WO2016133887A1 - Matériau hybride tissu décellularisé/nanofibres/hydrogel permettant d'optimiser la régénération tissulaire - Google Patents

Matériau hybride tissu décellularisé/nanofibres/hydrogel permettant d'optimiser la régénération tissulaire Download PDF

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WO2016133887A1
WO2016133887A1 PCT/US2016/018043 US2016018043W WO2016133887A1 WO 2016133887 A1 WO2016133887 A1 WO 2016133887A1 US 2016018043 W US2016018043 W US 2016018043W WO 2016133887 A1 WO2016133887 A1 WO 2016133887A1
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peg
hydrogel
cross
buffer solution
component
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PCT/US2016/018043
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English (en)
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Matthew L. Becker
Zachary K. ZANDER
Mary E. WADE
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The University Of Akron
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    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/26—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof

Definitions

  • the present invention generally relates to the use and method of making a hybrid material containing decellularized tissue.
  • the hybrid material comprises decellularized tissue that has been encased and stabilized in a PEG hydrogel matrix.
  • the PEG hydrogel matrix contains nanofibers which further assist in encasing and stabilizing the decellularized tissue.
  • Tissue engineering is a biomedical technique used to develop synthetic/biological scaffolds that can repair, maintain, or improve tissue function.
  • An important feature of the scaffolds is its ability to allow cellular attachment, proliferation, and differentiation, followed by the development of healthy tissues prior to the scaffold's degradation.
  • Human mesenchymal stem cells (hMSC) can sense their local environment and respond by altering their migration, signaling, and proliferation.
  • hMSC Human mesenchymal stem cells
  • the mechanical properties of scaffolds i.e. modulus/elasticity
  • tissue engineering is the mechanical properties of the scaffolds, which are governed by the target tissue.
  • the most commonly used material for many applications in soft tissue repair is a polypropylene mesh.
  • the polypropylene mesh is typically over engineered beyond native tissue parameters, primarily in strength.
  • the patient who uses the polypropylene mesh will have discomfort.
  • the polypropylene mesh elicits a strong immunogenic response that leads to inflammation and fibrous capsule formation that result in a plethora of undesired consequences for tissue repair.
  • the large water content and favorable elastic properties of hydrogels make them promising candidates for various biomedical applications, such as scaffolds for tissue engineering.
  • Hydrogels are materials that are primarily composed of hydrophilic cross-linked networks, containing as much as 99% water.
  • Synthetic polymers are the most preferred polymer to use in hydrogels, due to their reproducibility, high purity, and controlled molecular weight.
  • Polyethylene glycol- based hydrogels are most preferred because of their versatility in end group modification, hydrophilic chemical structure, and protein absorption resistance.
  • Polyethylene glycol is also referred to herein by the well-known PEG abbreviation.
  • PEG-based hydrogels with terminal acrylate functionality have been found to be quite useful in tissue engineered scaffolds. Upon irradiation with ultraviolet light, they can form a three-dimensional hydrogel network with tunable mechanical properties dependent on the cross-link density, and the mass fraction of the polymer precursors.
  • free radical polymerizations have limited control, which leads to multiple cross-linking reactions across the solution, which leads to the formation of micro-clusters that are tied together by the polymer chains.
  • Such networks are not favorable for incorporating biological cues due to their inhomogeneity and complex micro-cluster structural formations.
  • orthogonal chemistries Click chemistries
  • “Click” chemistries in general are defined as highly efficient, robust, and orthogonal reactions that form hydrogels having superior properties in comparison to in situ polymerized gels.
  • oxime chemistry employs a kinetically dependent reaction that varies with buffer pH and ionic strength. Oxime chemistry allows for very mild reaction conditions because no metal catalyst is required, the polymerization temperature is between 20-40 °C, the pH ranges from 2-7, and the only by-product produces is water.
  • hydrogel systems are capable of achieving the wide range of elasticities without altering the chemical makeup, and therefore the physical properties of the scaffold, and controlling the physical properties of the scaffold is essential for the application of hydrogels in tissue engineering. Therefore, there is a need in the art for a hydrogel system that allows for the modulus to be dialed in, using alternative means to control the mechanical properties, such as pH and buffer strength.
  • novel hydrogels and hydrogel formation methods herein are also employed to provide novel wound dressings utilizing decellularized extracellular matrix.
  • Decellularized extracellular matrix is generally known and forms an important component of the full hybrid wound dressings disclosed herein.
  • Decellularized tissue has shown enormous regenerative capabilities when used in wound healing applications, and studies are currently investigating the potential of decellularization in full organ transplantation. It is believed the reason for this material's success is derived from the numerous proteins that reside in the matrix allowing for excellent wound healing performance. These proteins are cleaved by metalloproteinases within the wound, and signal to incoming cells to migrate to the wound, proliferate, and to lay down more matrix.
  • the present invention provides further advances in the art by providing wound dressing better able to support and deliver the decellularized extracellular matrix to the wound site.
  • the present invention provides a wound dressing comprising: decellularized tissue and fibers at least partially surrounded by a PEG hydrogel, wherein the PEG hydrogel is structured by covalent bonds formed via an oxime ligation reaction of a polyethylene glycol (PEG) containing component and a cross-linking component.
  • PEG polyethylene glycol
  • the present invention provides a wound dressing as in the first embodiment, wherein the fibers are formed from polymers selected from the group consisting of poly(ester urea) (PEU), polycaprolactone (PCL), poly-L- lactide (PLLA), and polyf actic-co-glycolic acid) (PLGA), functionalized PEU, functionalized PCL, functionalized PLLA, and functionalized PLGA.
  • PEU poly(ester urea)
  • PCL polycaprolactone
  • PLLA poly-L- lactide
  • PLGA polyf actic-co-glycolic acid
  • the present invention provides a wound dressing as in either the first or second embodiment, wherein the PEG-containing component is tri- functional or greater containing ketone functionality, aldehyde functionality, or a combination thereof on the terminal end of the chains and having a molecular weight ranging from about 1,000 Da to about 50,000 Da.
  • the present invention provides a wound dressing as in any of the first through third embodiments, wherein the cross -linking component is selected from the group consisting of a 2 -arm cross-linking component, a 4-arm cross -linking component, or a mixture thereof.
  • the present invention provides a wound dressing as in any of the first through fourth embodiments, wherein the PEG hydrogel is formed using a buffer solution, wherein the buffer solution the buffer solution is selected from the group consisting of citric acid, disodium phosphate, and combinations thereof.
  • the present invention provides a wound dressing as in any of the first through fifth embodiments, wherein the pH of the buffer solution is from about 1.5 to about 7.5.
  • the present invention provides a wound dressing as in any of the first through sixth embodiments, wherein the pH of the buffer solution is from about 2.0 to about 7.6.
  • the present invention provides a wound dressing as in any of the first through seventh embodiments, wherein the concentration of the buffer solution is from about 10 mM to about 100 mM
  • the present invention provides a wound dressing as in any of the first through eighth embodiments, wherein the concentration of the buffer solution is from about 20 mM to about 50 mM.
  • the present invention provides a method of making a wound dressing comprising the steps of laying down decellularized tissue into a bath of a buffer solution and PEG-containing component, adding fibers to the top of the decellularized, and then adding a cross-linking component to the bath of buffer solution and the PEG-containing component to create a PEG hydrogel to at least partially surround the decellularized tissue and fibers.
  • the present invention provides a method of making a wound dressing as in the tenth embodiment, wherein the fibers are formed from polymers selected from the group consisting of poly(ester urea) (PEU), polycaprolactone (PCL), poly-L-lactide (PLLA), and poly(lactic-co-glycolic acid) (PLGA), functionalized PEU, functionalized PCL, functionalized PLLA, and functionalized PLGA.
  • PEU poly(ester urea)
  • PCL polycaprolactone
  • PLLA poly-L-lactide
  • PLGA poly(lactic-co-glycolic acid)
  • the present invention provides a method of making a wound dressing as in any of the tenth through eleventh embodiments, wherein the fibers are electrospun onto the decellularized tissue.
  • the present invention provides a method of making a wound dressing as in any of the tenth through twelfth embodiments, wherein the PEG-containing component is tri-functional or greater containing ketone functionality, aldehyde functionality, or a combination thereof on the terminal end of the chains and having a molecular weight ranging from about 1,000 Da to about 50,000 Da.
  • the present invention provides a method of making a wound dressing as in any of the tenth through thirteenth embodiments, wherein the cross-linking component is selected from the group consisting of a 2 -arm cross- linking component, a 4-arm cross-linking component, or a mixture thereof.
  • the present invention provides a method of making a wound dressing as in any of the tenth through fourteenth embodiments, wherein the buffer solution is selected from the group consisting of citric acid, disodium phosphate, and combinations thereof.
  • the present invention provides a method of making a wound dressing as in any of the tenth through fifteenth embodiments, wherein the pH of the buffer solution is from about 1.5 to about 7.5 and the concentration of the buffer solution is from about 10 mM to about 100 mM.
  • the present invention provides a method of making a wound dressing as in any of the tenth through sixteenth embodiments, wherein the pH of the buffer solution is from about 2 to about 7.6 and the concentration of the buffer solution is from about 20 mM to about 50 mM.
  • the present invention provides a method of making a hydrogel comprising the steps of mixing a polyethylene glycol (PEG) component with a cross-linking component in a buffer solution, the PEG component having multiple arms with terminal functionality selected from ketone functionality and aldehyde functionality, and the cross-linking component having multiple arms with terminal aminooxy functionality, the hydrogel forming in the buffer solution by oxime ligation reaction, wherein the pH of the buffer solution used to make the hydrogel has a pH range of from 1.5 to 7.6, and the concentration of the buffer solution is from about 10 mM to about 100 mM.
  • PEG polyethylene glycol
  • the present invention provides a method of making a hydrogel as in the eighteenth embodiment, wherein the buffer solution is a phosphate-citrate buffer solution.
  • the present invention provides a wound dressing comprising: decellularized tissue at least partially surrounded by a PEG hydrogel, wherein the PEG hydrogel is structured by covalent bonds formed via an oxime ligation reaction of a polyethylene glycol (PEG) containing component and a cross-linking component.
  • PEG polyethylene glycol
  • the present invention provides a wound dressing comprising: fibers at least partially surrounded by a PEG hydrogel, wherein the PEG hydrogel is structured by covalent bonds formed via an oxime ligation reaction of a polyethylene glycol (PEG) containing component and a cross-linking component, BRIEF DESCRIPTION OF THE DRAWINGS
  • PEG polyethylene glycol
  • FIG. 1A is a schematic perspective view of a generic embodiment of the present invention, showing regenerative material completely encased in hydrogel;
  • Fig. IB is a schematic perspective view of a generic embodiment of the present invention, showing regenerative material that is only partially encased or surrounded by the hydrogel such that the regenerative material is at least partially exposed;
  • FIG. 2 is a schematic side elevational view of a first embodiment of a wound dressing having only decellularized tissue as the regenerative material;
  • FIG. 3 is a schematic side elevational view of a second embodiment of a wound dressing having nanofibers as the regenerative material
  • FIG. 4 is a schematic side elevational view of a third embodiment of a wound dressing having decellularized material supported by nanofibers as the regenerative material;
  • Fig. 5 is an exemplary reaction scheme for the synthesizing of a particular PEG-containing component with ketone functionality
  • Fig. 6 is an exemplary reaction scheme for the synthesizing of a particular four-arm cross-linking component
  • Fig. 7 provides a general schematic for manufacturing a wound dressing such as that of Fig. 2;
  • Fig. 8 provides a general schematic for manufacturing a wound dressing such as that of Fig. 3;
  • Fig. 9 provides a general schematic for manufacturing a wound dressing such as that of Fig. 4;
  • Fig. 10 shows hydrogel storage modulus and loss modulus as a function of buffer strength
  • Fig. 11 shows hydrogel storage modulus and loss modulus as a function of pH
  • Fig. 12 shows hydrogel storage modulus and loss modulus as a function of the weight percentage of PEG-containing component
  • Fig. 13 is a graph showing a strain sweep performed to determine the linear viscoelastic regime
  • Fig. 14 is a graph showing a frequency sweep performed to determine the linear viscoelastic regime
  • Fig. 15 is a general schematic of a specific hydrogel formation in accordance with this invention, the hydrogel formation notably using a cross-linking reaction involving oxime formation, which produces water as a by-product and does not require a metal catalyst,
  • Fig. 16 is an exemplary reaction scheme for the synthesizing of a particular 4- arm PEG-containing component
  • Fig. 17 is an exemplary reaction scheme for the synthesizing of a particular 3- arm PEG-containing component
  • Fig. 18 is an exemplary reaction scheme for the synthesizing of a particular 2- arm PEG-containing component
  • Fig. 19 is an exemplary reaction scheme for the synthesizing of a particular four-arm cross-linking component
  • Fig. 20 is an exemplary reaction scheme for the synthesizing of a particular four-arm cross-linking component
  • Fig. 21 is an exemplary reaction scheme for the synthesizing of a particular three-arm cross-linking component; and [0055] Fig. 22 shows data for a hydrogel system of 4-arm aminooxy cross-linker made from 10k MW PEG and equimolar amounts of 10k 4-arm PEG-ketone.
  • the wound dressing 10 includes regenerative material 12 supported and at least partially surrounded by a polyethylene glycol (PEG) hydrogel 14.
  • PEG polyethylene glycol
  • the regenerative material 12 can be provided through different components.
  • FIG. 1A the description "at least partially surrounded” can be understood by comparison of Figs. 1A and IB, showing a wound dressing 10 and wound dressing 10'.
  • the regenerative material 12 is completed surrounded or encased in PEG hydrogel 14.
  • the regenerative material 12 is flush with the top surface of the PEG hydrogel 14 such that it is not fully surrounded or encased.
  • the regenerative material 12 is not immediately exposed, but will become so as the hydrogel biodegrades.
  • Fig. IB the regenerative material 12 is exposed at least partially.
  • Each embodiment might have preferred applications with different types of wounds.
  • a wound dressing 110 includes decellularized tissue 116 (also known as “decellularized extracellular matrix material”) as the regenerative material 112.
  • a wound dressing 210 includes fibers 218 as the regenerative material 212, wherein the fibers are functionalized to provide regenerative properties.
  • a wound dressing 310 the regenerative material 312 includes decellularized tissue 316 reinforced with fibers 318 as the regenerative material 112.
  • the regenerative properties can be provided by the decellularized tissue, alone, or both the decellularized tissue and the fibers.
  • the fibers will simply be biodegradable and will serve to provide mechanical support for the decellularized tissue for the time period prior to their biodegradation. It is know that decellularized tissue can degrade too quickly at a wound site, thus disappearing before full regeneration is achieved (i.e., before an acceptable level of healing of the wound), thus allowing for recurrence of the wound. This is noted particularly in applications such as hernia repair.
  • the fibers are provided to improve the performance of the wound dressing by degrading slower than the decellularized tissue and providing a structural integrity to the overall would dressing suitable to prohibit recurrence.
  • the PEG hydrogel 14 at least partially surrounds the regenerative material 112, 212, or 312.
  • at least partially surround it is meant that when the PEG hydrogel 14 is formed, the PEG hydrogel 14 either completely encapsulates the regenerative material 112, 212, or 313, or a portion of the regenerative material 112, 212, or 313 is exposed wherein the rest of the regenerative material 112, 212, or 313 is encapsulated by the PEG hydrogel 14.
  • the kinetics of cross-linking within the PEG hydrogel 14 By varying the kinetics of cross-linking within the PEG hydrogel 14, small changes in the structural features of the PEG hydrogel 14 can be achieved.
  • the kinetics of the network formation of the PEG hydrogel 14 are influenced by pH and buffer strength, and are utilized to intrinsically control the degree of heterogeneity within the microstructure of the PEG hydrogel 14.
  • the pH and buffer strength used in the formation of the PEG hydrogel 14 the properties of the PEG hydrogel can be controlled, but yet the chemical identity, concentration, and stoichiometry of the PEG hydrogel 14 is maintained.
  • the gelation is rapid but takes enough time to allow for manipulation and placement of the wound dressing (110, 210, or 310) at a desired wound site.
  • the PEG hydrogel 14 When properly implanted, the PEG hydrogel 14 will degrade into readily metabolized and safe byproducts in a matter of days, leaving the regenerative material 112, 212, or 312 exposed for optimal wound healing performance.
  • This PEG hydrogel 14 therefore acts mostly as a delivery vehicle and laminating agent for the regenerative material 112, 212, or 312.
  • the decellularized tissue (or decellularized extracellular matrix material) is selected to be non-toxic and biocompatible in its intended end application.
  • the decellularized extracellular matrix contains structural proteins such as: eiastin, laminin; functional proteins such as growth factors and cytokines, polysaccharides or mineral phases. This material is commercially available in different forms known to those of skill in the art to be useful for different applications. In some embodiments, any tissue that is void of cellular DMA or remnant components that illicit an immunogenic reaction would be acceptable in this application, and are to be understood as "decellularized tissue.”
  • the fibers can be formed from any fiber material that does not induce a prominent inflammatory reaction when used for the intended application at a wound site.
  • the fibers can be formed of polymers selected from the group consisting of poly(ester urea) (PEU), polycaprolactone (PCL), poly-L-lactide (PLLA), and poly(lactic-co-glycolic acid) (PLGA), functionalized PEU, functionalized PCL, functionalized PLLA, and functionalized PLGA, wherein the functionalization serves to induce regeneration of tissue at the wound site, i.e., the polymer is functionalized with functional groups that provide regenerative properties.
  • PEU poly(ester urea)
  • PCL polycaprolactone
  • PLLA poly-L-lactide
  • PLGA poly(lactic-co-glycolic acid)
  • functionalization serves to induce regeneration of tissue at the wound site, i.e., the polymer is functionalized with functional groups that provide regenerative properties.
  • the fibers are formed of polymers having functional groups that provide regenerative properties
  • the fibers can be formed of polymers selected from the group consisting of functionalized PEU, functionalized PCL, functionalized PLLA, and functionalized PLGA.
  • the polymers forming the fibers include functional groups
  • those functional groups can be selected from the group consisting of peptides, carbohydrates, proteins, oligonucleotides, small molecule drugs, an oxygen atom connected to a alkyl or aryl group containing an alkyne group, an alkene group, an azide group, a benzyl protected phenol group, a ketone group or a strained cyclooctyne.
  • the polymers include peptides that signal to cells to regenerate tissue.
  • the functional groups are peptides selected from the group consisting of RGD, BMP 2, BMP 7, BMP 9, and OGP.
  • the peptide is RGD, a component of fibronection that signals for cell attachment.
  • the fibers are first formed by electrospinning and then functionalized. In other embodiments, the fibers are first functionalized and then formed into fibers by electrospinning. In some embodiments, the fibers are functionalized post-formation as disclosed in Post-Electrospinning "Triclick” Functionalization of Degradable Polymer Nanofibers, by Becker M. L, et al., ACS Macro Lett., 2015, 4 (2), pp 207-213; and Postelectrospinning "Click” Modification of Degradable Amino Acid-Based Poly(ester urea) Nanofibers, by Becker M. L, et al, Macromolecules, 2013, 46 (24), pp 9515-9525, which are incorporated herein by reference.
  • the fibers are biodegradable.
  • the above mentioned polymers are biodegradable.
  • the fibers are biodegradable at a rate that matches the rate of tissue regeneration at the intended wound site.
  • the fibers are electrospun fibers.
  • the fibers are nanofibers.
  • the fibers are nanofibers that have been formed by electrospinning such that the polymer forming the nanofibers is chosen so as to be electrospinnable.
  • the polymer materials above are suitable for electrospinning.
  • a nanofiber is to be understood as a fiber having a diameter of less than 2 microns.
  • the nanofibers have a diameter of less than 1.5 microns, in other embodiments, less than 1.0 microns, in other embodiments, less than 750 nm, in other embodiments, less than 500 nm, in other embodiments, less than 400 nm, and, in other embodiments, less than 350 nm.
  • the fibers comprise hyperbranched amino acid- based poly(ester urea), such as those disclosed in U.S. Patent Application No. 14/939,216, which is herein incorporated by reference.
  • the fibers have the ability to have their surfaces derivatized with peptides and fluorescent probes using bio-orthogonal reaction strategies, such as those fibers disclosed in International Patent Application No. PCT/US14/58264, which is herein incorporated by reference.
  • the fibers comprise an amino acid-based poly(ester urea) with amino acid residues selected from L-leucine, L-isoleucine, L- valine or combinations thereof, such as those disclosed in International Patent Application No. PCT/US14/62888, which is herein incorporated by reference.
  • the fibers have a round, smooth, and consistent morphology. It has been found that electrospirmmg can provide such morphologies.
  • the fibers have a diameter greater than about 150 nm, in other embodiments the diameter is greater than about 400 nm, and in yet other embodiments the diameter is greater than about 700 nm. In some embodiments, the fibers have a diameter less than about 1500 nm, in other embodiments the diameter is less than about 1100 nm, and in yet other embodiments the diameter is less than about 900 nm. In some embodiments, the fibers have a diameter of from about 150 nm to about 1500 nm, in other embodiments the diameter is from about 400 nm to about 1100 nm, and in yet other embodiments the diameter is from about 700 nm to about 900 nm.
  • the fibers form a fabric structure having a pore size greater than about 1.0 ⁇ , in other embodiments the pore size is greater than about 20 ⁇ , and in yet other embodiments the pore size is greater than about 50 ⁇ . In some embodiments, the fibers form a fabric structure having a pore size less than about 125 urn, in other embodiments the pore size is less than about 100 ⁇ , and in yet other embodiments the pore size is less than about 75 ⁇ .
  • the fibers have form a fabric structure having a pore size of from about 1.0 ⁇ to about 125 ⁇ , in other embodiments the a pore size is from about 20 ⁇ to about 100 ⁇ , and in yet other embodiments the a pore size is from about 50 ⁇ to about 75 ⁇ .
  • the fibers have a rate of degradation of from 3 months to 2 years depending on application and need.
  • the fibers have a ceil viability of greater than 80%, in other embodiments greater than 90%, and in yet other embodiments greater than 95%. In some embodiments, the fibers have a cell viability of between about 80% and about 100%, in other embodiments the fibers have a cell viability of between about 90% and about 100%, and in yet other embodiments the fibers have a cell viability of between about 95% and about 100%.
  • the decellularized tissue (Fig. 2), the fibers (Fig. 3), or the decellularized tissue reinforced with fibers (Fig. 4) is at least partially surrounded and supported by a polyethylene glycol (PEG) hydrogel specifically taught herein.
  • PEG polyethylene glycol
  • the PEG hydrogel 14 component is formed via an oxime ligation reaction of a polyethylene glycol (PEG) containing component and a cross-linking component in a buffer solution.
  • the oxime ligation reaction produces water as a by-product, and is herein demonstrated to provide hydrogels with variable mechanical properties depending of the pH of the buffer.
  • the PEG component and the cross-linking component are chosen so as to form crosslinked networks wherein distal functional ends of the PEG component and cross-linking component react through the oxime ligation reaction to form the hydrogel system.
  • An example is shown in Fig. 15.
  • a significant portion of the components must be tri-functional or tetra-functional or even penta-functional, etc. This can also be considered in relation to the chain arms or "arms" of a given component. Tri-functional components will be seen to have three arms terminating in the appropriate functional group as taught below. Similarly, tetra-functional components will be seen to have four arms terminated appropriately.
  • a suitable number of the component must have tri-functionality and higher to create the covalently bonded network necessary for the formation of a suitably stable hydrogel. The ratios of each type of functional component necessary to form a suitable hydrogel will be appreciated to change with respect to the molecular weight of the components and the length of the arms serving to form the covalent network.
  • the PEG-containing component is selected to be tri- functional or greater, i.e., having three arms or four arms providing reactive terminal groups.
  • the cross-linker component has at least three functional sites for cross-linking, i.e., at least three arms providing reactive terminal groups, and the PEG component is di-functional, i.e., having only two arms.
  • the PEG-containing component is tetra-functional
  • the cross-linking component is tetra-functional, and they are mixed in equimolar ratios (Fig. 15). It is found that a good homogenous network is formed from this combination, and slight alterations of this system could be used to tailor the properties around a useful range such as those found in particular studies experimentally presented herein below.
  • the PEG-containing component is tri-functional (3 arms) or tetra-functional (4-arms; e.g. Fig. 15).
  • the PEG- containing component has a molecular weight (MW) greater than about 1,000 Dalton (Da), in other embodiments greater than about 2,000 Da, and in yet other embodiments greater than about 6,000 Da.
  • the PEG- containing component has a MW less than about 50,000 Da, in other embodiments less than about 25,000 Da, and in yet other embodiments less than about 10,000 Da.
  • the PEG-containing component has with a MW ranging from about 1,000 Da to about 50,000 Da (1 kDa to 50 kDa), in other embodiments from about 2 kDa to 25 kDa, and in yet other embodiments, from about 6 kDa to about 10 kDa.
  • the PEG-containing component includes ketone functionality or aldehyde functionality or a mixture thereof to participate in the oxime reaction disclosed herein for forming the hydrogel.
  • the ketone functionality is afforded via coupling with levulinic acid.
  • the PEG-containing component is selected from the group consisting of 10 kDa 4-arm PEG containing ketone functionality on the terminal end of the arms,
  • the PEG containing component is a 4- arm PEG containing ketone functionality at the terminal end of each arm, and is synthesized via DIC-coupling of levulinic acid as in Fig. 5.
  • Fig. 16 shows a 4-arm PEG- containing component
  • Fig. 17 which shows a 3-arm PEG-containing component
  • Fig. 18 which shows a 2-arm PEG-containing component.
  • the cross-linking component is selected to be tri- functional or greater, i.e., having three arms or four arms providing reactive terminal groups.
  • additional two-arm (di-functional) cross-linker is included in the creation of the hydrogel.
  • the cross-linking component contains either 4 arms or 2 arms.
  • the cross-linking components are functionalized with aminooxy groups to enable oxime crosslinking with the ketone or aldehyde functionality of the PEG-containing component.
  • the two arm cross-linking component contains an allyl group for peptide or other conjugations. This is particularly suitable for functionalization by a thiol-ene "click" reaction post gel formation.
  • Tuning the amount of 2-arm cross-linker that is incorporated also provides another handle for tuning the modulus of the gel.
  • the post-gelation functionalization through the allyl group is the primary desire for the 2-arm cross-linker. It is possible to "click" peptides to the surface of the gel that would provide favorable signals for cell migration and proliferation.
  • the 2 -arm cross-linker is made by coupling 3- allyloxy- 1,2 -propanediol and boc-protected aminooxy acetic acid followed by deprotection to afford the aminooxy functional group.
  • the allyl group (alkene) can be functionalized through a thiol-ene "click" reaction post gelation.
  • the cross-linking component is a 4 arm PEG molecule with MW from 2,000 - 10,000 containing aminooxy functional groups. In some embodiments, the cross-linking component is a 4-arm aminooxy cross-linking component synthesized from pentaerythritol and aminooxy acetic acid.
  • the cross-linking component is a 4-arm cross-linking component synthesized from tetra-PEG of various MW (2,000-10,000 Da) and aminooxy acetic acid.
  • 4-arm cross-linking components see Figs. 6, 19, and 20, and for an example of a 3-arm cross-linking component, see Fig. 21.
  • cross-linker The purpose of making the cross-linker larger is to make the hydrogel softer.
  • a larger cross-linker (lOkDa) gels with as low of modulus as ⁇ 5kPa and as high as ⁇ 30kPa have been achieved in practicing this invention. This large of a range could not be achieved with small cross-linker and a 10 kDa ketone-PEG.
  • the cross-linker is made identical in size to the main PEG-containing component to provide a structurally homogeneous network
  • the buffer solution when the PEG-containing component contains aldehyde functionality, has a pH range of from 1.5 to 7.5, in other embodiments the pH has a range of from 2.0 to 7.6, in other embodiments the pH has a range of from 5.0 to 7.5, and in yet other embodiments the pH has a range of from 5.0 to 7.6.
  • the buffer solution is water or a phosphate-citrate buffer solution. In some embodiments, the buffer solution is water or a phosphate- citrate buffer solution. The use of this buffer solution allows for more acidic pH levels to be attained than phosphate buffer alone.
  • the buffer solution has a pH range of 2.6-7.6.
  • the buffer solution is prepared by mixing various amounts (depending on desired pH and buffer strength) of citric acid monohydrate (CeHsOj ⁇ H 2 0) and sodium phosphate dibasic (Na 2 HP0 4 ) in water.
  • the concentration of the buffer solution is selected from about 10 mM to about 100 mM. In other embodiments the concentration of the buffer solution used was selected from about 20 mM to about 50 mM.
  • the hydrogel is formed upon introducing both the PEG containing component and a 2 -arm cross-linking component or a 4-arm cross-linking component or a mixture of 2 -arm and 4-arm cross-linking components to the buffer solution.
  • the PEG-containing component and the cross- linking component are mixed at equimolar ratios.
  • the final ratio of PEG-containing component: cross-linking component was still equimolar, but the ration of 4-arm crosslinking component: 2-arm cross-linking component is from 95:5, 90: 10, and/or 85: 15.
  • Buffer concentration plays a crucial role in this oxime ligation hydrogel system.
  • a notable rise in the storage modulus is observed when the buffer concentration was elevated from 10 mM to 20 mM, and a large drop appeared when the concentration was raised to 50 mM and greater.
  • the overall trend suggests that an increase in buffer strength may lead to an increase in the heterogeneity of the network, which will produce a mechanically weaker scaffold.
  • the ionization degree in the polymer gels plays a crucial role in determining the state of spatial inhomogeneity. The inversion phenomenon of a charged hydrogel network is expected when the degree of ionization exceeds a certain level.
  • Inversion here stands for the anomalous cross-linking dependence that takes place due to the competition between two effects of the cross-linking: one is the random distribution of cross-links in the network, and the other one is the suppressed tendency of a phase-segregated structure.
  • An increase of inversion may directly induce a weakness in strength of the formed hydrogels.
  • oxime ligation is a pH sensitive reaction
  • a change in pH values of the buffer should also influence the final strength of gels.
  • a drop in storage moduli is observed with a decrease in the pH.
  • a lower pH value accelerates the ligation rate. If the movement of the PEG molecules is limited within a confined space, the cross-linking between the ketone and the aminooxy groups may be inhibited or allow for more dangling ends and loops (network defects).
  • the wound dressing 110 is formed by first introducing decellularized tissue 116 into a bath 114 of the buffer solution used for the manufacture of the PEG hydrogel 14. Then, the PEG containing component and the cross-linking component used to create the PEG hydrogel 14 is added to the bath 114 to create the PEG hydrogel 14. In some embodiments, the cross-linking component is added after the PEG containing component. Depending upon where the decellularized tissue is held in the buffer solution, the formed PEG hydrogel 14 will either partially or fully surround the decellularized tissue 116 (e.g., Figs. 1A, IB). Network formation of the PEG hydrogel 14 occurs quickly; depending of the pH of the buffer solution, the gelation time may occur between 5-30 minutes.
  • the wound dressing 210 is formed by electrospinning nanofibers 218 into a bath 114 of the buffer solution used for the manufacture of the PEG hydrogel 14.
  • the electrospinning solution used to create the nanofibers 218 is loaded into a reservoir 220 that is mounted with a needle. High voltage is applied to the reservoir 220 so that the electrospinning solution experiences instability, and spins into the bath 114 of the buffer solution.
  • the PEG- containing component and the cross-linking component used to create the PEG hydrogel 14 are added to the bath 114 to create the PEG hydrogel 14, and the formed PEG hydrogel 14 at least partially surrounds the electrospun nanofibers 218.
  • the cross-linking component is added after the PEG containing component.
  • Network formation of the PEG hydrogel 14 occurs quickly; depending of the pH of the buffer solution, the gelation time may occur between 5-30 minutes.
  • the wound dressing 310 is formed by first introducing decellularized tissue 316 into a bath 114 of the buffer solution and the used for the manufacture of the PEG hydrogel 14. Then nanofibers 318 are electrospun into the bath 114 of buffer solution and onto the decellularized tissue 316.
  • the nanofibers tend to lie on the decellularized tissue and while there is some static interaction, the two components tend to delaminate from each other, particularly when submerged in fluid. This is the reason for including the hydrogel component.
  • the electrospinning solution used to create the nanofibers 318 is loaded into a reservoir 320 that is mounted with a needle. High voltage is applied to the reservoir 320 so that the electrospinning solution experiences instability, and spins onto the decellularized tissue 316, which is laying in the bath 114 of the buffer solution. Then, the PEG containing component and the cross-linking component used to create the PEG hydrogel 14 are added to the bath 114 to create the PEG hydrogel 14, and the formed PEG hydrogel 14 at least partially surrounds the regenerative material 312 which includes the decellularized tissue 316 reinforced with nanofibers 318. In some embodiments, the cross-linking component is added after the PEG containing component Network formation of the PEG hydrogel 14 occurs quickly; depending of the pH of the buffer solution, the gelation time may occur between 5-30 minutes.
  • the strength of the buffer solution selected changes the cross-linking kinetics of the reaction between the PEG-containing component and the cross-linking component. It is also theorized that the strength of the buffer solution affects the modulus of the hydrogel formed. The overall trend suggests that an increase in buffer strength may lead to an increase in the heterogeneity of the network, which will produce a mechanically weaker scaffold.
  • the inhomogeneity that results from cross-linking kinetics is a direct result of the pH of the buffer solution.
  • the oxime cross-linking reaction between the PEG-containing component and the cross-linking component is catalyzed in mildly acidic conditions, thus, the cross -linking is faster at lower pH and results in more defects/inhomogeneity. Therefore, gels formed under lower pH conditions have lower modulus than those formed at higher pH. The optimum pH is dependent on the target tissue.
  • hydrogels ranging from 80-90% water content with a storage modulus of 1.0-30 kPa using conditions that will not harm cells.
  • the present invention provided hydrogels with storage modulus of from 5.0 to 30.0 kPa.
  • the hydrogel is produced by simply mixing equimolar ratios of the two precursor components into the buffer of choice.
  • the precursors consist of a PEG containing component and 2 -arm and 4-arm cross-linking components as taught herein.
  • the entire composite material can be easily implanted to the site of injury during laparoscopic surgery (in the case of hernia repair) similar to current techniques for hernia mesh placement.
  • the hydrogel will degrade in a matter of days, leaving the electrospun nanofiber encapsulated tissue exposed for optimal wound healing performance. This hydrogel therefore acts mostly as a delivery vehicle and laminating agent for the nanofiber/ECM complex.
  • polymer nanofibers containing bioactive peptides will stimulate wound healing as they slowly degrade over time, allowing for tissue integration into the porous network.
  • the polymer nanofibers degrade into amino acids and diols, where the dials neutralize the acidic components resulting in less inflammation, and the byproducts are easily metabolized and eliminated from the body.
  • the full construct will degrade at a slower rate than conventionally used decellularized tissue, while maintaining the highly bioactive regenerative response seen when using decellularized tissue to heal wounds.
  • Hydrogels are materials that are primarily composed of hydrophilic cross- linked networks, containing as much as 99% water. Synthetic polymers are arguably more favorable in hydrogel systems due to their reproducibility, high purity, and controlled molecular weight. Polyethylene glycol (PEG)-based hydrogels, in particular, have become one of the most extensively studied systems because of their versatility in end group modification, hydrophilic chemical structure, and protein absorption resistance. In several studies, such as those done by Nanostructured PEG- Based Hydrogels with Tunable Physical Properties for Gene Delivery to Human Mesenchymal Stem Cells, Biomaterials, 2012, 33 (27), 6533-6541 by Li et al.
  • PEG polyethylene glycol
  • PEG-based hydrogels with terminal acrylate functionality have been examined widely as tissue engineered scaffolds. Upon irradiation with ultraviolet light, they can form a three dimensional hydrogel network with tunable mechanical properties dependent on the cross-link density, and the mass fraction of the polymer precursors.
  • free radical polymerizations have limited control, which leads to multiple cross-linking reactions across the solution.
  • the heterogeneous cross-linking leads to the formation of micro-clusters that are eventually tied together by the polymer chains.
  • Such networks are not favorable for incorporating biological cues due to their inhomogeneity and complex micro-cluster structural formation.
  • orthogonal chemistries Click" chemistries
  • “Click” chemistries are generally defined as highly efficient, robust and orthogonal reactions. With regards to hydrogel formation, some of the extensively investigated "click” reactions include the Michael addition, thiol-ene addition, tetrazine-norbornene addition, copper(I) catalyzed alkyne-azide cycloaddition, and strain-promoted alkyne-azide cycloaddition. In many instances, the hydrogels produced from "click” reactions show superior properties in comparison to in situ polymerized gels. As a result of their orthogonal nature, multiple "click” reactions can be carried out in one system, which allows for post-functionalization of the hydrogel. The PEG based hydrogels that will be discussed herein involves oxime "click” chemistry.
  • Oxime chemistry employs a kinetically dependent reaction that varies with buffer pH and ionic strength.
  • oxime reactions for hydrogel fabrication.
  • One such study Biocompatible Hydrogels by Oxime Click Chemistry, Biomacromolecules. 2012, 13 (10), 3013-3017, by Maynard, et al, which is herein incorporated by reference, developed an 8-aml aminooxy PEG cross-linked with glutaraldehyde to form hydrogels for supporting cell adhesion. They were able to tune the mechanical properties of their hydrogel based on the mass fraction of PEG, effectively altering the cross-linking ratio.
  • Another study Oxime Cross-Linked Injectable Hydrogels for Catheter Delivery, Advanced Materials.
  • Oxime chemistry has proven to be a truly robust method for hydrogel formation, and it allows for very mild conditions: no metal catalyst is required, 20-40 °C with pH ranges from 2-7 is suitable, and the by-product of the reaction is water.
  • the Oxime, PEG-based hydrogel system possesses widely tunable mechanical properties, without altering the chemistry of the network.
  • Previous research has incorporated multiple chemistries into their hydrogel systems, or utilized chemically different substrates altogether, to study the cell's response with varying mechanical properties.
  • very few hydrogel systems are capable of achieving a wide range of elasticities without altering the chemical makeup of the scaffold.
  • the advantage of such a system is to show, definitively, the effect of substrate elasticity on cell response. Therefore, a hydrogel system is needed that would allow for the modulus to be dialed in, using alternative means to control the mechanical properties, such as the pH and strength of the buffer.
  • pH and buffer strength influence the elasticity of an oxime hydrogel.
  • the 4-arm, 10K polyethylene glycol was purchased from Creative PEGWorks. All other commercial reagents and solvents were purchased from Sigma- Aldrich or Fisher Scientific and used as received unless noted otherwise. All synthesis reactions were performed under nitrogen unless noted otherwise.
  • FT-IR spectra were recorded by a DIGILAB EXCALIBUR Series FTS3000, with a scanned wavenumber range from 400 to 4000 cm-1. Samples were prepared by grinding KBr powder with dried sample powder into pellets and spectra were recorded after 64 scans. Baseline was deducted and normalized to the same reference peak intensity.
  • Mass spectrometry for the 4-arm keto-PEG was performed using a Bruker UltraFlex III MALDI tandem time-of-flight (TOF/TOF) mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a Nd:YAG laser emitting at 355 nm.
  • the matrix and cationization salt were DCTB (2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2- enylidenejmalonitrile) and sodium trifluoroacetate, respectively.
  • pH values of buffers were tested using an Orion® 350 PerpHecT® benchtop pH meter with an Orion® ROSS® Sure-Flow pH electrode at room temperature.
  • the PEG hydrogels were cast to be 2 mm thick in 25 mm diameter, and soaked in buffer overnight to reach swelling equilibrium.
  • the equilibrium moduli were determined using an ARES G2 (TA Instruments, New Castle, DE) with 25 mm serrated parallel plate geometry and a 6 N axial force. Hydrogels were immersed in buffer during testing to keep the gel from drying up.
  • the reaction was allowed to gradually warm to room temperature with stirring for 24 hrs.
  • the reaction mixture was filtered to remove the urea by-product, and rotary evaporated to remove solvent.
  • the crude product was re-dissolved in ethyl acetate, cooled in liquid nitrogen and centrifuged to further remove urea by-product.
  • the solution was concentrated and purified using silica column chromatography with a mobile phase of 5:3 (ethyl acetate : hexane). After rotary evaporation, a white solid intermediate was obtained.
  • the reaction flask was cooled to 0 °C in an ice bath for 15 min, followed by the injection of 2.03 mL (13.08 mmol, 3 eq) of DIC.
  • the reaction was allowed to gradually warm to room temperature with stirring for 24 hrs.
  • the reaction mixture was filtered to remove the urea by-product, and rotary evaporated to remove solvent.
  • the crude product was re-dissolved in ethyl acetate, cooled in liquid nitrogen and centrifuged to further remove urea by-product.
  • the solution was concentrated and purified using silica column chromatography with a mobile phase of 1:2 (ethyl acetate: hexane). After rotary evaporation, the white solid intermediate was obtained.
  • the reaction mixture was filtered to remove solid DIC-urea, and washed with CH2CI2.
  • the filtrate was concentrated via rotary evaporation and re-dissolved in a minimal amount of CH2CI2 for precipitation into cold methanol, followed by centrifugation.
  • the precipitate was then re-dissolved in a minimal amount of solvent again, and precipitated into cold diethyl ether, followed by centrifugation at 5000 RPM for 2 minutes.
  • the precipitate was then vacuum dried, and a white solid was obtained (7.10 g, 85.0% yield).
  • hydrogels were fabricated using a precursor mixing method.
  • Solutions of the 4-arm keto-PEG were prepared by dissolving a pre-weighed mass in 800 uL of the desired buffer.
  • Solutions of 4-arm cross-linker and 2 -arm cross-linker vinyl extender were prepared by dissolving the cross-linker precursors in 200 uL of the desired buffer.
  • the 4-arm keto-PEG solution was mixed with the 4-arm aminooxy cross-linker solution under stoichiometric balance (1: 1 aminooxy: ketone) with sufficient initial shaking to ensure thorough mixing of the precursors.
  • keto-PEG solution was mixed with 2-ann aminooxy alkene extender with sufficient shaking, and allowed to react for 30 minutes. Then, the remainder of the 4-ann aminooxy cross-linker was then added with additional shaking.
  • the hydrogel precursor mixtures were cast in silicone molds for at least 8 hours to ensure complete gelation. The hydrogels were then taken out of molds, soaked in the target buffer overnight to reach swelling equilibrium. The mass fraction of total precursors was calculated using the following equation:
  • Fig. 10 shows the differences in the hydrogel storage modulus with different buffer concentrations (10, 20, 50, 100 mM). Interestingly, a notable rise in the storage modulus is observed when the buffer concentration was elevated from 10 mM to 20 mM, and a large drop appeared when the concentration was raised to 50 mM and greater.
  • the buffer used in this experiment was citric acid/disodium phosphate with different concentrations. The overall trend suggests that an increase in buffer strength may lead to an increase in the heterogeneity of the network, which will produce a mechanically weaker scaffold. Alternatively, the existence of phosphate in the buffer may lead to a three component Kabachnik-Fields reaction.
  • oxime ligation is a pH sensitive reaction
  • a change in pH values of the buffer should also influence the final strength of gels.
  • Fig. 11 shows the differences between gel moduli with the corresponding buffer pH value of the buffer used; a drop in storage moduli is observed with a decrease in the pH.
  • a lower pH value would accelerate the ligation rate.
  • the cross-linking between the ketone and the aminooxy groups may be inhibited or allow for more dangling ends and loops (network defects).
  • Fig. 22 shows similar data for a hydrogel system of 4-arm aminooxy cross-linker made from 10k MW PEG and equimolar amounts of 10k 4-arm PEG- ketone.
  • the ratio of 4-arm: 2-arm cross-linking component shows a clear trend in terms of the resulting storage moduli. Testing showed that the storage modulus decreased when the ratio of 2-arm cross-linking component was increased. The highest storage modulus scaffold appears to be the system containing only 4-arm cross-linking component. This suggests that the incorporation of 2-arm cross-linking component effectively decreases the cross-linking density, and possibly introduces more sites for ineffective/incomplete cross-linking between the 4-arm aminooxy cross-linking component and the keto-PEG.
  • V2 is the swollen polymer volume fraction, is the specific volume of PEG (0.893 cm 1 g)
  • Vi is the molar volume of water (18 cm3 mol-1)
  • J M is the number-average molecular weight (10,330 g mol 1 )
  • ⁇ is the polymer-solvent interaction parameter (0.426 for PEG in water)
  • rj is the root-mean-square end to end distance
  • / is the bond length (1.46 A)
  • C n is the characteristic ratio for PEG (4.0).
  • Time sweeps were performed at 1% strain and 1 rad s-1 to determine the gelation time.
  • the precursor solutions were mixed and shaken for 5 seconds before injecting 400 uL of the hydrogel solution into the rheometer equipped with 25 mm parallel plates and a set gap height of 0.80 mm, and the response was measured
  • This experiment provides necessary guidance for the creation of covalently cross-linked systems that employ a kinetically controlled oxime ligation, and demonstrate variable mechanical properties by altering the pH and buffer strength.
  • SANS and rheology it has been demonstrated that the kinetics of the ligation reaction influence the development of the microstructure, which ultimately leads to changes in the rigidity of the gel.
  • This system holds promise to definitively demonstrate the effect of scaffold modulus on stem cell differentiation, because it does not rely on changing the precursor chemistry to change the scaffold elasticity.

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Abstract

La présente invention concerne un pansement pour plaies comprenant un tissu décellularisé et des fibres au moins partiellement entourées par un hydrogel de PEG, l'hydrogel de PEG étant structuré par des liaisons covalentes formées par l'intermédiaire d'une réaction de ligature d'oxime d'un composant contenant du polyéthylène glycol (PEG) et d'un composant de réticulation. Un procédé de fabrication d'un hydrogel comprend le mélange d'un composant de polyéthylène glycol (PEG) avec un composant de réticulation dans une solution tampon, le composant de PEG possédant des bras multiples avec une fonctionnalité terminale choisie parmi une fonctionnalité cétone et une fonctionnalité aldéhyde, et le composant de réticulation possédant des bras multiples avec une fonctionnalité amino-oxy terminale, l'hydrogel se formant dans la solution tampon par réaction de ligature d'oxime, le pH de la solution tampon utilisée pour fabriquer l'hydrogel ayant un pH compris entre 1,5 et 7,6, et la concentration de la solution tampon étant comprise entre environ 10 mM et environ 100 mM.
PCT/US2016/018043 2015-02-16 2016-02-16 Matériau hybride tissu décellularisé/nanofibres/hydrogel permettant d'optimiser la régénération tissulaire WO2016133887A1 (fr)

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CN109675107A (zh) * 2019-01-14 2019-04-26 华中科技大学同济医学院附属协和医院 一种应用peg水凝胶的复合瓣膜支架及其制备方法和其应用
WO2020060908A1 (fr) * 2018-09-17 2020-03-26 The Trustees Of Columbia University In The City Of New York Cétals et polycétals en tant qu'agents de libération

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WO2014039245A1 (fr) * 2012-09-07 2014-03-13 The Regents Of The University Of California Procédé d'élaboration d'hydrogels grâce à la formation de liaisons oximes
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US20060127873A1 (en) * 2002-07-16 2006-06-15 Caroline Hoemann Composition for cytocompatible, injectable, self-gelling chitosan solutions for encapsulating and delivering live cells or biologically active factors
US20100144902A1 (en) * 2007-07-06 2010-06-10 Bioregen Biomedical (Changzhou) Co., Ltd. Biocompatible rapid-gelating hydrogel and associated preparation method of spray
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020060908A1 (fr) * 2018-09-17 2020-03-26 The Trustees Of Columbia University In The City Of New York Cétals et polycétals en tant qu'agents de libération
CN109675107A (zh) * 2019-01-14 2019-04-26 华中科技大学同济医学院附属协和医院 一种应用peg水凝胶的复合瓣膜支架及其制备方法和其应用

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