Patent Application
20230310698
The present invention relates to a biocompatible, flexible, bone-adhesive sheet comprising:
The bone-adhesive sheet of the present invention is particularly suited for treatment of bone defects, e.g. by preventing fast-regenerating soft tissues from growing into more slowly regenerating bone tissue.
The invention further provides a method of preparing a bone-adhesive sheet, such as a barrier membrane, said method comprising:
The present method enables effective distribution of the polymer particles inside the fibrous carrier structure and the production of bone-adhesive sheets with excellent adhesive properties.
Barrier membranes are routinely applied in surgery to allow for regeneration of alveolar bone, while various fixation devices are used in general and trauma surgery to fixate soft tissue to bone (tendon rupture), bone tissue to bone (fractures) or foreign implants to bone tissue (hernia repair with meshes). The current generation of commercially available, degradable barrier membranes and fixation devices has serious shortcomings related to i) a poor control over degradation rates, ii) poor mechanical properties, iii) poor understanding of biological mechanisms governing soft/bone tissue reconstruction and infection, iv) poor clinical manageability and stability, and v) long-term dependence of rigid non-degradable materials.
Currently, no biomaterials are available which adhere specifically to bone. Consequently, the development of medical devices which adhere specifically to bone would solve one of the major problems in oral, general and trauma surgery, namely the facile repair of damaged bone and/or fixation of soft or hard tissues to bone.
US 2013/0149355 describes a method to prevent protein and cell adsorption, said method comprising:
The hydroxyapatite-targeting moiety may be selected from the group consisting of tetracycline, calcein, bisphosphonates, polyaspartic acid, polyglutamic acid, and aminophosphosugars. The water-soluble polymer may be selected from the group consisting of poly(alkyleneglycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrolidone), poly(hydroxypropylmethacrylamide poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, and copolymers and terpolymers thereof.
Ossipov (Bisphosphonate-modified biomaterials for drug delivery and bone tissue engineering, Expert Opin Drug Deliv. (2015); 12(9):1443-1458) reports that strategies of chemical immobilization of bisphosphonates in hydrogels and nanocomposites for bone tissue engineering have emerged that opened new applications of bisphosphonates in bone tissue engineering. Conjugates of bisphosphonates to different drug molecules, imaging agents, proteins and polymers are discussed in terms of specific targeting to bone and therapeutic effect induced. Conversion of these conjugates into hydrogel scaffolds is mentioned along with the application of the resulting materials for bone tissue engineering.
Zhang et al. (The interaction of cationic polymers and their bisphosphonate derivatives with hydroxyapatite, Macromol Biosci. (2007); 7(5), 656-670) describe the construction of a polymeric linker containing multiple copies of BPs for protein conjugation and targeting to bone. Poly(L-lysine) (PLL) and poly(ethylenimine) (PEI) were utilized as the polymeric backbones to incorporate a BP, namely 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP), by using N-hydroxysuccinimidyl polyethylene glycol maleimide and succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, respectively. In vitro and in vivo mineral affinity of the polymer-BP conjugates were determined in comparison with the unmodified polymers. The in vitro results indicated strong binding of the cationic polymers to HA in their unmodified form. BP conjugation did not enhance the inherent mineral affinity of the polymers; in contrast, certain modifications negatively affected the polymers' binding to the HA. In vivo results from a subcutaneous implant model in rats also showed no significant difference in mineral affinity of the BP modified and unmodified PEI.
Sánchez-Fernández et al. (Alendronate-functionalized poly(2-oxazoline)s with tunable affinity for calcium cations, Biomacromolecules (2019); 20(8):2913-2921) reported the synthesis of a library of alendronate-functionalized polyoxazolines with control over the polymerization and functionalization degrees. The binding affinity of these polymers for calcium cations was much higher in comparison with other calcium-binding polymers reported previously. Results showed that adjusting the alendronate content in the polymer produced robust gels with a strong capacity for self-healing. The tunable synthetic versatility and affinity for calcium render these polymers excellent candidates for various applications in biomedicine.
WO 2010/059280 describes an anhydrous fibrous sheet comprising a first component of fibrous polymer, said polymer containing electrophilic groups or nucleophilic groups, and a second component capable of crosslinking the first component when said sheet is exposed to an aqueous medium in contact with biological tissue to form a crosslinked hydrogel that is adhesive to the biological tissue; wherein:
U.S. Pat. No. 6,733,845 describes process for the electrostatic impregnation into a fibrous or filamentary network with powder, for producing a composite comprising a rigid or flexible matrix with which said network is in intimate contact, wherein the powder and said network of fibers or filaments are placed between two electrodes, said electrodes being electrically isolated insulated from each other and said electrodes being connected respectively to the oppositely charged poles of an AC voltage electrostatic generator so as to simultaneously subject said powder and said fibrous or filamentary network lying between said electrodes to an electrostatic field, the AC voltage of which is at least 5 kV, for a time of at least 2 seconds.
The inventors have developed a biocompatible, flexible, bone-adhesive sheet that is particularly suited for treatment of bone defects, e.g. by preventing fast-regenerating soft tissues from growing into more slowly regenerating bone tissue.
The bone-adhesive sheet according to the present invention comprises:
wherein calcium-binding group is a group that is capable of forming two or more separate coordinate bonds with Ca2+ ions.
The bone-adhesive sheet of the present invention comprises a cohesive fibrous carrier structure that absorbs fluids and that can easily be impregnated with polymer particles. Unlike impregnation with liquids, such dry impregnation does not affect the structural integrity or mechanical properties of the carrier structure.
When aqueous liquid is absorbed by the bone-adhesive sheet of the present invention, the polymer particles within the sheet start dissolving as soon as they are ‘wetted’, thereby releasing the water-soluble polymer carrying calcium-binding groups. In an alternative embodiment, the water-soluble polymer carrying calcium-binding groups is formed in situ upon wetting when the electrophilically activated water-soluble polymer in the reactive particles reacts with the nitrogenous bisphosphonate in the bisphosphonate particles.
The polymer carrying calcium-binding groups is capable of forming a hydrogel due to reversible cross-linking with Ca2+ ions. This hydrogel strongly binds to bone. Thus, when applied onto bone tissue, the bone-adhesive sheet of the present invention is rapidly glued to the bone tissue by the adhesive action of the water-soluble calcium-binding polymer.
The polymer particles or the combination of reactive particles and bisphosphonate particles may be distributed homogeneously throughout the bone-adhesive sheet, thereby minimising bending friction.
Due to its flexibility, the bone-adhesive sheet of the present invention can suitably be applied to irregularly shaped sites.
Another aspect of the present invention relates to a method of preparing the bone-adhesive sheet of the present invention, said method comprising:
A first aspect of the invention relates to a bone-adhesive sheet comprising:
wherein calcium-binding group is a group that is capable of forming two or more separate coordinate bonds with Ca2+ ions.
The term “interstitial space” as used herein refers to the void (“empty”) space within the fibrous carrier structure. The interstitial space within the fibrous carrier structure allows the introduction of polymer particles into the sheet. Also blood and other bodily fluids can enter the interstitial space, allowing the water-soluble calcium-binding polymer within the polymer particles to dissolve.
The “water-soluble calcium-binding polymer” that is employed in accordance with the present invention has a molecular weight of at least 1 kDa and a solubility in distilled water of 20° C. of at least 50 g/L.
The term “bisphosphonate” as used herein refers to a substance comprising two phosphonate groups that are interlinked by a central carbon atom by phosphoether bonds. The central carbon atom can carry two side chains referred to as R1 and R2.
The term “nitrogenous bisphosphonate” as used herein refers to a bisphosphonate wherein R1 and/or R2 contains nitrogen as part of an amine group.
The term “electrophilically activated water-soluble polymer” as used herein refers to a water-soluble polymer that comprises electrophilic groups that are capable of reacting with the amine group of the nitrogenous bisphosphonate under the formation of a covalent bond.
The term “water absorption capacity” as used herein is a measure of the capability of the bone-adhesive sheet to absorb water. The water absorption capacity is determined by weighing a sample of the dry sheet (weight=Wd) followed by immersion of the sample into distilled water (37° C.) for 45 minutes. Next, the sample is removed from the water and water clinging to the outside of the substrate is removed, following which the sample is weighed again (weight=Ww). The water absorption capacity=100%×(Ww−Wd)/Wd. The water absorption capacity is indicative of the porosity of the substrate as well as of its ability to swell in the presence of water.
The term “collagen” as used herein refers the main structural protein in the extracellular space of various connective tissues in animal bodies. Collagen forms a characteristic triple helix of three polypeptide chains. Depending upon the degree of mineralization, collagen tissues may be either rigid (bone) or compliant (tendon) or have a gradient from rigid to compliant (cartilage). Unless indicated otherwise, the term “collagen” also encompasses modified collagens other than gelatin.
The term “gelatin” as used herein refers to a mixture of peptides and proteins produced by partial hydrolysis of collagen extracted from the skin, bones, and connective tissues of animals such as domesticated cattle, chicken, pigs, and fish. During hydrolysis, the natural molecular bonds between individual collagen strands are broken down into a form that rearranges more easily.
The term “polyoxazoline” as used herein refers to a poly(N-acylalkylenimine) or a poly(aroylalkylenimine) and is further referred to as POx. An example of POx is poly(2-ethyl-2-oxazoline). The term “polyoxazoline” also encompasses POx copolymers.
The diameter distribution of the polymer particles, the reactive particles and the bisphosphonate particles may suitably be determined by means of laser diffraction using a Malvern Mastersizer 2000 in combination with the Stainless Steel Sample Dispersion Unit. The sample dispersion unit is filled with approx. 120 mL of cyclohexane, which is stabilized for 5 to 10 minutes at a stirring speed of 1800 rpm, followed by a background measurement (blanc measurement). The sample tube is shaken and turned horizontally for 20 times. Next, about 50 mg is dispersed in the sample dispersion unit containing the cyclohexane. After the sample is introduced in the dispersion unit, the sample is stirred for one and a half minute at 1800 rpm to ensure that all particles are properly dispersed, before carrying out the measurement. No ultrasonic treatment is performed on the dispersed particles. Mean particle size is expressed as D [4,3], the volume weighted mean diameter (ΣniDi4)/(ΣniDi3).
According to a particularly preferred embodiment, the fibrous carrier structure is water-resistant. Here the term “water-resistant” means that the fibrous carrier structure is not water soluble and does not disintegrate in water to form a colloidal dispersion, at neutral pH conditions (pH 7) and a temperature of 37° C.
According to a particularly preferred embodiment, the bone-adhesive sheet of the present invention is bio-degradable, meaning that the sheet, the particles contained therein and any other components of the bone-adhesive sheet are eventually degraded in the body. Biodegradation of the sheet and particles typically requires chemical decomposition (e.g. hydrolysis) of polymers contained therein. Complete bio-degradation of the bone-adhesive sheet by the human body is typically achieved in 1 to 16 weeks, more preferably in 4 to 14 weeks, most preferably in 8 to 12 weeks.
The bone-adhesive sheet of the present invention is preferably sterile.
The bone-adhesive sheet of the present invention typically has a mean thickness of 0.1-25 mm. More preferably, the mean thickness is in the range of 0.1-10 mm, most preferably in the range of 0.2-5 mm.
The dimensions of the bone-adhesive sheet preferably are such that the top and bottom of the sheet each have a surface area of at least 2 cm2, more preferably of at least 5 cm2 and most preferably of 5-50 cm2. Typically, the sheet is rectangular in shape and has a length of 25-200 mm, and a width of 20-200 mm.
The bone-adhesive sheet preferably has a density of less than 2 g/cm3, more preferably of less than 1 g/cm3 and most preferably of 0.2-0.8 g/cm3.
The bone-adhesive sheet of the present invention preferably is essentially anhydrous. Typically, the bone-adhesive sheet has a water content of not more than 5 wt. %, more preferably of not more than 2 wt. % and most preferably of not more than 1 wt. %.
The water absorption capacity of the bone-adhesive sheet preferably is at least 50%, more preferably lies in the range of 100% to 800%, most preferably in the range of 200% to 500%.
The fibres in the fibrous carrier structure preferably have a mean diameter of 1-500 μm, more preferably of 2-300 μm and most preferably of 5-200 μm. The mean diameter of the fibres can suitably be determined using a microscope.
Typically, at least 50 wt. %, more preferably at least 80 wt. % of the fibres in the fibrous carrier structure have a diameter of 1-300 μm and a length of at least 1 mm.
Preferably, at least 50 wt. %, more preferably at least 80 wt. % of the fibres in the fibrous carrier structure have an aspect ratio (ratio of length to diameter) of at least 1000.
The fibrous carrier structure that is employed in accordance with the present invention preferably is a felt structure, a woven structure or a knitted structure. Most preferably, the fibrous carrier structure is a felt structure. Here the term “felt structure” refers to a structure that is produced by matting and pressing fibres together to form a cohesive material.
The fibrous carrier structure preferably comprises at least 50 wt. %, more preferably at least 80 wt. % and most preferably at least 90 wt. % fibres containing gelatin, collagen, cellulose, modified cellulose, carboxymethyldextran, PLGA, sodium hyaluronate/carboxy methylcellulose, polyvinyl alcohol, chitosan or a combination thereof.
In an embodiment of the invention, the fibrous carrier structure does not comprise oxidised regenerated cellulose.
Preferred fibrous carrier structures have an open pore structure with a permeability to air of at least 0.1 L/min×cm2, more preferably of at least 0.5 L/min×cm2. The air permeability is determined in accordance with EN ISO 9237:1995 (Textiles—Determination of the permeability of fabrics to air).
The fibres in the fibrous carrier structure can be produced by means of methods known in the art, such as electrospinning, electro-blown spinning and high speed rotary sprayer spinning. Production of fibrous carrier structure by means of high speed rotary sprayer spinning is described in US 2015/0010612. It is also possible to use commercially available haemostatic fibrous sheets as the fibrous carrier structure.
In a preferred embodiment, the bone-adhesive sheet comprises, distributed within the interstitial space, a plurality of polymer particles comprising a water-soluble calcium-binding polymer, said water-soluble calcium-binding polymer carrying at least one calcium-binding group.
The polymer particles may be homogeneously distributed within the interstitial space of the fibrous carrier structure in the sense that the particle density is essentially the same throughout the sheet. The polymer particles may be unevenly distributed through the thickness of the sheet. For certain applications it may be advantageous if the polymer particle density shows a gradient, e.g. in that the density of polymer particles is lowest near the side of the sheet that is meant to applied onto a bleeding wound and highest near the other side of the sheet.
The bone-adhesive sheet of the present invention preferably comprises 5-90%, more preferably 10-80%, even more preferably 20-75% and most preferably 50-70% of the polymer particles, said percentage being calculated by weight of the fibrous carrier structure.
The polymer particles employed in the bone-adhesive sheet preferably contain at least 10 wt. % of the water-soluble calcium-binding polymer. More preferably, the polymer particles contain at least 50 wt. %, more preferably at least 90 wt. % of the water-soluble calcium-binding polymer.
In a very preferred embodiment, the polymer particles have a volume weighted mean diameter (D [4,3], (ΣniD4)/(ΣniD3)) in the range of 5-220 μm, more preferably 10-150 μm, even more preferably in the range of 15-120 μm and most preferably in the range of 20-100 μm.
According to a particularly preferred embodiment at least 80 vol. % of the polymer particles has a diameter in the range of 5-200 μm, more preferably in the range of 10-120 μm and most preferably in the range of 12-100 μm.
The polymer particles of the present invention may be prepared in various ways, e.g. by milling, by spray drying a polymeric solution, by freeze drying, by spray chilling a polymeric melt, by granulating a powder mixture, or by fluidised bed coating.
The water-soluble calcium-binding polymer that is contained in the polymer particles typically has a molecular weight of at least 2 kDa, more preferably of at least 5 kDa and most preferably of 10-100 kDa.
The water-soluble calcium-binding polymer preferably has a solubility in distilled water of 20° C. of at least 100 g/L, more preferably of at least 200 g/L.
The affinity of the water-soluble calcium-binding polymer to hydroxyapatite is dependent on the number of calcium-binding groups in the copolymer. Preferably, the polymer contains at least 4 calcium-binding groups, more preferably at least 6 calcium-binding groups, even more preferably at least 8 calcium-binding groups and most preferably at least 10 calcium-binding groups. Typically, the polymer contains not more than 200 calcium-binding groups.
On average the water-soluble calcium-binding polymer carries 0.05-5 calcium-binding groups, more preferably 0.1-3 calcium-binding groups, most preferably 0.2-2 calcium-binding groups per kDa.
The water-soluble calcium-binding polymer that is present in the polymer particles is preferably selected from polyoxazolines, polyethylene glycols, polyvinylpyrrolidones, polyurethanes (e.g. as described in WO 2017/171551) and combinations thereof. Even more preferably the calcium-binding polymer is selected from polyoxazolines, polyethylene glycols and combinations thereof. Most preferably the calcium-binding polymer is a polyoxazoline.
The polyoxazoline comprising calcium-binding groups is preferably derived from a polyoxazoline whose repeating units are represented by the following formula (I):
(CHR1)mNCOR2
wherein R2, and each of R1 are independently selected from H, optionally substituted C1-22 alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted aryl; and m being 2 or 3.
Preferably, R1 and R2 in formula (I) are selected from H and C1-3 alkyl, even more preferably from H and C1-4 alkyl. R1 most preferably is H. The integer m in formula (I) is preferably equal to 2.
According to a preferred embodiment, the polyoxazoline is a polymer, even more preferably a homopolymer of 2-alkyl-2-oxazoline, said 2-alkyl-2-oxazoline being selected from 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2-butyl-2-oxazoline and combinations thereof. Preferably, the polyoxazoline is a homopolymer of 2-propyl-2-oxazoline or 2-ethyl-oxazoline. Most preferably, the polyoxazoline is a homopolymer of 2-ethyl-oxazoline.
According to a particularly preferred embodiment, the water-soluble calcium-binding polymer comprises at least 20 oxazoline units, more preferably at least 30 oxazoline units and most preferably at least 80 oxazoline units. The calcium-binding polymer preferably comprises on average at least 0.05 calcium-binding groups per oxazoline residue. Even more preferably, the calcium-binding polymer comprises on average at least 0.1 calcium-binding groups per oxazoline residue. Most preferably, the calcium-binding polymer comprises on average 0.12-0.5 calcium-binding groups per oxazoline residue.
Oxazoline units containing a calcium-binding group preferably represent 3-50% of the monomeric units contained in the water-soluble calcium-binding polymer More preferably, the calcium-binding group containing oxazoline units represent 4-40%, most preferably 5-35% of the monomeric units contained in the water-soluble calcium-binding polymer.
According to a particularly preferred embodiment, the water-soluble calcium-binding polymer comprises:
repeating units A of formula —[N(Ra)CH2CH2]—; and
repeating units B of formula —[N(Rb)CH2CH2]—;
wherein:
Ra is CO—(CHR1)t—H
Rb is CO—(CHR1)t—CONH—R2—X—CO—(CHR3)u—CONR4—CO-ccr
R1 represents H or optionally substituted C1-5 alkyl;
R2 represents an optionally substituted C1-5 alkylene;
R3 represents H or optionally substituted C1-5 alkyl;
R4 represents H or CH3;
X represents O, NR11, S or *NR11(R12);
R11 and R12 represent H, methyl or ethyl;
t represents 1, 2 or 3;
u represents 1, 2 or 3;
ccr represents a calcium-binding group.
In a preferred embodiment, the calcium-binding group (ccr in the formula representing repeating units B) represents a group selected from bisphosphonate, citrate, ethylenediaminetetraacetic acid (EDTA) and combinations thereof. More preferably, the calcium-binding group comprises bisphosphonate. Even more preferably, the calcium-binding group (ccr) represents a group comprising bisphosphonate that is represented by formula (I):
-(L)C(Z)(PO3H2)2 (I)
or a pharmaceutically acceptable salt thereof;
wherein:
L represents an optionally substituted C1-5 alkylene;
Z represents H, OH, Cl, F or a methyl group.
In the above mentioned formula (I), L preferably represents C2-4 alkylene. Most preferably, L represents propylene.
In formula (I), Z preferably represents H, OH or a methyl group. Most preferably, Z represents OH.
According to a preferred embodiment, R1 in the abovementioned formulae of the repeating units A and B represents H.
The integer t in the formulae of the repeating units A and B preferably is 2 or 3.
In the formulae of the repeating units B, R2 preferably represents ethylene.
The integer u in the formula of the repeating unit B preferably is 2 or 3.
R3 in the formula of repeating unit B preferably represents H.
R4 in the formula of repeating unit B preferably represents H.
In the formulae of repeating units B, X preferably represents O.
According to preferred embodiment, the water-soluble calcium-binding polymer of the present invention has the following monomeric composition comprises 40-97 mol. % of repeating units A and 3-50 mol. % of repeating units B. Even more preferably, the water-soluble calcium-binding polymer comprises 50-95 mol. % of repeating units A and 4-40 mol. % of repeating units B.
Most preferably, the water-soluble calcium-binding polymer comprises 55-92 mol. % of repeating units A and 5-35 mol. % of repeating units B.
Together, the repeating units A and B preferably constitute at least 55%, more preferably at least 65% and most preferably at least 70% of the monomeric units present in the water-soluble calcium-binding polymer.
Another aspect of the present invention relates to the use of the water-soluble calcium-binding polymer of the present invention for imparting bone adhesiveness to a medical product.
Polyoxazoline can carry calcium-binding groups in its side chains (pendant calcium-binding groups), at its termini, or both. The polyoxazoline that is employed in accordance with the present invention advantageously contains one or more pendant calcium-binding groups. Typically, the polyoxazoline contains 0.03-0.5 pendant calcium-binding groups per monomer, more preferably 0.04-0.35 pendant calcium-binding groups per monomer, even more preferably 0.05-0.25 pendant calcium-binding groups per monomer.
The polyethylene glycol (PEG) comprising calcium-binding groups that is applied in accordance with the present invention preferably is a multi-arm PEG comprising at least 3 arms, more preferably at least 4 arms, most preferably at least 6 arms. Typically, the multi-arm PEG does not contain more than 20 arms.
Preferably, the multi-arm PEG comprises at least 3 arms that are terminated with one or two calcium-binding groups. Most preferably each arm of the multi-arm PEG is terminated with one or two calcium-binding groups.
An example of a multi-armed PEG having 4 arms each terminated with one calcium-binding group R and an example of a multi-armed PEG comprising 8 arms each terminated with one calcium-binding groups R are shown in FIG. 1.
In accordance with another preferred embodiment, the bone-adhesive sheet comprises, distributed within the interstitial space, a plurality of reactive particles comprising an electrophilically activated water-soluble polymer and a plurality of bisphosphonate particles comprising nitrogenous bisphosphonate.
The reactive particles and the bisphosphonate particles may be separately distributed within the interstitial space. According to a particularly preferred embodiment, however, the reactive particles and the bisphosphonate particles are combined in agglomerates and these agglomerates are distributed within the interstitial space. Combining the reactive particles and the bisphosphonate particles in agglomerates offers the advantage that the in situformation of water-soluble calcium-binding polymer proceeds very rapidly when the bone-adhesive sheet absorbs aqueous liquid.
The agglomerates of reactive particles and bisphosphonate particles preferably have a volume weighted mean diameter (D [4,3], (ΣniD4)/(ΣniD3)) in the range of 100-400 μm, more preferably in the range of 200-350 μm and most preferably 220-320-μm.
According to a preferred embodiment at least 80 vol. % of the agglomerates has a diameter in the range of 150-350 μm, more preferably in the range of 200-300 μm and most preferably in the range of 225-275 μm.
The bone-adhesive sheet of the present invention preferably comprises 10-140%, more preferably 20-130%, even more preferably 40-120% and most preferably 50-110% of the agglomerates of reactive particles, said percentage being calculated by weight of the fibrous carrier structure.
The bone-adhesive sheet of the present invention preferably comprises 1.6-37%, more preferably 3.2-34%, even more preferably 6.4-32% and most preferably 8.3-29% of the bisphosphonate particles, said percentage being calculated by weight of the fibrous carrier structure.
The reactive particles employed in the bone-adhesive sheet preferably contain at least 10 wt. % of the electrophilically activated water-soluble polymer. More preferably, the reactive particles contain at least 50 wt. %, more preferably at least 90 wt. % of the electrophilically activated water-soluble polymer.
The bisphosphonate particles employed in the bone-adhesive sheet preferably contain at least 10 wt. % of the nitrogenous bisphosphonate. More preferably, the reactive particles contain at least 50 wt. %, more preferably at least 90 wt. % of the nitrogenous bisphosphonate.
The nitrogenous bisphosphonate in the bisphosphonate particles is preferably selected from alendronate, pamidronate, neridronate, olpadronate, ibandronate and combinations thereof. More preferably, the nitrogenous bisphosphonate is selected from alendronate, pamidronate, neridronate and combinations thereof.
The electrophilically activated water-soluble polymer that is contained in the reactive particles preferably comprises one or more electrophilic groups selected from carboxylic acid esters, sulfonate esters, phosphonate esters, pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenyl esters, acid halide groups, anhydrides, ketones, aldehydes, isocyanato, thioisocyanato (isothiocyanato), isocyano, epoxides, activated hydroxyl groups, olefins, glycidyl ethers, carboxyl, succinimidyl esters, succinimidyl carbonates, succinimidyl carbamates, sulfosuccinimidyl esters, sulfosuccinimidyl carbonates, maleimido (maleimidyl), ethenesulfonyl, imido esters, aceto acetate, halo acetal, orthopyridyl disulfide, dihydroxy-phenyl derivatives, vinyl, acrylate, acrylamide, iodoacetamide and combinations thereof. More preferably, the electrophilic groups are selected from: carboxylic acid esters, acid chloride groups, anhydrides, ketones, aldehydes, isocyanato, thioisocyanato, epoxides, activated hydroxyl groups, olefins, carboxyl, succinimidyl ester, succinimidyl carbonate, succinimidyl carbamates, sulfosuccinimidyl ester, sulfosuccinimidyl carbonate, maleimido, ethenesulfonyl and combinations thereof. Even more preferably, the electrophilic groups are selected from aldehydes, isocyanato, thioisocyanato, succinimidyl ester, sulfosuccinimidyl ester, maleimido and combinations thereof. Most preferably, the electrophilic groups are selected from isocyanato, thioisocyanato, succinimidyl ester, sulfosuccinimidyl ester, maleimido and combinations thereof.
The electrophilically activated water-soluble polymer employed in the present method preferably contains on average at least 6, more preferably at least 12 and most preferably 15 to 30 electrophilic groups.
The electrophilically activated water-soluble polymer preferably contains on average at least 0.1 electrophilic groups per monomer, more preferably 0.12 to 2 electrophilic groups per monomer, most preferably 0.15 to 0.6 electrophilic groups per monomer.
The electrophilically activated water-soluble polymer that is contained in the reactive particles typically has a molecular weight of at least 2 kDa, more preferably of at least 5 kDa and most preferably of 10-100 kDa.
The electrophilicaly water-soluble polymer preferably has a solubility in distilled water of 20° C. of at least 100 g/L, more preferably of at least 200 g/L.
The electrophilically activated water-soluble polymer is preferably selected from polyoxazolines, polyethylene glycols, polyvinylpyrrolidones, polyurethanes (e.g. as described in WO 2017/171551) and combinations thereof. Even more preferably the electrophilically activated water-soluble polymer is selected from polyoxazolines, polyethylene glycols and combinations thereof. Most preferably electrophilically activated water-soluble polymer is a polyoxazoline.
The electrophilically activated polyoxazoline is preferably derived from a polyoxazoline as defined herein before.
According to another preferred embodiment, the bone adhesive sheet comprises an occlusive backing layer. Preferably, this backing layer is water-resistant. Here the term “water-resistant” means that the backing layer is not water soluble and does not disintegrate in water to form a colloidal dispersion, at neutral pH conditions (pH 7) and a temperature of 37° C.
The backing layer typically has an average thickness of 5-100 μm, more preferably an average thickness of 10-80 μm and most preferably of 20-60 μm.
The backing layer preferably contains at least 50 wt. % of one or more polymers selected from poly(L-lactide-co-caprolactone) (PLC), poly(D,L-lactic-co-glycolic acid) (PLGA), and poly(2-propyl-2-oxazoline) functionalized with pendant amine groups (P(PropOx-NH2).
Another aspect of the present invention relates to a method of preparing the bone-adhesive sheet as described herein before, said method comprising:
The fibrous carrier structure employed in the present method preferably is a fibrous carrier structure as described herein before. Likewise, the polymer particles, the reactive particles, the bisphosphonate particles, the water-soluble calcium-binding polymer, the electrophilically activated water-soluble polymer and the nitrogenous bisphosphonate preferably are as described herein before.
The reactive particles and bisphosphonate particles are preferably provided together in the form of an agglomerate containing both reactive particles and bisphosphonate particles.
According to a particularly preferred embodiment of the present method, the fibrous carrier structure is impregnated by:
According to an alternative preferred embodiment of the present method, the fibrous carrier structure is impregnated by:
The use of an electric field enables deep impregnation with, and a homogeneous distribution of the particles within the fibrous carrier structure. In addition, the present method offers the advantage that, unlike impregnation with liquids, it does not affect the structural integrity or mechanical properties of the fibrous sheet. Furthermore, in comparison to mechanical impregnation methods that make use of shaking or vibration, the present method does not impose mechanical stress and achieves a more effective impregnation, especially with very small (<100 μm) particles.
In the present method the fibrous carrier structure and the particles are preferably simultaneously subjected to an electric field of 0.5 to 30 kV/mm, more preferably to an electric field of 1 to 10 kV/mm.
According to a particularly preferred embodiment, the fibrous carrier structure and the particles are simultaneously subjected to an alternating electric field. Preferably, the electric field is alternated with a frequency of at least 10 s−1, more preferably with a frequency of at least 50 s−1 and most preferably with a frequency of 100 s−1.
Impregnation of the fibrous carrier structure may effectively be achieved in the present method by creating a layer of the particles adjacent to the fibrous carrier structure and by placing the fibrous carrier structure and the adjacent layer of particles between the two electrodes. Impregnation may also be achieved by creating a laminate containing two or more fibrous carrier structures separated by layers of particles and by placing this laminate between the two electrodes.
In a preferred embodiment of the present method, the fibrous carrier structure and the particles are placed between a lower electrode and an upper electrode, these electrodes being electrically insulated from each other by a dielectric and connected to the respective poles of an AC generator so as to simultaneously subject the fibrous carrier structure and the particles to the electric field.
The fibrous carrier structure and the particles are preferably passed between the lower electrode and upper electrode whilst simultaneously subjecting the fibrous carrier structure and the particles to the electric field. Thus, a roll of fibrous carrier structure may suitably be impregnated with the particles by the present method in a semi-continuous fashion.
In a preferred embodiment, the fibrous carrier structure and the particles are simultaneously exposed to the electric field for at least 0.1 second, more preferably for at least 5 seconds and most preferably for at least 30 seconds.
The invention is further illustrated by the following non-limiting examples.
Materials
Alendronate-functionalized poly(2-oxazoline)s were synthesized in five steps as shown in FIG. 2.
2-Methoxycarbonylethyl-2-oxazoline (MestOx) was copolymerized either with 2-ethyl-2-oxazoline (EtOx) to yield a randomly distributed statistical copolymer. Methyl-p-toluenesulfonate (1 equiv), EtOx (m equiv), MestOx (n equiv), and dry acetonitrile (4 M) were mixed under inert atmosphere in the desired ratios in microwave vials. The polymerization was carried out under microwave irradiation at 140° C. for 15 min following a cationic ring opening mechanism (CROP). After polymerization, the reaction was terminated by the addition of ethanolamine (10 equiv) stirring for 30 min at room temperature. Then, the solvent was evaporated in vacuo to afford P(EtOx-ran-MestOx) copolymer Pia.
The copolymer Pia (1 equiv) was modified by direct amidation with 2-ethanolamine (3.5 equiv) at 60° C. under reduced pressure (300 mbar) for 16 h. Then, the crude mixtures were purified by three consecutive precipitations in a mixture of acetone:ether v/v 3:1 and re-dissolved in DCM:MeOH v/v 8:2, followed by ion-exchange chromatography in MeOH. Finally, the solvents were evaporated under vacuum to afford P(EtOx-OH) copolymer P1b.
The hydroxyl side-functionalized polymer P1b (1 equiv) was either fully or partially converted to carboxylic acid moieties using succinic anhydride (1.1 equiv), 4-dimethylamino pyridine (DMAP) (1.1 equiv), and dissolved in DCM:DMF v/v 9:1 (2 M) under argon atmosphere for 16 h. The crude mixtures were purified by three consecutive precipitations in acetone and re-dissolved in DCM:MeOH v/v 8:2, followed by ion-exchange chromatography in MeOH. Finally, the solvent was evaporated under vacuum to afford either P(EtOx-COOH) or P(EtOx-OH—COOH) P1c and P2c.
These carboxylate side-functionalized copolymers were subsequently modified into reactive esters by carbodiimide coupling with N-hydroxysuccinimide. The functionalized polymers were dissolved in DCM:DMF v/v 95:5 (0.2 M), N-hydroxysuccinimide (1.1 equiv) and N,N′diisopropylcarbodiimide (1.2 equiv) as coupling agent were added, and stirred under argon atmosphere at room temperature for 16 h. They were purified by two precipitations in acetone:ether v/v 1:1 followed by other in ether, and re-dissolved in DCM. Finally, the solvents were evaporated under vacuum to afford P(EtOx-OH—NHS) or P(EtOx-NHS) P1d and P2d.
Alendronate moieties were incorporated in the polymer side chain by an amidation reaction. The NHS-activated copolymers P1d and P2d (1 equiv) were slowly added into a solution containing sodium alendronate trihydrate (2 equiv), N-hydroxysuccinimide (1 equiv) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1 equiv) in phosphate buffered saline (0.5 M), stirring at 3° C. for 4 h with the pH previously adjusted to 7.7 using NaOH. Afterwards, the mixtures were dialyzed for 16 h, follow by three precipitations in acetone:ether v/v 3:1, and re-dissolved in demi-water. Finally, they were lyophilized to afford either P(EtOx-Ale), P(EtOx-OH-Ale) copolymers P1e and P2e.
According to the general procedure, the reaction of a solution of methyl tosylate (0.46 mL, 3.01 mmol), 2-ethyl-2-oxazoline (24.32 mL, 240.94 mmol) and 2-methoxycarbonylethyl-2-oxazoline (8.31 mL, 60.24 mmol) in dry acetonitrile (43 mL, 4 M), terminated by the addition of 2-ethanolamine (1.82 mL, 30.12 mmol) afforded the desired polymer Pia (36.22 g, 2.93 mmol). 1H NMR [400 MHz, δ (ppm), CDCl3]: 3.65 (br, 3H, 5-CH3), 3.65-3.35 (br, 8H, 1-CH2), 2.75-2.50 (br, 4H, 4-CH2), 2.50-2.20 (br, 2H, 2-CH2), 0.95-1.15 (br, 3H, 3-CH3). Experimentally determined copolymer ratio: m/n 80:20. SEC: Mn 9.6 kDa, Ð: 1.17. MALDI-TOF: Mn 9.8 kDa. Yield: 97%.
According to the general procedure, the reaction of Pia (51 g, 4.35 mmol) with 2-aminoethanol (27.55 mL, 456.55 mmol) afforded the desired polymer P1b (43.98 g, 3.49 mmol). 1H NMR [400 MHz, δ (ppm), D2O]: 3.66 (br, 2H, 7-CH2), 3.75-3.45 (br, 8H, 1-CH2), 3.35-3.25 (br, 2H, 6-CH2), 2.75-2.60 (br, 2H, 5-CH2), 2.60-2.50 (br, 2H, 4-CH2), 2.45-2.25 (br, 2H, 2-CH2), 1.00-0.80 (br, 3H, 3-CH3). Experimentally determined copolymer ratio: m/n 70:30. MALDI-TOF: Mn 11.1 kDa. Yield: 80%.
According to the general procedure, the reaction of a solution of P1b (10 g, 0.79 mmol), 4-dimethylamino pyridine (0.58 g, 4.76 mmol) and succinic anhydride (2.86 g, 28.54 mmol) in DCM/ACN 9:1 (17 mL, 2 M) afforded the desired polymer P1c (9.85 g, 0.64 mmol). 1H NMR [400 MHz, δ (ppm), D2O]: 4.25-4.15 (br, 2H, 7-CH2), 3.80-3.45 (br, 10H, 1-CH2+6-CH2), 2.75-2.60 (br, 6H, 5-CH2+8-CH2), 2.60-2.45 (br, 2H, 4-CH2), 2.40-2.25 (br, 2H, 2-CH2), 1.15-1.00 (br, 3H, 3-CH3). Experimentally determined copolymer ratio: m/o 70:30. MALDI-TOF: Mn 13.5 kDa. Yield: 81%.
According to the general procedure, the reaction of a solution of P1c (61 g, 3.91 mmol), N-hydroxysuccinimide (14.85 g, 129.00 mmol) and N,N′-diisopropylcarbodiimide (21.79 mL, 140.72 mmol) in DCM/ACN 9:1 (1346 mL, 0.2 M) afforded the desired polymer P1d (57.06 g, mmol). 1H NMR [400 MHz, δ (ppm), DMSO-d6]: 4.00-3.90 (br, 2H, 7-CH2), 3.50-3.10 (br, 10H, 1-CH2+6-CH2), 2.90-2.85 (br, 2H, 8-CH2), 2.75-2.70 (br, 4H, 9-CH2), 2.65-2.60 (br, 2H, 8-CH2), 2.60-2.40 (br, 2H, 5-CH2), 2.30-2.10 (br, 4H, 2-CH2+4-CH2), 0.85-0.70 (br, 3H, 3-CH3). Experimentally determined copolymer ratio: m/o 70:30. SEC: Mn 15.6 kDa, Ð 1.25. MALDI-TOF: Mn 16.9 kDa. Yield: 79%.
According to the general procedure, the reaction of a solution of P1d (15 g, 0.81 mmol), sodium alendronate trihydrate (15.80 g, 48.61 mmol), N-hydroxysuccinimide (2.80 g, 24.31 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4.66 g, 24.31 mmol) in PBS (66 mL, 0.5 M) afforded the desired polymer P1e (5.81 g, 0.26 mmol). 1H NMR [400 MHz, δ (ppm), D2O]: 4.25-4.10 (br, 4H, 7-CH2), 3.80-3.30 (br, 16H, 1-CH2+6-CH2), 3.25-3.15 (br, 2H, 9-CH2), 2.75-2.45 (br, 16H, 4-CH2+5-CH2+8-CH2), 2.45-2.15 (br, 2H, 2-CH2), 2.15-1.70 (br, 4H, 10-CH2+11-CH2), 1.15-0.95 (br, 3H, 3-CH3). 31P NMR [400 MHz, δ (ppm), D2O]: 18.25. Experimentally determined copolymer ratio: m/o/p 70:2:28. MALDI-TOF: Mn 19.5 kDa. Yield: 32%.
According to the general procedure, the reaction of a solution of P1b (15 g, 1.19 mmol), 4-dimethylamino pyridine (0.58 g, 4.76 mmol) and succinic anhydride (2.86 g, 28.54 mmol) in DCM/ACN 9:1 (17 mL, 2 M) afforded the desired polymer P2c (17.33 g, 1.19 mmol). 1H NMR [400 MHz, δ (ppm), D2O]: 4.25-4.15 (br, 2H, 9-CH2), 3.80-3.45 (br, 16H, 1-CH2+7-CH2+8-CH2), 3.35-3.25 (br, 2H, 6-CH2), 2.75-2.60 (br, 8H, 5-CH2+10-CH2), 2.60-2.45 (br, 4H, 4-CH2), 2.40-2.25 (br, 2H, 2-CH2), 1.15-1.00 (br, 3H, 3-CH3). Experimentally determined copolymer ratio: m/n/o 70:12:18. MALDI-TOF: Mn 12.5 kDa. Yield: 94%.
According to the general procedure, the reaction of a solution of P2c (12 g, 0.82 mmol), N-hydroxysuccinimide (2.08 g, 18.07 mmol) and N,N′-diisopropylcarbodiimide (3.05 mL, 19.72 mmol) in DCM/ACN 9:1 (190 mL, 0.2 M) afforded the desired polymer P2d (11.95 g, 0.72 mmol). 1H NMR [400 MHz, δ (ppm), DMSO-d6]: 4.15-4.00 (br, 2H, 8-CH2), 3.75-3.40 (br, 16H, 1-CH2+6-CH2+7-CH2), 3.35-3.25 (br, 2H, 5-CH2), 3.05-2.95 (br, 2H, 9-CH2), 2.95-2.85 (br, 4H, 10-CH2), 2.85-2.70 (br, 2H, 9-CH2), 2.75-2.65 (br, 8H, 4-CH2), 2.40-2.10 (br, 2H, 2-CH2), 1.05-0.95 (br, 3H, 3-CH3). Experimentally determined copolymer ratio: m/n/o 70:12:18. SEC: Mn 14.7 kDa, Ð 1.25. MALDI-TOF: Mn 14.4 kDa. Yield: 88%.
According to the general procedure, the reaction of a solution of P2d (12 g, 0.74 mmol), sodium alendronate trihydrate (8.69 g, 6.74 mmol), N-hydroxysuccinimide (1.54 g, 13.37 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (2.56 g, 13.37 mmol) in PBS (108 mL, 0.5 M) afforded the desired polymer P2e (4.15 g, 0.23 mmol). 1H NMR [400 MHz, δ (ppm), D2O]: 4.30-4.10 (br, 4H, 8-CH2), 3.80-3.40 (br, 22H, 1-CH2+6-CH2+7-CH2), 3.40-3.30 (br, 2H, 5-CH2), 3.30-3.20 (br, 2H, 10-CH2), 2.75-2.15 (br, 22H, 2-CH2+4-CH2+9-CH2), 2.05-1.75 (br, 4H, 11-CH2+12-CH2), 1.15-0.95 (br, 3H, 3-CH3). 31P NMR [400 MHz, δ (ppm), D2O]: 18.25. Experimentally determined copolymer ratio: m/n/o/p 70:12:2:16. MALDI-TOF: Mn 14.6 kDa. Yield: 30%.
NHS-side chain activated poly[2-(ethyl/hydroxy-ethyl-amide-ethyl/NHS-ester-ethyl-ester-ethyl-amide-ethyl)-2-oxazoline] terpolymer containing 20% NHS-ester groups (=EL-POx, 20% NHS) was synthesized as follows: Poly[2-(ethyl/methoxy-carbonyl-ethyl)-2-oxazoline] copolymer (DP=+/−100) was synthesized by means of CROP using 60% 2-ethyl-2-oxazoline (EtOx) and 40% 2-methoxycarbonyl-ethyl-2-oxazoline (MestOx). A statistical copolymer containing 40% 2-methoxycarbonyl-ethyl groups (1H-NMR) was obtained. Secondly, the polymer containing 40% 2-methoxycarbonyl-ethyl groups, was reacted with ethanolamine yielding a copolymer with 40% 2-hydroxy-ethyl-amide-ethyl-groups (1H-NMR). After that, half of the 2-hydroxy-ethyl-amide-ethyl-groups was reacted with succinic anhydride yielding a terpolymer with 60% 2-ethyl groups, 20% 2-hydroxy-ethyl-amide-ethyl-groups and 20% 2-carboxy-ethyl-ester-ethyl-amide-ethyl-groups according to 1H-NMR. Lastly, the 2-carboxy-ethyl-ester-ethyl-amide-ethyl-groups were activated by N-hydroxysuccinimide (NHS) and diisopropylcarbodiimide (DIC), yielding EL-POx, 20% NHS. The NHS—POx contained 20% NHS-ester groups according to 1H-NMR. NHS—POx was dissolved between 2-8° C. in water (60 g in 300 mL), cooled at minus 80° C. for half an hour and freeze dried. The freeze dried powder so obtained was dried in a Rotavap at 40° C. until the water content was below 0.8% w/w as determined via Karl Fischer titration. This dry (white) powder was grinded using a ball mill (Retch MM400) until the average particle size was not more than 40 μm (D [4,3]) and vacuum sealed in alu-alu bags.
The degree of modification of the different substitutions in the polymers was determined by 1H NMR. Moreover, the presence of alendronate moieties by 31P NMR. NMR spectra was recorded on a Varian Inova 400 (400 MHz) or Bruker Avance III (400 MHz) spectrometer in the indicated solvent at 25° C. 1H NMR data is reported as: chemical shifts (given in parts per million (ppm) with respect to tetramethylsilane as standard), multiplicity (br=broad), integration, and assignment. The number average molecular weights (Mn) was recorded on a Bruker Microflex LRF Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) system. All mass spectra were obtained in positive ion mode. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as a matrix in tetrahydrofuran (10 mg/mL). Polymer samples were dissolved in THF:methanol v/v 1:1 (10 mg/mL) and analyte solutions were prepared by mixing 10 μL of matrix and 1 μL of polymer sample. Samples were applied using the dried droplet method. Size exclusion chromatography (SEC) was performed on an automated Shimadzu HPLC system, with a PLgel 5 μm MIXED-D column at 50° C., using N,N-dimethylacetamide (DMA) containing 50 mM LiCl as eluent at a flow rate of 0.6 mL/min. Polydispersity index values (PDI) were calculated against poly(methyl methacrylate) (PMMA) standards.
The results of these analyses are summarized in Table 1.
1H NMR (mol %)
Isothermal Titration Calorimetry (ITC)
Isothermal titration calorimetry was used to assess the affinity between the synthesized POx-Ale polymers and dissolved calcium cations in water. ITC experiments were carried out on a fully automated Microcal Auto-iTC200. Curve fitting was performed by Origin 6.0 using one set of sites binding model. Generally, 0.36 mM per unit of alendronate present in the polymer were titrated with 4 mM CaCl2 in Mili-Q water at 20° C. All polymers were titrated with the same batch of calcium. Each ITC titration consisted of 19 injections. All measurements were performed in triplicate.
The statistical analyses were performed using GraphPad InStat® software. Rheological results were analysed statistically using a one-way ANOVA test, followed by Tukey's multiple comparison test. The significance threshold was set at P<0.05.
The results of the ICT analyses are summarized in Table 2.
Preparation of Calcium-Crosslinked POx-Ale Gels
Gels were prepared mixing equal volumes of POx-Ale in phosphate buffer saline (PBS) at different polymer concentration (10, 20, 30 wt. %) with several CaCl2 solutions in miliQ water (1, 10, 20, 40 wt. %), with a total volume of 200 μL. The mixtures were stirred vigorously using vortex for 15 sec in order to get homogenous gels.
Visual Observation of Gelation
Mixing equal volumes of alendronate-functionalized polymers and Ca2+ solutions at final polymer concentrations of 10, 20 and 30 wt. %, and Ca2+ concentrations between 1 and 40 wt. % yielded either viscous solutions or self-healing hydrogels. Generally, increasing both polymer and calcium concentrations formed stronger gels.
Macroscopically, two different kind of gels were created: P(EtOx70-Ale30) produced a white stable transient network, whereas P(EtOx70-OH10-Ale20) formed transparent soft gels. When more than 20 mol % alendronate was present in the polymer, the binding affinity was so strong that the gelation process competed with polymer precipitation, such precipitation causing the gel to lose its transparency.
The self-healing properties of a hydrogel (30 wt. % P(EtOx70-OH10-Ale20) and 20 wt. % CaCl2) was evaluated visually. Two gels of identical composition were coloured using either blue (Brilliant) or red (Amaranth) dyes. They were cut transversally in half, and then two differently dyed halves colour were brought together without applying external force. After two minutes of contact, no border between them was observed, and their connexion was strong enough to allow for stretching.
In Vitro Stability of the Hydrogels
In order to study both the stability and the reversibility of the formed crosslinks, the gels were soaked in EDTA (100 mM, pH 6) and they were monitored for 48 h.
P(EtOx70-OH10-Ale20) was completely dissolved in EDTA within 3 h of immersion, which confirmed that the crosslinks between Ca2+ and alendronate were responsible for the network formation. However, P(EtO60-Ale30) remained stable even after 48 h of soaking time in EDTA, indicating that the crosslinks were strong enough not to be disrupted by the binding agent.
Preparation of Bone- and Mineral-Adhesive Sheets Adhesive sheets were prepared by dry deposition of adhesive polymers (P(EtOx70-Ale30) and P(EtOx70-OH10-Ale20) into commercially available fibrous gelatin carrier (GELITA TUFT-IT®, Germany) of 5×7 cm2, followed by application of a backing layer. Alendronate-free control sheets were prepared by dry deposition of P(EtOx) into the same fibrous gelatin carrier, again followed by application of a backing layer.
The fibrous gelatin carrier, which consisted of 8 cohesive layers, was delaminated to obtain carriers of 3 layers of approximately 300 mg and 0.3 mm thick.
The polymers were processed into fine powders by ball milling at 30 Hz for 10 min, followed by sieving below 63 μm. Next, the polymers were dried using a rotary evaporator at 40° C., 20 mbar for 16 h and the carrier was dried using a vacuum oven at 40° C., 5 mbar for 16 h.
The dry polymer powders were placed into a grid array of 5×7×0.4 cm3, consisting of 726 square wells, and the carriers were placed on top of the array, surrounded by a spacing frame of 1.5 mm thickness. The weight ratio polymer:carrier was 65:35. Next, the array was positioned between two dielectric plates and polymers were loaded into the carriers using a high voltage electrostatic impregnation system (Fibroline SL-Preg, France) at 40 kV, 100 Hz for 20 sec, with a loading efficacy of approximately 80%, obtaining a homogenous distribution of the polymer powders through the carriers.
After impregnation, a backing layer was placed on top of the impregnated carriers and anti-adhesive papers were placed underneath and on top of the combination before it was fixated to a heating plate with the backing layer facing the heating plate. Adhesion of the backing layer to the impregnated carrier was achieved by two cycles of heating while compressing at 150° C., 30 N for 3 sec, obtaining a final weight ratio of polymer/carrier/backing 42%:28%:30%.
The sheets so obtained were cut using a scalpel (carbon steel, blade 20) into the required dimensions for each experiment. Further, they were placed individually in aluminum bags and dried at 40° C., 5 mbar for 16 h. Finally, the bags were sealed at 2 mbar for 4 sec with a nitrogen flush of 600 mbar.
The physical properties of the sheets (1.5×2.5 cm2), i.e. thickness and density, are summarized in Table 3.
Preparation of Bone Samples
Porcine ribs were obtained and sectioned using a circular saw machine into rectangular pieces of approximately 2.5×0.7×1.2 cm3. Each piece of bone was polished (Struers TegraPol 35, USA) using carbon paper (P500) at 150 rpm until a flat surface of cortical bone was exposed. Thereafter, the samples were kept frozen at −20° C. until further use.
Flat bone specimens were immersed in 500 mL of Sakura reagent TDE™ 30 and decalcified using the Sakura TDE™ 30 electrode system for 24 h. Thereafter, they were kept frozen at −20° C. until further use.
Preparation of Apatite-Coated Titanium Plates
Commercially pure titanium plates (2.5×0.1×2.5 cm3, grade 2) were first grit-blasted to obtain an average roughness Ra of 1.5 μm. Subsequently, these substrates were ultrasonically cleaned with Milli-Q water, followed by isopropanol during 20 min. Next, the substrates were placed on a rotating holder and etched with argon plasma for 10 min prior to physical vapor deposition of a thin adherent coating of hydroxyapatite using a radiofrequent magnetron sputtering system (Edwards ESM 100), at a power of 400 W and an argon pressure of 5×10−3 mbar for 12 h. After deposition, the hydroxyapatite-coated plates were crystallized by a heating treatment in a furnace at 650° C. for 2 h, with a heating and cooling rate of 1.5° C./min, yielding an apatitic calcium phosphate coating of 1 μm.
Tensile Tests
Tensile tests were performed using a tensile bench (LS5, Lloyd Instruments, UK) equipped with a 100 N load cell. All measurements were performed in sextuplicate (n=6). The aforementioned sheets were cut into pieces of 1.5×2.5 cm2 (length×height).
Each end of the sheets was fixed to the tensile grips, and the sheets were subsequently tested in dry state at a crosshead displacement speed of 1 mm/min until breakage. The tensile force was recorded as a function of displacement, and the tensile force vs. sheet extension was acquired automatically. The tensile strength was calculated as the maximum load before breakage of the samples divided by their cross-sectional area. The tensile modulus of the sheets was calculated as the average slope of initial linear part of the stress-strain curves.
The tensile strength and the modulus of the different sheets are depicted in Table 4.
The application of hydroxyl-functionalized POx polymer onto the sheets composed of fibrous gelatin and polyester backing further reinforced these sheets. This phenomenon may be attributable to interactions between hydroxyl groups of the polyoxazoline and amines and carboxylic acid groups present in the carrier, but also with the amines present in the backing layer.
Lap-Shear Adhesion Tests to Apatite-Coated Substrates, Apatite-Free Control Substrates, Bone and Demineralized Bone Specimens
Lap-shear adhesion tests were performed using a tensile bench (LS5, Lloyd Instruments, UK) equipped with a 100 N load cell. All measurements were performed in sextuplicate (n=6). To measure the adhesion to apatite-coated substrates, the sheets were cut into pieces of 1.5×2.5 cm2. The sheets were cut into pieces of 1.5×1.0 cm2 for the adhesion tests onto bone, since the bone samples were smaller. Uncoated titanium and demineralized bone specimens were used as control model surfaces.
Sheets were glued to plastic holders. Next, substrates were moistened with PBS soaked gauze followed by controlled attachment of the sheets onto the substrates at a force of 10 N for 5 min. Next, lap-shear adhesion tests were carried out at a crosshead displacement speed of 0.5 mm/min. The shear strength was calculated by dividing the maximum load before breakage by their overlapping contact surface of 3.75 cm2 for Ti plates and 1.5 cm2 for bone specimens. The tensile modulus of the sheets was calculated as the average slope of initial linear part of the stress-strain curves.
The results of the lap-shear adhesion measurements are summarized in Table 5.
The polymer with the highest number of alendronate moieties (P(EtOx70-Ale30)) exhibited an adhesion to CaP coated-Ti plates that was 41 times higher, and an adhesion to bone that was 18 times higher than that of Bio-Gide®, a barrier membrane widely used for dental applications.
Underwater Adhesion Tests to Bone
Sheets of 1 cm2 were adhered to bone specimens for 5 minutes. Next, they were immersed in 200 mL of water, stirred at 150 rpm and monitored for 24 hours to evaluate their adhesion and swelling. Experiments were done in triplicate.
A summary of the results is shown in Table 6. Degree of swelling was scored as:
3=high swelling
2=medium swelling
1=low swelling
Degree of adhesion was scored as:
=no adhesion by any of the sheets
+/−=poor adhesion
+=good adhesion
++=excellent adhesion by all 3 sheets
Only the sheets containing POx-alendronate remained adhesive to bone after immersion in water for 24 hours. Moreover, these sheets showed less swelling than the other sheets.
Ex-Vivo Model
The Lung Assist (Organ Assist, Groningen, the Netherlands) perfusion device was used as the extra corporal organ perfusion system. This device, which is depicted in FIG. 3, consists of two blood reservoirs (1) and (2), an automatic centrifugal pump system (3) to provide arterial flow and pressure, a (de-)oxygenator (4), a heater/cooler unit with an inlet (5) and outlet (6) for cooling/heating medium, a flow sensor (7), temperature sensor (8) and warm water bath holding a porcine cadaveric head (9).
Heparinized full blood was used to prime the system. The system was running for several minutes to heat up and to stabilize the hemodynamic parameters. The porcine cadaveric head was placed in a box filled with warm water (37°) that was connected to a laboratory water bath. Subsequently, cannulation of both the common carotid arteries was performed. Cannulas were filled with blood to remove as much air as possible from the system, and the extracorporeal system was connected to the porcine head. One side of the porcine head was heated from the water from the water bath for approximately 30 minutes, thereafter the porcine head was turned to initiate the surgical procedure on the heated side. After performing the surgery, the head was turned again and the procedure was repeated on the other side of the mandibula. Heparinized blood was oxygenated using 95% oxygen and 5% carbon dioxide. Point-of-care (PoCT) hematology tests (pH, PCO2, PO2, BEecf, HCO3, TCO2, sO2, Hb Hematocrit, Na, K, iCa, Glu and Activated Clotting Time (ACT)) were performed using the i-STAT (Abbott Laboratories, Abbott Park, IL, USA) analyzer. Hemodynamic parameters (PoCT tests, pressures and flows) were measured every ˜1.5h.
Preparation of Bone- and Mineral-Adhesive Sheets
Adhesive sheets were prepared by dry deposition of adhesive polymers (P(EtOx70-Ale30), P(EtOx70-OH10-AIe20) and P(EtOx60-OH20—NHS20) into commercially available fibrous gelatin carrier (GELITA TUFT-IT®, Germany) of 5×7 cm2, followed by application of a backing layer.
The fibrous gelatin carrier, which consisted of 8 cohesive layers, was delaminated to obtain carriers of 3 layers of approximately 300 mg and 0.3 mm thick.
The polymers were processed into fine powders by ball milling at 30 Hz for 10 min, followed by sieving below 63 μm. Next, the polymers were dried using a rotary evaporator at 40° C., 20 mbar for 16 h and the carrier was dried using a vacuum oven at 40° C., 5 mbar for 16 h.
In order to prepare impregnated carriers containing a mixture of either P(EtO60-Ale30) and P(EtOx60-OH20—NHS20) 1:1 or P(EtOx70-OH10-Ale20) and P(EtOx60-OH20—NHS20) 1:1, these polymer mixtures were granulated by adding 3 wt % of acetone, followed by drying in a rotatory evaporator at 40° C., 20 mbar for 16 h.
The dry polymer powders were placed into a grid array of 5×7×0.4 cm2, consisting of 726 square wells, and the carriers were placed on top of the array surrounded with a spacing frame of the 1.5 mm thickness, in a weight ratio of polymer/carrier 65:35. Next, the array was positioned between two dielectric plates and polymers were loaded into the carriers using a high voltage electrostatic impregnation system (Fibroline SL-Preg, France) at 40 kV, 100 Hz for 20 sec, with a loading efficacy of approximately 80%, obtaining a homogenous distribution of the polymer powders through the carriers.
After impregnation, a backing layer was placed on top of the impregnated carriers and anti-adhesive papers were placed underneath and on top of the combination before it was introduced into a heating plate. Adhesion of the backing layer to the impregnated carrier was achieved by two cycles of heating while compressing at 150° C., 30 N for 3 sec, obtaining a final weight ratio of polymer/carrier/backing 42%:28%:30%.
The sheets so obtained were cut using a scalpel (carbon steel, blade 20) into the required dimensions for each experiment. Further, they were placed individually in aluminum bags and dried at 40° C., 5 mbar for 16 h. Finally, the bags were sealed at 2 mbar for 4 sec with a nitrogen flush of 600 mbar.
Ex-Vivo Adhesion Tests
Five cadaveric porcine heads were prepared for experiments. A submandibular incision was made, parallel to the inferior border of mandible. Subsequently, the periosteum was carefully elevated. In total, a maximum of five standardized bone defects (ø3.35 mm) were created using a dental drill, cooled with saline solution. All defects were filled with bone debris and, if necessary, supplemented with Bio-Oss (Geistlich Pharma AG, Wolhunsen, Switzerland) mixed with heparinized blood, and subsequently covered with a bone adhesive sheet (10×10 mm). The sheet was manually pressed for 30 seconds, and left for 4.5 minutes until the adhesive tests were performed.
Six sheets (see Example 7) were tested in sextuplicate (n=6) and all 36 sheets were randomized before testing.
Degree of adhesion was scored as: no adhesion, poor adhesion, moderate adhesion, and strong adhesion. A summary of the results is shown in Table 7. In the table, numbers represent the number of membranes tested.
Using the same procedure as used for the synthesis of alendronate-functionalized poly(2-oxazoline)s, alendronate-functionalized polyethylene glycol (2.97 g, 0.26 mmol) was prepared using 8-arm PEG-NHS (5.0 g, 0.5 mmol), sodium alendronate trihydrate (1.63 g, 6 mmol), N-hydroxysuccinimide (460 mg, 4 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (767 mg, 4 mmol) in PBS (25 mL, 0.5 M). 1H NMR [400 MHz, δ (ppm), D2O]: 4.10 (s, 16H), 3.96 (d, 6H) 3.87-3.58 (br, 1168H), 2.09-1.70 (br, 32H). 31P NMR [400 MHz, δ (ppm), D2O]: 18.04. Yield: 54%
Preparation of Granulate Comprising P(EtOx60-OH20—NHS20) and Alendronic Acid
P(EtOx60-OH20—NHS20) (4.00 g, 0.29 mmol) and alendronic acid (1.44 g, 5.78 mmol) were placed in a high-shear mill. While milling, acetone (0.8 mL) was added dropwise. Subsequently, the granulate was dried followed by drying in a rotatory evaporator at 40° C., 20 mbar for 16 h.
Preparation of Granulate Comprising PEG-NHS8 and Alendronic Acid
PEG-NHS8 (4.00 g, 0.40 mmol) and alendronic acid (0.79 g, 3.17 mmol) were placed in a high-shear mill. While milling, acetone (0.4 mL) was added dropwise. Subsequently, the granulate was dried followed by drying in a rotatory evaporator at 25° C., 20 mbar for 16 h.
Preparation of Bone- and Mineral-Adhesive Sheets
Adhesive sheets without a backing layer were prepared by dry deposition of adhesive polymers and adhesive granulates into commercially available fibrous gelatin carrier (GELITA TUFT-IT®, Germany) of 5×7 cm2 (see Example 7).
The average particle size (D[4,3]) of the adhesive polymers and adhesivie granulates used in the preparation of the adhesive sheets are shown in Table 8.
Lap-Shear Adhesion Tests to Apatite-Coated Titanium
Lap-shear adhesion tests to apatite-coated titanium were performed as in Example 7.
The results of the lap-shear adhesion measurements are summarized in Table 9.
The adhesion results for the sheet containing a granulate of P(EtOx60-OH20—NHS20) and alendronic acid can be improved considerably by including a carbonate/bicarbonate buffer (1:1 mole/mole, pH 8,5) in the granulate and/or by employing a coarser granulate.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/071317 | Jul 2020 | WO | international |
| 20206146.1 | Nov 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/071062 | 7/27/2021 | WO |
