Patent Application
20240424165
The present invention relates to an implant having a surface comprising a coating on at least a portion of the surface of the implant, wherein the coating comprises a first layer, the first layer comprising a polylactide and silver ions. The present invention further relates to a method of manufacturing the implant as well as to an implant obtainable by that method.
Implants and endoprostheses are always exposed to a risk of colonization by pathogens as germs, bacteria and biofilm formation after insertion into the human body, so that infections, usually mixed infections, occur in 0.5 to 2% of cases (Ong et al. 2009; Gbejuade et al. 2015). With the implant volume and depending on the implantation location, the risk of infection increases. This explains why the infection rate in tumor prostheses or megaprostheses for the reconstruction of a usually pronounced defect route is significantly higher risk of infection of up to 20% (Gosheger et al. 2006; Funovics et al. 2011). If there are further complications, the risk increases to about 40% with secondary revision procedures (Theil et al. 2019).
In tumor orthopedics, soft tissue reconstruction often includes knitted tubes, so-called connecting tubes, e.g., the Trevira (Mutars®, Implantcast Corp, Buxtehude, Germany) from polyethylene terephthalate. Due to their net-like structure, these result in a potentiation of the surface and thus of the attack surface. Infections can lead to amputations or sepsis with a lethal outcome in acute courses. Tumor patients are particularly affected by this risk, as they often have limited immune competence, e.g., due to necessary chemotherapy or radiotherapy.
The therapy of the prosthetic infection includes both surgical rehabilitation and anti-infective therapy. This usually takes place systemically over several weeks and is supplemented topically, e.g., by introducing antibiotic-bearing powders or placeholders made of bone cement and spherical chains as active ingredient carriers. Bone cement used is usually a plastic polymethyl-methacrylate (PMMA) mixed from powder and liquid activator, which becomes a malleable mass after mixing and cures completely within a few minutes. In everyday life, this material is known as plexiglass.
Antibiotic powders can cause tissue damage due to their high contact surface and the resulting locally high concentrations. This also applies to other substances such as virustatics and fungicides. The selection pressure on the pathogens is increased and resistance can potentially develop. Due to their side-effect profile, locally applied pharmaceuticals also carry the risk of temporary or even permanent organ damage. Antibiotic powders are often applied as additive to PMMA. The release from these PMMA carriers is very limited. The active ingredient is embedded into the carrier material, so to speak. Since this material is not absorbed in the human body, the concentration of the active ingredient drops quickly after introduction. Further release is only possible due to material damage, such as abrasion, on the carrier material. Other anti-infective substances include metals such as silver. These have a bactericidal and bacteriostatic effect. However, when introduced into the body as a powder or in high concentrations, it has potentially tissue-and cell-toxic effects. In order to take advantage of the positive properties, silver coatings on implants have been developed in the past (Gosheger and Sass 2002).
The development of anti-infective coatings, e.g., with antibiotics and compounds with other materials, have so far only been able to demonstrate short-term efficacy (Romanò et al. 2015). Even anti-infective metallic coatings such as e.g., with silver have already achieved a significant reduction in the infection rate of between 7 and 12% (Hardes et al. 2010; Schmidt-Braekling et al. 2017). However, control over the concentration and duration of release within the scope of elution kinetics is not yet possible (Scoccianti et al. 2016).
Thus, there is a need for further coatings for implants.
In a first aspect, the present invention relates to an implant having a surface comprising a coating on at least a portion of the surface of the implant, wherein the coating comprises a first layer, the first layer comprising a polylactide and silver ions.
In a second aspect, the present invention relates to a method of manufacturing an implant, preferably according to the first aspect, wherein the method comprises forming a first layer arranged on at least a portion of a surface of the implant, said forming of the first layer comprising contacting at a least a portion of the surface of the implant with a first coating composition comprising a polylactide and silver ions.
In a third aspect, the present invention relates to an implant, obtainable by a method as described above.
The present invention provides a coating for implants which allows the release of silver ions as antibiotic effect from the implant after implantation, in respect to the released concentration over time in a controlled manner. In one embodiment, the implanted implant may be subjected to a shock wave which triggers release of a high concentration of silver ions in a short period of time, in order to prevent or to treat a local infection near the implant. The shock wave application may be repeated.
In one embodiment, on the first coating, comprising the silver ions, a second coating may be present. In contrast to other developments, for example nanosilver particles, due to their precious metal properties, are released only in very small amounts from such layers when enclosed by a polymer-or quickly surround themselves with a poorly soluble silver chloride/protein layer. The second coating may comprise a further antibiotic compound. Preferably, the further antibiotic compound is released in a linear fashion with a constant concentration. Thus, initial high and toxic concentrations are avoided. Furthermore, the concentration of the antibiotic compound does not fall below the minimal inhibitory concentration up to months, and the development of pathogens resistant to the antibiotic compound may be avoided. Further application of multiple layers to combine the release kinetics is possible.
The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the Figures and reflected in the claims.
It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.
The term “less than” or in turn “more than” does not include the concrete number. For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, f.e. more than 80% means more than or greater than the indicated number of 80%.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of” excludes any element, step, or ingredient not specified.
The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
The invention is directed to an implant having a surface comprising a coating on at least a portion of the surface of the implant.
An intermediate may be arranged between the surface of the implant and the first layer. Preferably, the intermediate layer is a coating or pretreatment. More preferably, the intermediate layer is selected from the group consisting of PVD-layers (physical vapor deposition), TPS-layers (titanium plasma spray), calcium phosphate layers, layers caused by etching (oxidation, passivation, roughening), anodization, and a silver metal layer or any combination thereof.
The implant may be any implant suitable for implantation in the human or animal body. Preferably, the implant is a medical implant. More preferably, the implant is selected from a group comprising an implantable prosthesis, in particular a hip prosthesis, a shoulder prosthesis, an elbow prosthesis, a knee prosthesis, or an implant for trauma, maxillo-facial, or spinal surgery such as e.g. a screw or a plate, a nail or a rod, intervertebral spacers, as well as others including but not limited to cardiological and neurosurgical implants (shunts, electrodes, valves, stents, catheters, etc.), most preferably, the implant is selected from a group comprising an implantable prosthesis, in particular a hip prosthesis, a shoulder prosthesis, an elbow prosthesis, a knee prosthesis, or an implant for trauma surgery such as e.g. a screw or a plate.
The implant and/or the surface of the implant may comprise any material suitable for implants. The implant and/or the surface may comprise a metal, and/or a non-metal such as a polymer. Preferably, the implant and/or the surface comprise a metal. Preferably, the implant and/or the surface material comprise a metal such as 316 LVM implant steel, TIN coated Cr—Co—Mo, CoCrMo, titanium and/or a titanium alloy. More preferably, the titanium alloy is Ti6Al4V.
In one embodiment, at least the surface of the implant is made of metal. Preferably, the surface material comprises a metal such as 316 LVM implant steel, TIN coated Cr—Co—Mo, CoCrMo, titanium or a titanium alloy. More preferably, the titanium alloy is Ti6Al4V.
Preferably, the implant and/or the surface of the implant comprises at least 30 wt.-%, more preferably at least 60 wt.-%, most preferably at least 80 wt.-%, particular preferred at least 90 wt.-%, most particular preferred at least 95 wt.-% of a metal based on the overall weight of the implant and/or surface of the implant.
Preferably, the implant and/or the surface of the implant comprises less than 30 wt.-%, more preferably less than 60 wt.-%, most preferably less than 80 wt.-%, particular preferred less than 90 wt.-%, most particular preferred less than 95 wt.-% of a metal based on the overall weight of the implant and/or surface of the implant.
In one embodiment, at least the portion of surface of the implant which is coated is made of metal. Preferably, the surface material comprises titanium or a titanium alloy. Preferably, the titanium alloy is Ti6Al4V.
Preferably, at least 10%, more preferably at least 40%, most preferably at least 60%, particular preferred at least 90% and much more preferred 100% of the surface of the implant is coated.
In one embodiment, at least the surface of the implant is made of polymer. Preferably, the implant and/or the surface material comprises a polymer consisting of the group polyether ether ketone (PEEK), ultra-High molecular weight polyethylene (UHMWPE), low density polyethylene (LDPE), polymethylmethacrylate (PMMA), or polyphenylsulfone (PPSU). More preferably, the polymer is PEEK.
Preferably, the implant and/or the surface of the implant comprises at least 30 wt.-%, more preferably at least 60 wt.-%, most preferably at least 80 wt.-%, particular preferred at least 90 wt.-%, most particular preferred at least 95 wt.-% of a polymer based on the overall weight of the implant and/or surface of the implant.
Preferably, the implant and/or the surface of the implant comprises less than 30 wt.-%, more preferably less than 60 wt.-%, most preferably less than 80 wt.-%, particular preferred less than 90 wt.-%, most particular preferred less than 95 wt.-% of a polymer based on the overall weight of the implant and/or surface of the implant.
In one embodiment, at least the portion of surface of the implant which is coated is made of a polymer. Preferably, the surface material comprises PEEK. More preferably, the polymer is PEEK.
The coating comprises a first layer, the first layer comprising at least one polylactide and silver ions. Preferably, the silver ions are homogeneously dissolved (
Preferably, the first layer comprises at least one polylactide selected from the group consisting of poly(L-lactide) (CAS number: 33135-50-1), poly(D-lactide), poly(L-lactide-co-D,L-lactide) (CAS number: 52305-30-3), poly(D-lactide-co-glycolide), poly(L-lactide-co-glycolide) (CAS number: 30846-39-0), poly(L-lactide-co-caprolactone) (CAS number: 65408-67-5), and Poly(L-lactide-co-trimethylene carbonate) (CAS number: 113883-70-8), poly(D,L-lactide-co-glycolide) (CAS number: 1354955-03-5), more preferably poly(L-lactide).
Preferably, the first layer comprises at least 70%, more preferably at least 80%, most preferably at least 90%, particularly preferred at least 95%, more particularly preferred at least 99% of the at least one polylactide.
Preferably, the first layer comprises of from 0.01 to 20%, more preferably 0.1 to 15%, most preferably 0.5 to 10%, and particularly preferred 1.5 to 10%, particularly preferred 5 to 10%, by weight silver ions, based on 100% by weight of the first layer.
The counterion of the silver compounds may be selected from a group of silver compounds that are soluble and non-toxic in non-polar solvents. More preferably, the counterion is selected from the group consisting of diethyldithiocarbamate, nitrate, benzoate, and any combination thereof.
The silver ions may be provided in form of a salt or a silver complex compound. Preferably, the silver ions are provided in form of a salt. More preferably, the silver salt is selected from a group consisting of silver sulfadiazine, silver diethyldithiocarbamate, silver oxide, silver carbonate and silver nitrate, silver acetate, silver benzoate, silver iodate, silver laurate, silver protein, silver chloride, and silver palmitate and any combination thereof, most preferably silver diethyldithiocarbamate, particularly preferred silver nitrate.
The first layer may further comprise hydroxyapatite or another poorly soluble calcium compound e.g. calcium carbonate. The first layer may comprise 0.1 to 10% by weight hydroxyapatite, based on 100% by weight of the first layer.
The first layer may further comprise phages. Preferably, the first layer comprises phages-enzymes, more preferably the phage enzymes are endolysins, most preferably the endolysins are Staphefekt™ (Micreos, 3721 MA Bilthoven, Netherlands).
Preferably, the first layer comprises of from 0.01 to 20%, more preferably 0.1 to 15%, most preferably 0.5 to 10%, and particularly preferred 1.5 to 10%, particularly preferred 5 to 10%, by weight phage-enzymes, based on 100% by weight of the first layer.
Preferably, the first layer has a thickness of from 1 to 50 μm.
In a further embodiment, the coating further comprises a second layer arranged on the first layer, wherein the second layer comprises an antibiotic/antiinfective. The second layer comprises a poly(lactide-co-glycolide) and/or at least one lipid.
In a further embodiment,
Preferably, the poly(lactide-co-glycolide) in the second layer is poly(D-lactide-co-glycolide), or poly(L-lactide-co-glycolide) (CAS number: 30846-39-0), or poly(D,L-lactide-co-glycolide) (CAS number: 1354955-03-5).
Preferably, the at least one lipid is a monoacylglycerol, a diacyglycerol, a triacylglycerol, sterol, saturated and/or unsaturated fatty acid, saturated and/or unsaturated fatty acid-salts, and/or saturated fatty alcohol, and/or saturated fatty acid ester and/or a combination thereof. Wherein the monoacylglycerol comprises glycerol wherein one of the three available hydroxy groups are esterified with a saturated fatty acid. Wherein the diacyglycerol comprises glycerol wherein two of the three available hydroxy groups are esterified with a saturated fatty acid. Wherein the triacylglycerol comprises glycerol wherein three of the three available hydroxy groups are esterified with a saturated fatty acid.
The fatty acid is preferably R-COOH or the fatty acid salt the corresponding salt thereof, or a fatty alcohol R-OH, or a fatty acid ester R—OR wherein R is (C6-23) alkyl or (C6-23) alkenyl. More preferably, the fatty acid is selected from palmitic acid, CAS number: 57-10-3), the fatty alcohol is selected from 1-hexadecanol, CAS number: 36653-82-4 and the fatty acid ester is selected from cetylpalmitate, CAS number: 540-10-3.
Sterol is an organic compound with formula C17H28O; whose structure is derived from that of gonane by replacement of a hydrogen atom in position 3 by a hydroxyl group. Preferably, the sterol is cholesterol, CAS number: 26657-95-4.
More preferably, the monoglycerol is selected from glycerol a-monolaurate, CAS number: 142-18-7.
More preferably, the diacyglycerol is selected from dipalmitin, CAS number: 26657-95-4.
More preferably, the triacylycerol is selected from tristearin, CAS number: 555-43-1 or tripalmitin, CAS number: 55-44-2.
The term “alkyl” refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 6 to 23 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 carbon atoms, more preferably 8 to 23 carbon atoms, such as 10 to 23 or 12 to 20 carbon atoms. Exemplary alkyl groups include, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, and the like.
The term “alkenyl” refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 6 to 23, i.e., 1, 2, 3, or 4, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 6 to 23 carbon atoms, i.e. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 carbon atoms, more preferably 8 to 23 carbon atoms, such as 10 to 23 carbon atoms or 12 to 20 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 6 to 23 carbon atoms and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 carbon-carbon double bonds, more preferably it comprises 10 to 23 carbon atoms and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 carbon-carbon double bonds, such as 12 to 20 carbon atoms and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon-carbon double bonds may be in cis (Z) or trans (E) configuration. Exemplary 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom.
Preferably, the second layer comprises of from 0.1 to 60%, more preferably 1 to 50%, most preferably 10 to 25%, based on 100% by weight of the second layer. Preferably, the antibiotic is selected from a group consisting of beta-lactam antibiotics (β-lactams), penicillins, cephalosporins, monobactams, carbapenems, aminoglycoside antibiotics (aminoglycosides), quinolone antibiotics (quinolones), Glycopeptide antibiotics (glycopeptides), Lincosamide antibiotics (lincosamides), Macrolide antibiotics (macrolides), Ketolide antibiotics (ketolides), Nitroimidazole derivatives (nitroimidazoles), Polypeptide antibiotics (polypeptides), Sulfonamide antibiotics (sulfonamides), Diaminopyrimidines (trimethoprim), Tetracycline antibiotics (tetracyclines), Oxazolidinone antibiotics (oxazolidinones), lipopeptide antibiotics (lipopeptides), others such as rifampicin, chloramphenicol, tigecycline, mupirocin, fosfomycin and other anti-infective, antibiotic bactericidal, bacteriostatic substances more preferably vancomycin, daptomycin, rifampicin, fosfomycin, gentamycin, chloramphenicol, tigecyclin, mupirocin and any combination thereof.
The second layer may further comprise a saccharide, preferably the saccharide is at least one of maltotetraose, maltotriose, maltodextrine or a combination thereof. Preferably, the second layer comprises of from 0.1 to 10% by weight of the saccharide, based on 100% by weight of the second layer.
The second layer may further comprise phages. Preferably, the second layer comprises phages-enzymes, more preferably the phage-enzymes are endolysins, most preferably the endolysins are Staphefekt™ (Micreos, 3721 MA Bilthoven, Netherlands).
If phages-enzymes are comprised, a targeted therapy addressing at least one bacteria species would be possible. In this respect it is referenced to: Hamed Haddad Kashani, Mathias Schmelcher, et al. (20217)—Recombinant Endolysins as Potential Therapeutics against Antibiotic-Resistant Staphylococcus aureus: Current Status of Research and Novel Delivery Strategies Clin Microbiol Rev. 2017 Nov. 29;31 (1): e00071-17. doi: 10.1128/CMR.00071-17 and Lone, Ayesha Hany et Anany, al. (2016) Development of prototypes of bioactive packaging materials based on immobilized bacteriophages for control of growth of bacterial pathogens in foods; Int J Food Microbiol 2016Jan. 18;217:49-58, doi: 10.1016/j.ijfoodmicro.2015.10.011.Epub 2015 Oct. 22.
Preferably, the second layer comprises of from 0.01 to 20%, more preferably 0.1 to 15%, most preferably 0.5 to 10%, and particularly preferred 1.5 to 10%, particularly preferred 5 to 10%, by weight phage-enzymes, based on 100% by weight of the second layer.
Preferably, the second layer has a thickness of from 1 to 50 μm.
Preferably, the second layer in the implanted implant is biologically decomposed after at least 3 weeks, more preferably after at least 6 weeks, most preferably after at least 12 weeks.
The coating may further comprise a third layer arranged on the second layer, wherein the third layer comprises at least one lipid and at least one antibiotic and/or silver ions.
Preferably, the at least one lipid is a monoacylglycerol, diacyglycerol, triacylglycerol, sterol, saturated and/or unsaturated fatty acid, saturated and/or unsaturated fatty acid-salts, and/or saturated fatty alcohol, and/or saturated fatty acid ester and/or a combination thereof. Wherein the monoacylglycerol, diacyglycerol, triacylglycerol, sterol, saturated and/or unsaturated fatty acid, saturated and/or unsaturated fatty acid-salts, and/or saturated fatty alcohol, and/or saturated fatty acid ester is defined as above for the second layer.
Preferably, the third layer comprises of from 0.1 to 60%, more preferably 1 to 50%, most preferably 10 to 25%, by weight of the antibiotic, based on 100% by weight of the second layer. Preferably, the antibiotic is selected from a group consisting of beta-lactam antibiotics (β-lactams), penicillins, cephalosporins, monobactams, carbapenems, aminoglycoside antibiotics (aminoglycosides), quinolone antibiotics (quinolones), Glycopeptide antibiotics (glycopeptides), Lincosamide antibiotics (lincosamides), Macrolide antibiotics (macrolides), Ketolide antibiotics (ketolides), Nitroimidazole derivatives (nitroimidazoles), Polypeptide antibiotics (polypeptides), Sulfonamide antibiotics (sulfonamides), Diaminopyrimidines (trimethoprim), Tetracycline antibiotics (tetracyclines), Oxazolidinone antibiotics (oxazolidinones), lipopeptide antibiotics (lipopeptides), others such as rifampicin, chloramphenicol, tigecycline, mupirocin, fosfomycin and other anti-infective, antibiotic bactericidal, bacteriostatic substances more preferably vancomycin, daptomycin, rifampicin, fosfomycin, gentamycin, chloramphenicol, tigecyclin, mupirocin and any combination thereof.
The invention is further directed to a method of manufacturing an implant. Preferably, an implant as described above. The method comprises forming a first layer arranged on at least a portion of a surface of the implant, said forming of the first layer comprising contacting at a least a portion of the surface of the implant with a first coating composition comprising at least one polylactide and silver ions.
The implant and/or the surface of the implant may be made of any material suitable for implants. The implant and/or the surface of the implant may be made made of a metal or a non-metal such as a polymer.
Preferably, the implant and/or the surface is made of metal. Preferably, the implant and/or the surface material comprise 316 LVM implant steel, Co—Cr—Mo, TIN coated Cr—Co—Mo, titanium or a titanium alloy. Preferably, the titanium alloy is Ti6Al4V.
In one embodiment, at least the surface of the implant is made of metal. Preferably, the surface material comprises metal such as 316 LVM implant steel, Co—Cr—Mo, TIN, coated Cr—Co—Mo, titanium or a titanium alloy. Preferably, the titanium alloy is Ti6Al4V.
In one embodiment, at least the portion of surface of the implant, which is coated, is made of metal. Preferably, the surface material comprises a metal such as 316 LVM implant steel, Co—Cr—Mo, TiN coated Cr—Co—Mo, titanium or a titanium alloy. Preferably, the titanium alloy is Ti6Al4V.
In one embodiment, at least the surface of the implant is made of polymer. Preferably, the implant and/or the surface material comprises a polymer consisting of the group polyether ether ketone (PEEK), ultra-High molecular weight polyethylene (UHMWPE), low density polyethylene (LDPE), polymethylmethacrylate (PMMA), or polyphenylsulfone (PPSU). More preferably, the polymer is PEEK.
Preferably, the implant and/or the surface of the implant comprises at least 30 wt.-%, more preferably at least 60 wt.-%, most preferably at least 80 wt.-%, particular preferred at least 90wt.-%, most particular preferred at least 95 wt.-% of a polymer based on the overall weight of the implant.
Preferably, the implant and/or the surface of the implant comprises less than 30 wt.-%, more preferably less than 60 wt.-%, most preferably less than 80 wt.-%, particular preferred less than 90 wt.-%, most particular preferred less than 95 wt.-% of a polymer based on the overall weight of the implant.
In one embodiment, at least the portion of surface of the implant which is coated is made of a polymer. Preferably, the surface material comprises PEEK. More preferably, the polymer is PEEK.
A silver metal layer may be arranged between the surface of the implant and the first layer.
A calcium oxalate layer, may be arranged between the surface of the implant and the first layer.
The surface of the implant to be coated may be treated with glass bead blasting.
If the implant and/or the surface material is made of metal, preferably titanium or a titanium alloy, the implant and/or the surface material may be pretreated and/or etched, preferably to achieve osteointegration/osteoinduction with at least with one of the following hydrochloric acid, sulfuric acid, hydrogen peroxide, hydrofluoric acid, nitric acid, oxalic acid, potassium hydroxide, calcium lactate and dihydrogen phosphate. In this respect it is referred to Souza, Julio C. M.; Sordi, Mariane B.; Kanazawa, Miya; Ravindran, Sriram; Henriques, Bruno; Silva, Filipe S. et al. (2019): Nano-scale modification of titanium implant surfaces to enhance osseointegration. In: Acta Biomaterialia 94, S. 112-131. DOI: 10.1016/j.actbio.2019.05.045.
Preferably, the first layer comprises at least one polylactide selected from the group consisting of poly(L-lactide) (CAS number: 33135-50-1), poly(D-lactide), poly(L-lactide-co-D,L-lactide) (CAS number: 52305-30-3), poly(D-lactide-co-glycolide), poly(L-lactide-co-glycolide) (CAS number: 30846-39-0), poly(L-lactide-co-caprolactone) (CAS number: 65408-67-5), and Poly (L-lactide-co-trimethylene carbonate) (CAS number: 113883-70-8), poly(D,L-lactide-co-glycolide) (CAS number: 1354955-03-5), more preferably poly(L-lactide).
Preferably, the first layer comprises at least 70%, more preferably at least 80%, most preferably at least 90%, particularly preferred at least 95%, more particularly preferred at least 99% of the at least one polylactide.
Preferably, the silver ions are provided in form of a silver salt or a silver complex compound. More preferably, the silver ions are provided in form of a silver salt. Most preferably, the silver salt is selected from the group consisting of silver sulfadiazine, silver diethyldithiocarbamate, silver oxide, silver carbonate and silver nitrate, silver acetate, silver benzoate, silver iodate, silver laurate, silver protein, silver chloride, silver palmitate or combination thereof. Even more preferred silver diethyldithiocarbamate, particularly preferred silver nitrate.
Preferably, the first coating composition may comprise hydroxyapatite, or another poorly soluble calcium compound e.g. calcium carbonate.
Preferably, the first coating composition may comprise endolysins.
The first coating composition may comprise a solvent. Preferably, the solvent is chloroform or pyridine, more preferably pyridine. The solvent can be completely removed by heating in the manufacturing process.
Preferably, at least a portion of the surface of the implant is contacted with the first coating composition by dip coating or spray coating.
Preferably, forming of the first layer comprises drying.
The method may further comprise forming a second layer arranged on the first layer, said forming of the second layer comprising contacting the first layer with a second coating composition comprising at least one poly(lactide-co-glycolide) and at least one antibiotic/antiinfective and/or silver ions.
Preferably, the poly(lactide-co-glycolide) in the second layer is poly(D-lactide-co-glycolide), or poly(L-lactide-co-glycolide) (CAS number: 30846-39-0), or poly(D,L-lactide-co-glycolide) (CAS number: 1354955-03-5).
Preferably, the antibiotic is selected from a group consisting of beta-lactam antibiotics (β-lactams), penicillins, cephalosporins, monobactams, carbapenems, aminoglycoside antibiotics (aminoglycosides), quinolone antibiotics (quinolones), Glycopeptide antibiotics (glycopeptides), Lincosamide antibiotics (lincosamides), Macrolide antibiotics (macrolides), Ketolide antibiotics (ketolides), Nitroimidazole derivatives (nitroimidazoles), Polypeptide antibiotics (polypeptides), Sulfonamide antibiotics (sulfonamides), Diaminopyrimidines (trimethoprim), Tetracycline antibiotics (tetracyclines), Oxazolidinone antibiotics (oxazolidinones), lipopeptide antibiotics (lipopeptides), others such as rifampicin, chloramphenicol, tigecycline, mupirocin, fosfomycin and other anti-infective, antibiotic bactericidal, bacteriostatic substances more preferrably vancomycin, daptomycin, rifampicin, fosfomycin, gentamycin, chloramphenicol, tigecyclin, mupirocin and any combination thereof.
The second coating composition may further comprise a saccharide. Preferably, the saccharide is at least one of maltotetraose, maltotriose, maltodextrine or combinations thereof.
Preferably, the second coating composition may comprise endolysins.
Preferably, the second coating composition comprises a solvent for poly(lactide-co-glycolide) (e.g. acetone, dimethyl sulfoxide, formic acid) and for lipid (e.g. pentane, heptane). More preferably for poly(lactide-co-glycolide), the solvent is dimethyl sulfoxide, for TAG is pentane and cholesterol is tert-butyl methyl ether. The solvent can be completely removed by heating in the manufacturing process.
The first layer may be contacted with the second coating composition by dip coating or spray coating.
Preferably, forming of the second layer further comprises drying.
The method may further comprise forming a second layer arranged on the first layer, said forming of the second layer comprising contacting the first layer with a second coating composition comprising at least one lipid and/or at least one polylactide and at least one antibiotic and/or at least one silver salt.
In a further embodiment, the method comprises forming a first layer arranged on at least a portion of a surface of the implant, said forming of the first layer comprising contacting at a least a portion of the surface of the implant with a coating composition comprising at least one poly(lactide-co-glycolide) and/or at least one lipid and at least one antiinfective/antibiotic, preferably a coanting composition as referred to above as “second coating composition”. This means that the second coating composition is arranged directly on the implant's surface in this embodiment forming the first layer instead of the first coating composition.
Preferably, the at least one lipid is a monoacylglycerol, diacyglycerol, triacylglycerol, sterol, saturated and/or unsaturated fatty acid, saturated and/or unsaturated fatty acid-salts, and/or saturated fatty alcohol, and/or saturated fatty acid ester and/or a combination thereof as further defined above for the first layer. More preferably, the at least one lipid is a triacyglycerol.
Preferably, the antibiotic is selected from a group consisting of beta-lactam antibiotics (β-lactams), penicillins, cephalosporins, monobactams, carbapenems, aminoglycoside antibiotics (aminoglycosides), quinolone antibiotics (quinolones), Glycopeptide antibiotics (glycopeptides), Lincosamide antibiotics (lincosamides), Macrolide antibiotics (macrolides), Ketolide antibiotics (ketolides), Nitroimidazole derivatives (nitroimidazoles), Polypeptide antibiotics (polypeptides), Sulfonamide antibiotics (sulfonamides), Diaminopyrimidines (trimethoprim), Tetracycline antibiotics (tetracyclines), Oxazolidinone antibiotics (oxazolidinones), lipopeptide antibiotics (lipopeptides), others such as rifampicin, chloramphenicol, tigecycline, mupirocin, fosfomycin and other anti-infective, antibiotic bactericidal, bacteriostatic substances more preferably vancomycin, daptomycin, rifampicin, fosfomycin, gentamycin, chloramphenicol, tigecyclin, mupirocin, and any combination thereof.
The second coating composition may further comprise a saccharide. Preferably, the saccharide is at least one of maltotetraose, maltotriose, maltodextrine or combinations thereof.
Preferably, the second coating composition may comprise endolysins.
Preferably, the second coating composition may comprise a solvent, more preferably the solvent is pentane.
The first layer may be contacted with the second coating composition by dip coating or spray coating.
Preferably, forming of the second layer further comprises drying.
The method may further comprise forming a third layer arranged on the second layer, said forming of the third layer comprising contacting the second layer with a second coating composition comprising at least one lipid and/or at least one polylactide and at least one antiinfective/antibiotic and/or a silver salt.
Preferably, the at least one lipid is a monoacylglycerol, diacyglycerol, triacylglycerol,, sterol, saturated and/or unsaturated fatty acid, saturated and/or unsaturated fatty acid-salts, and/or saturated fatty alcohol, and/or saturated fatty acid ester and/or a combination thereof and as further defined above for the first layer. More preferably, the at least one lipid is a triacyglycerol.
Preferably, the antibiotic is selected from a group consisting of beta-lactam antibiotics (β-lactams), penicillins, cephalosporins, monobactams, carbapenems, aminoglycoside antibiotics (aminoglycosides), quinolone antibiotics (quinolones), Glycopeptide antibiotics (glycopeptides), Lincosamide antibiotics (lincosamides), Macrolide antibiotics (macrolides), Ketolide antibiotics (ketolides), Nitroimidazole derivatives (nitroimidazoles), Polypeptide antibiotics (polypeptides), Sulfonamide antibiotics (sulfonamides), Diaminopyrimidines (trimethoprim), Tetracycline antibiotics (tetracyclines), Oxazolidinone antibiotics (oxazolidinones), lipopeptide antibiotics (lipopeptides), others such as rifampicin, chloramphenicol, tigecycline, mupirocin, fosfomycin and other anti-infective, antibiotic bactericidal, bacteriostatic substances more preferably vancomycin, daptomycin, rifampicin, fosfomycin, gentamycin, chloramphenicol, tigecyclin, mupirocin, and any combination thereof.
The third coating composition may further comprise a saccharide. Preferably, the saccharide is at least one of maltotetraose, maltotriose, maltodextrine or combinations thereof.
Preferably, the third coating composition may comprise endolysins.
Preferably, the third coating composition comprises a solvent for polylactide (e.g. dimethyl sulfoxide, formic acid) and for lipid (e.g. pentane, heptane). More preferably for polylactide, the solvent is dimethyl sulfoxide, for TAG is pentane and cholesterol is tert-butyl methyl ether. The solvent can be completely removed by heating in the manufacturing process.
The second layer may be contacted with the third coating composition by dip coating or spray coating.
Preferably, forming of the third layer further comprises drying.
The invention is further directed to an implant, obtainable by a method as described above.
The invention is further directed to a coating of an implant as defined above for use in a method for preventing or treating a local infection with a pathogen near the implanted implant exhibiting said coating as defined above, wherein the method comprises exposure of the first layer of the coating of the implanted implant to shock waves resulting in an increased release of silver ions from the first layer of the coating.
Wherein local infection means that the effect of the released silver anions is local in the subject and not systemic.
Silver ions exhibit a broad-spectrum activity against both gram-positive and gram-negative bacteria, fungi, protozoans, and viruses and are less likely to induce bacterial resistance than antibiotics (Brennan et al. 2015, Chaloupka et al. 2010).
Wherein the pathogen's growth may be inhibited or killed by silver anions. Preferably the pathogen is selected from bacteria and fungi, protozoans and viruses, more preferably from bacteria.
Preferably, the bacteria are gram-positive or gram-negative bacteria, more preferably selected from the group consisting of staphylococcus aureus, coagulase negative staphylococcus, streptococcus, enterococcus, anaerobic bacteria.
Preferably, the fungi is Candida albicans.
Acoustic shock waves are commonly applied in medicine for the treatment of several indications. Thus, the person skilled in the art may use commercially available equipment and established procedures to apply the shock waves to expose the implanted implant to the shock waves in order to induce the increased release of silver ions from the first layer.
Preferably, the shock waves are acoustic waves
In one embodiment, the shock waves exhibit at least one of the following characteristics a)-h):
A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.
An overview of a coating variant with activation is summarized in a graphical abstract
A durable layer (approx. 10 μm) of poly-L-lactide (Resomer® L 206 S, Evonik Health Care, Darmstadt) with a soluble silver compound is applied to the implant made of a frequently used titanium alloy (Ti6Al4V Grade 5). Resomer L 206 S is a very slowly (>10 years) absorbable biocompatible layer that releases homogeneously dissolved silver ions when colonized with bacteria. Three different processes have been developed, from which result different silver release.
Variant 1: 100 mg Resomer L206 S are dissolved in 900 μL 0.5% Ag-diethyldithiocarbamate in chloroform in a 3 mL PFA (perfluoroalkoxy-polymer) vessel. The implant specimen is briefly immersed and dried hanging under an S2 workbench for 1 hour at room temperature. Ag-diethyldithiocarbamate is a nontoxic silver compound homogeneously soluble in nonpolar polymers such as L206 S or chloroform. By drying, the solvent chloroform (boiling point 61° C.) is completely removed.
Variant 2: In a 3 mL PFA vessel, mix 25 μL of 10% AgNO3 solution (shake 100 mg AgNO3 (powdered)+1 ml chloroform+100 μl pyridine at room temperature for several hours until crystals are dissolved) with 1 mL of 10% Resomer (100 mg) L 206 S in chloroform. The implant specimen is briefly immersed. The test specimen is then heated to 150° C. for 3 min. Silver nitrate is also soluble in organic solvents in small quantities, as a silver pyridine complex well soluble. After homogeneous solution in the polymer, the pyridine (boiling point 115° C.) is removed completely by heating to 150° C.
Variant 3: In a 3 mL PFA vessel, mix 25 μl of 10% AgNO3 solution (shake 100 mg AgNO3(powdered)+1 ml chloroform+100 μl pyridine at room temperature for several hours until crystals are dissolved) with 1 mL of 10% Resomer (100 mg) L 206 S in chloroform. The implant specimen is briefly immersed. The test specimen is then heated to 200° C. for 1-2 min until the polymer is melted clear. The increased temperature to 200° C. promotes adhesion to the titanium substrate and the homogeneity of the surface of the layer. In the case of real implants, the amount of solution can be scaled accordingly and the PFA vessels can be replaced by glass vessels of an appropriate size (glass blowing).
Typical metallic biomaterials in medical technology are Co—Cr—Mo alloys, pure titanium and titanium alloys (Karacan et al. 2018) as well as Co—Cr—Mo alloys with surface modifications like TiN and others. Typical polymers in medical technology are PEEK, UHMWPE, LDPE, PMMA, PPSU. The selection of these materials results from the requirements regarding corrosion resistance, biocompatibility, bio-adhesion and favorable mechanical properties (Peters et al. 2002). Due to its extensive use as an implant material in orthopedics, the titanium alloy Ti6Al4V Grade 5 was chosen as preferred substrate material for the present coating system. The surface was prepared using glass bead blasting (dk=150-250 μm, Eisenwerk Würth GmbH, Bad Friedrichshall, Germany) in accordance with recommendations by Guo, Matinlinna et al. with a pressure of p=2.5 bar for 12 s in a distance of 65 mm. The substrate geometry was defined as disc with a diameter of 14 mm, a thickness of 1.5 mm and a centric hole with a diameter of 2 mm.
Standardized titanium alloy substrates were used for the evaluation of the coatings (Ti6Al4V Grade 5). To characterize the mechanical properties of the coating system on these, surface texture analysis was performed. In a first step glass bead blasting was used in accordance with data from the literature (Byrne et al. 2013; Guo et al. 2019). In a second step a novel, very promising method was conceived: A oxalic acid (H2C2O4) etching followed by rinsing with 1 m calcium lactate solution results in a finer structure (Ra 0.25 μm) compared to glass bead blasting (Ra 0.67 μm). The resulting compound calcium oxalate is extremely stable and insoluble and thus resists absorption in the body. The homogeneity of this coating was validated by an energy dispersive X-ray spectroscopy (EDX-analysis) using a scanning electron microscope (SEM) and XFLASHr® 6|10 detector (Bruker Co., Billerica, Massachusetts, USA) (
Pull-off tensile tests revealed a 2.3-fold increased bond strength between the coating and the Ti6Al4V substrate of 10 MPa (without) to 23 MPa with CaC2O4 etching. This complies with the ASTM F1147-05 (2017) e1 standard requirement for coatings on medical implants of 22 MPa. Hydrophobicity and a low surface free energy comparable to silver were determined from contact angle measurement, whereas Ti6Al4V was hydrophilic with a higher surface free energy indicating a lowered risk for bacterial adhesion for the etched surface (
The first layer with activatable properties consisting of a Poly(L-lactide) (PLLA—Resomer L206 S, Evonik Health Care, Darmstadt, Germany) The PLLA is soluble in chloroform, and 1 mL of 10% PLLA were loaded with 25 μL homogeneously dissolved silver ions (Ag+) in concentrations of 2%/6%/8%.
All steps were performed under sterile conditions in a S2 workbench (HERAsafe HS12—Thermo Scientific GmbH, Dreieich-Germany). The substrate was sterilized by immersion in 70% ethanol and after evaporation manually dip-coated by immersion/emergence within 5 seconds. The substrate is then heated to 200° C. for 1-2 min until the polymer is melted clear, which completely removes the solvent chloroform. After cooling sterilization with a short immersion in 70% ethanol and subsequent evaporation was then repeated. The coated substrates were stored in sterile bags (SteriBag, 60 ml, 127×76 mm Bürkle GmbH, Germany) until testing at the next day. The anti-infective effect of this coating is the release of incorporated silver ions induced by an activation with a extracorporeal shock wave (ESW) which results in a local detachment of coating fragments.
The second coating layer is designed for a loading with anti-infectives and for resorption within weeks to reduce the initial risk of infection after implantation on the one hand, and in case of an infection to locally support the systemic therapy on the other hand. Different lipids (triacylglycerol (TAG) tristearin CAS number: 555-43-1 and tripalmitin CAS number: 55-44-2 (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany), cholesterol (CAS number: 57-88-5, Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany), diacylglycerol dipalmitin (CAS number: 26657-95-4 Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany), monoacylglycerol α-monolaurate (CAS number: 142-18-7, Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany)) were included. The preferred lipid coating consisted of triacylglycerol (TAG).
The TAG is applied by dipping the substrate in a 10% triacylglycerol solution in the solvent pentane with a finely powdered (mortared and sieved) or dissolved antibiotic. Alternatively, the antibiotic was mixed in a 20% RG 502 (PLGA) (Evonik Health Care, Darmstadt, Germany) solution in dimethyl sulfoxide or formic acid alone or with 100 μl 20% lauricidin (monolaurin) in formic acid with 100 mg cholesterol in 800 μl TBME, under sonication. The solvent is removed by drying at room temperature in a hanging position and followed by sterilization with a short immersion in 70% ethanol and subsequent evaporation. The coated substrates were stored until testing in sterile bags until next day. All steps were performed under sterile conditions in a S2 workbench. The coating degrades with defined kinetics, constantly releasing anti-infectives within the first 6 weeks and 3 months, respectively.
Samples were transferred to a 500 μL PFA vessel (AHF-Analysetechnik, Tübingen, Germany). 100 μL HNO3 (0.16 mol/L) was added and incubated overnight at room temperature. Before measurement, the samples were incubated at 70° C. for 90 min, cooled, and diluted 10-fold with H2O. A volume of 20 μL of the solution was added directly to the graphite furnace atomic absorption spectrometry (GF-AAS) (AAS-6300, Shimadzu, Kyoto, Japan). Triplicate measurements were performed for each determination. The recommended operating conditions for the spectrometer were: Lamp current of the hollow cathode lamp: 12 mA; absorption wavelength: 328.1 nm; BGC-D2 mode; slit width: 0.7 nm. Working standard solutions containing 0, 0.5, 1, 2, and 4 μg/L Ag (Standard Merck, Germany) were used. The intra-and inter-day precision of the assay, expressed as coefficient of variation (CV %), ranged from 2.8 to 6.5%. The recommended operating conditions for the GF-AAS nebulizer are listed in Table 1.
Ag-Elution in water for untreated variants 1 to 3 has been determined and the results listed in Table 2.
Depending on the preparation method, only a negligible amount of silver can be eluted in recipes 1 and 3.
The prepared samples according to variant 1 to 3, as prepared above, have been treated with acoustic shock waves and Ag-elution has been determined. The results are listed in Table 3.
The silver content in the supernatant (aqua) measured by graphite furnace atomic absorption spectrometry changes depending on the coating production method and the intensity of the applied shock wave.
In order to be able to detect and analyze the possibility of mechanical activation of the RESOMER® L coating, the test bench shown in
Due to the comparable acoustic impedance Z of the media water (Zwater=1.48×10E6 Ns/m3), adipose tissue (Zfat=1.33×10E6 Ns/m3) and muscle tissue (Zmuscle=1.67×10E6 Ns/m3) an appropriate transferability to the ratios in vivo can already be ensured. Following the filling of the interior with water, the DUOLITH SD1 shock wave device with C-ACTOR handpiece (STORZ MEDICAL AG, Tägerwilen, Switzerland) was positioned at a distance of 35 mm from the sample surface. The following parameters were selected for the subsequent shock wave application:
Low intensity
Following the test execution, the liquid was pippet from the sample bags and again sterilely packaged in a centrifuge tube (Safe-Lock Tubes, Eppendorf AG, Hamburg) in order to examine the amount of released silver by atomic absorption spectrometry (GF-AAS) (see above). Furthermore, the specimen was used for further scanning electron microscopic analysis of the surface degradation.
For the analysis of the shattering effect of the RESOMER® L coating achieved by the shock wave exposure, the scanning electron microscope (SEM) including the energy dispersive X-ray spectroscopy (EDX) was used. The REM Zeiss EVO MA10 (Carl Zeiss Microscopy GmbH, Jena, Germany) was used in conjunction with the XFLASH® 6|10 detector (Bruker Co., Billerica, Massachusetts, USA). In order to generate an electrical conductivity of the sample surface, it was first vaporized with a gold layer. For this purpose, DC sputtering was carried out with a current of I=10 mA and a voltage of U=1.17 kV for a duration of 40 seconds. The SEM analysis was carried out with an acceleration voltage of EHT=20 kV and a working distance of WD=10 mm using an SE detector in up to 5000× magnification.
The EDX analysis was performed for both the element titanium (Ti) and the element silver (Ag). In addition to the qualitative statement via SEM, the measurement of the Ti content at the surface allows a quantitative statement about the area of the coating shattered by the shock wave treatment. The measurement of Ag content on the surface also provides quantitative information on the release of silver by measuring an area without fhESWT (1.54% Ag before shock wave) and an area after fhESWT (0.17% Ag after shock wave) of the sample surface (
Based on the 6-week to 3-month clinical anti-infective therapy period, the drug concentration in the eluate is quantified and the microbiological activity of the fractions is tested in a continuous flow chamber assay.
The release of the antibiotic from the layer of the test specimen (P) was studied over 6 weeks using a continuous flow chamber experiment (
In the flow chamber (AKTA Ettan LC FPLC System, Amersham Pharmacia Biotech, Uppsala, Sweden), sample body is continuously passed through the sample chamber by a buffer liquid (PBS buffer) at a rate of 1 mL/h at 37° C. using a fast protein liquid chromatography (FPLC) pump. The eluate with PBS buffer is collected once an hour using a fraction collector (Fraction Collector FRAC-950, Pharmacia, Uppsala, Sweden). Using a Kontron Uvikon photometer (Kontron, Neufahrn, Germany), the concentration of rifampin in 500 μL of the samples was quantified at 237 nm (EmM (max absorbance, PBS, pH 7.38:33.20) and the other part was used for microbiological activity determination.
The aim is to obtain the best possible linear effective level above the minimum inhibitor concentration (MIC), and a subsequent adequate drop in the effective level after 6 weeks.
The release of active ingredients as antibiotics, as rifampin, incorporated in the lipid coating is shown in
The rifampin-eluates were also tested from a series with different concentrations of maltotriose in an inhibition test shown in
Cell cultivation is required to assess cell viability in direct contact with the coating, as well as by indirect contact with eluates or components dissolved after shock wave application.
Human dermal fibroblasts from healthy individuals were obtained from biopsies proliferated in primary cultures and then sub cultured. They are non-specific connective tissue cells with an irregular shape, whose function is to generate components of the extracellular matrix. Their isolation and proliferation in cultures is uncomplicated.
SaOs-2 cells: Osteosarcoma cells, “osteoblast-like-cells,” are immortalized bone cells and are highly favored for cell culture methods because they can be easily and rapidly cultured and are free of transformation due to their sarcomatous origin as a cell line.
Human osteoblasts and fibroblasts were cultured at 37° C. in a 5% CO2-95% atmospheric incubator. As growth medium, DMEM (Dulbecco's modification of Eagle's medium) with an addition of 10% (v/v) fetal calf serum, 2 mM L-glutamine and 1% antibiotic-antimycotic solution was used. When this medium was used, the culture wells (75 cm2) were filled with 1 ml of sterile 0.1% fibronectin solution (0.1% fibronectin in PBS) and the solution was dispersed homogeneously on the bottom of the well. After 2 h incubation at 37° C., the cells could be seeded.
Two buffer systems were used to preserve the pH range of 7.4: HEPES (N 2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 20 mM) and sodium hydrogen carbonate (3.7 g/l). Changes in pH could be detected by the change in color of phenol red.
Cells grew as monolayer culture in tissue culture flasks (75 cm2, 25 cm2), or multidishes with six wells (each 10 cm2).
The intervals of medium replacement have varied with the different used cells. A medium change was required as soon as a pH decrease was indicated by the color indicator. In addition, for thawed or subcultured cells, a medium change was routinely performed the following day to remove unattached cells or cell debris.
All solutions used for subcultivation were warmed to 37° C. The medium was removed from culture flasks (75 cm2) with confluent cell lawn for passaging. The monolayer was then briefly rinsed with 10 mL PBS/0.1% EDTA to remove calcium, magnesium (essential cations for cell adhesion) and traces of the medium. The latter may interfere with the effect of subsequent trypsin treatment and significantly prolong the time of exposure to the cells. After addition of 2 mL of trypsin (0.05%)-EDTA (0.02%) solution, the process of detachment of cells was observed under the light microscope. When the cells began to become round (approximately 1 min), the solution was aspirated, and the cells were incubated at 37° C. for 5-10 min until complete detachment. Using a pipette, the cells were rinsed with fresh medium from the bottom of the vessel, stopping the protease action of trypsin. Cells were seeded in 20 ml of medium at a dilution factor of 1:3 to 1:7, depending on requirements, and multidishes were loaded with 3 mL of medium. The amount of wash and trypsin-containing solutions could be varied depending on the contamination and size of the culture vessels.
Cell storage was performed in liquid nitrogen (−196° C.), thus guaranteeing storage for extended periods without loss of viability. It was used to store less frequently used cell lines or excess cell quantities, to protect them from variability due to repeated subculturing and from microbial contamination.
Cells from one culture flask (75 cm2) were suspended in 1 mL of medium and placed in specifically designed vessels (“cryotubes”), resulting in a concentration of approximately 1×107 cells per ml of medium. After addition of 100 μL glycerol, the cryotubes were sealed tightly and mixed briefly until phase separation disappeared. Then the cells were cooled for 1 h in an ice bath. The tubes were then placed in a Styrofoam container and stored in the freezer at −80° C. overnight. They were then transferred to a liquid nitrogen container. To thaw the cells, the cryotubes were warmed to 37° C. in a water bath and immediately after the solid phase disappeared, they were placed in a new culture flask (75 cm2) filled with culture medium.
Analysis of viability and cytotoxicity by determining the cell count: An electronic counter (CASY® Cell Counter 1, Schärfer System GmbH, Germany) was used to determine the cell count. This is a semi-automatic cell counter that works according to the resistance measuring principle. The voltage applied between two large-area platinum electrodes experiences an electrical resistance change when a cell is located between the electrodes and triggers an electrical pulse. The number of electrical pulses is equal to the number of cells. The size distribution of the cells can be recorded and evaluated by means of a measuring amplifier based on a proportional relationship to the pulse strength. Larger pulses represent vital cells, the smaller pulses represent cell debris or dead cells. Avital cells cause a smaller pulse because their cell membrane is more conductive. By shifting the cell boundaries these could be excluded from the vital cell count. For this purpose, a portion of the cell suspension was added to 10 mL of physiological saline (0.9%, pH 7.4), the dilution factor was included, and the solution was measured three times.
Additionally, cell number was determined using a hemocytometer (Neubauer counting chamber). To distinguish between dead and living cells, vital staining with trypan blue was performed. For this purpose, after the trypsinization procedure, 0.1 mL of the cell suspension was taken up in 3.6 ml PBS and 2.7 mL of a pre-warmed 0.5% trypan blue solution was added. The mixture was mixed and incubated at 37° C. for 2 min. In contrast to living cells, the dead cells took up the dye. This process was followed under the light microscope. The cell numbers from the two measurement methods were compared and agreed.
Cell number determination by the above method of osteoblasts in contact with lipid compounds (such as triacylglycerol-TAG) was performed after one, three and five days (
The aim of this experiment is to determine the sensitivity of cultures of S. aureus to supernatants containing silver ions after shock wave application (3 Hz, n=1000, 1.24 mJ/mm2) to the polylactide coating (Resomer L206 S). For this purpose, the inhibition host test, also called agar diffusion test, was performed following EUCAST (European Committee for Antimicrobial Susceptibility Testing). On the agar plate, 2 mL of the bacterial suspension S. aureus 6850 was added and inoculated by carefully circling the plate on the laboratory bench, distributed over the entire agar surface. The supernatant of the bacterial suspension was removed with the pipette and discarded. Test slides are placed on the agar plate and gently pressed. 100 μL of the supernatants after shock wave application are added to the test leaflets. The agar plate was incubated with the bottom side up overnight at 37° C. Test plates inoculated with supernatants of the coating (L206 S) according to recipe 1 to 3 showed an inhibition zone and thus bactericidal effect as shown in
To analyze the effect of released silver from the coating (Recipe 3) after shock wave application (3 Hz, n=1000, 1.24 mJ/mm2), supernatants were transferred to suspension cultures. Bacteria (Staphylococcus aureus 680, Escherichia coli TG1) from an overnight culture in Mueller Hinton II-medium (a nutrient-poor growth medium) were adjusted to approximately an optical density (OD) 600 nm of 0.1 and then pipetted 100 μL per well into a 96 well plate. In addition to an untreated control, 10 μL of sample (supernatant after shock wave/eluate solution) was added to each well. The plate was incubated at 37° C. OD was determined hourly for 24 hours in an automated manner and transferred to growth curves. Growth or inhibition of growth can be determined from an increase or decrease in OD (
For the assessment of possible cytotoxicity, the study of cell proliferation of fibroblasts in contact with the supernatants was performed after shock wave application (3 Hz, n=1000, 1.24 mJ/mm2) of the coating (L206 S, according to recipe 3) loaded with different concentrations of silver. Cell proliferation was determined by WST-1 assay:
A stable tetrazolium salt (WST-1) can be cleaved into a soluble formazan by a bio-reductive cellular mechanism that occurs on the surface of the cell membrane. This depends on the glycolytic formation of NAD (P) H in viable cells. The amount of formazan dye formed correlates directly with the number of metabolically active cells in the cell culture. Cells grown in a 6-well tissue culture plate were incubated with WST-1 reagent for 4 hours. For this purpose, 100 μL/well of WST-1 cell proliferation reagent was added to the cells (culture medium 1000 uL/well DMEM/5% FCS) under sterile conditions. (1:10 final dilution). After this incubation period, the formazan dye formed is measured and quantified using a spectrophotometer (Uvikon Kontron, Neufahrn, Germany). The measured absorbance at 450 nm (reference wavelength 630nm) correlates directly with the number of viable cells.
Cell proliferation of fibroblasts under the influence of different Ag concentrations was determined by WST-1 assay. The results are shown in
The topography of the coating(s) according to 1.2.2 and 1.2.3 was studied qualitatively and quantitatively. For a qualitative analysis regarding surface texture and coating coverage integrity, a scanning electron microscope (SEM) (Zeiss EVO MA10, Carl Zeiss Microscopy GmbH, Jena, Germany) in combination with the energy dispersive X-ray spectroscopy (EDS) (XFLASH® 6|10 Detector, Bruker Co., Billerica, Massachusetts, USA) with an accelerating voltage of EV=20 kV, working distance WD=11 mm and 500×, 1000×, 2000× and 5000× magnifications was used. To reduce charging of the non-conductive coatings, a thin layer of gold was previously applied on the sample surfaces using DC-sputtering (Polaron E5000, Polaron Equipment Ltd, Watford, UK) for t=40 s with a current of I=10 mA and a voltage of U=1.17 kV. In an SEM environment, the specimens must be stable under vacuum. For coating materials that do not fulfill this requirement, a light microscope (LM) (Axio Imager, Carl Zeiss Microscopy GmbH, Jena) was used to qualitatively analyze the topography in bright field mode with 100×, 200× and 500× magnification, respectively. For a quantitative analysis, contactless surface roughness measurements were conducted using a confocal microscope (CM) (MarSurf CM mobile, Mahr GmbH, Göttingen, Germany) with a 20× lens, an evaluation length of 4 mm and a cutoff wavelength of λc=0.8 mm in accordance with the ASTM F2791-15 standard (n=5). The post processing was done using the MountainsMap® software (Digital Surf®, Besançon, France) and the widely used mean roughness indexes Ra (2D) (ISO 21920) and Sa (3D) (EN ISO 25178) was calculated. To improve the quality when testing the transparent PLLA coating, a DC-sputtering was conducted additionally for t=120 s.
Both the analysis of the morphology and the layer thickness were performed microscopically using a cross-section polish with an SEM and LM according to the ISO 2808 standard. The required metallographic preparation was conducted according to the method listed in Table 4.
To detect changes in the layer thickness over the cross-section, measurements were performed 18 times along each cross-section polish. Additionally, the thickness was determined by an eddy-current gauge (Fischerscope MMS PC2 ETA3.3, Helmut Fischer GmbH, Sindelfingen, Germany) with 18 measurements randomly distributed along the coating surface.
The qualitative analysis regarding surface texture and coating coverage integrity for both coatings is shown in
Regarding the effect of topography on bacterial adhesion, defined surface features (e.g. patterned or line-like grooves) decrease the bacterial adhesion if their dimension is smaller than a single bacterium and vice versa (Vasudevan et al. 2014; Helbig et al. 2016). Therefore, this coating shows a favorable topography in order to prevent bacterial adhesion.
The component surface roughness of the coating system determined by confocal microscope is shown in
To investigate the coating's morphology, cross-section polishes were analyzed via LM and SEM (
The cross-section polishes (
The cytotoxicity has been investigated by determining the cell count analysis for the lipid coating (
Cell viability was additionally investigated from a WST-1 assay with fibroblasts and a silver concentration series in PLLA. The results are shown
The antibacterial effect of the silver released by ESW was first tested for its biological activity in a suspension culture and inhibitory test (
The antibacterial effect of the vancomycin loaded TAG coating on S. epidermidis after incubation for 24 h is shown in
The Invention further comprises the following items:
| Number | Date | Country | Kind |
|---|---|---|---|
| 21193637.2 | Aug 2021 | EP | regional |
| 22168980.5 | Apr 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/073799 | 8/26/2022 | WO |
