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WO2025223272A1 - Membranes mimétiques de tissu pour guider la régénération d'interface fibrocartilagineuse dans la réparation de coiffe de rotateur - Google Patents

Membranes mimétiques de tissu pour guider la régénération d'interface fibrocartilagineuse dans la réparation de coiffe de rotateur

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Publication number
WO2025223272A1
WO2025223272A1 PCT/CN2025/089215 CN2025089215W WO2025223272A1 WO 2025223272 A1 WO2025223272 A1 WO 2025223272A1 CN 2025089215 W CN2025089215 W CN 2025089215W WO 2025223272 A1 WO2025223272 A1 WO 2025223272A1
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WO
WIPO (PCT)
Prior art keywords
nanogel
membrane
polymer
scaffold
multilayer membrane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/089215
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English (en)
Inventor
To Ngai
Yuwei ZHU
Jiankun Xu
Ling Qin
Bingyang DAI
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Chinese University of Hong Kong CUHK
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Chinese University of Hong Kong CUHK
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Publication date
Application filed by Chinese University of Hong Kong CUHK filed Critical Chinese University of Hong Kong CUHK
Publication of WO2025223272A1 publication Critical patent/WO2025223272A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products

Definitions

  • Rotator cuff tear is one of the most common musculoskeletal disorders, occurring at a high prevalence ( ⁇ 34%) and placing a large burden on both healthcare and socioeconomic systems. Rotator cuff tears often occur at the tendon–bone insertion (i.e., enthesis) site, resulting in substantial pain and impairment of daily activities. Surgical repair is usually required to restore shoulder function in clinics, such that more than 1.1 million/year rotator cuff tendon surgeries are performed globally. However, despite remarkable improvements in surgical techniques over the past decades, the rate of re-tears after surgical intervention continues to range between 20%and 94%.
  • bio-scaffold-mediated regenerative strategies in particular receiving immense attention.
  • Surgical procedures for the treatment of large rotator cuff tears generally use bio-scaffolds having the form of a film interposed between tendon and bone (i.e., the enthesis site) , or a patch that bridges a massive defect to offer a structural support and a biomimicry microenvironment.
  • the enthesis is a transitional region that exhibits gradients in structural, bio-functional, and mechanical properties to allow the appropriate dissipation of force from soft tissue (tendon) to hard tissue (bone) .
  • biomimetic scaffolds involve a wide variety of biomaterials, including decellularized extracellular matrix (ECM) , and natural-/synthetic-based materials, some of which are supplemented with growth factors, stem cells, or other bioactive compounds.
  • ECM extracellular matrix
  • bio-scaffolds used for the augmentation of rotator cuff reconstruction are commercially available, including those with a biological base (allogenic/xenogenic acellular ECM) , those with a synthetic base (degradable/nondegradable) , and those with a combination of the two.
  • a biological base allogenic/xenogenic acellular ECM
  • synthetic base degradable/nondegradable
  • collagen-rich xenogenic orthobiologic augmentation patch (DePuy Orthopaedics, USA) is derived from porcine small intestine submucosa and has been used for tendon reinforcement. Unfortunately, no significant difference could be found in patients with the augmentation and those without.
  • regenerative tissue matrix (LifeCell, Wright Medical Technology, USA) is a kind of allogenic ECM scaffold made from human dermis and shown to provide effective augmentation of rotator cuff repair, with healing outcomes comparable to those of autogenic tendon, a lower surgical failure rate (19%) , an elevated load-to-failure, and improved tendon-to-bone healing.
  • the existence of residual DNA from the allogenic source means that inflammatory responses cannot be absolutely eliminated in allogenic materials, and their elasticity remains notably weaker than that of autologous tendon, which can induce adverse effects in the healing process.
  • biomaterials ranging from natural (e.g., collagen, chitosan) to nondegradable synthetic (e.g., polyurethane, polyethylene terephthalate) to degradable synthetic (e.g., poly glycolic acid [PGA] , poly lactic acid [PLA] , polycaprolactone [PCL] ) polymeric materials, have been developed to augment tendon-to-bone repair.
  • natural e.g., collagen, chitosan
  • nondegradable synthetic e.g., polyurethane, polyethylene terephthalate
  • degradable synthetic e.g., poly glycolic acid [PGA] , poly lactic acid [PLA] , polycaprolactone [PCL]
  • dual-phase/multi-phase scaffolds show unparalleled superiority to single-phase scaffolds for use in interface regeneration.
  • Each of the separate phases can be designed with different compositions and topographies to recapitulate the region-specific properties of the natural enthesis.
  • Numerous nanomaterials and nanofabrication technologies have been investigated to achieve a highly precisive simulation of the interface tissues and further facilitate tendon–bone healing.
  • bioceramics like nano-sized hydroxyapatite (nHAp) , can be incorporated into a hierarchical scaffold to mimic the mineralized gradient from tendon to bone.
  • the micro-/nano-structures of a scaffold can be tailored to reproduce the architecture of native tissue, as well as provide topographical cues to guide cellular behaviors and spur tissue formation.
  • bio-scaffolds reported with elaborate structures and advanced biomimetic properties, their application in vivo remains limited.
  • few studies have simultaneously addressed the problem of stem cells differentiating into cartilage and bone lineages at the transitional defected area, which is a critical issue for treating chronic rotator cuff tears.
  • compositions and methods related to multiphasic tissue mimetic membranes having several beneficial advantages for use in simultaneously promoting soft and hard tissue healing in tendon–bone defects.
  • the present disclosure generally relates to a formulation and fabrication strategy for constructing a tissue mimetic membrane for guiding fibrocartilaginous interface regeneration (e.g., regeneration in rotator cuff repair) and facilitating the restoration of normal interface function (e.g., normal shoulder function) .
  • fibrocartilaginous interface regeneration e.g., regeneration in rotator cuff repair
  • normal interface function e.g., normal shoulder function
  • the disclosure provides a nanogel conjugated with a chondro-promotive agent.
  • the nanogel includes a thermoresponsive polymer.
  • the nanogel is also coated with a cationic polymer.
  • the disclosure provides a method of producing a multilayer membrane.
  • the method includes forming a dispersion of a nanogel in a polymer solution.
  • the polymer solution includes a pre-scaffold polymer dissolved in a mixture of a solvent and a non-solvent.
  • the method further includes casting the dispersion to produce a wet layer having a target thickness.
  • the method further includes evaporating the mixture of the solvent and the non-solvent to yield a microporous layer.
  • the yielded microporous layer has a target concentration of the nanogel.
  • the method further includes fabricating a fibrous layer on the microporous layer, thereby producing the multilayer membrane.
  • the fibrous layer includes core-shell fibers generated on the microporous layer by co-axial electrospinning of a core solution and a shell solution.
  • the disclosure provides a method of repairing a defect at a tendon–bone interface of a subject.
  • the method includes implanting a multilayer membrane as disclosed herein in the subject proximate to the defect.
  • FIG. 1 presents a schematic illustration of the biphasic structure of the tissue mimetic membrane and its application in a rotator cuff tear model for augmenting fibrocartilaginous interface regeneration.
  • FIG. 2 presents TEM images of synthesized nGel-KGN particles.
  • FIG. 3 presents a graph plotting the size distribution of synthesized nGel-KGN particles at dry state.
  • FIG. 4 presents graphs plotting the hydrodynamic size distribution of synthesized nGel-KGN particles in 2.5 mM HEPES buffer solution at 25 °C and 37 °C.
  • Insets video frames of the nGel-KGN particles in 2.5 mM HEPES buffer solution at 25 °C and 37 °C obtained from nanoparticle tracking analysis (NTA) measurements.
  • NTA nanoparticle tracking analysis
  • FIG. 5 presents graphs plotting the UV-vis spectra of KGN, nGel, and nGel-KGN dissolved in dimethyl sulfoxide (DMSO) .
  • FIG. 6 presents a graph plotting the zeta potential of synthesized nGel-KGN particles in 2.5 mM HEPES buffer solution.
  • FIG. 7 presents a series of images showing surface morphologies of fabricated microporous and fibrous layers for five groups of membranes: 500 ⁇ m casting, 500 ⁇ m casting/5%nGel-KGN, 1000 ⁇ m casting/5%nGel-KGN, ES, and ES/4%St.
  • FIG. 8 presents a high-angle annular dark-field image of St. nanowires.
  • FIG. 9 presents a series of EDS maps of the compositional elements (P, Mg, O, and N) corresponding to the image of FIG. 8.
  • FIG. 10 presents a high-angle annular dark-field image of a single PCL/St. -GelA core-shell fiber.
  • FIG. 11 presents a series of EDS maps of the compositional elements (P, Mg, O, and N) corresponding to the image of FIG. 10.
  • FIG. 12 presents a series of images showing cross-sectional morphologies of three groups of fabricated tissue mimetic membranes.
  • FIG. 14 presents a graph plotting representative strain-stress curves with data obtained from tensile tests of three fabricated membranes at wet state. Insets: representative photographs of the Grp 2 membrane during tensile test.
  • the data are presented as the mean ⁇ SD. *P ⁇ 0.05.
  • One-way ANOVA with Tukey’s post hoc test was used.
  • the data are presented as the mean ⁇ SD. *P ⁇ 0.05.
  • One-way ANOVA with Tukey’s post hoc test was used.
  • the data are presented as the mean ⁇ SD. *P ⁇ 0.05.
  • One-way ANOVA with Tukey’s post hoc test was used.
  • the data are presented as the mean ⁇ SD.
  • the data are presented as the mean ⁇ SD.
  • the data are presented as the mean ⁇ SD.
  • FIG. 21 presents a series of images showing surface morphologies of the tissue mimetic membranes after 56-day degradation in PBS solution.
  • FIG. 22 presents representative 3D and 2D cross-sectional fluorescent images of MC3T3-E1 cells cultured on the fibrous layers of the tissue mimetic membranes for 3 days.
  • FIG. 23 presents representative SEM images of MC3T3-E1 cells cultured on the fibrous layers of the tissue mimetic membranes for 3 days.
  • the pseudocolor was added to the cells using ImageJ software.
  • FIG. 24 representative SEM images of the surfaces of the microporous and fibrous layer in each membrane after a 5-day culture of NIH3T3 cells on the microporous surfaces of the membranes.
  • FIG. 26 presents images showing Alcian blue staining of the samples after a 21-day induction.
  • FIG. 28 presents images showing alkaline phosphatase (ALP) staining of the samples after a 7-day induction.
  • ALP alkaline phosphatase
  • FIG. 29 presents images showing Alizarin Red S (ARS) staining of the samples after a 10-day induction.
  • ARS Alizarin Red S
  • FIG. 30 presents a series of photographs illustrating the RCT surgical procedure and membrane implantation in a rat model.
  • FIG. 31 presents a series of representative macroscopic images of the anatomical plane in the membrane implantation at week 8 post-RCT surgery.
  • the red arrow indicates the membrane implantation site.
  • FIG. 32 presents a series of representative X-ray images of the humerus in rats at week 8 post-RCT surgery.
  • FIG. 33 presents a series of reconstructed 3D micro-CT images of cross-sections in the proximal humerus at week 8 post-RCT surgery. Scale bar: 1 mm.
  • FIG. 34 presents a series of representative SEM images of ingrowth cells and well-aligned collagen formation on the implanted membranes at week 8 post-RCT surgery. Scale bar: 10 ⁇ m.
  • FIG. 35 presents output from a catwalk gait analysis system showing automatic footprint and gaits capture measurements.
  • FIG. 36 presents a series of graphs plotting data from representative catwalk gait analysis across groups at week 8 post-RCT surgery using the system described in FIG. 35.
  • n 5 rats per group.
  • LF Left forelimb; RF: right forelimb; LH: left hindlimb; RH: right hindlimb.
  • Max Contact Area Maximal contact area; Max Contact AT: maximal contact AT; Max Intensity At: maximal intensity AT. All data are presented as mean ⁇ SD. One-way ANOVA with Tukey’s post hoc test was used.
  • the present disclosure provides a tissue mimetic membrane particularly useful in generating an in-situ co-delivery of both osteo-and chondro-promotive cues for functional fibrocartilaginous interface regeneration.
  • the formulation and fabrication strategies disclosed herein can be used, for example, to construct tissue mimetic membranes for guiding the fibrocartilaginous interface regeneration in rotator cuff repair and facilitating the restoration of normal shoulder function.
  • a non-solvent induced phase separation (NIPS) strategy followed by a co-axial electrospinning procedure can be used to construct a biphasic membranous matrix including a microporous layer and a mineralized core-shell nanofibrous layer, where the chondro-and osteo-promotive agents are incorporated in a region-specific manner.
  • the microporous layer can be laden with a cationic kartogenin (KGN) -conjugated nanogel (nGel-KGN) for chondrogenic induction, while the osteoinductive struvite nanowires can be encapsulated into the core of core-shell fibers.
  • KGN cationic kartogenin
  • nGel-KGN cationic kartogenin
  • the nGel-KGN-functionalized microporous layer is adjacent to the tendon, thereby suppressing scar tissue formation at the lesion and simultaneously heightening chondrogenesis.
  • a struvite-containing fibrous layer can cover the tubercula minus to enhance stem cell aggregation and biomineralization.
  • tissue-specific features and spatiotemporal release behaviors contribute to an effective guidance of specific defect-healing events at the transitional region, further leading to remarkably promoted fibrocartilaginous interface regeneration (FIG. 1) .
  • the provided biomimetic membrane is thus a promising material for use in, for example, clinical rotator cuff repair, presenting new avenues for developing improved tendon–bone healing strategies.
  • the terms “about” and “approximately, ” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ⁇ 20%, ⁇ 10%, or ⁇ 5%, are within the intended meaning of the recited value.
  • the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ( “or” ) .
  • the terms “including, ” “comprising, ” “having, ” “containing, ” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited.
  • the phrase “consisting of” is closed and excludes any element, step, or ingredient not explicitly specified.
  • the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.
  • bioceramic refers to a biocompatible inorganic and nonmetallic material.
  • a “biocompatible” material is one that does not have toxic or injurious effects on biological systems. In some applications, a biocompatible material does not have toxic or injurious effects on a treated subject.
  • chondro-promotive agent refers to a substance that induces or otherwise promotes the proliferation, growth, differentiation, orientation, production and/or other maturation of fibroblasts, chondrocytes, chondroprogenitor cells, and/or cartilage tissue.
  • the term “nanogel” refers to a three-dimensional hydrogel particle, or a population of such particles, where the average equivalent spherical diameter of the particles of the nanogel is less than 1 ⁇ m, i.e., between 1 and 999 nm.
  • the term “hydrogel” refers to a highly-interdependent, biphasic matrix comprising a solid component (usually a polymer, and more commonly a highly cross-linked polymer) that has both hydrophilic and hydrophobic character, and a liquid dispersion medium (e.g., water) that is retained in the matrix by intermolecular forces.
  • hydrophobic character provides the matrix with a degree of water insolubility while the hydrophilic character affords water permeability.
  • hydrogels useful in the methods of the present invention. Being polymer networks that have high water-absorbing capacity, hydrogels often closely mimic native extracellular matrices. Hydrogels also tend to possess a degree of flexibility very similar to natural tissues, due to the relatively high water content. In some cases, hydrogels may contain well over 90%water.
  • osteo-promotive agent refers to a substance that induces or otherwise promotes the proliferation, growth, differentiation, orientation, production and/or other maturation of osteoprogenitor cells, osteoblasts, osteoclasts, osteocytes, lining cells, and/or bone tissue.
  • the term “subject” refers to a vertebrate, and preferably to a mammal.
  • Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets.
  • the subject is human.
  • the subject is male.
  • the subject is female.
  • the subject is an adult.
  • the subject is an adolescent.
  • the subject is a child.
  • the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age. III. NANOGELS
  • the present disclosure provides various nanogels that are advantageously effective in delivering a chondro-promotive agent to stimulate repair of soft tissue, e.g., cartilage and/or tendon, at a site of an injured tendon–bone interface (e.g., tear) within a subject.
  • a chondro-promotive agent to stimulate repair of soft tissue, e.g., cartilage and/or tendon
  • the particular composition and configuration of the nanogel provides the material with several beneficial properties, including high biocompatibility and favorable stability (e.g., low shrinkage) under physiological conditions such as average body temperatures and pH levels.
  • the provided nanogel is conjugated with a chondro-promotive agent.
  • Chondro-promotive agents suitable for use with the provided nanogel include, for example, small-molecule drugs, such as kartogenin, that mimic natural ligands involved in cell differentiation; growth factors such as transforming growth factor-beta (TGF- ⁇ ) , bone morphogenetic proteins (e.g., BMP-2 or BMP-7) , or insulin-like growth factor-1 (IGF-1) ; anti-inflammatory agents such as non-steroidal anti-inflammatory drugs (e.g., ibuprofen or diclofenac) or corticosteroids; cartilage extracellular matrix components such as collagen type II or glycosaminoglycans (GAG) ; nutritional supplements such as glucosamine, chondroitin, or omega-3 fatty acids; and derivatives and combinations thereof.
  • the chondro-promotive agent conjugated with the provided nanogel includes or consists of kartogenin.
  • the polymer forming the provided nanogel includes or consists of a thermoresponsive polymer.
  • a thermoresponsive polymer in the nanogel can provide the nanogel with several beneficial characteristics including an enhanced ability to release bioactive substances in a controlled and/or sustained manner, an improved ease of fabrication, and favorable mechanical properties and biocompatibility appropriate for in vivo applications.
  • Thermoresponsive polymers suitable for use with the provided nanogel include, for example, poly (N-isopropylmethacrylamide) (PNIPMAM) , poly (N-isopropylacrylamide) (PNIPAM) , poly (ethylene glycol) (PEG) , poly (N-vinylcaprolactam) (PNVCL) , poly (caprolactone) (PCL) , poly (propylene fumarate) (PPF) , poly (vinyl methyl ether) (PVME) , and derivatives and combinations thereof.
  • the thermoresponsive polymer of the provided nanogel includes or consists of PNIPMAM.
  • the polymer forming the provided nanogel can typically be crosslinked with any suitable crosslinker.
  • the crosslinker is selected based on the chemistry of the nanogel polymer and/or chondro-promotive agent, and to provide the nanogel with its advantageous mechanical, hydrophilic, and drug-releasing properties.
  • the nanogel polymer includes or consists of a polymer (e.g., PNIPMAM) composed of acrylate monomers or derivatives thereof
  • the crosslinker can include or consist of N, N′-methylenebis (acrylamide) (BIS) .
  • the provided nanogel is coated with a cationic polymer, giving the nanogel a net positive charge.
  • the positive charge of the nanogel can advantageously assist the ability of the nanogel to introduce its chondro-promotive agent payload to cells.
  • the nanogel can have a positive zeta potential that is between about 1 mV and about 10 mV, e.g., between about 1 mV and about 6.4 mV, between about 1.9 mV and about 7.3 mV, between about 2.8 mV and about 8.2 mV, between about 3.7 mV and about 9.1 mV, or between about 4.6 mV and about 10 mV.
  • the positive zeta potential of the nanogel can be, for example, no more than about 10 mV, no more than about 9.1 mV, no more than about 8.2 mV, no more than about 7.3 mV, no more than about 6.4 mV, no more than about 5.5 mV, no more than about 4.6 mV, no more than about 3.7 mV, no more than about 2.8 mV, or no more than about 1.9 mV.
  • the positive zeta potential of the nanogel can be, for example, no less than about 1 mV, e.g., no less than about 1.9 mV, no less than about 2.8 mV, no less than about 3.7 mV, no less than about 4.6 mV, no less than about 5.5 mV, no less than about 6.4 mV, no less than about 7.3 mV, or no less than about 8.2 mV.
  • Cationic polymers suitable for use in coating the provided nanogels include, for example, poly (ethyleneimine) (PEI) , poly-L-lysine (PLL) , poly (allylamine hydrochloride) (PAH) , chitosan, poly (dimethylaminoethyl methacrylate) (PDMAEMA) , Poly (vinylamine) (PVAM) , cationically modified poly (ethylene glycol) (PEG) , and derivatives and combinations thereof.
  • the cationic polymer coating of the provided nanogel includes or consists of PEI. IV. MEMBRANES
  • the present disclosure further provides various membranes, e.g., multilayer membranes (such as bilayer membranes) or multiphasic membranes (such as biphasic membranes) .
  • the membranes generally include or consist of two distinct layers: a microporous layer that is particularly configured for the efficient delivery of a chondro-promotive agent to a soft tissue (e.g., cartilage or tendon) adjacent or proximate to the tendon, and a fibrous layer that is particularly configured for the efficient delivery of an osteo-promotive agent to a hard tissue (e.g., bone) adjacent or proximate to the bone.
  • a microporous layer that is particularly configured for the efficient delivery of a chondro-promotive agent to a soft tissue (e.g., cartilage or tendon) adjacent or proximate to the tendon
  • a fibrous layer that is particularly configured for the efficient delivery of an osteo-promotive agent to a hard tissue (e.g., bone) adjacent or prox
  • the microporous layer can beneficially stimulate the unmineralized interfacial region at a location (e.g., a location of a tendon–bone defect) where the membrane is implanted.
  • the fibrous layer can beneficially stimulate the mineralized region at this implantation location.
  • the microporous layer of the provided membrane generally includes a nanogel conjugated with a chondro-promotive agent.
  • the nanogel can be, for example, any of the nanogels described in Section III.
  • the nanogel within the microporous layer is conjugated with kartogenin.
  • the nanogel includes a thermoresponsive polymer, e.g., poly (N-isopropylmethacrylamide) (PNIPMAM) .
  • the polymer of the nanogel is crosslinked with N, N′-methylenebis (acrylamide) (BIS) .
  • the nanogel within the microporous layer is coated with a cationic polymer, e.g., poly (ethyleneimine) (PEI) .
  • PEI poly (ethyleneimine)
  • the concentration of the nanogel in the microporous layer of the provided membrane can be selected to provide the membrane with desired advantageous properties.
  • increased nanogel concentrations can correlate with an increased ability of the membrane to promote chondrogenic differentiation at a membrane implantation site (e.g., a site of an injured tendon–bone interface) .
  • Excessive nanogel concentrations can, however, impair the stem cell differentiation, proliferation, and/or aggregation that otherwise assists the intended desired tissue repair process.
  • the nanogel concentration in the microporous layer can be, for example, between about 1.5 wt%and about 15 wt%, e.g., between about 1.5 wt%and about 6 wt%, between about 1.9 wt%and about 7.5 wt%, between about 2.4 wt%and about 9.5 wt%, between about 3 wt%and about 12 wt%, or between about 3.8 wt%and about 15 wt%.
  • the nanogel concentration in the microporous layer can be, for example, no more than about 15 wt%, e.g., no more than about 12 wt%, no more than about 9.5 wt%, no more than about 7.5 wt%, no more than about 6 wt%, no more than about 4.7 wt%, no more than about 3.8 wt%, no more than about 3 wt%, no more than about 2.4 wt%, or no more than about 1.9 wt%.
  • the nanogel concentration in the microporous layer can be, for example, no less than about 1.5 wt%, e.g., no less than about 1.9 wt%, no less than about 2.4 wt%, no less than about 3 wt%, no less than about 3.8 wt%, no less than about 4.7 wt%, no less than about 6 wt%, no less than about 7.5 wt%, no less than about 9.5 wt%, or no less than about 12 wt%.
  • Higher nanogel concentrations e.g., greater than about 15 wt%
  • lower nanogel concentrations e.g., less than about 1.5 wt%, are also contemplated.
  • the microporous layer of the provided membrane includes a polymer scaffold, such that the nanogel is dispersed, e.g., substantially homogenously dispersed, within the polymer scaffold.
  • the polymer scaffold can be formed from, for example, a polyester (i.e., a scaffold polyester) and a gelatin (i.e., a scaffold gelatin) .
  • the identities of the scaffold polyester and the scaffold gelatin can be selected to provide the microporous layer with its beneficial properties, such as a porosity that is advantageous for the controlled and/or sustained release of the nanogel, and a balance of mechanical strength and flexibility that is advantageous for in vivo membrane implantation procedures.
  • the scaffold polyester includes or consists of poly (caprolactone) (PCL) .
  • the scaffold gelatin includes or consists of gelatin A (GelA) .
  • the polymer scaffold includes both PCL and GelA.
  • the amounts, e.g., relative amounts, of a scaffold polyester and a scaffold gelatin in the microporous layer of the provided membrane can also be selected or configured to provide the microporous layer with its desired nanogel-release and mechanical properties.
  • the weight ratio of the scaffold polyester to the scaffold gelatin in the polymer scaffold can be, for example, between about 0.2: 1 and about 5: 1, e.g., between about 0.2: 1 and about 1.4: 1, between about 0.28: 1 and about 1.9: 1, between about 0.38: 1 and about 2.6: 1, between about 0.53: 1 and about 3.6: 1, or between about 0.72: 1 and about 5: 1.
  • the weight ratio of the scaffold polyester to the scaffold gelatin in the polymer scaffold can be, for example, no more than about 5: 1, e.g., no more than about 3.6: 1, no more than about 2.6: 1, no more than about 1.9: 1, no more than about 1.4: 1, no more than about 1: 1, no more than about 0.72: 1, no more than about 0.53: 1, no more than about 0.38: 1, or no more than about 0.28: 1.
  • the weight ratio of the scaffold polyester to the scaffold gelatin in the polymer scaffold can be, for example, no less than about 0.2: 1, e.g., no less than about 0.28: 1, no less than about 0.38: 1, no less than about 0.53: 1, no less than about 0.72: 1, no less than about 1: 1, no less than about 1.3: 1, no less than about 1.9: 1, no less than about 1.9: 1, no less than about 2.6: 1, or no less than about 3.6: 1. Higher weight ratios, e.g., greater than about 5: 1, and lower weight ratios, e.g., less than about 0.2: 1, are also contemplated.
  • the microporous layer is configured to have a thickness that provides the membrane with desired mechanical and cargo delivery properties.
  • microporous layers that are thicker are capable of being loaded with additional chondro-promotive agents for transfer to a subject.
  • microporous layers thicknesses beyond a certain point can negatively impact the mechanical properties of the membrane, for example by reducing the yield strength and/or Young’s modulus of the membrane.
  • the thickness of the microporous layer can be, for example between about 100 ⁇ m and about 200 ⁇ m, e.g., between about 100 ⁇ m and about 160 ⁇ m, between about 110 ⁇ m and about 170 ⁇ m, between about 120 ⁇ m and about 180 ⁇ m, between about 130 ⁇ m and about 190 ⁇ m, between about 140 ⁇ m and about 200 ⁇ m.
  • the microporous layer thickness can be, for example, no less than about 100 ⁇ m, e.g., no less than about 110 ⁇ m, no less than about 120 ⁇ m, no less than about 130 ⁇ m, no less than about 140 ⁇ m, no less than about 150 ⁇ m, no less than about 160 ⁇ m, no less than about 170 ⁇ m, no less than about 180 ⁇ m, or no less than about 190 ⁇ m. Larger microporous layer thicknesses, e.g., greater than about 200 ⁇ m, and smaller microporous layer thicknesses, e.g., less than about 100 ⁇ m, are also contemplated.
  • the fibrous layer of the provided membrane generally includes core-shell fibers, where the cores of the fibers encapsulate an osteo-promotive agent.
  • Osteo-promotive agents suitable for use with the provided membrane include, for example, bioceramic materials (e.g., calcium-and/or magnesium-containing materials) , bone morphogenetic proteins (e.g., BMP-2 or BMP-7) , platelet-derived growth factor (PDGF) , transforming growth factor-beta (TGF- ⁇ ) , insulin-like growth factor-1 (IGF-1) , drugs such as bisphosphonates or strontium ranelate, parathyroid hormone (PTH) , and derivatives and combinations thereof.
  • bioceramic materials e.g., calcium-and/or magnesium-containing materials
  • bone morphogenetic proteins e.g., BMP-2 or BMP-7
  • PDGF platelet-derived growth factor
  • TGF- ⁇ transforming growth factor-beta
  • IGF-1 insulin-like growth factor
  • the osteo-promotive agent encapsulated in the fibrous layer includes or consists of a magnesium-containing bioceramic material. In some embodiments, the osteo-promotive agent includes or consists of struvite, e.g., struvite nanowires.
  • the amount of the osteo-promotive agent can also be selected or configured to provide the membrane with advantageous properties.
  • the concentration of the osteo-promotive agent in the fibrous layer can be, for example, between about 1 wt%and about 10 wt%, e.g., between about 1 wt%and about 6.4 wt%, between about 1.9 wt%and about 7.3 wt%, between about 2.8 wt%and about 8.2 wt%, between about 3.7 wt%and about 9.1 wt%, or between about 4.6 wt%and about 10 wt%.
  • the osteo-promotive agent concentration in the fibrous layer can be, for example, no more than about 10 wt%, e.g., no more than about 9.1 wt%, no more than about 8.2 wt%, no more than about 7.3 wt%, no more than about 6.4 wt%, no more than about 5.5 wt%, no more than about 4.6 wt%, no more than about 3.7wt%, no more than about 2.8 wt%, or no more than about 1.9 wt%.
  • the osteo-promotive agent concentration in the fibrous layer can be, for example, no less than about 1 wt%, e.g., no less than about 1.9 wt%, no less than about 2.8 wt%, no less than about 3.7 wt%, no less than about 4.6 wt%, no less than about 5.5 wt%, no less than about 6.4 wt%, no less than about 7.3 wt%, no less than about 8.2 wt%, or no less than about 9.1 wt%.
  • Higher osteo-promotive agent concentrations e.g., greater than about 10 wt%
  • lower osteo-promotive agent concentrations e.g., less than about 1 wt%, are also contemplated.
  • the core of the core-shell fibers in the fibrous layer of the provided membrane includes a polymer, i.e., a core polymer.
  • the core polymer of the fibers can include or consist of, for example, a polyester, i.e., a core polyester.
  • the core polyester includes or consists of poly (caprolactone) (PCL) .
  • the shell of the core-shell fibers in the fibrous layer of the provided membrane includes or consist of a gelatin, i.e., a shell gelatin.
  • the shell gelatin includes or consists of gelatin A (GelA) .
  • composition and configuration of the provided membrane can be designed or selected as described herein to provide the membrane with advantageous mechanical properties including a high yield strength.
  • the yield strength of the membrane can influence the load-bearing capacity, stability, and durability of the membrane, each of which can be particularly important properties when the membrane is implanted in an area (e.g., proximate to a rotator cuff) subject to significant stresses.
  • the membrane can exhibit a yield strength that is, for example, between about 1.75 MPa and about 2.75 MPa, e.g., between about 1.75 MPa and about 2.35 MPa, between about 1.85 MPa and about 2.45 MPa, between about 1.95 MPa and about 2.55 MPa, between about 2.05 MPa and about 2.65 MPa, or between about 2.15 MPa and about 2.75 MPa.
  • the membrane yield strength can be, for example, no more than about 2.75 MPa, e.g., no more than about 2.65 MPa, no more than about 2.55 MPa, no more than about 2.45 MPa, no more than about 2.35 MPa, no more than about 2.25 MPa, no more than about 2.15 MPa, no more than about 2.05 MPa, no more than about 1.95 MPa, or no more than about 1.85 MPa.
  • the membrane yield strength can be, for example, no less than about 1.75 MPa, e.g., no less than about 1.85 MPa, no less than about 1.95 MPa, no less than about 2.05 MPa, no less than about 2.15 MPa, no less than about 2.25 MPa, no less than about 2.35 MPa, no less than about 2.45 MPa, no less than about 2.55 MPa, or no less than about 2.65 MPa.
  • Higher membrane yield strengths e.g., greater than about 2.75 MPa
  • lower membrane yield strengths e.g., less than about 1.75 MPa, are also contemplated.
  • the provided membrane can also be configured or designed as described herein to have a Young’s modulus that is advantageously high.
  • the Young’s modulus is a measure of the elasticity of a material, and a membrane with a high Young’s modulus can better provide flexibility, tissue compatibility, and mechanical support when applied in vivo, e.g., for repairing a tendon–bone defect.
  • the membrane can exhibit a Young’s modulus that is, for example, between about 8 MPa and about 18 MPa, e.g., between about 8 MPa and about 14 MPa, between about 9 MPa and about 15 MPa, between about 10 MPa and about 16 MPa, between about 11 MPa and about 17 MPa, or between about 12 MPa and about 18 MPa.
  • the membrane Young’s modulus can be, for example, no more than about 18 MPa, e.g., no more than about 17 MPa, no more than about 16 MPa, no more than about 15 MPa, no more than about 14 MPa, no more than about 13 MPa, no more than about 12 MPa, no more than about 11 MPa, no more than about 10 MPa, or no more than about 9 MPa.
  • the membrane Young’s modulus can be, for example, no less than about 8 MPa, e.g., no less than about 9 MPa, no less than about 10 MPa, no less than about 11 MPa, no less than about 12 MPa, no less than about 13 MPa, no less than about 14 MPa, no less than about 15 MPa, no less than about 16 MPa, or no less than about 17 MPa.
  • Higher membrane Young’s modulus values e.g., greater than about 18 MPa
  • lower membrane Young’s modulus values e.g., less than about 8 MPa, are also contemplated.
  • composition and configuration of the membrane can also be configured or designed as disclosed herein to provide the membrane with a beneficial elongation rate.
  • a high elongation rate for the membrane is indicative of good deformability, which can positively influence the ease with which the membrane can be implanted in a subject, for example during a surgical procedure.
  • the membrane can exhibit an elongation rate that is, for example, between about 40%and about 140%, e.g., between about 40%and about 100%, between about 50%and about 110%, between about 60%and about 120%, between about 70%and about 130%, or between about 80%and about 140%.
  • the membrane elongation rate can be, for example, no more than about 140%, e.g., no more than about 130%, no more than about 120%, no more than about 110%, no more than about 100%, no more than about 90%, no more than about 80%, no more than about 70%, no more than about 60%, or no more than about 50%.
  • the membrane elongation rate can be, for example, no less than about 40%, e.g., no less than about 50%, no less than about 60%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 100%, no less than about 110%, no less than about 120%, or no less than about 130%.
  • Higher membrane elongation rates, e.g., greater than about 140%, and lower membrane elongation rates, e.g., less than about 40%, are also contemplated.
  • the present disclosure also provides methods for producing a membrane, e.g., any of the membranes described in Section IV.
  • the particular combinations of steps of the methods have been shown to impart the membranes with the advantageous features described in Sections I and IV, including an improved ability to simultaneously provide both osteo-and chondro-promotive cues for functional fibrocartilaginous interface regeneration.
  • the provided methods benefit from a surprisingly effective combination of a non-solvent induced phase separation process used to generate a chondro-promotive microporous layer of the membrane, and a co-axial electrospinning process used to generate an osteo-promotive fibrous layer of the membrane.
  • the provided method generally includes a step of forming a dispersion of a nanogel in a polymer solution.
  • the nanogel can be, for example, any of the nanogels described in Section III.
  • the nanogel is conjugated with a chondro-promotive agent, e.g., kartogenin.
  • the nanogel includes a thermoresponsive polymer, e.g., poly (N-isopropylmethacrylamide) (PNIPMAM) .
  • the polymer of the nanogel is crosslinked with N, N′-methylenebis (acrylamide) (BIS) .
  • the nanogel within the microporous layer is coated with a cationic polymer, e.g., poly (ethyleneimine) (PEI) .
  • PEI poly (ethyleneimine)
  • the polymer solution in which the nanogel is dispersed includes a polymer, i.e., a pre-scaffold polymer.
  • the pre-scaffold polymer can, for example, have the composition of any of the scaffold polymers described in Section IV,
  • the pre-scaffold polymer can include a scaffold polyester (e.g., poly (caprolactone) (PCL) ) and a scaffold gelatin (e.g., gelatin A (GelA) ) .
  • the weight ratio of the scaffold polyester to the scaffold gelatin in the polymer solution can be, for example, between about 0.2: 1 and about 5: 1, or any of the related weight ratios described in Section IV.
  • the pre-scaffold polymer is dissolved in a mixture of a solvent and a non-solvent.
  • the solvent and non-solvent can be selected for their compatibility with the particular pre-scaffold polymer components, and for their suitability in a non-solvent induced phase separation process.
  • the solvent can be an alcoholic organic solvent that dissolves the pre-scaffold polymer
  • the non-solvent can be a non-alcoholic solvent that does not dissolve the pre-scaffold polymer.
  • the solvent includes or consists of 1, 1, 1, 3, 3, 3-hexafluoro-2-propanal (HFIP) .
  • the non-solvent includes or consists of dimethylformamide (DMF) .
  • the mixture includes both HFIP and DMF.
  • the volume ratio of the solvent to the non-solvent in the mixture can be, for example, between about 5: 1 and about 100: 1, e.g., between about 5: 1 and about 30: 1, between about 6.7: 1 and about 41: 1, between about 9.1: 1 and about 55: 1, between about 12: 1 and about 74: 1, or between about 17: 1 and about 100: 1.
  • the volume ratio of the solvent to the non-solvent in the mixture can be, for example, no more than about 100: 1, e.g., no more than about 74: 1, no more than about 55: 1, no more than about 41: 1, no more than about 30: 1, no more than about 22: 1, no more than about 17: 1, no more than about 12: 1, no more than about 9.1: 1, or no more than about 6.7: 1.
  • the volume ratio of the solvent to the non-solvent in the mixture can be, for example, no less than about 5: 1, e.g., no less than about 6.7: 1, no less than about 9.1: 1, no less than about 12: 1, no less than about 17: 1, no less than about 22: 1, no less than about 30: 1, no less than about 41: 1, no less than about 55: 1, or no less than about 74: 1.
  • Higher volume ratios e.g., greater than about 100: 1, and lower volume ratios, e.g., less than about 5: 1, are also contemplated.
  • the concentration of the pre-scaffold polymer in the polymer solution can be, for example, between about 5 wt%and about 25 wt%, e.g., between about 5 wt%and about 17 wt%, between about 7 wt%and about 19 wt%, between about 9 wt%and about 21 wt%, between about 11 wt%and about 23 wt%, or between about 13 wt%and about 25 wt%.
  • the pre-scaffold polymer concentration in the polymer solution can be, for example, no more than about 25 wt%, e.g., no more than about 23 wt%, no more than about 21 wt%, no more than about 19 wt%, no more than about 17 wt%, no more than about 15 wt%, no more than about 13 wt%, no more than about 11 wt%, no more than about 9 wt%, or no more than about 7 wt%.
  • the pre-scaffold polymer concentration in the polymer solution can be, for example, no less than about 5 wt%, e.g., no less than about 7 wt%, no less than about 9 wt%, no less than about 11 wt%, no less than about 13 wt%, no less than about 15 wt%, no less than about 17 wt%, no less than about 19 wt%, no less than about 21 wt%, or no less than about 23 wt%.
  • Higher pre-scaffold polymer concentrations e.g., greater than about 2550 wt%
  • lower pre-scaffold polymer concentrations e.g., less than about 5 wt%, are also contemplated.
  • the provided method generally further includes a step of casting the dispersion to produce a wet layer having a target thickness.
  • the target thickness can be pre-determined, and can be selected to yield a microporous layer having the desired thickness-associated properties described in Section IV.
  • the target thickness of the wet layer can be between about 150 ⁇ m and about 850 ⁇ m, e.g., between about 150 ⁇ m and about 570 ⁇ m, between about 220 ⁇ m and about 640 ⁇ m, between about 290 ⁇ m and about 710 ⁇ m, between about 360 ⁇ m and about 780 ⁇ m, or between about 430 ⁇ m and about 850 ⁇ m.
  • the wet layer thickness can be, for example, no more than about 850 ⁇ m, e.g., no more than about 780 ⁇ m, no more than about 710 ⁇ m, no more than about 640 ⁇ m, no more than about 570 ⁇ m, no more than about 500 ⁇ m, no more than about 430 ⁇ m, no more than about 360 ⁇ m, no more than about 290 ⁇ m, or no more than about 220 ⁇ m.
  • the wet layer thickness can be, for example, no less than about 150 ⁇ m, e.g., no less than about 220 ⁇ m, no less than about 290 ⁇ m, no less than about 360 ⁇ m, no less than about 430 ⁇ m, no less than about 570 ⁇ m, no less than about 640 ⁇ m, no less than about 710 ⁇ m, or no less than about 780 ⁇ m. Larger wet layer thicknesses, e.g., greater than about 850 ⁇ m, and smaller wet layer thicknesses, e.g., less than about 150 ⁇ m, are also contemplated.
  • the provided method generally further includes a step of evaporating the mixture of the solvent and the non-solvent to yield a microporous layer.
  • the microporous layer can have the desired nanogel concentration-associated properties described in Section IV.
  • the concentration of the nanogel in the microporous layer following the evaporation step can be, for example, between about 1.5 wt%and about 15 wt%, or any of the other nanogel concentrations described in Section IV.
  • the provided method generally further includes a step of fabricating a fibrous layer on (e.g., directly on) the microporous layer to produce the membrane (i.e., the multilayer membrane) .
  • a fibrous layer on (e.g., directly on) the microporous layer to produce the membrane (i.e., the multilayer membrane) .
  • co-axial electrospinning of a core solution and a shell solution is used to generate core-shell fibers.
  • the core solution and the shell solution can be configured such that the core-shell fibers generated by the co-axial electrospinning are, for example, any of the core-shell fibers described in Section IV.
  • the core solution includes an osteo-protective agent, e.g., a magnesium-containing bioceramic material such as struvite (e.g., struvite nanowires) .
  • the concentration of the osteo-promotive agent in the fibrous layer generated by the co-axial electrospinning can be, for example, between about 1 wt%and about 15 wt%., or any of the other osteo-promotive agent concentrations described in Section IV.
  • the core solution includes a polyester (i.e., a core polyester) , e.g., poly (caprolactone) (PCL) .
  • the shell solution includes a gelatin (i.e., a shell gelatin) , e.g., gelatin A (GelA) .
  • the provided method further includes one or more additional steps including, for example, drying the membrane, crosslinking the membrane, and/or administering the membrane to a subject as described in Section VI. VI. METHODS FOR ENTHESIS REPAIR
  • Another aspect of the present disclosure relates to methods for repairing a defect at a tendon–bone interface (i.e., at an enthesis) in a subject.
  • the particular features of the methods have been shown to provide a subject with treatments benefiting from the enhanced characteristics of the membranes disclosed herein.
  • the methods generally include implanting a membrane in a subject.
  • the membrane can be, for example, any of those described in Sections IV or V.
  • the membrane can be implanted in the subject at a location proximate to an interfacial defect of the subject, e.g., at the site of the tendon–bone interface.
  • the membrane is implanted in a subject in an orientation that maximizes the different benefits of the separate chondro-and osteo-promotive layers of the membrane.
  • the membrane can be implanted with the microporous layer of the membrane adjacent to soft tissue (e.g., tendon and/or cartilage) of the subject proximate to the defect site, and with the fibrous layer of the membrane adjacent to hard tissue (e.g., bone) of the subject proximate to the defect site.
  • the defect of the subject can be the result of, for example, a trauma, an injury, overuse, a degenerative disease, aging, or a genetic disorder.
  • the provided membrane can be implemented in treatments related to, for example, sports injuries or elder care.
  • the defect site includes a rotator cuff tear.
  • Defects that can be repaired with the provided graft material include defects involving the supraspinatus tendon.
  • Exemplary defects include a diseased, degenerated, or damaged, e.g., torn, enthesis.
  • the method further includes evaluating the subject to determine the nature of the defect that requires repair, and the characteristics of the membrane appropriate to treat the subject.
  • the evaluating of the subject can include medical imaging, such as X-ray imaging, MRI scans, or CT scans, which can provide dimensions of the defect site, and can be utilized for determining the desired configuration, such as size and/or shape, of the membrane to be implanted.
  • Embodiment 1 A multilayer membrane comprising: a microporous layer comprising a nanogel conjugated with a chondro-promotive agent; and a fibrous layer comprising core-shell fibers having a core that encapsulates an osteo-promotive agent.
  • Embodiment 2 An embodiment of embodiment 1, wherein the chondro-promotive agent comprises kartogenin.
  • Embodiment 3 An embodiment of embodiment 1 or 2, wherein the nanogel comprises a thermoresponsive polymer.
  • Embodiment 4 An embodiment of embodiment 3, wherein the thermoresponsive polymer comprises poly (N-isopropylmethacrylamide) (PNIPMAM) .
  • PNIPMAM poly (N-isopropylmethacrylamide)
  • Embodiment 5 An embodiment of any one of embodiments 1-4, wherein the nanogel is crosslinked with N, N′-methylenebis (acrylamide) (BIS) .
  • BIOS N, N′-methylenebis (acrylamide)
  • Embodiment 6 An embodiment of any one of embodiments 1-5, wherein the nanogel is coated with a cationic polymer.
  • Embodiment 7 An embodiment of embodiment 6, wherein the cationic polymer comprises poly (ethyleneimine) (PEI) .
  • PEI poly (ethyleneimine)
  • Embodiment 8 An embodiment of any one of embodiments 1-7, wherein the nanogel has a concentration in the microporous layer about 1.5 wt%and 15 wt%.
  • Embodiment 9 An embodiment of any one of embodiments 1-8, wherein the microporous layer comprises a polymer scaffold, and wherein the nanogel is dispersed in the polymer scaffold.
  • Embodiment 10 An embodiment of embodiment 9, wherein the polymer scaffold comprises a scaffold polyester and a scaffold gelatin.
  • Embodiment 11 An embodiment of embodiment 10, wherein the scaffold polyester comprises poly (caprolactone) (PCL) .
  • PCL poly (caprolactone)
  • Embodiment 12 An embodiment of embodiment 10 or 11, wherein the scaffold gelatin comprises gelatin A.
  • Embodiment 13 An embodiment of any one of embodiments 10-12, wherein the weight ratio of the scaffold polyester to the scaffold gelatin in the polymer scaffold is between about 0.2: 1 and 5: 1.
  • Embodiment 14 An embodiment of any one of embodiments 1-13, wherein the osteo-promotive agent comprises a magnesium-containing bioceramic material.
  • Embodiment 15 An embodiment of embodiment 14, wherein the magnesium-containing bioceramic material comprises struvite.
  • Embodiment 16 An embodiment of any one of embodiments 1-15, wherein the osteo-promotive agent has a concentration in the fibrous layer between about 1 wt%and about 10 wt%.
  • Embodiment 17 An embodiment of any one of embodiments 1-16, wherein the core of the core-shell fibers comprises a core polyester.
  • Embodiment 18 An embodiment of embodiment 17, wherein the core polyester comprises poly (caprolactone) (PCL) .
  • PCL poly (caprolactone)
  • Embodiment 19 An embodiment of any one of embodiments 1-18, wherein the core-shell fibers have a shell comprising a shell gelatin.
  • Embodiment 20 An embodiment of embodiment 19, wherein the shell gelatin comprises gelatin A (GelA) .
  • GelA gelatin A
  • Embodiment 21 An embodiment of any one of embodiments 1-20, wherein the microporous layer has a thickness between about 100 ⁇ m and about 200 ⁇ m.
  • Embodiment 22 An embodiment of any one of embodiments 1-21, wherein the multilayer membrane exhibits a yield strength between about 1.75 MPa and about 2.75 MPa.
  • Embodiment 23 An embodiment of any one of embodiments 1-22, wherein the multilayer membrane exhibits a Young’s modulus between about 8 MPa and about 18 MPa.
  • Embodiment 24 An embodiment of any one of embodiments 1-23, wherein the multilayer membrane exhibits an elongation rate between about 40%and about 140%.
  • Embodiment 25 A nanogel conjugated with a chondro-promotive agent, wherein the nanogel comprises a thermoresponsive polymer and is coated with a cationic polymer.
  • Embodiment 26 An embodiment of embodiment 25, wherein the chondro-promotive agent comprises kartogenin.
  • Embodiment 27 An embodiment of embodiment 25 or 26, wherein the thermoresponsive polymer comprises poly (N-isopropylmethacrylamide) (PNIPMAM) .
  • PNIPMAM poly (N-isopropylmethacrylamide)
  • Embodiment 28 An embodiment of any one of embodiments 25-27, wherein the nanogel is crosslinked with N, N′-methylenebis (acrylamide) (BIS) .
  • BIOS N, N′-methylenebis (acrylamide)
  • Embodiment 29 An embodiment of any one of embodiments 25-28, wherein the cationic polymer comprises poly (ethyleneimine) (PEI) .
  • PEI poly (ethyleneimine)
  • Embodiment 30 An embodiment of any one of embodiments A method of producing a multilayer membrane, the method comprising forming a dispersion of a nanogel in a polymer solution, the polymer solution comprising a pre-scaffold polymer dissolved in a mixture of a solvent and a non-solvent; casting the dispersion to produce a wet layer having a target thickness; evaporating the mixture of the solvent and the non-solvent to yield a microporous layer, the microporous layer having a target concentration of the nanogel; and fabricating a fibrous layer on the microporous layer, thereby producing the multilayer membrane, wherein the fibrous layer comprises core-shell fibers generated on the microporous layer by co-axial electrospinning of a core solution and a shell solution.
  • Embodiment 31 An embodiment of embodiment 30, wherein the method further comprises: crosslinking the multilayer membrane.
  • Embodiment 32 An embodiment of embodiment 30 or 31, wherein the nanogel is conjugated with a chondro-promotive agent.
  • Embodiment 33 An embodiment of embodiment 32, wherein the chondro-promotive agent comprises kartogenin.
  • Embodiment 34 An embodiment of any one of embodiments 30-33, wherein the core solution comprises an osteo-promotive agent.
  • Embodiment 35 An embodiment of embodiment 34, wherein the osteo-promotive agent comprises a magnesium-containing bioceramic material.
  • Embodiment 36 An embodiment of embodiment 35, wherein the magnesium-containing bioceramic material comprises struvite.
  • Embodiment 37 An embodiment of any one of embodiments 34-36, wherein the osteo-promotive agent has a concentration in the fibrous layer between about 1 wt%and about 10 wt%.
  • Embodiment 38 An embodiment of any one of embodiments 30-37, wherein the target thickness of the wet layer is between about 150 ⁇ m and about 850 ⁇ m.
  • Embodiment 39 An embodiment of any one of embodiments 30-38, wherein the target concentration of the nanogel is between about 1.5 wt%and 15 wt%.
  • Embodiment 40 An embodiment of any one of embodiments 30-39, wherein the pre-scaffold polymer has a concentration in the polymer solution that is between about 5 wt%and about 25 wt%.
  • Embodiment 41 An embodiment of any one of embodiments 30-40, wherein the volume ratio of the solvent to the non-solvent in the mixture is between about 5: 1 and about 100: 1.
  • Embodiment 42 An embodiment of any one of embodiments 30-41, wherein the solvent comprises an alcoholic organic solvent, and the non-solvent comprises a non-alcoholic organic solvent.
  • Embodiment 43 An embodiment of embodiment 42, wherein the alcoholic organic solvent comprises 1, 1, 1, 3, 3, 3-hexafluoro-2-propanal (HFIP) .
  • HFIP hexafluoro-2-propanal
  • Embodiment 44 An embodiment of embodiment 42 or 43, wherein the non-alcoholic organic solvent comprises dimethylformamide (DMF) .
  • DMF dimethylformamide
  • Embodiment 45 An embodiment of any one of embodiments 30-44, wherein the nanogel comprises a thermoresponsive polymer.
  • Embodiment 46 An embodiment of embodiment 45, wherein the thermoresponsive polymer comprises poly (N-isopropylmethacrylamide) (PNIPMAM) .
  • PNIPMAM poly (N-isopropylmethacrylamide)
  • Embodiment 47 An embodiment of any one of embodiments 30-46, wherein the nanogel is crosslinked with N, N′-methylenebis (acrylamide) (BIS) .
  • BIOS N, N′-methylenebis (acrylamide)
  • Embodiment 48 An embodiment of any one of embodiments 30-47, wherein the nanogel is coated with a cationic polymer.
  • Embodiment 49 An embodiment of embodiment 48, wherein the cationic polymer comprises poly (ethyleneimine) (PEI) .
  • PEI poly (ethyleneimine)
  • Embodiment 50 An embodiment of any one of embodiments 30-49, wherein the pre-scaffold polymer comprises a scaffold polyester and a scaffold gelatin.
  • Embodiment 51 An embodiment of embodiment 50, wherein the weight ratio of the scaffold polyester to the scaffold gelatin in the mixture is between about 0.2: 1 and 5: 1.
  • Embodiment 52 An embodiment of embodiment 50, wherein the scaffold polyester comprises poly (caprolactone) (PCL) .
  • PCL poly (caprolactone)
  • Embodiment 53 An embodiment of embodiment 50 or 52, wherein the scaffold gelatin comprises gelatin A.
  • Embodiment 54 An embodiment of any one of embodiments 30-53, wherein the core solution comprises a core polyester.
  • Embodiment 55 An embodiment of embodiment 54, wherein the core polyester comprises poly (caprolactone) (PCL) .
  • PCL poly (caprolactone)
  • Embodiment 56 An embodiment of any one of embodiments 30-55, wherein the shell solution comprises a shell gelatin.
  • Embodiment 57 An embodiment of embodiment 56, wherein the shell gelatin comprises gelatin A (GelA) .
  • Embodiment 58 An embodiment of any one of embodiments 30-57, wherein the microporous layer has a thickness between about 100 ⁇ m and about 200 ⁇ m.
  • Embodiment 59 A method of repairing a defect at a tendon–bone interface of a subject, the method comprising implanting the multilayer membrane of any one of claims 1-24 in the subject proximate to the defect.
  • Embodiment 60 An embodiment of embodiment 59, wherein the implanting comprises positioning the microporous layer of the multilayer membrane adjacent to a tendon of the subject, and the fibrous layer of the multilayer membrane adjacent to a bone of the subject.
  • Embodiment 61 An embodiment of embodiment 59 or 60, wherein the defect comprises a rotator cuff tear.
  • kartogenin (KGN) -conjugated poly (N-isopropylmethacrylamide) (PNIPMAM) nanogel (nGel) particles with weak positive surface charge were synthesized for use as chondro-inductive factors showing enhanced solubility and biocompatibility compared to single KGN molecules.
  • PNIPMAM N-isopropylmethacrylamide
  • nGel nanogel particles with weak positive surface charge
  • the biphasic tissue mimetic membrane included a nGel-KGN-laden polycaprolactone (PCL) -gelatin A (GelA) microporous layer and a mineralized PCL-GelA core-shell nanofibrous layer with struvite encapsulated in the PCL core.
  • PCL polycaprolactone
  • GalA polycaprolactone
  • the fabricated membrane was implanted between tendon and bone in a rat rotator cuff tear model, where the upper nGel-KGN-laden microporous layer simulated the interfacial unmineralized region while the lower struvite-containing fibrous layer mimicked the mineralized region (FIG. 1) .
  • this tissue mimetic membrane provided region-specific compositional (i.e., mineralized gradient) and topographical cues to guide the healing process.
  • region-specific compositional i.e., mineralized gradient
  • topographical cues to guide the healing process.
  • In vitro and in vivo observations revealed the promotive effect of this tissue mimetic membrane and the beneficial role of the co-release of nGel-KGN/struvite on augmenting fibrocartilaginous tissue regeneration, indicating promising clinical curative efficacy in interface tissue repair.
  • Example 1 Synthesis of positively charged nGel-KGN particles
  • N-isopropylmethacrylamide N-isopropylmethacrylamide
  • NIPMAM N-isopropylmethacrylamide
  • BIOS N′-methylenebis (acrylamide)
  • SDS sodium dodecyl sulfate
  • the synthesized nanogel core particles were then utilized as the seeds for the growth of an amino-functionalized polymer shell.
  • 48.75 mM NIPMAM, 1 mM BIS, and 0.25 mM N- (3-aminopropyl) methacrylamide hydrochloride (AMPA) were dissolved in 39.5 mL D. I. H 2 O to obtain a monomer solution.
  • AMPA N- (3-aminopropyl) methacrylamide hydrochloride
  • AMPA N- (3-aminopropyl) methacrylamide hydrochloride
  • the reaction was terminated via cooling.
  • the product was dialyzed (MWCO: 12–14 kDa) in D. I. water for 3–5 days to remove any unreacted molecules and short-chain polymers. Following dialysis, the solution was lyophilized to obtain the dry fluorescent amino-functionalized nGel particles.
  • KGN molecules were conjugated to the surface of nGel particles via EDC/NHS coupling reaction.
  • 88.3 mg nGel particles were dissolved in 35 mL N, N-dimethyl formamide (DMF) to prepare a homogeneous suspension.
  • 1.4 mg KGN, 0.84 mg 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC ⁇ HCl) , and 0.51 mg N-hydroxysuccinimide (NHS) were dissolved in 3 mL DMF and shaken for 2 h at room temperature. Then, this solution was added to the nGel suspension and reacted at room temperature on a shaker overnight.
  • the product was dialyzed (MWCO: 12–14 kDa) in D. I. H 2 O for 3–5 days for purification.
  • the purified solution was then lyophilized to obtain the dry nGel-KGN particles.
  • the nanogel-KGN particles were coated with poly (ethyleneimine) (PEI) polymer by simple mixing and sonication.
  • PEI poly (ethyleneimine)
  • 4 mg/mL nGel-KGN particles and 1 mg/mL PEI (branched, Mw. ⁇ 25,000) were mixed in D. I. H 2 O and sonicated for 5 min.
  • the mixture was dialyzed (MWCO: 50 kDa) in D. I. H 2 O to remove any free PEI polymers.
  • the product was lyophilized to obtain the positive-charged nGel-KGN particles.
  • Struvite nanowires were synthesized by a controlled crystallization method. Briefly, a solution A containing 1 mg/mL magnesium chloride hexahydrate (MgCl 2 ⁇ 6H 2 O) and a solution B containing 2.3 mg/mL ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ) and 2.1 mg/mL ammonium chloride (NH 4 Cl) were prepared. Then, 10 mL solution B was added to 40 mL solution A under continuous stirring to obtain a homogeneous solution. After that, 1.75 g sodium chloride (NaCl) was dissolved into the as-prepared solution. Then, 10 M sodium hydroxide (NaOH) was used to adjust the pH value of the solution to around 11.0. White precipitation gradually appeared as the pH increase and the suspension was stirred for 24 h at room temperature. Then, the product was purified using absolute ethanol, and subsequently vacuum dried at room temperature for 3 days. Example 3. Fabrication of the dual-layer tissue mimetic membrane
  • a tissue mimetic membrane was fabricated using a non-solvent induced phase separation (NIPS) strategy followed by co-axial electrospinning (ES) .
  • NIPS non-solvent induced phase separation
  • ES co-axial electrospinning
  • a microporous layer was prepared using a polymer/nGel-KGN mixed solution.
  • the 15%w/v polymer blend solution was prepared by dissolving polycaprolactone (PCL) and gelatin A (GelA) at a weight ratio of 1: 1 into a mixed solvent of 1, 1, 1, 3, 3, 3-hexafluoro-2-propanal (HFIP) and DMF at a 20: 1 volume ratio, in which nGel-KGN particles were homogeneously dispersed.
  • the prepared solution was then casted onto a silicon paper with a casting blade, where the wet film thickness was set to 500 ⁇ m or 1000 ⁇ m.
  • NIPS occurred during evaporation and eventually formed the microporous layers with particle concentrations of 0 wt%and 5 wt%.
  • the obtained microporous layers were designated as 500 ⁇ m casting, 500 ⁇ m casting/5%nGel-KGN, and 1000 ⁇ m casting/5%nGel-KGN.
  • the mineralized core-shell fibrous layer composed of a PCL or PCL/St. core and a GelA shell was produced directly on the prepared microporous layer through co-axial electrospinning.
  • the core solution was prepared by dissolving 10%w/v PCL into HFIP, in which the St. nanowires were homogeneously dispersed.
  • the sheath solution was prepared by dissolving 10%w/v GelA in HFIP.
  • the co-axial electrospinning was performed using a co-axial spinneret consisting of an inner needle (20 G) and an outer needle (14 G) .
  • the core and sheath solutions were delivered to the inner and outer needles using two syringe pumps at the flow rate of 0.4 mL/h and 0.6 mL/h, respectively, and then electrospun for 10 h at a working distance of 12 cm and an applied voltage of 15–17 kV to produce core-shell fibrous layers with St. concentrations of 0 wt%and 4 wt%.
  • the fabricated fibrous layers were designated as ES and ES/4%St.
  • the fabricated dual-layer tissue mimetic membranes were crosslinked in EDC/NHS solution (25 mM EDC and 10 mM NHS in absolute ethanol) for 15–20 min. After crosslinking, the membranes were washed with absolute ethanol, and dried at room temperature. Eventually, three tissue mimetic membranes were prepared by tuning the composition. These were designated as Grp 1 (500 ⁇ m casting + ES) , Grp 2 (500 ⁇ m casting/5%nGel-KGN + ES/4%St. ) , and Grp 3 (1000 ⁇ m casting/5%nGel-KGN + ES/4%St. ) .
  • Example 4 Characterization of the biphasic structures and mineral gradients of the tissue mimetic membranes
  • nGel-KGN particle morphology was examined using transmission electron microscopy (TEM) at the accelerated voltage of 120 kV. Further, the size (diameter) distribution of the dry nGel-KGN particles was analyzed based on TEM images using ImageJ software. The hydrodynamic size distribution of the nGel-KGN particles was determined via a dynamic light scattering analyzer at 25 °C and 37 °C to examine the phase stability in the physiological environment. Particle zeta potential was measured using the same instrument at 37 °C.
  • Nanoparticle tracking analysis (NTA) measurements of the nGel-KGN particles in 2.5 mM HEPES buffer at 1.25 mg/mL were performed with a NanoSight LM10 instrument at 25 °C and 37 °C.
  • the video frame of nGel-KGN particles was extracted via the ImageJ software.
  • the ultraviolet-visible (UV-Vis) spectrum of KGN, nGel, and nGel-KGN was measured using a UV-Vis spectrometer (UV-3600 Plus, Shimadzu, Japan) .
  • the KGN, nGel, and nGel-KGN were dispersed in DMSO to prepare a transparent suspension.
  • the UV-Vis spectrum was recorded from 450 nm to 220 nm at a scanning rate of 0.5 nm/s.
  • TEM scanning transmission electron microscopy
  • EDS energy X-ray dispersive spectroscopy
  • TEM Transmission electron microscopy
  • nGel-KGN particles displayed a spherical shape with an average size of 44.9 ⁇ 11.6 nm in the dry state (FIGS. 2 and 3) .
  • a significant increase of the hydrodynamic size (73.1 ⁇ 0.8 nm at 25 °C) was observed for the nGel-KGN particles after being swollen by HEPES buffer (FIG. 4) .
  • the lower critical solution temperature (LCST) of the thermoresponsive PNIPMAM polymer is ⁇ 44 °C
  • the nGel-KGN particles remained highly swollen with negligible shrinkage (67.9 ⁇ 0.1 nm) when the temperature was increased to 37 °C, implying satisfactory phase stability in the body (FIG.
  • the small portion of the non-solvent i.e., DMF
  • DMF non-solvent
  • the subsequent coagulation of secondary particles contributed to a highly interconnected microporous structure in the resulting dry membrane.
  • the surface porosity decreased, leading to the formation of a targeting selective-permeable microporous layer with homogenously dispersed nGel-KGN particles (FIG. 7) , which can exclude the scar tissue interference and deliver chondro-inductive cues in vivo.
  • Struvite nanowires with nano-sized diameter and high phase purity were successfully synthesized through a controlled crystallization strategy (FIG. 8) .
  • These nanowire-like particles were then encapsulated into the core-shell polymer fibers via co-axial electrospinning.
  • the core-shell structures of the PCL/St. -GelA nanofibers were visualized by TEM imaging.
  • the struvite nanowire was embedded within the PCL core along the fiber orientation (FIG. 10) , as further verified by the strong signal of the characteristic compositional elements, Mg and P, in EDS analysis (FIG. 11) .
  • tissue mimetic membranes with different structures, thickness, and compositions were fabricated: 500 ⁇ m casting + ES (Grp 1) , 500 ⁇ m casting/5%nGel-KGN + ES/4%St (Grp 2) , and 1000 ⁇ m casting/5%nGel-KGN + ES/4%St (Grp 3) (FIGS. 7, 12, 13) .
  • the cross-sectional morphological analysis revealed the good cohesion of the two layers in three groups, demonstrating the well-constructed biphasic structures (FIG. 12) .
  • Example 5 Mechanical performance, controllable release behavior, and biodegradation tendency of the tissue mimetic membranes
  • the mechanical properties of the membranes at wet state were examined with tensile tests using a material test system.
  • a release test was used to determine in vitro release and degradation behaviors of the membranes.
  • the testing was conducted on a shaker with an agitation rate of 100 rpm, in a 37 °C incubator, for 14 days. At predetermined time intervals, the soaking liquid was partially collected and replaced by an equal volume of fresh PBS.
  • Concentrations of magnesium ions were determined using an Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES) . Concentrations of released nGel-KGN particle were determined using a fluorescence spectrometer (F-7000, Hitachi, Japan) . The cumulative released concentration of Mg 2+ and nGel-KGN particles from a 2 cm ⁇ 2 cm membrane was plotted against the time point to obtain the release profile of each membrane. The concentration of Mg 2+ and nGel-KGN particles released at an indicated time point was presented as the concentration of Mg 2+ released from a 2 cm ⁇ 2 cm membrane in 5 mL PBS. This data was plotted against time to obtain the release level at each indicated time point for each membrane.
  • the weight loss ratio (wt%) at the indicated time point was calculated according to Equation 1, in which m 0 represents the initial weight of the sample, and m 1 denotes the sample weight after degradation for the prescribed period.
  • the weight loss ratio was plotted against time point for each membrane to determine a degradation profile. The data is expressed as the mean ⁇ SD.
  • Weight loss ratio (wt. %) [ (m 0 -m 1 ) /m 0 ] ⁇ 100% (Equation 1)
  • SEM Quanta-400, FEI, USA
  • nGel-KGN and struvite can easily be transferred from the membrane matrix to the surrounding microenvironment during implantation.
  • the release tendency of nGel-KGN was similar in Grp 2 and Grp 3, where a quick release occurred in the first day, followed by a more controllable release rate thereafter (FIG. 18) .
  • the increased thickness of the nGel-KGN-laden microporous layer contributed to a higher loading amount of nGel-KGN in Grp 3, so the cumulative concentration of nGel-KGN was significantly higher in Grp 3 ( ⁇ 3.29 mg/mL) than that in Grp 2 ( ⁇ 1.29 mg/mL) after the 14-day release.
  • the difference between nGel-KGN concentration level released from the two membranes became smaller after the third day (FIG. 18) .
  • Mg ions Mg 2+
  • concentration of the major degradation products were measured, where these degradation products also play a potentially key role in promoting osteogenesis.
  • a rapid increase of Mg 2+ level was observed in both groups (Grp 1: ⁇ 0.35 mM; Grp 2: ⁇ 0.46 mM) after 1 day of release, following which the release rate gradually slowed (FIG. 19) .
  • the release levels of Mg 2+ continued to be comparable in Grp 2 and Grp 3, where these comparable levels corresponded to the similarity of the configuration of the struvite-incorporated fibrous layer.
  • membranes were cut into squares having a size of 15 mm ⁇ 15 mm and sterilized by being immersed in 75%ethanol for 30 min and being exposed to ultraviolet (UV) radiation for 2 h.
  • UV radiation ultraviolet
  • the sterile samples were mounted onto CELLCROWN TM transwells and fixed into a 24-well plate.
  • MC3T3-E1 cells were then seeded onto the membrane at a density of 10,000 cells/cm 2 . After a 3-day cultivation, the samples were rinsed and then fixed with 3.7%formaldehyde.
  • the samples were stained with Alexa Fluor 546 phalloidin and DAPI and then imaged with a Nikon Eclipse Ti inverted microscope at exciting wavelengths of 543 nm (red, actin) and 408 nm (blue, nucleus) under a Z-stack mode with a step size of 1 ⁇ m.
  • the Z-scanning started when the cells on the membrane first appeared and ended when the cells disappeared within the frame.
  • the sequence of images (512 pixel ⁇ 512 pixel) was then processed with ImageJ software.
  • MC3T3-E1 cells were seeded on the fibrous surfaces of sterilized samples as described above, at a density of 10,000 cells/cm 2 . On day 3, samples were rinsed and then fixed with 2.5%glutaraldehyde overnight at 4 °C. After fixation, the samples were washed with PBS (10 ⁇ , pH 7.40) to remove the residual crosslinking agent, and then subjected to gradient dehydration with 30, 50, 70, 90, 95, and 100 vol%ethanol. Finally, the samples were dried in the air overnight.
  • PBS 10 ⁇ , pH 7.40
  • the samples were then imaged through SEM at 10 kV after being sputtered with Au nanoparticles. Pseudocolor was added to the cells in the SEM images using ImageJ software. Cell spreading areas were then measured based on the SEM images using ImageJ software. The measurements were performed on 10–12 random cells from each sample.
  • the barrier function of membranes was evaluated using NIH3T3 fibroblasts. After sterilization, a membrane with a size of 15 mm ⁇ 15 mm was mounted on a CELLCROWN TM transwell and fixed in a 24-well culture plate. NIH3T3 cells were carefully seeded on the microporous surface of the membrane at a density of 10,000 cells/cm 2 . The cell culture medium was refreshed every 2 days. After culturing for 5 days, the two sides of the membrane were observed by SEM after fixation, following the same protocol described in the cell adhesion test.
  • rBMSCs rat bone marrow mesenchymal stem cells
  • ALP alkaline phosphatase
  • ARS Alizarin Red S
  • ALP staining was performed after induction for 7 days according to the manufacturer’s instructions, and the stained samples were then imaged with a digital camera and an optical microscope. After induction for 10 days, ARS staining was performed according to the manufacturer’s instructions, and the stained samples were then imaged. In both ALP and ARS assays, three parallel samples were used for each type of membrane.
  • Chondrogenic differentiation of rBMSCs on the prepared membranes was evaluated through Alcian blue staining.
  • a membrane with a size of 15 mm ⁇ 15 mm was mounted on a CELLCROWN TM transwell and fixed in a 24-well culture plate.
  • Cells at passage 2 were seeded on the membranes at a density of 75,000 cells per membrane and cultured for 2 days to allow for optimal attachment.
  • the cell culture medium was then replaced by the chondrogenic induction medium for further induction.
  • the medium was refreshed every 2 days.
  • Alcian blue/nuclear fast red staining was performed according to the manufacturer’s instructions, and the stained samples were then imaged.
  • rBMSCs For determining the in vitro expression of fibrocartilage markers, the chondrogenic differentiation of rBMSCs was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) to detect the expression of fibrocartilage marker, SRY-box transcription factor 9 (Sox9) .
  • RT-qPCR quantitative real-time polymerase chain reaction
  • Sox9 SRY-box transcription factor 9
  • a membrane with a size of 35 mm ⁇ 35 mm was mounted on a CELLCROWN TM transwell and fixed in a 6-well culture plate.
  • rBMSCs at passage 2 were seeded on the membranes at a density of 150,000 cells per membrane and cultured for 2 days to allow for optimal attachment.
  • the cell culture medium was replaced by the chondrogenic induction medium for further induction.
  • the medium was refreshed every 2 days.
  • the microporous layer is put near the tendon during in vivo repair to exclude invasive fibrovascular scar tissue at early stage.
  • the NIH3T3 fibroblasts grew well on all the three microporous surfaces, while no cell was found on the opposite fibrous surfaces, demonstrating the robust barrier effect (FIG. 24) .
  • the attached fibroblasts displayed flattened and spread morphologies with a cellular layer formation, especially in Grp 2 (FIG. 25) . Because normal tendon is populated by elongated fibroblasts interspersed within aligned collagen fibrils, the unmineralized microporous layer can guide tendon-to-membrane ingrowth at the late stage of implantation.
  • nGel-KGN may induce an adverse effect, as evidenced by observed suppressed expression of Sox9, and the lower cell density in Grp 3 as compared to Grp 2 (FIGS. 18, 26, and 27) .
  • ALP alkaline phosphatase
  • significantly enhanced alkaline phosphatase (ALP) activity of the rBMSCs in Grp 2 after the 7-day incubation was confirmed by the most intense purple stain in comparison with the other two groups (FIG. 28) .
  • Alizarin Red S (ARS) staining was applied to visualize ECM mineralization, a late-stage osteogenic biomarker, after a 10-day incubation.
  • Grp 2 displayed the darkest red stain along with the most calcium nodule formation among the three groups, thereby demonstrating substantially optimized osteogenesis, which is likely attributed to the osteoinductive struvite (FIG. 29) . Similar to the chondrogenesis, suppressed osteogenesis also appeared in Grp 3 as compared to Grp 2. This may be due to a negative effect on the stem cell proliferation and aggregation from excessive nGel-KGN particles.
  • nGel-KGN and struvite can contribute to a bio-inductive microenvironment and drive the bi-lineage differentiation of stem cells, in a manner that can be dependent on specific dosing of nGel-KGN.
  • Example 7 Regenerative capacity of tissue mimetic membranes for the repair of tendon- bone interface in rotator cuff tear (RCT) rats
  • mice Animal surgeries were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong (Ref. No.: 22-214-GRF) .
  • the rats were randomly allocated into four RCT groups: control group (Ctrl, without membrane implantation) , group 1 (echoing to Grp 1) , group 2 (echoing to Grp 2) , and group 3 (echoing to Grp 3) .
  • the rats were anesthetized by intraperitoneal injection of 75 mg/kg ketamine and 10 mg/kg xylazine. Rats were placed on a warming operating table while respiration and heart rates were monitored. A deltoid splitting incision with the length of 1.5–2 cm was made.
  • the supraspinatus tendon was cut off at the bone insertion of the greater tuberosity. All soft tissues and fibrocartilage at the tendon–bone interface were debrided. Two non-absorbable 5-0 sutures were passed through the supraspinatus tendon in Mason-Allen fashion and bone tunnels drilled using a 25-gauge needle into the greater tuberosity. The membrane (3 mm ⁇ 3 mm, width ⁇ length) was inserted between the supraspinatus tendon and bone, with the microporous layer facing the tendon. The wound was subsequently sutured. All procedures were performed under aseptic conditions. Rats were sacrificed by intraperitoneal injection of an overdose of sodium pentobarbital at weeks 4 and 8 post-surgeries.
  • Gait analysis was performed to evaluate gait abnormality using the Catwalk XT 9.0 system. Each rat was trained to be familiar with the glass walkway where the rat walked ad libitum for paw print picking. Paw prints were automatically recorded when the rat entered the region of interest (ROI) . Paw prints were set as left forelimb, right forelimb, left hindlimb, and right hindlimb by the built-in software. Successful records for each rat included three times of one crossing that allowed a maximum 60%speed variation without any interruption. The paw prints were manually checked in the system to maintain correctness of the classifications. Gait parameters measured at week 8 post-surgery included the stand, maximal contact area, maximal contact AT, maximal intensity AT, swing, stride length, single distance, and duty cycle.
  • the humeral joints were scanned by a vivaCT40 micro-computed tomography (micro-CT) imaging system with a resolution of 19 ⁇ m per voxel size.
  • the scanner was set at a voltage of 70 kVp and a current of 114 ⁇ A. Twenty slices of the humeral subchondral bone around the growth plate were used to reconstruct a 3D image using the built-in software.
  • the biocompatibility of the implanted membranes was evaluated by SEM observations. Briefly, paraffin sections (5 ⁇ m thick) were chronologically dewaxed for 10 min twice using absolute xylene, and then immersed in absolute ethanol for 5 min. After air drying, the sections were imaged with SEM at 10 kV after sputtering with Au nanoparticles.
  • Rats were euthanized at weeks 4 and 8 post-surgeries.
  • the isolated humeral joints were fixed in 4%paraformaldehyde (PFA) for 48 hours. Then the joints were decalcified in 12.5%ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 21 days at room temperature. The EDTA solution was changed every four days.
  • the joints were embedded in paraffin and sectioned to 5 ⁇ m thickness for histological analysis. Sections were stained with hematoxylin and eosin (H&E) to observe the healing progress of tendon–bone interface. Safranin O/Fast green and Picro-Sirius Red staining were respectively performed for evaluating the fibrocartilaginous interface regeneration according to the established protocols.
  • X-ray and micro-CT imaging of the humerus indicated enhanced bone regeneration along with superior tissue–material integration in Grp 2 as compared to the other groups, implying high efficiency in stimulating the tendon–bone reattachment (FIGS. 32 and 33) .
  • SEM analysis showed extensive fibrous and cellular proliferation within the membrane layers, with cells displaying adaptability across all membrane-treated groups, especially for those in Grp 2 (FIG. 34) .
  • the significant differences in relative Stand, Max Contact Area, and Swing demonstrated substantially improved functional behaviors of the rats in Grp 2, in comparison with the control group (FIGS. 35 and 36) .
  • the Grp 2 membrane provided a particularly effective augmentation for restoring the normal function of the rotator cuff, by mimicking the heterogeneous compositional and structural features of the native enthesis.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
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  • Prostheses (AREA)

Abstract

L'invention concerne des nanogels chondro-promoteurs, et des membranes multicouches avec une couche comprenant une dispersion des nanogels chondro-promoteurs, et une autre couche comprenant des agents ostéo-promoteurs encapsulés. Les matériaux fournis sont particulièrement utiles pour guider la régénération d'interface fibrocartilagineuse chez un sujet ayant une interface tendon-os lésée. L'invention concerne également des procédés de production des membranes multicouches divulguées, et des procédés d'utilisation des membranes multicouches pour la réparation d'interfaces tendon-os.
PCT/CN2025/089215 2024-04-24 2025-04-16 Membranes mimétiques de tissu pour guider la régénération d'interface fibrocartilagineuse dans la réparation de coiffe de rotateur Pending WO2025223272A1 (fr)

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