WO2023040853A1 - Complexe périoste-os pour la reconstruction d'un environnement de réparation immunitaire de tissu mou-os et son procédé de préparation - Google Patents

Complexe périoste-os pour la reconstruction d'un environnement de réparation immunitaire de tissu mou-os et son procédé de préparation Download PDF

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WO2023040853A1
WO2023040853A1 PCT/CN2022/118561 CN2022118561W WO2023040853A1 WO 2023040853 A1 WO2023040853 A1 WO 2023040853A1 CN 2022118561 W CN2022118561 W CN 2022118561W WO 2023040853 A1 WO2023040853 A1 WO 2023040853A1
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bone
periosteum
pbs buffer
complex
hours
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PCT/CN2022/118561
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English (en)
Chinese (zh)
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林贤丰
赵晨晨
范顺武
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浙江大学医学院附属邵逸夫医院
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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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention mainly relates to the biological field for bone tissue regeneration and repair, specifically a periosteum-bone complex and its preparation method aimed at rebuilding the soft tissue-bone immune repair environment.
  • the impact of bone repair rational use of the advantages of spatial composite mode scaffolds to promote bone regeneration and repair.
  • Bone defects caused by trauma, tumors and infections are common clinical diseases that affect human health, especially multiple complex bone defects, which often require repeated treatment, causing heavy psychological pressure and economic burden on individuals, and affecting social stability.
  • the potential impact is an urgent problem to be solved in clinical orthopedics.
  • Natural bone is a highly vascularized hard tissue covered by soft periosteum.
  • the periosteum can serve as a cellular barrier against excessive penetration of inflammatory cells in a traumatic environment, and it is also a physical barrier that blocks muscle and fibrous tissue from filling the defect.
  • the periosteum provides sufficient blood supply and nutrition for the cortical bone, and also provides a supporting microenvironment for the differentiation and maturation of mesenchymal stem cells.
  • hydrogels derived from periosteal extracellular matrix can enhance the M2 polarization of macrophages in the early stage of bone injury and play an immunomodulatory role.
  • the present invention obtains a space-patterned periosteum-bone complex through an innovative extracellular matrix preparation technology, proving that it has Regulate the performance of the immune microenvironment in the early stage of soft tissue-bone injury, coordinate subsequent angiogenesis and osteogenesis events, and promote bone healing.
  • periosteum-bone complex there is no report on the preparation of periosteum-bone complex.
  • the purpose of the present invention is to fill in the prior art, single-phase bone repair biological scaffolds are difficult to effectively avoid soft tissue-derived inflammatory infiltration, which aggravates the lack of immune regulation in the early stage of bone defect repair, and therefore provides a space pattern periosteum for rebuilding the soft tissue-bone immune repair environment - Bone complex and method for its preparation.
  • the preparation method of the space pattern periosteum-bone complex of natural animal origin comprises the following steps:
  • steps (5)-(9) rinse with double distilled water for 3-6 hours after each step is completed.
  • the mass concentration of EDTA-Na 2 is 5%-20%;
  • PBS buffer containing protease inhibitors the concentration of protease inhibitors is 10-50K IU/ml;
  • the PBS buffer containing Triton X is Triton X-100 PBS buffer with a mass concentration of 0.01%-5%;
  • the PBS buffer solution containing SLES is the PBS buffer solution of SLES with a mass concentration of 0.01%-5%;
  • the PBS buffer solution containing DNase I is the PBS buffer solution with a concentration of 1-2mg/mL;
  • Tris-HCl buffer solution Containing Tris-HCl buffer solution, the concentration of Tris-HCl is 5-20mM;
  • the concentrations of penicillin and streptomycin in the mixed antibacterial solution were 20 U/ml and 20 ⁇ g/ml respectively; the ratio of penicillin and streptomycin was 1:1, and the volume ratio of the added mixed antibacterial solution to the original solution was 5:1.
  • the present invention aims at the problems existing in clinical substitutes for soft tissue combined with bone injury, and establishes a preparation method of a periosteum-bone complex scaffold derived from natural allogeneic tissue. , PBS buffer containing protease inhibitors, PBS buffer containing Triton X-100, PBS buffer containing SLES, PBS buffer containing DNase I and Tris-HCl buffer after detoxification treatment.
  • PBS buffer containing protease inhibitors PBS buffer containing Triton X-100
  • PBS buffer containing SLES PBS buffer containing DNase I
  • Tris-HCl buffer after detoxification treatment.
  • the periosteum-bone scaffold used in the present invention is a space-patterned periosteum-bone complex obtained from an allogeneic natural source using an innovative method combining physical freeze-thaw, ultrasonic decalcification, and combined decellularization techniques.
  • the material of the present invention is detoxified by repeated rinsing with Tris-HCl buffer and sterile saline, the scaffold has low cytotoxicity, has good biocompatibility and main bioactive components, and has a natural complex structure that is difficult to replicate .
  • the material prepared by the present invention can be used as an immune barrier to participate in the transformation of macrophage phenotype in the early stage of bone repair.
  • the material prepared by the present invention has the ability to coordinate early immune regulation and subsequent osteogenesis-angiogenesis events, and has the ability to promote bone repair.
  • Fig. 1 is material general figure and CT scanning figure of the present invention
  • Fig. 2 is material H&E staining of the present invention, DAPI staining figure and DNA quantitative detection figure;
  • Fig. 3 is material Sirius red staining of the present invention, polarized light observation, collagen and GAGs quantitative detection figure;
  • Fig. 4 is scanning electron micrograph of each part microstructure of material of the present invention.
  • Fig. 5 is the atomic force microscope diagram, pressure-strain curve diagram and stiffness calculation value of the material of the present invention
  • Figure 6 is a diagram showing that the material of the present invention inhibits the polarization of macrophage M1 and inhibits the expression of inflammation-related genes;
  • Fig. 7 is a graph showing that the material of the present invention promotes the polarization of macrophage M2 and promotes the expression of anti-inflammatory related genes;
  • Figure 8 is the material of the present invention promotes the expression of osteogenesis-related markers
  • Fig. 9 is a diagram showing that the material of the present invention promotes blood vessel migration
  • Fig. 10 is a CT scan and histological evaluation of bone repair promoted by the material of the present invention.
  • Embodiment 1 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
  • the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
  • Example 2 After each step in Example 1, a periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and a spatial complex structure is finally obtained.
  • the collagen arrangement, bone crystal structure and periosteum-bone interface connection structure of the present invention were observed by a scanning electron microscope, as shown in FIG. 3 .
  • the atomic force microscope observes the microscopic surface topography of the present invention, as shown in Fig. 4 .
  • Mechanomechanical test evaluation records the pressure-strain curve, and calculates the stiffness of the periosteal phase and bone phase of the periosteal bone scaffold in the space mode, as shown in Figure 5.
  • the periosteum-bone complex scaffold affects the phenotype of macrophages.
  • the periosteum phase inhibits the polarization of macrophages to the M1 direction and inhibits the expression of inflammation-related genes; the bone phase inevitably induces the polarization of macrophages to the M1 direction, as shown in Figure 6;
  • the periosteum phase of the scaffold of the periosteum-bone complex induces the polarization of macrophages in the M2 direction and promotes the expression of anti-inflammatory related genes, as shown in Figure 7.
  • Example 1 the periosteum-bone complex scaffold promotes osteogenesis performance verification
  • the periosteum-bone complex scaffold promoted the expression of osteogenesis-related genes (Runx2, ALP, Col 1a1, OPN, OCN), as shown in Figure 8.
  • Example 7 the periosteum-bone complex scaffold promotes angiogenesis performance verification
  • the periosteum-bone complex scaffold promotes blood vessel formation and migration, as shown in Figure 9.
  • Rats with bone defects were divided into four groups, namely the simple defect group, the softened acellular bone treatment group, and the periosteum-bone complex treatment group.
  • the corresponding scaffolds were filled in the bone defects respectively. After 4 weeks and 8 weeks of treatment, it can be found that the effect of the periosteum-bone complex treatment group is significantly better than that of the other three groups.
  • Figure 10 The effect of the periosteum-bone complex treatment group is significantly better than that of the other three groups.
  • Embodiment 2 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
  • the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
  • Embodiment 3 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
  • the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
  • Embodiment 4 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
  • the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
  • the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
  • the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
  • Example 7 The periosteum-bone complex and its preparation method aimed at rebuilding the soft tissue-bone immune repair environment
  • the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
  • Examples 2-7 all can prepare periosteum-bone complex decellularized materials with complete decellularization, well-retained active ingredients and complete three-dimensional structure, and the corresponding results are basically consistent with those of Example 1.

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  • Animal Behavior & Ethology (AREA)
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Abstract

L'invention concerne un complexe périoste-os pour reconstruire un environnement de réparation immunitaire de tissu mou-os et son procédé de préparation. Le procédé de préparation comprend : la coupe et l'apprêt, le rinçage répété avec de l'eau désionisée, la congélation et la décongélation avec de l'azote liquide, la réalisation d'une décalcification par ultrasons et le traitement avec une solution tampon PBS contenant un inhibiteur de protéase, une solution tampon PBS contenant du Triton X-100, une solution tampon PBS contenant du SLES, une solution tampon PBS contenant de la DNase I et une solution tampon Tris-HCl pour obtenir un matériau complexe périostum-os exempt de cellules. Le complexe conserve une bonne structure spatiale périoste-os et des principes actifs extracellulaires, fournit une interface biphasique « perichondrium-sclérotine », bloque efficacement l'infiltration excessive d'inflammation d'une source de tissu mou, participe au remodelage immunitaire précoce d'une lésion de tissu mou-os, et forme un microenvironnement de réparation immunitaire bénigne en combinant ses avantages d'ostéogenèse et de vascularisation propres du complexe. Une nouvelle manière de penser est fournie pour favoriser la régénération et la réparation de tissu osseux à l'aide d'un matériau biologique.
PCT/CN2022/118561 2021-09-18 2022-09-13 Complexe périoste-os pour la reconstruction d'un environnement de réparation immunitaire de tissu mou-os et son procédé de préparation WO2023040853A1 (fr)

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CN202111127749.9A CN113975464B (zh) 2021-09-18 2021-09-18 重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法
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CN113975464B (zh) * 2021-09-18 2022-07-22 浙江大学医学院附属邵逸夫医院 重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法

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