobutyrate, Methyl 3-bromopropionate, tert-Butyl 3-bromopropionate, Ethyl 2- bromopropionate.
  • the initiator of step (b) is independently selected from the list consisting of 1 ,Tazobis(cyclohexanecarbonitrile) (ACHN), 2,2’-azobis (2-methylpropionamidine) 2,2’- dihydrochloride (AAPH), 4,4’-azobis(4-cyanovaleric acid) (ACVA), tert-butyl hydroperoxide, cumene hydroperoxide, 2,5-di(tert-butylperoxide)-2,5-dimethyl-3- hexyne, dicumyl peroxide and 2,5-bis(tert-butylperoxide)-2,5-dimethylhexane.
  • ACVA 4,4’-azobis(4-cyanovaleric acid)
  • the weight percentage of the initiator in step (a) and step (b) is between 0.00001 % and 0.2%.
  • the solvent used with the metal catalytic system of step (a) and step (b) is independently selected from the list consisting of acetone, dimethyl formamide, polyethylene glycol), dimethyl sulfoxide, 1 -4 Dioxane, ethanol, propanol, hexane, water, carbon dioxide , ionic liquid, and a combination thereof.
  • the weight percentage of the solvent in step (a) and step (b) is below 90%; preferably the weight percentage of the solvent in step (a) and step (b) is between 40% and 60%.
  • step (a) and step (b) are carried out without solvent since monomers are liquids and miscible to each other.
  • the metal catalytic system of step (a) uses Cu°/Cu 2+ as transition metal, Tris(2-dimethylaminoethyl)amine as ligand, Methyl 2-bromopropionate as initiator, and dimethyl sulfoxide as solvent.
  • Step (c) of the process of the invention refers to the preparation of a nanofibers membrane comprising a blend, said blend comprising the first copolymer obtained in step (a) and the second copolymer obtained in step (b) by electrospinning.
  • step (c) is performed in the presence of an additive capable of increasing the conductivity of the solution blend/solvent. More preferably, in the presence of hydrochloric acid (1HCI), wherein the weight percent of HCI in step (c) is between 0.0001 % and 0.2%.
  • HCI hydrochloric acid
  • the solvent of step (c) is selected from the list consisting of acetone, dimethyl formamide, polyethylene glycol), dimethyl sulfoxide, 1 -4 Dioxane, ethanol, propanol, hexane, water, carbon dioxide , ionic liquid, and a combination thereof. More preferably, the solvent of step (c) is dimethyl sulfoxide.
  • the weight percent of the solvent used in step (c) is ranging between 20% and 98%.
  • Step (d) refers to a heat treatment of the nanofibers membrane obtained in step (c), wherein the heat treatment is applied in the form of hot water at a temperature range between 30 e C and 80 e C and wherein the nanofibers membrane obtained in step (c) is kept tensioned by means of a frame.
  • the aim of this step (d) is to convert the nanofibers membrane obtained in step (c) from hydrophobic to hydrophilic; a visual transformation of the membrane is observed when wet thermal treatment of step (d) is performed for at least 4 hours. Please note that the membrane lasts hydrophilic for days, even years.
  • a third aspect of the invention refers to a process of preparation of the hydrophilic nonwoven nanofibers membrane comprising S1O2 nanoparticles, wherein said S1O2 nanoparticles are homogenously dispersed in the membrane, characterised in that it comprises all steps of the process of the invention:
  • step (c) preparation of a nanofibers membrane comprising a blend, said blend comprising the first copolymer obtained in step (a) and the second copolymer obtained in step (b) by electrospinning, and d) heat treatment of the nanofibers membrane obtained in step (c), wherein the heat treatment is applied in the form of hot water at a temperature range between 30 e C and 80 e C, for instance for at least 4 hours, and wherein the nanofibers membrane obtained in step (c) is kept tensioned by means of a frame.
  • step (c) comprises S1O2 nanoparticles.
  • Another aspect of the invention refers to a process of preparation of the hydrophilic nonwoven nanofibers membrane comprising S1O2 nanoparticles, wherein said S1O2 nanoparticles are physically adsorbed, characterised in that it comprises all steps of the process of the invention:
  • a nanofibers membrane comprising a blend, said blend comprising the first copolymer obtained in step (a) and the second copolymer obtained in step (b) and optionally comprises S1O2 nanoparticles, by electrospinning, and
  • step (c) heat treatment of the nanofibers membrane obtained in step (c), wherein the heat treatment is applied in the form of hot water at a temperature range between 30 e C and 80 e C, for instance for at least 4 hours, and wherein the nanofibers membrane obtained in step (c) is kept tensioned by means of a frame.
  • hydrolysed hydrophilic nonwoven nanofibers membrane characterised in that it comprises the hydrophilic nonwoven nanofibers membrane comprising carboxyl groups, wherein the concentration of carboxyl groups in the membrane is ranging between 20 pmol/g of the membrane and 3000 pmol/g of the membrane.
  • hydrolysed hydrophilic nonwoven nanofibers membrane refers herein to the hydrophilic nonwoven nanofibers membrane mentioned above which has been partially hydrolysed and now comprises carboxyl groups (COOH) and dried afterwards at room temperature (18-28 e C).
  • the number of accessible COOH groups in the membrane is ranging between 20 pmol/g of the membrane and 3000 pmol/g of the membrane. It was found that a hydrolysis time longer than 1 hour, produced a high rigidity in the membranes, making them fragile and brittle.
  • said membrane is functionalised with a divalent cation selected from Zn +2 , Ca +2 , Mg +2 and Sr +2 , an antibacterial agent and/or any of the combinations thereof. More preferably, the hydrophilic nonwoven nanofibers membrane is functionalised with Zn +2 , Ca +2 and doxicycline.
  • the functionalization of the hydrolysed membrane of the present invention with a divalent cation selected from Zn +2 , Ca +2 , Mg +2 and Sr +2 comprises a step of soaking the hydrolysed with a solution of a divalent cation selected from Zn +2 , Ca +2 , Mg +2 and Sr +2 , and a step of drying at room temperature (18-28 e C).
  • the functionalization of the hydrolysed membrane of the present invention with an antibacterial agent comprises a step of soaking the hydrolysed with a solution of an antibacterial agent and a step of drying at room temperature (18-28 e C).
  • the concentration of Ca 2+ and Zn 2+ loaded in the membrane as (COO )2 is ranging between 0.0125 pmol/g of the membrane and 1500 pmol/g of the membrane.
  • a concentration of Calcium and Zinc higher than 1500 pmol/g of the membrane can be loaded in the hydrolysed hydrophilic nonwoven nanofibers membrane, when all the accessible COOH groups are coordinated.
  • the excess of Zn 2+ or Ca 2 is physically adsorbed on the membrane ' s surface in the form of their respective salts (ZnC and CaCh) during the drying of the membrane
  • Doxycycline (DOX) was bound non-covalently into membrane by acid-base interactions between amine groups of DOX and carboxyl groups of the membrane as well as by hydrogen bonds between the hydroxyl groups of the membrane.
  • DOX Doxycycline
  • the concentration of DOX is ranging between 0.01 mg/mg of the membrane and 1 mg/mg of the membrane.
  • Another aspect of the invention refers to a non-resorbable membrane for promoting bone regeneration characterised in that it comprises the hydrophilic nonwoven nanofibers membrane mentioned above.
  • Another aspect of the invention refers to a non-resorbable periodontal membrane characterised in that it comprises the hydrophilic nonwoven nanofibers membrane mentioned above.
  • Resorbable and non-resorbable barrier membranes are commercially available, being non-resorbable PFTE membranes the Standard of care in Guided Bone Regeneration.
  • the main disadvantage of resorbable membranes is the unpredictable resorption time and toxic substances liberated during degradation, affecting bone formation.
  • the main disadvantage of non-resorbable barrier membranes is that they do not osseointegrate.
  • it is necessary a second surgical intervention to remove them after regeneration that may result in injury of the regenerated tissue. Their poor efficacy results in a high degree of relapse.
  • the non-resorbable membrane of the invention is a breakthrough bioactive membrane which allows:
  • the last aspect of the invention refers to a coating for an implant characterised in that it comprises the hydrophilic nonwoven nanofibers membrane mentioned above which may provide an advantage in osseointegration.
  • Osseointegration involves direct contact between for instance titanium implant and bone.
  • Most metal transcutaneous implants have failed, primarily owing to infection. Titanium alloy implants produce corrosion particles and fail by mechanisms generally related to surface interaction on bone to promote an inflammation with fibrous aseptic loosening or infection that can require implant removal. Further, lowered oxygen concentrations from poor vasculature at a foreign metal surface interface promote a build-up of host-cell-related electrons as free radicals and proton acid that can encourage infection and inflammation to greatly influence implant failure. Covering the implant with the hydrophilic nonwoven nanofibers membrane mentioned above is an efficient way for avoiding the mentioned risks.
  • FIG. 1 Theoretical modelling of co-polymerization of MA and HEA, F a vs conversion (A) and F a vs f a (B).
  • FIG. 2 Chromatographic profile of HEA/MA-10/90 (A), HEA/MA-15/85 (B), HEA/MA- 25/75 (C), HEA/MA-35/65 (D), HEA/MA-45/55 (E).
  • FIG. 3 FTFIMN spectra of HEA/MA-10/90 (A), HEA/MA-15/85 (B), HEA/MA-25/75 (C), HEA/MA-35/75 (D)
  • FIG. 4 Theoretical modelling of co-polymerization of MMA and HEMA, F a vs conversion (A) and F a vs f a (B).
  • FIG. 5 Chromatographic profile of MMA-co-HEMA (A), and H 1 RMN spectra of MMA- co-HEMA (B).
  • FIG. 6. Electrospinning set up: injection pump (1 ), injection needle (2), Drum collector (3), high voltage sources (4), Taylor cone display (5) and mechanical axis with transversal movement (6).
  • FIG. 7 Nonwoven mat produced with the blends: (A)/(B) 0:100 (A), 100:0 (B), 25:75 (C), 50:50 (D), 75:25 (E).
  • FIG. 8 Nonwoven mats produced with the blend (A)/(B)75:25 (A), and with the blend (A)/(B)50:50 (B)
  • FIG.9. Tiss-OH before (A) and after heating (B).
  • FIG.10 Connective collagen network (A), and nonwoven nanofibers mat (Tiss-OH) (B).
  • FIG. 11 FESEM micrographs of membranes after 7 days of immersion in SBFS; TissHYD (A), Tiss-Ca 2+ (B), Tiss-Zn 2+ (C).
  • FIG. 12 Bone histomorphometry obtained after using Tiss-Zn 2+ , by coloration with von Kossa silver nitrate to visualize mineralized bone, at six weeks of follow up: histology section including the bone defect and the region of interest (ROI) showing a large formation of dense bone (A).
  • FIG. 13 Bone histomorphometry obtained by coloration with von Kossa silver nitrate to visualize mineralized bone, at six weeks of follow up, after using no membrane- control (A) and Tiss-Ca 2+ (B). Trabecular bone formation were observed along the margin of calvarial defect (arrow head), and within the defect. Mbr: membrane, NB: new bone and OB: old bone (pointers show scattered bone islands, in correspondence with new bone).
  • FIG. 14 Bone histology obtained after using Tiss-Zn 2+ (A) and Tiss-Ca 2+ (B) membranes by coloration with toluidine blue to visualize mineralized bone, at six weeks of healing time.
  • Single arrows indicate the presence of osteoblasts; double arrows indicate the presence of osteocytes; faced arrows mean blood vessels; pointers indicate fibrous connective tissue.
  • NB new bone
  • Os osteoid tissue.
  • FIG.15 Field Emission scanning electron microscopy (FE-SEM) of F. nucleatum, S. oralis, A. naeslundii, V. parvula, A. actinomycetemcomitans and P. gingivalis grown as multi-species biofilm in vitro at 12 hours of incubation on, PTFE (control) (A), hydroxyapatite (FIAp) discs (B) TissFIYD (C), Tiss-Ca 2+ (D),Tiss-Zn 2+ (E) and Tiss-DOX (F).
  • FE-SEM Field Emission scanning electron microscopy
  • qPCR quantitative real-time polymerase chain reaction
  • Fig.17 FESEM micrographs of tissues after silicon dioxide doping and SBFS immersion for 7 days: Tiss-Si0 2 -COOH (A), Tiss-Si0 2 -Ca 2+ (B), Tiss- Si0 2 -Zn 2+ (C) and Tiss- Si0 -DOX (D).
  • MC-LRP Metal Catalysed Living Radical Polymerization
  • Cu°-MC-LRP Copper-mediated Living Radical Polymerization
  • MA-co-HEA hydrophilic acrylate lineal copolymer with statistical topology and high molecular weight (above 1 x10 6 Da).
  • the Cu°-LRP system used was: Methyl 2-bromopropionate as initiator, Tris(2- dimethylaminoethyl)amine as ligand, Copper/Copper(ll) as transition metal: MBP/M 6 - TREN/Cu°/BrCu2, and dimethyl sulfoxide (DMSO) was used as solvent.
  • the monomers selected were: methyl acrylate (MA), and hydroxyethyl acrylate (HEA).
  • Fig. 1 shows the theoretical modelling of co-polymerization of MA and HEA: F a vs conversion ( F a is the molar fraction of MA in the copolymer along the Polymerization) for different initial molar fractions feed of MA (f 0a ), and F a vs f a (f a is the molar fraction of MA in the feed along the Polymerization).
  • Cu°-LRP is very sensitive to any trace of impurities: mainly the inhibitor that contains both monomers, and di-acrylates that are formed in monomer HEA by condensation of HEA molecules.
  • impurities mainly the inhibitor that contains both monomers, and di-acrylates that are formed in monomer HEA by condensation of HEA molecules.
  • the presence of impurities at very low concentration provides low yields, low molecular weights and crosslinked polymer, and thus it is strictly necessary to properly purify the monomers.
  • First HEA was purified in a basic alumina column.
  • the required volume of MA was passed through a column of basic alumina.
  • (MA-co-HEA)_ copolymers with different molar % of HEA and MA in the feed were synthesised by Cu°-LRP .
  • the six different HEA/MA molar % were: a) HEA/MA 10/90 b) HEA/MA 15/85, c) HEA/MA 25/75, d) HEA/MA 34/66, e) HEA/MA 45/55, f) HEA/MA 55/45.
  • Table 1 shows the % wt of each component in the final polymerization mixture for each molar % of HEA and MA.
  • Table 1 % wt of each component in the mixture for each molar % of HEA and MA.
  • the total mass of the monomers (MA + HEA) 59.2700 g was added into 50 ml Schlenk flasks, and then were added: 59.2700 g of DMSO, 0.0020 g of Cu°, 0.0160 g of Tris[2- (dimethylamino)ethyl]amine (M 6 -TREN), 0.0012 g of CuBr 2 , and 0.0060 g of methyl 2- bromopropionate (MBP).
  • M 6 -TREN Tris[2- (dimethylamino)ethyl]amine
  • MBP methyl 2- bromopropionate
  • the sealed flask was placed in a thermostatic oil bath at 25 °C during 24h. Then the copolymers were purified by dissolving in acetone and precipitating them in distilled water (two times). After purification copolymers were dried in a vacuum at 80 e C to a constant weight.
  • the copolymers a), b), c), d) had a white colour and rubber texture, and the final conversion was between 90-95%wt in all the cases.
  • the co polymerization of e) and f) (Table 1 ) did not occur properly: the yield was below 40%, and the copolymer had not a rubber texture. Therefore the optimal range of molar % of HEA in the feed was between 10% and 34%.
  • (MA-co-HEA) copolymers were characterised by GPC (Viscotek 270max of Malvern) and by H 1 RMN (Bruker Avance 400 MHz spectrometer). The samples for GPC were prepared dissolving 1 mg of copolymers in 10 ml 1 -methyl-2-pyrrolidinone (NMP) and they were analysed in triplicate.
  • Fig. 2 shows a chromatographic profile of each copolymers, and Table 2 shown the molecular weight ( M w and M n ) and M w /M n .
  • Fig. 3 shows the H 1 RMN spectra of said copolymers.
  • Table 3 shows the real molar % of HEA in each copolymer: it was calculated by the intensity ratio between the signals a (CH 3 of MA) and b (CH 2 -CH 2 of HEA) of H 1 RMN spectra.
  • H 1 RMN The analysis of H 1 RMN shows that the concentrations of HEA in the copolymers are practically the same as the feed concentrations.
  • the solubility of synthesised acrylate copolymers was tested in acetone, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), 1 -4 dioxane and NMP.
  • the copolymers were totally soluble in all the tested solvents up to 6% wt: above 6% the viscosity of the solutions was extremely high.
  • the 6% wt solution with lower viscosity was the DMF solution, which indicates that DMF is the best solvent for these copolymers.
  • the selected acrylate copolymer for the blend formulations was (MA) 3 -CO-(HEA) 2 (Table 2).
  • Fa vs conversion wherein Fa is the molar fraction of MMA in the copolymer along the polymerization for different initial molar fractions feed of MMA ( fo a ), and F a vs 4 wherein 4 is the molar fraction of MMA in the feed along the Polymerization.
  • the reverse-ATRP system used was: 2,2'-azobis(2-methylpropionitrile) (AIBN) as initiator, A/,A/,/V,A/",A/"-pentamethyldiethylenetriamine (PMDETA) as ligand, copper(ll) as transition metal, and a mixture of dimethyl sulfoxide (DMSO)/xilene was used as solvent.
  • AIBN 2,2'-azobis(2-methylpropionitrile)
  • PMDETA A/,A/,/V,A/",A/"-pentamethyldiethylenetriamine
  • DMSO dimethyl sulfoxide
  • MMA methyl methacrylate
  • HEMA 2- hydroxyethylmethacrylate
  • the reaction was carried out at 80 °C in an oil bath for 6 hour. After polymerization, the copolymer was purified by dissolving in acetone and precipitating it in distilled water three times. Then the solid copolymer was washing with distilled water 3 times, and dried in a vacuum at 80 e C to a constant weight.
  • the methacrylate copolymer had a white colour and a hard and brittle texture. The conversion was 70%.
  • MMA-co-HEMA copolymer was characterised by GPC (Viscotek 270 max of Malvern) and by H 1 RMN (Bruker Avance 400 MHz spectrometer). The samples for GPC were prepared dissolving 1 mg of copolymers in 10 ml 1 -Methyl-2-pyrrolidinone (NMP) and they were analysed in triplicate. Fig. 5 shows the chromatographic profile and H 1 RMN spectrum of the prepared MMA-co-HEMA copolymer.
  • Table 4 shows molecular weights M w and M n and M w /M n calculated by GPC.
  • Table 5 shows the real molar % of HEMA in the copolymer calculated by the intensity ratio between the signals a (CH 3 of MMA) and b (CH 2 -CH 2 of HEMA) of the 1 H-NMR spectra.
  • the selected blends; (A)/(B) w/w to be process by electrospinning were: 0/100, 25:75, 50:50, 75:25 and 100/0, and blend/solvent, w/w was 3/97.
  • the solutions were loaded into 20 cm 3 teflon syringes (Becton & Dickinson) and extruded through a stainless-steel capillary tube with outer and inner diameters of 1 .5 mm and 1 .1 mm, respectively.
  • the injection system was coupled to a mechanical system with axial movement, the flow rates and voltages were selected in order to allow the collection of dry fibers in nonwoven mats, and the fibers were collected on a rotary drum collector.
  • Fig. 6 shows the electrospinning set up, and Table 6 shows the electrospinning processing parameters.
  • Fig. 7 shows a SEM analysis of nonwoven nanofibers mats obtained with the blends (A)/(B) 0:100 (A), 100:0 (B), 25:75 (C), 50:50 (D), 75:25 (E).
  • the copolymer (B) pure provided a very elastic gummy materials, in which the fibers are 100% fused together forming a film (Fig. 7A).
  • the blend (A)/(B) 50:50 w/w (from now called Tiss-OH; Fig. 7D) provided a compact nonwoven mats with excellent mechanical properties: very high abrasion resistance, high flexibility, high elasticity, high stress resistance, and thus it is easily to manipulated: can be cut, bend, twist ... etc.
  • the optimal solution blend/solvent for scaling the electrospinning process was prepared as follows: 5.000g of (MA) 3 -co-(FIEA)2 (6.246% wt) and 5.000 g of (MMA)i-co- (FIEMA)i (6.246% wt) were dissolve in 70.000g of DMF (87.4479% wt), when the copolymers were completely dissolve, 0.048g of hydrochloric acid (HCI) (0.059% wt) were added to the solution.
  • HCI hydrochloric acid
  • the electrospinning set up was the same as the one shown in Fig. 6, but in order to increase the production the injection system of a single needle of Fig. 6 was replaced by a ten-needle head.
  • Tiss-OH was easily controlled from a few microns to hundreds of microns, by controlling the processing time (2h of processing « 45 pm in thickness).
  • Tiss-OH is hydrophobic; to convert it into hydrophilic material is necessary to carry out a further thermal treatment.
  • the thermal treatment was done by introducing Tiss-OH, in hot water (40 e C) for 5 hours. To prevent the shrinking of the materials during the thermal treatment, they were kept tensioned using frames.
  • the thermal treatment produces an irreversible reorientation of the hydrophobic and hydrophilic domains present on the fibers surface, causing the material to go from being completely hydrophobic to being highly hydrophilic: the OH groups of the fibers are reordered in order to interact by hydrogen bonding with the water molecules, while the hydrophobic groups hide from the water.
  • Tiss-OH allowed passing particles from 800nm to 3000nm of diameter.
  • the thermal resistance of Tiss-OH was studied by immersing it in water at 100°C during 24h.
  • the internal structure, mechanical properties and the mass of Tiss-OH were exactly the same before and after heating (Fig. 9).
  • Natural collagen mesh that forms the connective tissue of bones is composed of nanofibrils of approximately 50nm that are grouped to form fibers of approximately 500nm with a similar morphology, mechanical and physical-chemical properties to those of Tiss-OH.
  • FIG. 10 SEM pictures of connective collagen network (A) and Tiss-OH (B), that showing the very similar morphological structure.
  • the membranes (TissHYD) were washed 3 times with distillated water and dry at room temperature.
  • the assay includes the incubation of carboxilated matrixes with toluidine blue O in alkaline buffer with subsequent washing, followed by elution and quantification of eluted TBO via UV-Vis spectrometry.
  • the number of accessible carboxyl groups was 560 ⁇ 50 pmol/g of the membrane. After the hydrolysis the Q calculated was 3.06 ⁇ 0.20.
  • TissHYD was functionalised with Zn +2 (Tiss-Zn 2+ ) and Ca 2+ (Tiss-Ca 2+ ). The ability of carboxyl groups to complex divalent cations was used to functionalise TissHYD (Tiss- membranes) with Zn +2 and Ca 2+ . TissHYD was soaked with a Zn 2+ and Ca 2+ solution, and then the water was evaporated in a vacuum at constant temperature: by this way TissHYD was loaded with 1 .1 pg/mg, of Ca 2+ and Zn 2+ .
  • Doxycycline (DOX) was bound non -covalently into TissHYD by acid-base interactions between amino groups of DOX and carboxyl groups of TissHYD.
  • TissHYD was soaked with twice its mass of a DOX solution of 40 mg/ml, and then the water was evaporated in a vacuum at constant temperature: by this way TissHYD was loaded with 0.8 mg of DOX/mg Tiss.
  • Membranes should enhance bone formation trough bioactivity, therefore for said application analysis proposed by Kokubo has been performed (ISO 23317:2012. Implants for surgery. In vitro evaluation for apatite-forming ability of implant materials).
  • SBFS simulated body fluid solution
  • SBFS are fluids with ion concentrations nearly equal to those of human blood plasma and are employed for evaluating the bioactivity of biomaterials for hard tissue repair.
  • Zinc complexation on tissues facilitated phosphate groups binding. These phosphate groups, at the surface, have under-coordinated oxygens, which lead to reactive surfaces that will attract calcium ions from SBFS.
  • This biomimetic deposition of Ca/P is considered as a coating method inspired by the natural process of biomineralization. Moreover, it should be considered that crystalline HAp is very slow to resorb, and most bone substitutes based on HAp do not resorb or resorb extremely slowly. However, if HAp or nano-HAp is precipitated onto the surfaces, it does resorb, facilitating hard tissue regeneration. Biomimetic remineralization of the tested tissues will facilitate bone regeneration. HAp facilitates formation of other bone apatite-like materials as carbonate HAp and it is able to stimulate cells, leading to the formation of bone. Moreover, HAp promotes osteoconductivity. Osteoblasts stimulated with extracellular Ca 2+ and PO4 2 increased bone morphogenetic protein-2 mRNA expression. Fibroblast growth factor-2 (FGF-2) gene and protein expression levels are also augmented by increases in extracellular Ca 2+ concentration.
  • FGF-2 Fibroblast growth factor-2
  • the experiment was developed in accordance with the guidelines of the US National Institute of Health (NIH) and European Directive 86/609/EEC regarding the care and use of animals for experimentation.
  • the study also complied with the European Directive 2010/63/EU about the protection of animals used for scientific purposes and with all local laws and regulations.
  • the researchers obtained the approval of the Ethics Committee of the Institution.
  • the minimum number of animals was used for ethical reasons. Comparable models have been published concerning the histological and animal experimentation methods.
  • a Prichard periosteotome With a Prichard periosteotome, the epithelial, connective, and muscular tissues were separated from the operation field and the skull surface was washed with a sterile saline solution.
  • Six non-critical bone defects (diameter: 6 mm; depth: 3 mm) were created on the parietal bone, on each side of the skull midline, 3 mm apart, using a trephine (Helmut-Zepf Medical Gmbh, Seitingen, Germany) mounted on an implant micromotor operating at 2000 rpm under saline irrigation.
  • the trephine had an external diameter of 6 mm, a length of 30 mm, and teeth of 2.35 mm.
  • Piezosurgery was used to remove the inner table and the medullary bone in every defect. The depth was controlled with a periodontal probe. A randomly assigned membrane was used to cover each bone defect, leaving a naked defect in each animal. The randomisation sequence was generated using specific software (Research Randomizer, V. 4.0, Urbaniak GC & Pious S, 2013). The membranes were fixed with the fibrin tissue adhesive Tissucol (Baxter, Hyland S.A. Immuno, Rochester, Ml, USA), which was placed on the bone rims adjacent to the defects. Proper adhesion and limited mobility of the membranes were confirmed when the flaps were moved back to their initial positions.
  • Sutures were made on the following planes using resorbable material: periosteal (4/0), sub-epidermal (4/0) and skin (2/0). Simple stitches were used as close as possible to the edge.
  • the wound was carefully cleaned with a sterile saline solution.
  • Anti-inflammatory analgesia (buprenorphine 0.05 mg/kg and carprofen 1 mL/12.5 kg) was administered.
  • the animals were sacrificed six weeks after surgery using an intravenous overdose of potassium chloride solution. Samples were obtained from the skull of each specimen, cutting them in an anatomical sagittal plane. After the brain mass was separated and the skull was washed with a sterile saline solution, the tissue samples were cut and marked individually.
  • Specimens in cranial blocks were recovered and stored in a 5% formaldehyde solution (pH 7) and blocks were retrieved from the regenerated bone defect using an oscillating autopsy saw (Exakt, Kulzer, Wehrheim, Germany).
  • the dissected specimens were immediately immersed in a solution of 4% formaldehyde and 1% calcium and processed for ground sectioning following the Donath and Bruener method.
  • For histological staining and rapid contrast tissue analysis Merck Toluidine Blue-Merck, Darmstadt, Germany), a metachromatic dye was used to assess the percentage of new bone formation.
  • VK von Kossa
  • OS osteoid surface
  • OS/TS percentage of osteoid surface
  • BPm bone perimeter
  • BTh bone thickness
  • a 1 % toluidine blue (TB) solution with a pH of 3.6 was chosen and adjusted with HCI 1 N. The samples were exposed to the dye for 10 minutes at RT, rinsed with distilled water, and air-dried. Osteocytes, osteoblasts and blood vessels were analysed in TB stained sections.
  • the implanted membranes were well tolerated by the surrounding soft tissues, with no evidence of necrosis, allergy symptoms, immune reactions, or incompatibility. All specimens showed no signs of inflammation or infection induced by the use of biomaterials.
  • Fig. 12 shows bone a bone defect with an implanted Tiss-Zn 2+ membrane stained with von Kossa silver nitrate to visualize mineralized bone, at six weeks of follow up.
  • the Fig. 12A is a histology section including the bone defect and the region of interest (ROI), showing a large formation of dense bone.
  • the Fig. 12B is the total surface (TS) at ROI; asterisks ( * ) show the presence of marrow and adipose-like tissue. Bony bridging (BB) images are observed. At Fig. 12C, bone perimeter (BPm) at ROI is observed, and in Fig. 12D, bone thickness (BTh) with the traced measurements at ROI is measured.
  • an osteoid surface (OS)
  • the membranes have higher BTh than control (Ctr), and thus they produced more osteoid surface (OS), in comparison with the control group (see the ratio OS/TS in Tables 9 and 10).
  • BS Bone Surface
  • OS Osteoid Surface
  • TS Total Surface
  • BPm Bone Perimeter
  • BTh Bone Thickness
  • Ctr control.
  • Tiss-Zn 2+ achieved higher bone perimeter (BPm) than that produced by TissHYD
  • BS Bone Surface
  • OS Osteoid Surface
  • TS Total Surface
  • BPm Bone Perimeter
  • BTh Bone Thickness
  • Ctr control.
  • Fig. 13 For comparison in Fig. 13 are shown bone sections stained by the von Kossa silver nitrate technique at six weeks of follow up, with no membrane -control (Fig. 13A)- and Tiss-Ca 2+ membrane (Fig. 13B). Trabecular bone formation were observed along the margin of calvarial defect (arrow head), and within the defect. The pointers of Fig. 13 (Mbr: membrane, NB: new bone and OB: old bone) show scattered bone islands, in correspondence with new bone. The bone defect in the control group was found to be filled with connective tissue and a few immature bone trabeculae (Fig. 13A). Areas of trabecular bone formation could also be identified in the defects treated with either type of the membrane (Figs. 12B, 13B). Table 11. Bone cells and blood vessels detected within the new bone formed in the region of interest (ROI) (Mean ⁇ Standard Deviation SD).
  • ROI region of interest
  • Tiss-Zn 2+ and Tiss-Ca 2+ promoted higher number of osteoblasts than the control group.
  • the number of osteoblasts was higher in subjects treated with Tiss-Zn 2+ membranes than with unloaded membranes (Table 12). In some fields of all samples, osteoblasts were observed in the process of opposing bone directly on the membrane surface (Figs. 14A and 14B).
  • Tiss-Ca 2+ did not produce greater number of osteoblasts than the rest of the membranes but originated higher number of blood vessels than the control group (Table 12).
  • Tiss-Ca 2+ showed dense and neat collagen fibers that run parallel to the bone defect and membrane.
  • the control group promoted lower amount of blood vessels than Tiss-Ca 2+ .
  • Fig. 14A Many large vessels could be detected in samples treated with Tiss-Zn 2+ membranes (Fig. 14A). Small blood vessels were shown in close proximity to the new bone and the Tiss-Ca 2+ biomaterial. Images obtained with TB also permitted to observe that Tiss-Zn 2+ and Tiss-Ca 2+ membranes promoted the formation of bond matrix (Fig. 14) over the membrane, outside the surgical defect. No inflammatory cells or multinuclear giant cells were present at the interface with bone in animals treated with Tiss-Zn 2+ (Fig. 14A).
  • BS bone surface
  • BTh osteoid and bond thickness
  • Tiss-Zn 2+ , Tiss-Ca 2+ and TissHYD membranes induced significant changes in remodeling and structural indices of bone. This increase remodeling might result in the replacement of older, overly mature bone with younger and more resilient bone (Rubin et al., 2018). Osteoid or bone matrix that will be, but not yet, mineralized showed higher surface than in the control group when membranes were used, typical of young bone (La Monaca et al., 2018).
  • HAp Hydroxylapatite
  • TissHYD Tiss-Ca 2+ (loaded with 1 .1 pg(Ca 2+ )/mg Tiss)
  • Tiss-Zn 2+ loaded with 1 .1 pg(Zn 2+ )/mg Tiss
  • Tiss-DOX loaded with 0.8 mg(DOX)/mg Tiss. Naked HAp discs and HAp discs covered with a PTFE membrane were used as a control.
  • Biofilms from 12 to 72 hours of evolution were observed by Scanning Electron Microscopy (SEM).
  • SEM Scanning Electron Microscopy
  • the specimens were fixed in a solution at 4% paraformaldehyde and 2.5% glutaraldehyde for 4h at 4 e C. After that, specimens were critical point dried, sputter-coated with gold and analysed.
  • the quantitative Polymerase Chain Reaction (qPCR) amplification was performed in a total reaction mixture volume of 10 pL. Analyses were performed with a LightCycler ® 480 II thermocycler (Roche). The plates used in the study was FramStar 480 of natural frame and white wells (4titude; The North Barn; Damphurst Lane, UK), sealed by QPCR Adhesive Clear Seals (4titude). Each DNA sample was analyzed in duplicate. Quantification cycle (Cq) was determined using the provided software package (LC 480 Software 1 .5; Roche). Quantification of cells by qPCR was based on standard curves. The correlation between Cq values and CFU mL -1 were automatically generated through the software (LC 480 Software 1 .5; Roche).
  • CFU mL 1 Bacterial counts (CFU mL 1 ) for the six species at 72 h incubation time in the tested specimens are shown in Fig. 16. With time, the dynamics of bacterial growth were similar independent from the specimen. Biofilms on HAp discs coated with Tiss-DOX reached the lowest numbers of bacteria, when is compared with the rest of the groups (p ⁇ 0.01 ).
  • Silicon dioxide (S1O2) is able to improve not only bioactivity of materials but also cell adhesion and proliferation on artificial tissues, facilitating osteogenic cells differentiation.
  • S1O2 is considered to be osteoinductive and a catalyst for bone formation. Therefore, in order to improve the bioactivity of membranes they were doped with S1O2 nanoparticles (NPs-SiC>2) by two different ways:
  • NPS-S1O2 1 g was added to the optimum electrospinning scaling solution (see section 6), and the solution was prepared as follows: 1 .000g of NPS-S1O2 (1 .219 % wt) was dispersed in 70.000 g of DMF (85.316% wt) by 20 min of sonication, then 5.000g of (MA) 5 -co-(HEA) 5 (6.094 % wt), and 6.000 g of (MMA) 3 -co- (HEMA)2 (7.313% wt) were dissolved in the NPS-S1O2/DMF suspension.
  • TissHYDSi The ability of carboxyl groups to complex divalent cations was used to functionalise TissHYDSi with Zn +2 and Ca 2+ .
  • TissHYDSi was soaked with a Zn 2+ and Ca 2+ solution, and then the water was evaporated in a vacuum at constant temperature: by this way TissHYDSi was loaded with 1 .1 pg/mg, of Ca 2+ and Zn 2+ .
  • Doxycycline (DOX) was bound non -covalently into TissHYDSi by acid-base interactions between amine groups of DOX and carboxyl groups of TissHYDSi as well as by hydrogen bonds between the hydroxyl groups of membrane and amine groups of DOX.
  • TissHYDSi was soaked with a DOX solution, and then the water was evaporated in a vacuum at constant temperature: by this way TissHYDSi was loaded with 0.8 mg of DOX/mg Tiss.

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