CN109675108B - Method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold material - Google Patents

Method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold material Download PDF

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CN109675108B
CN109675108B CN201910119844.0A CN201910119844A CN109675108B CN 109675108 B CN109675108 B CN 109675108B CN 201910119844 A CN201910119844 A CN 201910119844A CN 109675108 B CN109675108 B CN 109675108B
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tissue
adipose tissue
culture
adipose
induction
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CN109675108A (en
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黄如林
李青峰
王文进
李佳奇
傅娆
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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    • 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
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    • 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
    • A61L27/38—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 containing added animal cells
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/3843—Connective tissue
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    • 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
    • A61L27/38—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 containing added animal cells
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    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
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    • 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
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus

Abstract

The invention provides a method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and a scaffold material, which comprises the following steps: (1) adding a scaffold material into the particulate adipose tissue; (2) carrying out in-vitro proliferation culture to form a composite tissue block which is rich in adipose-derived mesenchymal stem cells (ASC) and still has multidirectional differentiation potential; (3) in vitro construction of hypertrophic cartilage tissue. The scaffold material is added in the invention, and the added scaffold material has the following functions: (1) provides an attachment surface for cells in adipose tissues, can promote the proliferation and migration of ASC, and increases the number of ASC; (2) promote the mineralization of hypertrophic cartilage tissue and further promote the regeneration of bones; (3) promote the chemotaxis of osteoclast, and further promote the bone reconstruction and the bone marrow regeneration; (4) as a bone graft substitute, clinical controllability is increased.

Description

Method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold material
Technical Field
The invention relates to the technical field of bone tissue regeneration, in particular to a method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and a scaffold material.
Background
The traditional bone tissue engineering technology is that stem cells are planted on a biological material, after in vitro culture is carried out for a period of time, the stem cells are differentiated into osteocytes on the biological material, ossein is secreted, and finally, a new bone tissue is regenerated and then transplanted to a bone defect part to repair the defects of function and appearance. At present, two major challenges are faced in transforming and applying the bone tissue engineering technology of the laboratory to the clinic: on the one hand, it is difficult to obtain stem cells with easily available materials, small damage and strong bone regeneration capability. Currently, stem cell-based tissue engineeringThe process technology is mainly used for obtaining a sufficient number of stem cells from a limited clinical tissue sample by an in vitro amplification method and then is used for laboratory bone tissue construction. However, the osteogenic differentiation capacity of stem cells gradually decreases with the in vitro expansion by passage2The ability of the stem cells after multiple passages to induce bone regeneration in vivo is remarkably reduced. On the other hand, the bone tissue constructed in vitro lacks effective vascular system, and the large volume of tissue engineered bone tissue is difficult to revascularize with the receiving area after being transplanted to the receiving area, thereby causing the failure of the transplantation operation3,4. Effectively solving these two problems will clear the barrier for clinical transformation of tissue engineering technology. Because Adipose tissue is present in large amounts in the human body, is easy to obtain, and has little damage to the body, more and more researchers are beginning to use Adipose-derived stem cells (ASCs) for bone regeneration research. Studies have shown that ASC can differentiate into osteoblasts and chondrocytes under different in vitro induction conditions5,6(ii) a In animal experiments, the ASC-loaded biological material is implanted to the bone defect part after in-vitro osteogenic induction culture, and can promote the bone regeneration of the defect part7,8The repairing effect is similar to the same type of biological material loaded with Bone marrow mesenchymal stem cells (BMSC)9,10. Although ASCs have been shown to be effective in inducing bone regeneration, one problem that is not negligible is that ASCs also gradually lose their multipotentiality as Mesenchymal Stem Cells (MSCs) due to increased number of passages.
The current method has the following problems: 1) after long-time in vitro passage, the stem cells gradually lose multidirectional differentiation potential, including bone regeneration capacity; 2) the traditional periosteal osteogenic differentiation scheme has insufficient bone regeneration capacity; 3) bone grafts constructed from a single stem cell lack hemangiogenic capacity.
Because ASC has the function of differentiating into various cells, research shows that ASC can differentiate into osteoblasts and chondrocytes under different in vitro induction conditions, thereby achieving the purpose of treating bone defects. Researches find that the Extracellular matrix (ECM) can provide a three-dimensional living space for stem cells, regulate various biological signal molecules to enter stem cell nests, and play an important role in regulating the biological functions and differentiation fate of the stem cells.
Reference documents:
1.Campana V,Milano G,Pagano E,Barba M,Cicione C,Salonna G,Lattanzi W,Logroscino G.Bone substitutes in orthopaedic surgery:from basic science to clinical practice.J Mater Sci Mater Med.2014；25(10):2445-2461.
2.Di Maggio N,Martella E,Frismantiene A,Resink TJ,Schreiner S,Lucarelli E,Jaquiery C,Schaefer DJ,Martin I,Scherberich A.Extracellular matrix and alpha5beta1integrin signaling control the maintenance of bone formation capacity by human adipose-derived stromal cells.Sci Rep.2017；7:44398.
3.Mercado-Pagan AE,Stahl AM,Shanjani Y,Yang Y.Vascularization in bone tissue engineering constructs.Ann Biomed Eng.2015；43(3):718-729.
4.Liu Y,Chan JK,Teoh SH.Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems.J Tissue Eng Regen Med.2015；9(2):85-105.
5.Mellor LF,Mohiti-Asli M,Williams J,Kannan A,Dent MR,Guilak F,Loboa EG.Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells:A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source.Tissue Eng Part A.2015；21(17-18):2323-2333.
6.Ge W,Liu Y,Chen T,Zhang X,Lv L,Jin C,Jiang Y,Shi L,Zhou Y.The epigenetic promotion of osteogenic differentiation of human adipose-derived stem cells by the genetic and chemical blockade of histone demethylase LSD1.Biomaterials.2014；35(23):6015-6025.
7.Hung BP,Hutton DL,Kozielski KL,Bishop CJ,Naved B,Green JJ,Caplan AI,Gimble JM,Dorafshar AH,Grayson WL.Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells.Stem Cells.2015；33(9):2773-2784.
8.Park HJ,Yu SJ,Yang K,Jin Y,Cho AN,Kim J,Lee B,Yang HS,Im SG,Cho SW.Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering.Biomaterials.2014；35(37):9811-9823.
9.Hiwatashi N,Hirano S,Mizuta M,Tateya I,Kanemaru S,Nakamura T,Ito J.Adipose-derived stem cells versus bone marrow-derived stem cells for vocal fold regeneration.Laryngoscope.2014；124(12):E461-469.
10.Han DS,Chang HK,Kim KR,Woo SM.Consideration of bone regeneration effect of stem cells:comparison of bone regeneration between bone marrow stem cells and adipose-derived stem cells.J Craniofac Surg.2014；25(1):196-201.
disclosure of Invention
The invention provides a method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and a scaffold material, which solves the problems that the multidirectional differentiation capacity of the ASC as mesenchymal stem cells is weakened after long-time in-vitro passage in the prior art and the like.
The technical scheme of the invention is realized as follows:
a method for directly regenerating hypertrophic cartilage tissue using adipose tissue, comprising:
(1) in vitro proliferation culture of mixed tissues
Adding a scaffold material into the particulate adipose tissue, and uniformly stirring to obtain a mixed tissue;
after the mixed tissue is subjected to in vitro proliferation culture, a tissue block which is rich in ASC and still has multidirectional differentiation potential is formed;
(2) in vitro construction of hypertrophic cartilage tissue
Taking the sample after the in-vitro proliferation culture in the step (1), and directly differentiating into hypertrophic cartilage tissue through in-vitro endochondral osteogenesis induction culture;
wherein, the in vitro endochondral osteogenesis induction culture comprises two stages of chondrogenesis induction culture and hypertrophy induction culture; the induction culture is performed by using a chondrogenic induction medium, and then the induction culture is performed by using a hypertrophic induction medium.
The preparation method of the mixed tissue comprises the following steps:
(a) after the adipose tissues obtained from the liposuction operation are repeatedly rinsed by normal saline, the upper adipose tissues are collected;
(b) shearing the upper layer adipose tissue by scissors, centrifuging for 3-5min at 2000g and 1000-;
(c) connecting the two syringes by using a three-way pipe, and pushing the two syringes back and forth for 20-40 times to obtain the particulate adipose tissues;
(d) centrifuging the granular adipose tissues for 3-5min at the temperature of 1000-2000g, removing an upper grease layer, and removing a lower purified granular adipose tissue;
(e) adding HA/beta-TCP support materials into the particle adipose tissues, and uniformly stirring to obtain an adipose-material mixture;
(f) the 6-well plate was coated with 1% agarose gel, and the prepared fat-material mixture was seeded in the 6-well plate at 1.5mL per well.
As a preferable technical scheme, the volume of the scaffold material and the corpuscular adipose tissue is 1:4-1: 20.
As a preferred technical scheme, the scaffold material is HA/beta-TCP.
As a preferred technical solution, the in vitro proliferation medium is:
alpha-MEM + 10% FBS + 1% PSG + 1% HEPES + dexamethasone (10)-7mol/L) + ascorbic acid (10)-5mol/L) + FGF-2(2.5-10ng/mL) + PDGF (5-20 ng/mL). Wherein the alpha-MEM is in volume ratio with FBS, PSG and HEPES.
Preferably, the in vitro proliferation culture is carried out for 2-4 weeks, and the culture is replaced 2-3 times per week.
Preferably, the chondrogenic induction medium is cultured for 3-5 weeks under induction; inducing and culturing for 2-3 weeks by using a hypertrophy inducing culture medium; the induction medium was changed 2-3 times per week.
Preferably, the sample in the step (2) is an emulsified and particulate adipose tissue.
As a preferred embodiment, the chondrogenic induction phase isThe culture medium used was: SFM + BMP-6(5-20ng/mL) + TGF-. beta.3(5-20ng/mL) + dexamethasone (10)-7mol/L) + ascorbic acid (10)-5mol/L)；
The media used during the hypertrophy induction phase were: SFM + beta-Glycerol disodium phosphate (10)-2mol/L) + dexamethasone (10)- 8mol/L) + ascorbic acid (10)-5mol/L)。
Wherein SFM is: DMEM culture solution + 1% HSA + 1% PSG + 1% HEPES + (0.5-2)% ITS + (0.3-1.2)% linoleic acid.
The application range of the invention is as follows:
for patients with bone defects, autologous adipose tissue and scaffold materials are used for constructing tissue engineering hypertrophic cartilage tissue, and then the tissue engineering hypertrophic cartilage tissue is transplanted to a bone defect part to promote bone regeneration of the defect part.
For a patient with large-volume bone defect, autologous adipose tissues and scaffold materials are used for constructing tissue engineering hypertrophic cartilage tissues, and then a bone tissue flap capable of being freely transplanted is pre-constructed by using a vascular pedicle, so that the large-volume bone defect is repaired by remote free transplantation.
For patients who have difficulty in obtaining adipose tissues and urgently need bone transplantation operations, acellular hypertrophic cartilage matrixes constructed by allogeneic bone adipose tissues are implanted into bone defect parts of the tracts for repairing bone defects.
The tissue engineering hypertrophic cartilage constructed by using the adipose tissue can form bone tissue rich in bone marrow in a mode of endochondral ossification after the subcutaneous ectopic implantation of a nude mouse, and is used for constructing a tissue engineering bone marrow niche model.
Advantageous effects
(1) The scaffold material is added, and the added scaffold material provides an attachment surface for cell proliferation, can promote proliferation and migration of ASC, and increases the number of ASC; the bracket material promotes mineralization of adipose tissue at an adipose tissue hypertrophy induction stage, so that bone regeneration is promoted; promote the chemotaxis of osteoclast, and further promote the bone reconstruction and the bone marrow regeneration; as a bone graft substitute, clinical controllability is increased.
(2) The hypertrophic cartilage induced by the invention can be transplanted into a mouse body subsequently, and after undergoing an endochondral ossification process, a complete bone tissue with a bone marrow niche is formed, the bone forming efficiency is higher compared with the traditional intramembranous bone forming mode, and the added scaffold material promotes chemotaxis and migration of macrophages, so that the reconstruction of bone and cartilage is promoted, the formation of the bone marrow niche is more favorable, and the clinical transformation is expected to be realized so as to achieve the purpose of treating bone defect.
(3) The invention directly plants the obtained particulate adipose tissues and the scaffold material on a culture medium for culture, but not adopts the traditional SVF cells which are subjected to subculture, the method reserves the ECM among cells, provides a favorable stem cell nest for the differentiation of the ASC, and reserves the multidirectional differentiation and angiogenesis potential of the stem cells.
(4) The particulate adipose tissue and scaffold material of the present invention, after undergoing 2-4 weeks of external proliferation culture, still has a cellular phenotype and a multi-directional differentiation potential similar to freshly prepared ASCs.
(5) The induction culture medium adopted by the invention can effectively induce the proliferation of the granular adipose tissues into hypertrophic cartilage tissues.
(6) The method is simple and easy to operate, and avoids the traditional complicated steps of obtaining, proliferating, planting and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive exercise.
FIG. 1 is a flow chart of examples 1 to 6.
FIG. 2 is a graph of HE staining of examples 1-3 (fat-material mixture) and comparative example 1 (fine particle adipose tissue alone) after 3 weeks in vitro proliferation culture.
FIG. 3 is a histological staining pattern after 4 weeks in vitro chondrogenic differentiation-inducing culture of examples 1 to 3 and comparative example 1.
FIG. 4 is a histological staining pattern after 2 weeks in vitro hypertrophy induction culture of examples 1 to 3 and comparative example 1.
FIG. 5 is a MicroCT scan and a mineralized tissue quantitation image after 2 weeks in vitro hypertrophy induction culture of examples 1-3 and comparative example 1.
FIG. 6 is a graph of HE staining of hypertrophic cartilage tissue of example 1 and comparative example 1 at 12 weeks after subcutaneous implantation in nude mice. Wherein: A. adding HA/beta-TCP granular material; B. no HA/beta-TCP particulate material.
FIG. 7 TRAP staining pattern of hypertrophic cartilage tissue of example 1 and comparative example 1 after subcutaneous implantation in nude mice for 12 weeks. Wherein: A. adding HA/beta-TCP granular material; B. no HA/beta-TCP particulate material.
FIG. 8 is a graph of the microcT scan and the quantitative analysis of mineralized tissue of examples 1-3 and comparative example 1 after 12 weeks of subcutaneous implantation in nude mice.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the method of the present invention, a scaffold material is added to the particulate adipose tissue. The experimental result proves that the added scaffold material promotes the proliferation and migration of ASC in adipose tissues, does not influence the chondrogenic differentiation of the ASC, and promotes the mineralization of adipose tissue at the hypertrophy induction stage; in vivo experiments also prove that the added scaffold material promotes bone regeneration and chemotaxis and migration of macrophages, thereby being more beneficial to the formation of bone marrow niche.
See the examples for details. The materials used in the following examples are all commercially available.
Example 1
A method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold material comprises the following steps (see figure 1):
(1) preparation of particulate adipose tissue
(a) Human adipose tissues obtained from liposuction surgery are rinsed for multiple times by normal saline, and then upper adipose tissues are collected;
(b) shearing the upper layer adipose tissue with scissors, centrifuging at 1200g for 5min, removing upper layer oil and lower layer swelling liquid, and collecting the middle adipose layer in a 20mL syringe;
(c) connecting two 20mL syringes by using a three-way pipe, and pushing the two syringes back and forth for 30 times to obtain the particulate adipose tissues;
(d) centrifuging the particulate adipose tissue at 1500g for 5min to remove the upper lipid layer and the lower purified particulate adipose tissue.
(e) Adding HA/beta-TCP support materials into the particle adipose tissues, and uniformly stirring to obtain an adipose-material mixture; wherein the volume of HA/beta-TCP scaffold material to particulate adipose tissue can be selected within the ratio of 1:4 to 1:20, in this example 1: 4.
(2) In vitro propagation culture of the fat-material mixture;
(a) coating a 6-well plate with 1% agarose gel, and inoculating the above prepared fat-material mixture into the 6-well plate at 1.5mL per well;
(b) adding proliferation culture medium 3-5mL per well, placing in CO2Culturing in cell culture box for 2-4 weeks, and changing liquid 2-3 times per week; this example was cultured for 3 weeks;
wherein the proliferation culture medium is: the proliferation culture medium is: alpha-MEM + 10% FBS + 1% PSG + 1% HEPES + FGF-2(5ng/mL) + PDGF (10ng/mL) + dexamethasone (10ng/mL)-7mol/L) + ascorbic acid (10)-5mol/L)。
(c) After the fat-material mixture is subjected to in vitro proliferation culture, stem cells in the mixed tissue proliferate in a large amount, and loose mixed tissue gradually gathers to form a tissue block which is rich in ASC and still has multidirectional differentiation potential.
(3) In vitro construction of hypertrophic cartilage tissue
(a) After the in vitro proliferation culture in the step (2) is finished, rinsing the fat-material mixture for 2 times by PBS, and drilling a small block sample with the diameter of 4mm on the fat-material mixture subjected to the proliferation culture by using a biopsy drill with the diameter of 4mm for the next experiment;
(b) transferring the small sample of the obtained fat-material mixture block to a 12-hole plate, adding 2mL of chondrogenic induction culture medium into 1 particle of each hole, and carrying out chondrogenic induction culture for 4 weeks; then, a hypertrophy induction medium was added thereto, and the hypertrophy induction culture was carried out for 2 weeks. Change the solution 2-3 times per week.
Wherein the culture medium can be as follows:
the media for the chondrogenic phase include:
SFM + dexamethasone (10)-7mol/L) + ascorbic acid (10)-5mol/L)+BMP-6(10ng/mL)+TGF-β3(10ng/mL)；
The medium for the hypertrophy induction phase includes:
SFM + beta-Glycerol disodium phosphate (10)-2mol/L) + dexamethasone (10)-8mol/L) + ascorbic acid (10)-5mol/L)。
Wherein the SFM: DMEM medium + 1% HSA + 1% PSG + 1% HEPES + 1% ITS + 0.56% linoleic acid.
(4) Quality control of hypertrophic cartilage tissue
(a) After the hypertrophy induction culture is finished, fixing by 4% paraformaldehyde, embedding by paraffin and slicing;
(b) safranin O staining, detecting the expression of GAG in cartilage matrix of the samples in the cartilage induction stage and the hypertrophy induction stage;
(c) detecting the content of GAG in the culture medium and hypertrophic cartilage tissue specimen of the last time before the end of the culture for inducing cartilage and inducing hypertrophy by using an ELISA method;
(d) the level of differentiation into cartilage and the level of induction of hypertrophy of cartilage matrix were evaluated by detecting the cartilage matrix-specific protein type II collagen and the hypertrophic cartilage matrix-specific protein type X collagen in the samples at the cartilage induction stage and the hypertrophic induction stage, respectively, using an immunohistochemical method.
(e) The constructed hypertrophic cartilage tissue is implanted into a nude mouse in an ectopic mode, taken out after 12 weeks, stained by Safranin O or HE, and the regeneration level of bones and bone marrow in a specimen is observed.
Example 2
This example differs from example 1 in that: the volume of HA/beta-TCP scaffold material and particulate adipose tissue was 1:8, and the other steps were the same as in example 1.
Example 3
This example differs from example 1 in that: the volume of HA/beta-TCP scaffold material and particulate adipose tissue was 1:16, and the other steps were the same as in example 1.
Example 4
This example differs from example 1 in that: the volume of HA/beta-TCP scaffold material and particulate adipose tissue was 1:20, and the other steps were the same as in example 1.
Example 5
The media used in this example consisted of:
chondrogenic induction medium: SFM + BMP-6(5ng/mL) + TGF-. beta.3(5ng/mL) + dexamethasone (10)-7mol/L) + ascorbic acid (10)-5mol/L)；
Hypertrophy induction medium: SFM + beta-Glycerol disodium phosphate (10)-2mol/L) + dexamethasone (10)-8mol/L) + ascorbic acid (10)-5mol/L)；
Wherein the SFM: DMEM medium + 1% HSA + 1% PSG + 1% HEPES + 2% ITS + 1.2% linoleic acid.
The above-mentioned medium was applied to the method in example 1, and the same requirements were satisfied.
Example 6
The media used in this example consisted of:
chondrogenic induction medium: SFM + BMP-6(20ng/mL) + TGF-. beta.3(20ng/mL) + dexamethasone (10)-7mol/L) + ascorbic acid (10)-5mol/L)；
Hypertrophy induction medium: SFM + beta-Glycerol disodium phosphate (10)-2mol/L) + dexamethasone (10)-8mol/L) + ascorbic acid (10)-5mol/L)；
Wherein the SFM: DMEM medium + 1% HSA + 1% PSG + 1% HEPES + 0.5% ITS + 0.3% linoleic acid.
The above-mentioned medium was applied to the method in example 1, and the same requirements were satisfied.
Comparative example 1
In this example, the particulate adipose tissue was used without adding a scaffold material, and the rest was the same as in example 1.
Comparative example 1
In the embodiment, hypertrophic cartilage tissue constructed by SVF cells and Ultrafoam materials (collagen type I sponges) is used as a control, and except for the technical difference in construction of SVF/Ultrafoam composite carriers, the scheme and the time length of in-vitro endochondral ossification induction of the composite carriers are consistent with the scheme of constructing hypertrophic cartilage tissue by using adipose tissue.
Construction of the SVF/Ultrafoam composite vector:
(1) rinsing the freshly obtained adipose tissues by normal saline, centrifuging for 3-5min at 2000g of 1000-;
(2) adipose tissues were mixed with collagenase type II at a concentration of 1.5% in equal proportion and digested in a 37 ℃ constant temperature shaker for 1 hour. The obtained chylified adipose tissue after 1 hour is centrifuged for 3-5min by 1000-2000g, the upper undigested adipose tissue and lipid layer are removed, and the mixture of the residual tissue and cells in the lower layer forms single cell suspension through a cell filter screen of 40 um. Centrifuging the single cell suspension for 3-5min at the temperature of 1000-2000g, removing a liquid part, and suspending the cell part in a red blood cell lysate to lyse red blood cells in the cell suspension. The cells obtained were finally SVF.
(3) Count 1 × 106Individual SVF cells were seeded on type I (Ultrafoam) 4mm in diameter and 2mm in thickness using the same endochondral osteogenesis induction culture protocol as a control group of small clumps of particulate adipose tissue.
Experimental conclusions for examples 1-4, comparative example 1 and comparative example 1:
1. the addition of the granular scaffold material HA/beta-TCP can provide an attachment surface for ASC proliferation in the granular adipose tissues and promote the ASC proliferation and migration.
2. The granular scaffold material HA/beta-TCP did not affect the chondrogenic differentiation of ASC in the granular adipose tissue, but made the distribution of chondrogenic differentiation more uniform.
3. The scaffold material HA/beta-TCP promotes the mineralization of hypertrophic cartilage tissue in vitro, and further can promote the bone regeneration and bone marrow regeneration of the hypertrophic cartilage tissue in vivo.
4. The scaffold material HA/beta-TCP can promote bone resorption and further promote bone marrow regeneration.
FIG. 2 cell proliferation of HA/β -TCP particulate material-adipose tissue mixture
After 3 weeks of proliferation culture, cell proliferation was seen in all 4 groups of material-fat mixtures. The proliferating cells in the adipose tissues without the added HA/beta-TCP granular material are mainly positioned at the periphery of the tissues, while the proliferating cells are also visible in the adipose tissues added with the HA/beta-TCP granular material, and the proliferating cells are the most in the 1:16 material-fat mixture.
FIG. 3 chondrogenic differentiation of HA/β -TCP particulate material-adipose tissue mixture
After 4 weeks of chondrogenic differentiation, positive staining of Safranin O, strong positive expression of type II collagen and weak positive expression of type X collagen were observed in all the 4 groups of material-fat mixtures. The expression of the 3 markers in the 1:16 material-fat mixture was most homogeneous.
FIG. 4 hypertrophy Induction of HA/β -TCP particulate material-adipose tissue mixture
After 2 weeks of induction of hypertrophy, positive staining of Safranin O, strong positive expression of type II collagen and strong positive expression of type X collagen were observed in all of the 4 groups of material-fat mixtures. The expression of the 3 markers in the 1:16 material-fat mixture was most homogeneous.
FIG. 5 MicroCT scan after hypertrophic induction of HA/β -TCP granular material-adipose tissue mixture
After 2 weeks of hypertrophy induction, 4 groups of material-fat mixture specimens were scanned by microCT. Mineral salt deposition was observed in all 4 specimens. With the most mineral salt deposition in the 1:8 material-fat mixture.
FIG. 6 HE staining of post-bone-regeneration specimens of HA/β -TCP particulate material-adipose tissue mixture in vivo
Bone tissue and bone marrow tissue formation was observed in both adipose tissue specimens mixed with HA/β -TCP particulate material and adipose tissue specimens not mixed with material. However, the bone tissue and adipose tissue of the adipose tissue specimens mixed with the HA/β -TCP particulate material were significantly greater than those of the adipose tissue specimens not mixed with the material, and the non-mixed adipose tissue specimens still had unrepaired hypertrophic cartilage tissue visible therein.
FIG. 7 TRAP staining of HA/β -TCP particulate material-adipose tissue mixture
Osteoclasts stained positively by TRAP were found in both adipose tissue specimens mixed with HA/β -TCP particulate material and adipose tissue specimens not mixed with material. In the adipose tissue specimen mixed with the HA/beta-TCP granular material, TRAP positive cells are fewer and are mainly positioned at the periphery of the HA/beta-TCP granular material, and the suggestion is that the HA/beta-TCP granular material can promote chemotaxis of osteoclasts. TRAP-stained positive cells were located mainly on the margins of new bone and hypertrophic cartilage tissue in adipose tissue specimens without mixed material, suggesting that hypertrophic cartilage tissue is still in the remodeling stage.
FIG. 8 MicroCT scan of HA/β -TCP particulate material-adipose tissue mixture after bone regeneration in vivo
After 12 weeks of subcutaneous implantation in nude mice, 4 groups of material-fat mixture specimens were scanned by micct. Regeneration of bone mass was observed in all 4 specimens. With the largest amount of bone in a 1:8 material-fat mixture.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (7)

1. A method for directly regenerating hypertrophic cartilage tissue using adipose tissue and scaffold material, comprising:
(1) in vitro proliferation culture of fat-material mixtures
Adding a support material into the particle adipose tissue, and stirring and uniformly mixing to obtain an adipose-material mixture; after the mixture is subjected to in vitro proliferation culture, a composite tissue block which is rich in ASC and still has multidirectional differentiation potential is formed; the volume of the support material and the particle adipose tissue is 1:4-1: 20; the stent material is HA/beta-TCP;
(2) in vitro construction of hypertrophic cartilage tissue
Taking the sample after the in-vitro proliferation culture in the step (1), and directly differentiating into hypertrophic cartilage tissue through in-vitro endochondral osteogenesis induction culture;
wherein, the in vitro endochondral osteogenesis induction culture comprises two stages of chondrogenesis induction culture and hypertrophy induction culture; the induction culture is performed by using a chondrogenic induction medium, and then the induction culture is performed by using a hypertrophic induction medium.
2. The method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold according to claim 1, wherein the in vitro proliferation medium is:
alpha-MEM + 10% FBS + 1% PSG + 1% HEPES + dexamethasone 10-7mol/L + ascorbic acid 10-5mol/L+FGF-2 2.5-10ng/mL+PDGF 5-20ng/mL；
Wherein the alpha-MEM is in volume ratio with FBS, PSG and HEPES.
3. The method for directly regenerating hypertrophic cartilage tissue from adipose tissue and scaffold according to claim 1 or 2, wherein the in vitro proliferation culture is performed for 2-4 weeks with 2-3 weekly replacement.
4. The method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold material according to claim 1, wherein said chondrogenic induction medium is induced for 3-5 weeks, and said hypertrophic induction medium is induced for 2-3 weeks; the induction medium was changed 2-3 times per week.
5. The method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold according to claim 1, wherein the adipose tissue is human adipose tissue.
6. The method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold material according to claim 1, wherein the sample in step (2) is a mass of microparticulate adipose tissue subjected to proliferation culture.
7. The method for directly regenerating hypertrophic cartilage tissue by using adipose tissue and scaffold according to claim 1, wherein the culture medium used in the chondrogenic induction stage is: serum-free medium SFM + dexamethasone 10-7mol/L + ascorbic acid 10-5mol/L+BMP-6(5-20)ng/mL+TGF-β3(5-20)ng/mL；
The media used during the hypertrophy induction phase were: SFM + beta-Glycerol disodium phosphate 10-2mol/L + dexamethasone 10-8mol/L + ascorbic acid 10-5mol/L；
Wherein SFM is: DMEM culture solution + 1% HSA + 1% PSG + 1% HEPES + (0.5-2)% ITS + (0.3-1.2)% linoleic acid.
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