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WO2013044078A2 - Composites de polymères à teneur élevée en minéraux, élastomères et dégradables - Google Patents

Composites de polymères à teneur élevée en minéraux, élastomères et dégradables Download PDF

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WO2013044078A2
WO2013044078A2 PCT/US2012/056641 US2012056641W WO2013044078A2 WO 2013044078 A2 WO2013044078 A2 WO 2013044078A2 US 2012056641 W US2012056641 W US 2012056641W WO 2013044078 A2 WO2013044078 A2 WO 2013044078A2
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composition
hydroxyapatite
pela
polymer
block
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PCT/US2012/056641
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WO2013044078A3 (fr
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Jie Song
Artem KUTIKOV
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University Of Massachusetts Medical School
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Priority to EP12834037.9A priority Critical patent/EP2768896A4/fr
Priority to CA2849891A priority patent/CA2849891A1/fr
Publication of WO2013044078A2 publication Critical patent/WO2013044078A2/fr
Publication of WO2013044078A3 publication Critical patent/WO2013044078A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/32—Phosphorus-containing compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F1/00—General methods for the manufacture of artificial filaments or the like
    • D01F1/02—Addition of substances to the spinning solution or to the melt
    • D01F1/10—Other agents for modifying properties
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
    • D01F6/78—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from copolycondensation products
    • D01F6/84—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from copolycondensation products from copolyesters
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
    • D01F6/78—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from copolycondensation products
    • D01F6/86—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from copolycondensation products from polyetheresters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/16—Materials with shape-memory or superelastic properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/32—Phosphorus-containing compounds
    • C08K2003/321—Phosphates
    • C08K2003/325—Calcium, strontium or barium phosphate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K2201/00—Specific properties of additives
    • C08K2201/018—Additives for biodegradable polymeric composition

Definitions

  • the invention generally relates to polymer compositions. More particularly, the invention relates to compositions of hydroxyapatite and block co-polymers, methods of their preparation and uses thereof, wherein the co-polymers have hydrophilic and biodegradable hydrophobic blocks for stable interfacing with HA, resulting in stable polymer- hydroxyapatite suspensions suitable for a variety of applications.
  • HA hydroxyapatite
  • HA provides the necessary mechanical strength, enhances the material's osteoconductivity, and serves an important source for calcium and phosphate ions.
  • HA also plays an important role in retaining a variety of proteins on its surfaces as it has been shown to support bone cell attachment and growth factor binding and release, and to expedite healing of bone defects in vivo.
  • HA has been incorporated with synthetic polymers to form 2- or 3 -dimensional, dense or porous composite scaffolds using a wide range of fabrication techniques including electrospinning, injection molding, and 3 -dimensional prototyping.
  • a widely used fabrication technology for generating porous thin membrane scaffolds (or fibrous meshes) is electrospinning, where a grounded surface collects a charged polymer jet of nano and/or micro-sized fibers.
  • electrospinning where a grounded surface collects a charged polymer jet of nano and/or micro-sized fibers.
  • Previously reported co-electrospinning of various polymers with hydroxyapatite suffers from a variety of limitations, such as material defects, settling of the hydroxyapatite, poor integration and brittleness, low strength and inferior surgical handling properties.
  • beneficial effects occur when blending HA with hydrophilic polymers such as poly(hydroxyethyl methacrylate), for example improved toughness, elastic modulus and osteoblast adhesion, unfortunately poly(hydroxyethyl methacrylate) is not biodegradable.
  • Biodegradable polyesters such as poly(lactic acid) (PLA) are readily electrospinable with established in vitro and in vivo degradation profiles.
  • the intrinsic hydrophobicity of PLA results in its poor mixing and adhesion with hydrophilic HA, making it difficult to achieve adequate structural and mechanical properties in electrospun HA-PLA composite meshes.
  • HA-PLA composites often exhibit inferior handling properties (e.g., brittleness) and inconsistent biological performance.
  • Approaches for addressing the lack of interfacial adhesion include the addition of amphiphilic surfactants or modifying HA with surface-grafted polymers to improve interactions with hydrophobic polyesters.
  • HA mixing and adhesion can be achieved through favorable hydrophilic interactions.
  • hydrophilic polymers such as poly(hydroxyethyl methacrylate) (pHEMA) and poly(ethylene glycol) (PEG) have been reported. (Gaharwar, et al. 2011 Biomacromolecules 12, 1641-50; Song 2003 J.
  • the invention provides novel compositions of hydroxyapatite and block copolymers, methods of their preparation and uses thereof, wherein the co-polymers have hydrophilic and biodegradable hydrophobic blocks for stable interfacing with HA, resulting in stable polymer-HA suspensions.
  • the super-hydrophilicity, strengthened mechanical integrity, and retained structural integrity of the HA-poly(ethylene glycol-co-lactic acid) (PELA) composite in aqueous environment represent major advantages over the HA- poly(lactic acid) (PLA) composites for various skeletal tissue engineering applications.
  • the invention generally relates to a composition that includes hydroxyapatite and a block co-polymer.
  • the block co-polymer includes hydrophilic blocks and degradable hydrophobic blocks.
  • the composition exhibits hydrophilic surface properties, elasticity and retains mechanical integrity in aqueous environment.
  • the composition possesses a stable structural interface between the co-polymer and the hydroxyapatite.
  • the composition of the invention is characterized by the properties of biodegradability, aqueous stability and eletrospinability.
  • the composition may be electrospun into fibrous composite mesh.
  • the block co-polymer is crosslinked to form a three-dimensional polymer-hydroxyapatite network.
  • the block co- polymer/HA composite is extruded into an un-crosslinked three-dimensional scaffold by rapid prototyping (or 3-D printing).
  • the invention generally relates to a medical implant that includes a composition comprising hydroxyapatite and a block co-polymer, wherein the block copolymer comprises hydrophilic blocks and degradable hydrophobic blocks.
  • the implant may be a 3 -dimensional filler for bony defects or a repair material for bone, cartilage, osteochondral, tendon or ligament damage.
  • the invention generally relates to a biodegradable composite scaffold prepared from a fibrous composite mesh electrospun from a suspension of hydroxyapatite with an amphiphilic block poly(ethylene gly col-co-lactic acid).
  • the invention generally relates to a biodegradable, three- dimensional composite scaffold prepared by crosslinking a suspension of hydroxyapatite with an amphiphilic block poly(ethylene glycol-co-lactic acid).
  • the invention generally relates to a biodegradable, three- dimensional composite scaffold, prepared by rapid prototyping from a suspension of hydroxyapatite with an amphiphilic block poly(ethylene glycol-co-lactic acid).
  • FIG. 1 shows representative GPC spectrum and peak analysis for as-synthesized PELA.
  • FIG. 2 shows exemplary 1H NMR spectrum for purified PELA.
  • FIG. 3 shows exemplary photographs of stable PELA-HA (20% w/w HA) suspension over 1 week.
  • FIG. 4 shows an exemplary electrospinning setup.
  • FIG. 5 shows exemplary Von Kossa stain of PELA (A) PELA-HA (20% w/w HA) and (B) meshes. 100X magnification. 1 mL of Von Kossa stain (3% silver nitrate) was added to each mesh (1 cm 2 ) and placed in a UV crosslinker for 10 min. Samples were then washed 3 times with MilliQ water and imaged under a bright-field light microscope. [0021] FIG. 6 shows exemplary X-ray powder diffraction of PELA-HA mesh (33% w/w/
  • FIG. 7 shows exemplary SEM of PELA (A), PELA-HA (33% w/w HA) (B), and
  • FIG. 8 shows exemplary EDX spectra of PELA-HA (33% w/w HA) meshes.
  • FIG. 9 shows (A) qualitative depiction of recoverable strain for PELA-HA (33% w/w HA) mesh, before (1), during (2), and after (3) manual elongation. (B) Tensile testing of electrospun composite meshes.
  • FIG. 10 shows exemplary strain sweep data for PELA (A), PELA-HA (33% w/w HA) (B), and PELA-HA (50% w/w HA) (C) meshes.
  • Mechanical testing was performed on a TA Instruments DMA Q800 dynamic mechanical analyzer under dry conditions at room temperature. Samples were cut into 5 mm X 15 mm strips and ramped at 1 Hz from 10 ⁇ to 200 ⁇ strain amplitude.
  • FIG. 11 shows exemplary reaction scheme for the synthesis of PELA (1) and PLA (2).
  • FIG. 12 shows exemplary microstructural, surface and compositional properties of electrospun PELA-HA and PLA-HA composite scaffolds.
  • C TGA plots of electrospun scaffolds with HA powder as a control.
  • FIG. 13 shows exemplary surface, mechanical and microstructural property changes of electrospun PELA-HA and PLA-HA composite scaffolds upon hydration.
  • C SEM micrographs of scaffolds after 1-week incubation in PBS (pH 7.4, Ca 2+ /Mg 2+ -free) at 37 °C. Scale bar: 50 mm.
  • FIG. 15 shows exemplary effect of scaffold composition (chemistry and HA content) on lineage commitment of rMSC under un-stimulated culture condition as
  • FIG. 16 shows exemplary stability of HA (33% w/w) suspension in 1 :4
  • FIG. 17 shows exemplary degradation of HA-PELA and HA-PLA scaffolds in PBS at 37 °C as monitored by mass reduction over 12 weeks.
  • FIG. 18 shows exemplary macroporous 3-D PELA and HA-PELA composites prepared by 3-D prototyping.
  • B Computer-assisted design (CAD) of the macroporous scaffold.
  • FIG. 19 shows exemplary scanning electron microscopy (SEM) images of 3D- printed 10% HA-PELA scaffold. Top view (left) and cross section (right).
  • This invention provides a novel biodegradable composite material: a stable suspension of HA with a block copolymer of poly(ethylene glycol) and poly(lactic acid).
  • a block copolymer of poly(ethylene glycol) and poly(lactic acid) for example, disclosed herein is an electrospun, biodegradable amphiphilic block
  • the block co-polymer fulfills key requirements, including HA integration, ease of processing (such as electrospinnability), aqueous stability, and biodegradability.
  • the length of the PLA and PEG segments can be varied to modify the properties (mechanical, hydrophobicity, degradability) of the final polymer.
  • PLA-PEG-PLA or PEG-PLA-PEG block copolymers can be synthesized depending on the application.
  • This novel approach combines the degradability and aqueous stability of the poly(D,L-lactic acid) (PLA) block with the HA-binding capability of the poly(ethylene glycol) (PEG) block and the electrospinability of both.
  • Electrospun PELA-HA composites exhibit a more uniform fiber morphology than those of electrospun PLA-HA composites.
  • the HA- PELA composites are super-hydrophilic and compliant, enabling easy cell seeding and potential surgical manipulations. Equally important, the HA-PELA composite scaffolds more readily promote early osteochondral lineage commitment while suppressing the adipogenesis of bone marrow stromal cells in a HA-dose dependent manner than the HA-PLA composites.
  • a key aspect of this invention is the use of a block co-polymer comprised of hydrophilic and degradable hydrophobic blocks for stable interfacing with HA, resulting in stable polymer-HA suspensions.
  • the hydrophilic blocks are important for HA binding while the hydrophobic blocks allow for degradability and aqueous stability as well as
  • This formulation can be crosslinked into a degradable three-dimensional (3- D) scaffold for filling bony defects or repairing bone, cartilage, osteochondral, tendon or ligament (such as anterior cruciate ligament) damage. It can also be extruded into fibers to serve as a degradable suture or as a material for fused deposition modeling machines, allowing for the printing of custom scaffolds.
  • 3- D three-dimensional
  • the example below describes one format (an electrospun 2-D fibrous composite mesh) for potential application as synthetic periosteum to expedite the healing (tissue integration) of structural bone allografts or 3-D tissue engineered bone constructs. This would function through the delivery of exogenous therapeutic agents (e.g., growth factors), enriching endogenous factors, and/or enabling the attachment and differentiation of stem or progenitor cells (endogenous or exogenous) at the implant site.
  • exogenous therapeutic agents e.g., growth factors
  • enriching endogenous factors e.g., growth
  • the invention generally relates to a composition that includes hydroxyapatite and a block co-polymer.
  • the block co-polymer includes hydrophilic blocks and degradable hydrophobic blocks.
  • the composition exhibits hydrophilic surface properties, elasticity and retains mechanical integrity in aqueous environment.
  • the composition possesses a stable structural interface between the co-polymer and the hydroxyapatite.
  • Hydrophilic surface properties here refer to properties that are characteristic of and associated with hydrophilic surfaces, for example, water contact angle below about 100°.
  • retaining mechanical integrity in aqueous environment it is meant that the storage modulus of hydrated composite is comparable or better than the storage modulus of the dry composite.
  • elasticity refers to the ability of a material, when prepared in an electrospun membrane format with dimensions complying to ASTM D882-97, to undergo tensile deformations of at least about 20% (e.g., preferably at least about 50%>, 75%, 100%, 150% or 200%) prior to failure.
  • the hydroxyapatite may be present in any suitable percentage depending on the application at hand.
  • the hydroxyapatite is present in a weight percentage of at least 1 % (e.g., at least 5, at least 10%>, at least 20%>, from about 20%> to about 50%), from about 20%> to about 60%>, from about 20%> to about 70%>, from about 1% to about 70%).
  • the block co-polymer includes blocks of poly(ethylene glycol) and polyesters. In certain preferred embodiments, the block co-polymer comprises blocks of poly(ethylene glycol) and poly(lactic acid).
  • the composition of the invention is characterized by the properties of biodegradability, aqueous stability and eletrospinability.
  • the composition may be electrospun into fibrous composite mesh.
  • the block co-polymer is crosslinked forming a 3-D polymer-hydroxyapatite network.
  • the composition is a 3-D network prepared rapid prototyping.
  • the composition is characterized by a shape-memory property.
  • Shape-memory property refers to the ability of a material to return from a deformed state (temporary shape) to its original (permanent) shape induced by an external stimulus (trigger), such as a temperature change.
  • the invention also features an article of manufacture made from the composition disclosed herein.
  • the invention generally relates to a medical implant that includes a composition comprising hydroxyapatite and a block co-polymer, wherein the block copolymer comprises hydrophilic blocks and degradable hydrophobic blocks.
  • the implant may be a 3-D filler for bony defects or a repair material for bone, cartilage, osteochondral, tendon or ligament damage.
  • the implant is a degradable fibrous membrane wrapped around one or more structural allografts or one or more 3-D synthetic scaffolds to augment tissue repair function.
  • the implant is biodegradable. Additionally, it is preferred that the implant is capable of supporting attachment of cells and/or supporting attachment of a biological agent. Any suitable cells may be employed depending on the desired application. In certain preferred embodiment, the cells are stem or progenitor cells.
  • the biological agent may be a therapeutic, diagnostic or imaging agent.
  • the biological agent attached to the implant is a growth factor or an antibiotic agent.
  • the invention generally relates to a biodegradable composite scaffold prepared from a fibrous composite mesh electrospun from a suspension of hydroxyapatite with an amphiphilic block poly(ethylene gly col-co-lactic acid).
  • the invention generally relates to a biodegradable, three- dimensional composite scaffold prepared by crosslinking a suspension of hydroxyapatite with an amphiphilic block poly(ethylene glycol-co-lactic acid).
  • the invention generally relates to a biodegradable, three- dimensional composite scaffold, prepared by rapid prototyping from a suspension of hydroxyapatite with an amphiphilic block poly(ethylene glycol-co-lactic acid).
  • the present invention describes a rational block co-polymer design consisting of hydrophilic and degradable hydrophobic segments. Such a design allows for stable suspension of HA (or similar bioceramics) at high mineral concentrations be made in such a block co-polymer solution, which can be used to develop a variety of scaffold architectures, from 2-D electrospun composite fibrous meshes to 3-D composite scaffolds, all with excellent structural integration between the polymer and mineral component.
  • the invention simultaneously addresses several major limitations in prior biomaterial designs, including (1) the lack of degradable yet hydrophilic materials for musculoskeletal applications; (2) the lack of adequate structural integration, thus poor mechanical properties, of organic-inorganic structural composites for orthopedic applications; (3) the difficulty of incorporating high percentage of osteoconductive mineral in synthetic bone substitute without making the composites brittle.
  • the stable suspension of a high weight percentage of HA (content close to those in human bone), critical to recapitulate the structure and biological activity of bone, can be achieved without the addition of any surfactants or potentially harmful chemicals.
  • Such materials are particularly suitable for load-bearing applications (e.g., tensile and compressive) such as synthetic bone grafts or ligaments.
  • the material is thermoplastic and suspends the HA without crosslinking, it can be extruded for use as degradable sutures or other applications (i.e., fused deposition modeling).
  • the polymer/HA mixture can also be electrospun into high HA concentration membranes for the augmentation of bone repair in combination with structural allografts or 3-D tissue engineered constructs.
  • PEG (20,000 Da) was used to initiate the ring-opening polymerization of cyclic D,L-Lactide forming the block copolymer PLA600-PEG454-PLA600 (PELA) or a composition of 80% w/w PLA and 20% w/w PEG.
  • the reaction was performed at 130 °C under inert gas and catalyzed by 500 ppm of Sn(Oct) 2 .
  • the resulting polymer was characterized by gas permeation chromatography (GPC) and nuclear magnetic resonance (NMR), representative GPC and NMR data are shown in FIG. 1 and FIG. 2.
  • the PELA was dissolved in chloroform and purified by precipitation in methanol.
  • HA from 0%> to 50%> w/w
  • PELA 50% w/v
  • the HA remained stably dispersed in the electrospinning solution for at least 1 week with minimal settling detected which represents a major improvement over previous report (FIG. 3).
  • the suspensions were electrospun through an 18G needle at a flow rate of 1.7 mL/hr, a voltage of 12kV, and a working distance of 15 cm (FIG. 4).
  • FIG. 5 Representative bright- field images of a HA- free mesh and of a mesh containing 20 w/w HA are shown in FIG. 5.
  • XRD confirmed no change in the crystal structure of the hydroxyapatite during the spinning process (FIG. 6).
  • Scanning electron microscopy demonstrated that fiber dimensions were relatively uniform. Even with the incorporation of up to 50% w/w HA, HA remained embedded within the fibers with roughened fiber surfaces (FIG. 7). The presence of HA was further confirmed by energy-dispersive x-ray
  • the composite meshes exhibit highly elastic properties with recoverable strains of over 100% (FIG. 9).
  • 25% HA-PELA composite meshes exhibit ultimate tensile strains of over 200%, as opposed to 25% HA-PLA with an ultimate tensile strain below 40%.
  • Dynamic mechanical analysis strain sweep, 1 Hz, 0-200 ⁇ amplitude
  • Meshes with 50% HA w/w exhibited a decreased storage modulus but still had high failure strains and excellent handling
  • the well-controlled molecular weight and polydispersity allows us to investigate the effect of polymer chemistry on the composite scaffold properties without additional confounding factors.
  • Racemic D,L-lactide was chosen over L-lactide due to the accelerated degradation profile of the former and the concern over undesired crystalline degradation by-products of the latter which can elicit adverse responses in vivo.
  • DSC Differential scanning calorimetry
  • HA Prior to electrospinning, HA was sonicated in 1 :4 dimethylformamide/chloroform to disrupt HA aggregates. PELA or PLA was then added and stirred overnight to produce a polymer/HA suspension. The PELA-HA suspension was stable for over 1 week whereas the HA tended to quickly settle from the PLA solution without immediate electrospinning (FIG. 16). The HA-PELA scaffolds were electrospun without noticeable interference from the HA component while periodic blockage of the needle tip was observed during the electrospinning of HA-PLA composite.
  • HA-PELA and HA-PLA composite scaffolds (average thickness of 0.1-0.2 mm) with 0 to 25 wt% HA were obtained after 2 hr and dried in a vacuum oven for 48 hours to remove residual solvent.
  • the HA-PELA composites exhibited a narrow distribution of fiber dimensions and fewer defects than HA-PLA (FIGs. 12A & B). While there was no significant difference in fiber diameter between 0% HA-PELA and 25% HA- PELA, the diameter of the 25% HA-PLA fibers increased over two-fold from 0% HA-PLA.
  • Thermogravimetric analysis was used to determine the copolymer composition and actual HA content in the electrospun composites (FIG. 12C).
  • the 0% HA- PELA TGA curve was characterized with a transition at approximately 83% weight loss, which closely correlated with the weight percentage of PLA in the PELA scaffolds.
  • the PLA blocks, with a lower decomposition temperature than PEG, were burnt away first. This transition was not observed in the 10 and 25% HA-PELA composite, likely due to an increase in the decomposition temperature of the composite beyond those associated with the PLA and PELA blocks upon the HA incorporation.
  • the HA appears to have a thermal insulating effect on the scaffolds.
  • the percentage of HA that remained after PELA thermal decomposition as determined by TGA matched precisely with their weight percentages in the electrospinning solution (10%> and 25%), supporting excellent uniformity and stability of the PELA-HA suspension throughout the electrospinning.
  • the actual HA content in the 25% HA-PLA composite as determined by TGA is 28.4%, likely resulting from the inhomogeneous dispersion of HA with PLA and some level of settling/aggregation of HA during the electrospinning.
  • the wettability or hydrophilicity of a synthetic tissue scaffold is important for facilitating cell seeding, growth factor loading, and surgical handling.
  • the hydrophilicity of the HA-PELA and HA-PLA electrospun composites was assessed by water contact angle measurements (FIG. 13A).
  • the hydrophilic PEG block in PELA significantly reduced the water contact angle (increased the wettability) of the scaffolds when compared to PLA.
  • HA incorporation slightly increased the contact angle of the vacuum-dried as-spun PELA while exerted no significant effect on the wettability of PLA. It is possible that the preferential interaction between the PEG blocks with HA has made them less exposed to the surface, contributing to the slight reduction in wettability of HA-PELA.
  • MSCs Mesenchymal stem cells residing in the bone marrow
  • osteoblasts chondrocytes, adipocytes, and myoblasts.
  • chondrocytes chondrocytes
  • adipocytes adipocytes
  • myoblasts a variety of cell types including osteoblasts, chondrocytes, adipocytes, and myoblasts.
  • An effective synthetic tissue scaffold should support the attachment and guide lineage-specific differentiations of MSCs, allowing for effective regeneration of tissues of interest.
  • rMSC rat bone marrow stromal cell
  • the non-fouling PEG block and the osteoconductive HA component of the HA-PELA composites are expected to exhibit opposite effects on protein and cell adhesion.
  • Un- mineralized di-block or tri-block PELA have been used as anti-adhesion membranes, with their low protein adsorption characteristics attributed to the non-fouling PEG exposed on the surface in the aqueous environment. (Yang, et al. 2009 Acta Biomater. 5, 2467-74;
  • HA is known for its ability to absorb a wide range of proteins due to its dynamic surface properties ⁇ e.g., pH-dependent zeta potential) and large surface area (for HA nanocrystals) and promote cell attachment.
  • dynamic surface properties e.g., pH-dependent zeta potential
  • large surface area for HA nanocrystals
  • HA dose-dependent increases in viable adherent cells at 24 h were observed, supporting that HA promoted MSC attachment to the amphiphilic polymer.
  • MSCs adhered on all substrates were able to proliferate well as indicated by MTT cell viability at 96 hr, irrespective of the chemical environment and mineralization status.
  • rMSCs To examine the effects of dose-dependent HA incorporation in PELA and PLA on the spontaneous lineage commitment of MSCs, we cultured rMSCs on 0-25% HA-PELA scaffolds along with 25% HA-PLA control in expansion media without differentiation inducing supplements. After 7, 14, or 21 days, total RNA was isolated from cells adhered to each scaffold and the gene expression of typical osteoblast, chondrocyte, and adipocyte markers as a function of the scaffold environment and time was quantified by qPCR (FIG. 15). Data were normalized to those obtained from the rMSCs prior to seeding on the various substrates (time 0).
  • HA incorporation resulted in dose-dependent increases in the expression of chondrogenic marker Sox9 and osteogenic marker osteocalcin as early as 7 days, with the HA dose-dependent trend persisting even when the overall expression of these markers started to decline at later time points.
  • the decline of the overall expression of these phenotypical markers at later time points can be attributed to both the temporal nature of the expression, observed with other tissue-engineering scaffolds, and the un-stimulated culture condition that may be insufficient to drive potent and persistent expression of these markers.
  • osteochondral lineage markers was significantly higher for MSCs cultured on 25% HA- PELA scaffolds than for those cultured on 25% HA-PLA, suggesting that the osteochondro- inductive properties of HA are more effectively manifested on HA-PELA where HA were more homogeneously dispersed.
  • adipogenic marker PPARG significantly decreased upon the addition of HA to PELA in a dose-dependent manner at all time points examined. Furthermore, the expression of PPARG in MSCs cultured on the 25% HA-PLA scaffold is significantly higher than those cultured on the 25% HA-PELA scaffold, and the adipogenic lineage commitment promoted by the more hydrophobic HA-PELA peaked at 21 days. Overall, these data show that the PELA-HA composites promote early osteochondral lineage commitment while suppress adipogenic lineage commitment of MSCs under un-stimulated culture conditions. By contrast, such effects of HA incorporation were not manifested on the PLA-HA composite, where significantly lower osteochondral gene expressions and more elevated adipocyte maker expression were observed instead.
  • a CAD of a macroporous cylindrical scaffold was designed using 3-matics
  • the CAD file was processed into g-code for 3-D printing by ReplicatorG.
  • a MakerBot Thing-O-Matic 3D printer (MakerBot Industries) was used to rapid prototype the PELA and PELA-HA scaffolds by fused deposition modeling. The printer built the scaffolds layer-by-layer by thermal extrusion. Extruder temperatures of 140 °C, 160 °C, and 185 °C were used for 0% HA-PELA, 10% HA-PELA, and 25% HA-PELA scaffolds, respectively (FIG. 18A; FIG. 19).
  • Hydroxyapatite was purchased from Alfa Aesar. All other solvents and reagents were purchased from Sigma- Aldrich and used as received.
  • Poly(ethylene glycol-co-lactic acid), PELA was synthesized by melt ring opening polymerization. Briefly, poly(ethylene glycol) (4 g, 0.2 mmol) was heated to 100 °C in a shlenk flask and stirred under vacuum for 1 hr to remove residual water. The melt was cooled to room temperature before Tin(II) 2- ethylhexanoate (24.18 mg, 0.06 mmol) in anhydrous toluene was introduced by syringe. The toluene was removed by heating the mixture under vacuum at 100 °C for 15 min.
  • Molecular weight and polydispersity of PELA and PLA was determined by gel permeation chromatography (GPC) on a Varian Prostar HPLC system equipped with two 5- mm PLGel MiniMIX-D columns (Agilent) and a PL-ELS2100 evaporative light scattering detector (Polymer Laboratories). THF was used as an eluent at 0.3 mL/hr at room temperature. Molecular weight and polydispersity was calculated based on EasiVial polystyrene standards (Agilent).
  • Thermal transitions of PELA and HA-PELA composites were determined by conventional differential scanning calorimetry (DSC) on a Q200 MDSC (TA Instruments). Samples ( ⁇ 6 mg) were scanned twice from -90°C to 250°C (20°C/min). A constant nitrogen flow of 50 mL/min was applied. Temperature was calibrated with indium, gallium, and tin standards. T g was defined as the midpoint of the inflection tangent from the second heating curve.
  • Electrospining PELA-HA and PLA-HA composite scaffolds with 0-25 wt% HA were prepared by electrospinning. HA was bath-sonicated in 5-mL 1 :4
  • As-spun scaffolds were sputter coated in Au (4nm) and imaged on a Quanta 200 FEG MKII SEM (FEI Inc.) under high vacuum at 5 kV. Fiber diameter was quantified from the SEM micrographs by measuring 100 random fibers with ImageJ software (National Institutes of Health).
  • TGA was used to determine the actual percentage of HA in the as-spun scaffolds.
  • the samples were heated at a rate of 20 °C/min. from room temperature to 500°C and the mass change was recorded on a TGA Q50 (TA Instruments). HA powder was used as a control.
  • the wettability of the scaffolds was examined by the sessile drop technique. 5- ⁇ , droplets of deionized water were deposited onto as-spun scaffolds or scaffolds freeze-dried following 24 hr equilibration in 37°C deionized water. The water contact angle was recorded using a CAM 200 goniometer (KSV Instruments). The droplet was imaged after 30 sec. and the average contact angle from the left and right side of the drop was recorded. Five randomly selected areas per scaffold were used for each water contact angle measurement.
  • the tensile storage modulus of dry and hydrated (deionized water) scaffolds was determined on a Q800 DMA (TA Instruments). Specimens (5.3 mm x 20 mm) were cut with a parallel blade cutter and loaded onto a film tension fixture with a grip separation of 10 mm. Dry and hydrated samples were applied with a 0.001 N and 0.05 N pre-load force, respectively. Samples were equilibrated at 37°C and held isothermal for 10 min. prior to initiating 0.02% strain at a frequency 1 Hz. The 0.02% strain was chosen as it falls within the linear viscoelastic region of the scaffolds. The storage modulus at 0.02%> strain was recorded.
  • Rat bone marrow stromal cells were isolated from the long bones of a 4- week old male Charles River SASCO SD rat as previously described. Briefly, whole bone marrow was flushed from the femur with minimal essential medium (aMEM without ascorbic acid) and the red blood cells were lysed with sterile water. The cells were
  • Non-adherent cells were aspirated 4 days after platting and remaining adherent cells were cultured until 70% confluence before being trypsinized and seeded on various scaffolds.
  • rMSCs were cultured on the scaffolds in expansion media to determine the effect of scaffold composition on un-stimulated lineage commitment. Scaffolds were sterilized under UV for 1 h each side and equilibrated in MSC expansion media at 37°C overnight. The scaffolds were placed in ultra-low attachment 24 well plates (Corning) and seeded with passage 1 rMSCs (50,000 /cm 2 ). Following 7, 14, or 21 days in culture, total RNA from the MSCs adhered on the scaffolds and from P0 rMSCs (prior to seeding on scaffolds, time 0) was isolated using TRIzol (Invitrogen) and purified by Direct-Zol miniprep (Zymo Research). RNA was reverse transcribed into cDNA with Superscript III Reverse Transcriptase

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Abstract

L'invention propose de nouvelles compositions d'hydroxyapatite et de copolymères à blocs, des procédés de leur préparation et des utilisations de ceux-ci, les copolymères ayant des blocs hydrophobes dégradables et des blocs hydrophiles dégradables pour réaliser une interface stable avec l'hydroxyapatite, conduisant à des suspensions polymère-hydroxyapatite stables. La super-hydrophilicité, l'intégrité mécanique renforcée et l'intégrité structurale conservée du composite HA-PELA dans un environnement aqueux représentent des avantages majeurs par rapport aux composites HA-PLA pour des applications d'ingénierie de tissu squelettique.
PCT/US2012/056641 2011-09-23 2012-09-21 Composites de polymères à teneur élevée en minéraux, élastomères et dégradables WO2013044078A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014204634A1 (fr) * 2013-05-31 2014-12-24 University Of Massachusetts Medical School Échafaudages composites, minéraux, polymères, dégradables et élastomères
WO2016181402A1 (fr) 2015-05-14 2016-11-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Traitement de polymères thermodurcis à mémoire de forme pour obtenir des formes 3d complexes
WO2018120560A1 (fr) * 2016-12-29 2018-07-05 中国科学院深圳先进技术研究院 Composite à base de polymère comprenant un réseau tridimensionnel de particules de charge et procédé de préparation associé
CN109646715A (zh) * 2019-01-25 2019-04-19 上海交通大学医学院附属第九人民医院 电纺3d打印制备含细胞因子微球的多层软骨复合体

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* Cited by examiner, † Cited by third party
Title
See references of EP2768896A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014204634A1 (fr) * 2013-05-31 2014-12-24 University Of Massachusetts Medical School Échafaudages composites, minéraux, polymères, dégradables et élastomères
EP3003224A4 (fr) * 2013-05-31 2016-11-23 Univ Massachusetts Medical Échafaudages composites, minéraux, polymères, dégradables et élastomères
WO2016181402A1 (fr) 2015-05-14 2016-11-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Traitement de polymères thermodurcis à mémoire de forme pour obtenir des formes 3d complexes
WO2018120560A1 (fr) * 2016-12-29 2018-07-05 中国科学院深圳先进技术研究院 Composite à base de polymère comprenant un réseau tridimensionnel de particules de charge et procédé de préparation associé
CN109646715A (zh) * 2019-01-25 2019-04-19 上海交通大学医学院附属第九人民医院 电纺3d打印制备含细胞因子微球的多层软骨复合体

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