Method for directly preparing acellular hypertrophic cartilage matrix from adipose tissue

A technology of adipose tissue and cartilage matrix, applied in the direction of bone/connective tissue cells, animal cells, tissue regeneration, etc., can solve the problems of low osteogenesis efficiency and low osteoinductive performance, and achieve high differentiation potential and reduced immunogenicity Effect

Active Publication Date: 2021-08-27
SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0003] The present invention proposes a method for directly preparing acellular hypertrophic cartilage matrix by using adipose tissue, which solves the problems of low osteoinductive performance and low osteogenesis efficiency in vivo of acellular allogeneic bone graft materials in the prior art

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  • Method for directly preparing acellular hypertrophic cartilage matrix from adipose tissue
  • Method for directly preparing acellular hypertrophic cartilage matrix from adipose tissue
  • Method for directly preparing acellular hypertrophic cartilage matrix from adipose tissue

Examples

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Embodiment 1

[0045] A method for directly preparing acellular hypertrophic cartilage matrix from adipose tissue, comprising the following steps:

[0046] (1) Preparation of particulate adipose tissue;

[0047] The preparation of particulate adipose tissue is a mature technique in the art, and the steps in this example are as follows:

[0048] (a) Human adipose tissue obtained from liposuction operation, after repeated rinsing with saline, the upper layer of adipose tissue was collected;

[0049] (b) Cut the upper layer of fat tissue with scissors, centrifuge at 1000-2000g for 3-5min, remove the upper layer of fat and the lower part of swelling fluid, and collect the middle fat layer in a 20mL syringe;

[0050] (c) Use a three-way tube to connect two 20mL syringes, inject the two syringes back and forth 30 times, and obtain particulate adipose tissue;

[0051] (d) The particulate adipose tissue is centrifuged at 1000-2000 g for 3-5 minutes to remove the upper layer of fat layer, and the l...

Embodiment 2

[0077] The culture medium used in this embodiment consists of the following:

[0078] Serum-free basal medium (SFM):

[0079] DMEM culture fluid + 1% HSA + 1% PSG + 1% HEPES + 2% ITS + 1.2% linoleic acid;

[0080] Hypertrophy Inducing Medium:

[0081] SFM+β-glycerophosphate disodium salt (10 -2 mol / L)+dexamethasone (10 -8 mol / L)+ascorbic acid (10 -5 mol / L).

[0082] Chondrogenic induction medium: SFM+dexamethasone (10 -7 mol / L)+ascorbic acid (10 -5 mol / L)+BMP-6(20ng / mL)+TGF-β 3 (5ng / mL);

[0083] Applying the above medium to the method in Example 1 also meets the requirements.

Embodiment 3

[0085] The culture medium used in this embodiment consists of the following:

[0086] Chondrogenic induction medium:

[0087] SFM+BMP-6(5ng / mL)+TGF-β 3 (20ng / mL)+dexamethasone (10 -7 mol / L)+ascorbic acid (10 -5 mol / L);

[0088] Hypertrophy Inducing Medium:

[0089] SFM+β-glycerophosphate disodium salt (10 -2 mol / L)+dexamethasone (10 -8 mol / L)+ascorbic acid (10 -5 mol / L).

[0090] Serum-free basal medium (SFM):

[0091] DMEM culture fluid+1%HSA+1%PSG+1%HEPES+0.5%ITS+0.3%linoleic acid;

[0092] Applying the above medium to the method in Example 1 also meets the requirements.

[0093] Our study found that ASC in adipose tissue can exist in the form of stem cell niche under the protection of extracellular matrix (ECM), and ASC in its protection can form endochondral bone , directly induce differentiation into hypertrophic cartilage tissue. Moreover, the hypertrophic cartilage tissue can form ectopic bone even under the skin of nude rats, regenerating bone tissue rich i...

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Abstract

The present invention proposes a method for directly preparing acellular hypertrophic cartilage matrix from adipose tissue, which includes: (1) after in vitro proliferation and culture of particulate adipose tissue, adipose-rich mesenchymal stem cells (ASC) are formed and still have multiple (2) Construction of hypertrophic cartilage tissue in vitro; (3) Preparation of decellularized hypertrophic cartilage matrix. The in vitro endochondral osteogenic induction culture includes two stages of chondrogenic induction culture and hypertrophy induction culture; firstly use chondrogenic induction medium to induce culture, and then use hypertrophy induction medium to induce culture; the above hypertrophic cartilage tissue is decellularized, namely Acellular hypertrophic cartilage matrix was obtained. The immunogenicity of the decellularized hypertrophic cartilage matrix prepared by the invention is greatly reduced, and can be used for allogeneic transplantation.

Description

technical field [0001] The invention relates to the technical field of bone tissue regeneration, in particular to a method for directly preparing decellularized hypertrophic cartilage matrix from adipose tissue. Background technique [0002] At present, the stem cell-based tissue engineering bone construction method is to inoculate freshly obtained stem cells on biological materials after multiple passages and expansions in vitro, and then after a period of in vivo and in vitro cultivation, regenerate bone cells that can be used for bone repair. Defective bone graft. However, the current method has the following problems: 1) Stem cells gradually lose their multi-directional differentiation potential, including bone regeneration ability after long-term passage and proliferation in vitro; 2) The traditional intraperiosteal osteogenesis induction differentiation protocol and insufficient vascularization ability; 3) The bone graft constructed from single stem cells lacks angiog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077A61L27/36A61L27/38
CPCA61L27/3604A61L27/365A61L27/3654A61L27/3834A61L27/3847A61L27/3852A61L2430/02A61L2430/06
Inventor 黄如林李青峰王文进李佳奇傅娆
Owner SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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