CN109675108A - Utilize the method for adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue - Google Patents

Utilize the method for adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue Download PDF

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CN109675108A
CN109675108A CN201910119844.0A CN201910119844A CN109675108A CN 109675108 A CN109675108 A CN 109675108A CN 201910119844 A CN201910119844 A CN 201910119844A CN 109675108 A CN109675108 A CN 109675108A
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tissue
timbering material
hypertrophic cartilage
adipose tissue
mol
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CN109675108B (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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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Abstract

The invention proposes a kind of methods using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue, comprising: (1) timbering material is added in micronised fat tissue；(2) in-vitro multiplication culture forms to be rich in and enriches adipose-derived mescenchymal stem cell (ASC) and still with the Complex tissue mass of multi-lineage potential；(3) external structure of hypertrophic cartilage tissue.It joined timbering material in the present invention, the timbering material being added has following effect: (1) providing attachment surface for the cell in adipose tissue, can promote proliferation, the migration of ASC, increase the number of ASC；(2) promote hypertrophic cartilage tissue mineralization, and then promote osteanagenesis；(3) promote osteoclast chemotactic, and then promote bone remoulding and bone marrow regeneration；(4) it is used as bone graft substitute, is increased clinical handling.

Description

Utilize the method for adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue
Technical field
The present invention relates to bone tissue regeneration technical field, particularly relate to it is a kind of using adipose tissue and timbering material directly again The method of raw hypertrophic cartilage tissue.
Background technique
Traditional bone tissue engineer technology is by stem cell plantation on biomaterial, through in vitro culture after a period of time Afterwards, stem cell breaks up osteoblast on biomaterial, secretes ossein, finally regenerates freshman bone tissue, then transplant in At bone defect, the defect of repair function and shape.Currently, the bone tissue engineer technical transform in laboratory is applied to clinical main Face two big challenges: on the one hand, it is difficult to get that materials are convenient, damage stem cell small, that bone regeneration capability is strong.Currently, being based on The method that the tissue engineering technique of stem cell mainly passes through amplification in vitro obtains sufficient amount from limited clinical tissue sample Stem cell, be subsequently used for laboratory bone tissue building.But the Osteoblast Differentiation ability of stem cell can expand with external passage Increase and gradually decreases2, the regenerated ability of stem cell its inductor in-seam repeatedly passed on significantly reduces.On the other hand, external structure The bone tissue built lacks effective vascular system, the tissue-engineered bone of large volume be transplanted to after by area be difficult with by area again Vascularization, and transfer operation is caused to fail3,4.Efficiently solving this two hang-up will be the clinical conversion of tissue engineering technique Removing obstacles.Since adipose tissue is human body storage is big, acquisition is convenient, small to body injury, more and more researchers start It is studied using adipose-derived stem cells (Adipose-derived stem cell, ASC) for osteanagenesis.Studies have shown that ASC Osteoblast and cartilage cell can be broken up under the conditions of different external evoked5,6；In animal experiments, the biology of ASC will be loaded Material is implanted at bone defect after external osteogenic induction culture, can promote defect osteanagenesis7,8, repairing effect Similar to having loaded mesenchymal stem cell (Bone marrow-derived mesenchymal stem cell, BMSC) Same type biomaterial9,10.Although ASC be proved can effective bone induction and regeneration, cannot be neglected a problem is ASC Also it can gradually be lost as the more of mescenchymal stem cell (mesenchymal stem cell, MSC) because passage number increases To differentiation potential.
There are the following problems for current method: 1) stem cell is gradually lost multidirectional after long-time subculture in vitro separately Differentiation potential, including bone regeneration capability；2) Osteoblast Differentiation scheme bone regeneration capability is insufficient in traditional periosteum；3) single dry thin The bone graft of born of the same parents' building lacks into vessel patency.
Since ASC has the function of that various kinds of cell can be divided into, studies have shown that ASC it is different it is external evoked under the conditions of Osteoblast and cartilage cell can be broken up, to achieve the purpose that treat bone defect.Research finds extracellular matrix (Extracellular matrix, ECM) can provide three-dimensional living space for stem cell, adjust different kind organism signaling molecule into Enter Stem Cell Niche, the biological function and differentiation destiny to stem cell play important adjustment effect.
Bibliography:
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.
Summary of the invention
The present invention proposes a kind of method using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue, solves ASC is after long-time subculture in vitro separately in the prior art, the problems such as mescenchymal stem cell Multidirectional Differentiation reduced capability.
The technical scheme of the present invention is realized as follows:
A method of utilizing adipose tissue Direct Regeneration hypertrophic cartilage tissue, comprising:
(1) the in-vitro multiplication culture of line and staff control
Timbering material is added in micronised fat tissue, stirs and evenly mixs, obtains line and staff control；
The line and staff control forms after in-vitro multiplication culture and is rich in abundant ASC and still has multi-lineage potential Tissue block；
(2) external structure of hypertrophic cartilage tissue
Sample after taking step (1) in-vitro multiplication culture is directly divided into fertilizer by osteogenic induction culture in chondrocytes in vitro Big cartilaginous tissue；
Wherein, osteogenic induction culture includes into two ranks of chondrocyte induction culture and loose Fiber differentiation in the chondrocytes in vitro Section；Chondrocyte induction culture medium Fiber differentiation is first used into, then using loose induced medium Fiber differentiation.
It is above-mentioned line and staff control the preparation method is as follows:
(a) adipose tissue obtained from liposuction collects upper-layer fat tissue after physiological saline repeatedly rinses；
(b) above-mentioned upper-layer fat tissue is shredded with scissors, 1000-2000g is centrifuged 3-5min later, removes upper layer grease With lower layer's Tumescent fluid part, intermediate fat deposit is collected in syringe；
(c) two syringes are connected using tee tube, inject back and forth two syringe 20-40 times, obtain particle Adipose tissue；
(d) micronised fat tissue is centrifuged 3-5min through 1000-2000g, removes the oil layer on upper layer, and lower layer is the micro- of purifying Grain adipose tissue；
(e) HA/ β-TCP timbering material is added in micronised fat tissue, stirs and evenly mixs, obtains fat-material blends；
(f) 6 orifice plates are coated with using 1% Ago-Gel, fat-material blends of preparation is inoculated in 6 orifice plates, every hole 1.5mL。
The volume of the timbering material and micronised fat tissue is 1:4-1:20 as a preferred technical solution,.
The timbering material is HA/ β-TCP as a preferred technical solution,.
In-vitro multiplication culture medium as a preferred technical solution, are as follows:
α-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 α-MEM and FBS, PSG, HEPES are volume ratio.
The time of the in-vitro multiplication culture is 2-4 weeks as a preferred technical solution, is replaced 2-3 times weekly.
It is described at chondrocyte induction culture medium Fiber differentiation 3-5 weeks as a preferred technical solution,；Loose induced medium lures Lead culture 2-3 weeks；The induced medium is changed 2-3 times weekly.
Sample in the step (2) as a preferred technical solution, is chylosis micronised fat tissue.
As a preferred technical solution, wherein at chondrocyte induction stage used medium are as follows: SFM+BMP-6 (5-20ng/ mL)+TGF-β3(5-20ng/mL)+dexamethasone (10-7Mol/L)+ascorbic acid (10-5mol/L)；
Loose induction period used medium are as follows: SFM+ β-phosphoglycerol disodium salt (10-2Mol/L)+dexamethasone (10- 8Mol/L)+ascorbic acid (10-5mol/L)。
Wherein SFM are as follows: DMEM culture solution+1%HSA+1%PSG+1%HEPES+ (0.5-2) %ITS+ (0.3-1.2) % Linoleic acid.
Application range of the invention is as follows:
For bone defect patient, organizational project hypertrophic cartilage tissue is constructed using autologous adipose tissue and timbering material, so Hui Zhi is to bone defect position afterwards, to promote defect osteanagenesis.
For the patient of large volume bone defect, organizational project hypertrophic cartilage is constructed using autologous adipose tissue and timbering material Tissue, then using the pre- structure of vessel pedicle can free grafting bone tissue valve, amphi position free grafting repair large volume bone defect.
Patient that is difficult, and being badly in need of bone collection operation is obtained for adipose tissue, uses homogeneous allogenic bone adipose tissue structure The de- cellular mast cartilage matrix built, implantation road bone defect position are used for the reparation of bone defect.
The organizational project hypertrophic cartilage constructed using adipose tissue can pass through cartilage after the implantation of nude mice by subcutaneous dystopy The mode of ossification forms the bone tissue for being rich in marrow, for constructing engineered marrow tabernacle model.
Beneficial effect
(1) it joined timbering material in the present invention, the timbering material being added provides attachment surface for cell Proliferation, can promote Proliferation, migration into ASC, increase the number of ASC；Timbering material promotes its mineralising in adipose tissue hyperplasia induction period, into And promote osteanagenesis；Promote osteoclast chemotactic, and then promotes bone remoulding and bone marrow regeneration；As bone graft substitute, It increases clinical handling.
(2) in the subsequent portable to Mice Body of hypertrophic cartilage that the present invention induces, the mistake of endochondral ossification is undergone Journey forms the complete, bone tissue with marrow tabernacle, and skeletonization efficiency is higher for comparing traditional intramembranous ossification mode, and The timbering material being added promotes the chemotactic of macrophage, migration, so that the reconstruction of bone and cartilage is promoted, to formation marrow Tabernacle is more advantageous, is expected to realize clinical conversion to achieve the purpose that treat bone defect.
(3) the micronised fat tissue that will acquire of the present invention and timbering material are directly planted in being cultivated on culture medium, and It is non-using traditional SVF cell by secondary culture, the method remains intercellular ECM, has provided for the differentiation of ASC " Stem Cell Niche " of benefit remains stem cell Multidirectional Differentiation and at blood vessel potential.
(4) after the outer Multiplying culture that micronised fat tissue of the invention and timbering material undergo 2-4 week, still with it is fresh The ASC of preparation similar cell phenotype and multi-lineage potential.
(5) micronised fat hyperblastosis induction can be more effectively loose soft by induced medium of the present invention Bone tissue.
(6) method of the invention is simple, easily operated, avoids the tedious steps such as traditional acquisition, proliferation, plantation.
Detailed description of the invention
It, below will be to embodiment in order to illustrate more clearly of embodiment of the present invention or technical solution in the prior art Or attached drawing needed to be used in the description of the prior art is briefly described, it should be apparent that, the accompanying drawings in the following description is only It is some embodiments of the invention, for those of ordinary skill in the art, in the premise of not making the creative labor property Under, it is also possible to obtain other drawings based on these drawings.
The flow chart of Fig. 1 embodiment 1-6.
Fig. 2 embodiment 1-3 (fat-material blends) and comparing embodiment 1 (simple micronised fat tissue) pass through 3 weeks HE colored graph after in-vitro multiplication culture.
Fig. 3 embodiment 1-3 is with comparative example 1 by 4 weeks in vitro at the histological stain figure after cartilage differentiation Fiber differentiation.
The histological stain figure of Fig. 4 embodiment 1-3 and comparative example 1 after 2 weeks external loose Fiber differentiations.
The microCT scanning figure and mineralising of Fig. 5 embodiment 1-3 and comparative example 1 after 2 weeks external loose Fiber differentiations Quantitative Analysis of Microstructure figure.
Fig. 6 embodiment 1 and the hypertrophic cartilage group of comparative example 1 are woven in the HE colored graph of nude mice by subcutaneous implantation 12 weeks after operation.Its In: A, add HA/ β-TCP granular materials；B, without HA/ β-TCP granular materials.
Fig. 7 embodiment 1 and the hypertrophic cartilage group of comparative example 1 are woven in the TRAP colored graph of nude mice by subcutaneous implantation 12 weeks after operation. Wherein: A, adding HA/ β-TCP granular materials；B, without HA/ β-TCP granular materials.
The microCT scanning figure and mineralising group of Fig. 8 embodiment 1-3 and comparative example 1 after nude mice by subcutaneous is implanted into 12 weeks after operation Knit quantitative analysis figure.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects It encloses.
In method of the invention, timbering material joined in micronised fat tissue.The results show, the bracket of addition Material promotes the proliferation of ASC in adipose tissue, migration, does not influence ASC into cartilage differentiation, and promote adipose tissue hyperplasia The mineralising of induction period；Experiment in vivo be also demonstrated that the timbering material of addition promote osteanagenesis, macrophage chemotactic, move It moves, to be more advantageous to the formation of marrow tabernacle.
Referring specifically to embodiment.Substance used in following embodiments is commercially available.
Embodiment 1
A method of utilizing adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue, including following steps (see Fig. 1):
(1) preparation of micronised fat tissue
(a) human fat tissue obtained from liposuction collects upper-layer fat group after physiological saline repeatedly rinses It knits；
(b) above-mentioned upper-layer fat tissue is shredded with scissors, 1200g is centrifuged 5min, removes upper layer grease and lower layer's swelling Intermediate fat deposit is collected in 20mL syringe in liquid part；
(c) two 20mL syringes are connected using a tee tube, injects two syringes back and forth 30 times, obtained Micronised fat tissue；
(d) micronised fat tissue is centrifuged 5min through 1500g, removes the oil layer on upper layer, and lower layer is purifying micronised fat group It knits.
(e) HA/ β-TCP timbering material is added in micronised fat tissue, stirs and evenly mixs, obtains fat-material blends； Wherein the volume of HA/ β-TCP timbering material and micronised fat tissue can select in 1:4-1:20 ratio, be 1 in the present embodiment: 4。
(2) fat-material blends in-vitro multiplication culture；
(a) 6 orifice plates are coated with using 1% Ago-Gel, fat-material blends of above-mentioned preparation are inoculated in 6 orifice plates, Every hole 1.5mL；
(b) every hole adds proliferated culture medium 3-5mL, is placed in CO2It cultivates 2-4 weeks, changes weekly liquid 2-3 times in cell incubator； It has been cultivated in the present embodiment 3 weeks；
Wherein, proliferated culture medium are as follows: proliferated culture medium are as follows: α-MEM+10%FBS+1%PSG+1%HEPES+FGF-2 (5ng/mL)+PDGF (10ng/mL)+dexamethasone (10-7Mol/L)+ascorbic acid (10-5mol/L)。
(c) fat-material blends are after in-vitro multiplication culture, and stem cell is largely proliferated in line and staff control, loose Line and staff control gradually assembles, and forms one piece rich in abundant ASC and still with the tissue block of multi-lineage potential.
(3) external structure of hypertrophic cartilage tissue
(a) after the in-vitro multiplication culture of step (2), with PBS rinsing fat-material blends 2 times, diameter is used The biopsy punch of 4mm drills through the small agglomerate sample of diameter 4mm on fat-material blends by Multiplying culture, is used for It tests in next step；
(b) fat-material blends agglomerate small sample obtained is transferred in 12 orifice plates, and cartilage is first added into 1, every hole Induced medium 2mL, at chondrocyte induction culture 4 weeks；Then loose induced medium, loose Fiber differentiation 2 weeks is added.It changes weekly Liquid 2-3 times.
Wherein culture medium can be such that
Include: at the culture medium of cartilage phase
SFM+ dexamethasone (10-7Mol/L)+ascorbic acid (10-5mol/L)+BMP-6(10ng/mL)+TGF-β3(10ng/ mL)；
The culture medium of loose induction period includes:
SFM+ β-phosphoglycerol disodium salt (10-2Mol/L)+dexamethasone (10-8Mol/L)+ascorbic acid (10-5mol/ L)。
Wherein SFM:DMEM culture solution+1%HSA+1%PSG+1%HEPES+1%ITS+0.56% linoleic acid.
(4) the quality control of hypertrophic cartilage tissue
(a) after loose Fiber differentiation, paraffin embedding, slice solid through 4% paraformaldehyde；
(b) Safranin O is dyed, and detection is in the cartilage matrix of chondrocyte induction stage and the sample of loose induction period The expression of GAG；
(c) before using ELISA method, detection to terminate at chondrocyte induction and loose Fiber differentiation in the culture medium of last time and The content of GAG in hypertrophic cartilage tissue specimen；
(d) immunohistochemical method is used, is detected respectively soft in the sample of chondrocyte induction stage and loose induction period Bone matrix differential protein II Collagen Type VI and hypertrophic cartilage Medium Culture differential protein X-type collagen, assessment is at cartilage differentiation level and soft Bone matrix hypertrophy induced levels.
(e) the hypertrophic cartilage tissue constructed by, dystopy are implanted into nude mice by subcutaneous, take out after 12 weeks, Safranin O or HE Dyeing, observes sample in-seam and bone marrow regeneration is horizontal.
Embodiment 2
The present embodiment the difference from embodiment 1 is that: the volume of HA/ β-TCP timbering material and micronised fat tissue is 1: 8, other steps and embodiment 1 are same.
Embodiment 3
The present embodiment the difference from embodiment 1 is that: the volume of HA/ β-TCP timbering material and micronised fat tissue is 1: 16, other steps and embodiment 1 are same.
Embodiment 4
The present embodiment the difference from embodiment 1 is that: the volume of HA/ β-TCP timbering material and micronised fat tissue is 1: 20, other steps and embodiment 1 are same.
Embodiment 5
The composition of culture medium used in this implementation column is as follows:
At chondrocyte induction culture medium: SFM+BMP-6 (5ng/mL)+TGF-β3(5ng/mL)+dexamethasone (10-7mol/L)+ Ascorbic acid (10-5mol/L)；
Loose induced medium: SFM+ β-phosphoglycerol disodium salt (10-2Mol/L)+dexamethasone (10-8Mol/L)+anti- Bad hematic acid (10-5mol/L)；
Wherein SFM:DMEM culture solution+1%HSA+1%PSG+1%HEPES+2%ITS+1.2% linoleic acid.
Above-mentioned culture medium is applied in the method in embodiment 1, is equally met the requirements.
Embodiment 6
The composition of culture medium used in this implementation column is as follows:
At chondrocyte induction culture medium: SFM+BMP-6 (20ng/mL)+TGF-β3(20ng/mL)+dexamethasone (10-7mol/ L)+ascorbic acid (10-5mol/L)；
Loose induced medium: SFM+ β-phosphoglycerol disodium salt (10-2Mol/L)+dexamethasone (10-8Mol/L)+anti- Bad hematic acid (10-5mol/L)；
Wherein SFM:DMEM culture solution+1%HSA+1%PSG+1%HEPES+0.5%ITS+0.3% linoleic acid.
Above-mentioned culture medium is applied in the method in embodiment 1, is equally met the requirements.
Comparing embodiment 1
It is not add timbering material in the present embodiment, directly uses micronised fat tissue, rest part and embodiment 1 Together.
Comparative examples 1
The present embodiment uses the hypertrophic cartilage tissue constructed using SVF cell and Ultrafoam material (type i collagen sponge) As control, except the building of SVF/Ultrafoam complex carrier is there are in addition to technological disparity, the chondrocytes in vitro ossification of complex carrier Induction scheme, duration, detection means are consistent with using the adipose tissue building scheme of hypertrophic cartilage tissue.
The building of SVF/Ultrafoam complex carrier:
(1) adipose tissue of fresh acquisition, after physiological saline rinses, 1000-2000g is centrifuged 3-5min, removes upper layer Grease and lower liquid, obtain intermediate fat deposit, obtain SVF cell for collagenase digesting；
(2) adipose tissue is mixed with the II Collagenase Type equal proportion of 1.5% concentration volume, is placed in 37 DEG C of constant-temperature tables and is digested 1 hour.The chylosis adipose tissue obtained after 1 hour is centrifuged 3-5min through 1000-2000g, removes the indigested fat in upper layer Tissue and oil layer, the remaining tissue of lower layer and the mixture of cell form single cell suspension through the cell strainer of 40um.It is slender Born of the same parents' suspension is centrifuged 3-5min through 1000-2000g, removes liquid portion, and cellular portions are resuspended in erythrocyte cracked liquid, lytic cell Red blood cell in suspension.Final acquired cell is SVF.
(3) 1 × 10 is counted6A SVF cell is inoculated in the I type (Ultrafoam) of diameter 4mm, thickness 2mm, and use is identical Entochondrostosis Fiber differentiation scheme, the control group as the small agglomerate of micronised fat tissue.
The experiment conclusion of embodiment 1-4, comparing embodiment 1 and comparative examples 1:
1. addition particle timbering material HA/ β-TCP can be proliferated for the ASC in micronised fat tissue and provide attachment surface, promote Into ASC proliferation, migration.
2. particle timbering material HA/ β-TCP does not influence the ASC in micronised fat tissue into cartilage differentiation, but makes it at soft The distribution of bone differentiation more homogenizes.
3. timbering material HA/ β-TCP promotes mineralising outside hypertrophic cartilage organizer, and then can promote hypertrophic cartilage tissue Internal osteanagenesis and bone marrow regeneration.
4. timbering material HA/ β-TCP can be by promoting bone resorption, and then promotes bone marrow regeneration.
Fig. 2 .HA/ β-TCP granular materials-adipose tissue mixture cell Proliferation
After Multiplying culture 3 weeks, equal visible cell proliferation in 4 groups of material-fat blends.No added HA/ β-TCP granular material Increment cell is predominantly located at tissue periphery in the adipose tissue of material, and adds inside the adipose tissue of HA/ β-TCP granular materials also It can be seen that increment cell, and it is most with proliferative cell in 1:16 material-fat blend.
Fig. 3 .HA/ β-TCP granular materials-adipose tissue mixture is at cartilage differentiation
After cartilage differentiation 4 weeks, visible Safranin O positive staining, II Collagen Type VI in 4 groups of material-fat blends Strong positive expression and the expression of X-type collagen weakly positive.It is wherein the most equal with 3 group mark objects expression in 1:16 material-fat blend Matter.
Fig. 4 .HA/ β-TCP granular materials-adipose tissue mixture hypertrophy induction
After hypertrophy induction 2 weeks, visible Safranin O positive staining, II Collagen Type VI are strong in 4 groups of material-fat blends Positive expression and the expression of X-type collagen strong positive.Wherein with 3 group mark objects expression homogeneous the most in 1:16 material-fat blend.
MicroCT is scanned after Fig. 5 .HA/ β-TCP granular materials-adipose tissue mixture hypertrophy induction
After hypertrophy induction 2 weeks, microCT scans 4 groups of materials-fat blend sample.Visible rock salt is heavy in 4 groups of samples Product.It is wherein most with rock salt deposition in 1:8 material-fat blend.
The HE of sample is dyed after osteanagenesis in Fig. 6 .HA/ β-TCP granular materials-adipose tissue mixture body
It is mixed in the adipose tissue sample of HA/ β-TCP granular materials and the adipose tissue sample of non-mixing material See that bone tissue and myeloid tissue generate.But it is mixed with the bone tissue and fat group of the adipose tissue sample of HA/ β-TCP granular materials Knit the adipose tissue sample of significantly more than not mixing material, not in the adipose tissue sample of mixing material still it is visible have do not rebuild Hypertrophic cartilage tissue.
Fig. 7 .HA/ β-TCP granular materials-adipose tissue mixture TRAP dyeing
It is mixed in the adipose tissue sample of HA/ β-TCP granular materials and the adipose tissue sample of non-mixing material See the osteoclast of TRAP stained positive.In the adipose tissue sample for being mixed with HA/ β-TCP granular materials, TRAP is positive thin Born of the same parents are less, are predominantly located at HA/ β-TCP granular materials periphery, prompt HA/ β-TCP granular materials that may promote becoming for osteoclast Change.TRAP staining positive cells are predominantly located at newborn sclerotin and hypertrophic cartilage tissue in the adipose tissue sample of not mixing material Edge, prompt hypertrophic cartilage tissue still in rebuild the phase.
MicroCT scanning in Fig. 8 .HA/ β-TCP granular materials-adipose tissue mixture body after osteanagenesis
After nude mice by subcutaneous is implanted into 12 weeks, microCT scans 4 groups of materials-fat blend sample.It is visible in 4 groups of samples Inostosis.It is wherein most with bone amount in 1:8 material-fat blend.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Claims (9)

1. a kind of method using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue characterized by comprising
(1) fat-material blends in-vitro multiplication culture
Timbering material is added in micronised fat tissue, after stirring and evenly mixing, obtains fat-material blends；The mixture warp After crossing in-vitro multiplication culture, formed rich in ASC and still with the Complex tissue mass of multi-lineage potential；
(2) external structure of hypertrophic cartilage tissue
Sample after taking step (1) in-vitro multiplication culture is directly divided into loose soft by osteogenic induction culture in chondrocytes in vitro Bone tissue；
Wherein, osteogenic induction culture includes into chondrocyte induction culture and loose two stages of Fiber differentiation in the chondrocytes in vitro； Chondrocyte induction culture medium Fiber differentiation is first used into, then using loose induced medium Fiber differentiation.
2. a kind of side using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue according to claim 1 Method, which is characterized in that the volume of the timbering material and micronised fat tissue is 1:4-1:20.
3. a kind of side using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue according to claim 1 Method, which is characterized in that the timbering material is HA/ β-TCP.
4. a kind of side using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue according to claim 1 Method, which is characterized in that in-vitro multiplication culture medium are as follows:
α-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 α-MEM and FBS, PSG, HEPES are volume ratio.
5. according to claim 1 or 2 a kind of using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue Method, which is characterized in that the time of the in-vitro multiplication culture is 2-4 weeks, is replaced 2-3 times weekly.
6. a kind of side using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue according to claim 1 Method, which is characterized in that described at chondrocyte induction culture medium Fiber differentiation 3-5 weeks, loose induced medium Fiber differentiation 2-3 weeks； The induced medium is changed 2-3 times weekly.
7. a kind of side using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue according to claim 1 Method, which is characterized in that the adipose tissue is human fat tissue.
8. a kind of side using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue according to claim 1 Method, which is characterized in that the sample in the step (2) is the micronised fat tissue agglomerate by Multiplying culture.
9. a kind of side using adipose tissue and timbering material Direct Regeneration hypertrophic cartilage tissue according to claim 1 Method, which is characterized in that wherein at chondrocyte induction stage used medium are as follows: serum free medium (SFM)+dexamethasone (10- 7Mol/L)+ascorbic acid (10-5mol/L)+BMP-6(5-20ng/mL)+TGF-β3(5-20ng/mL)；
Loose induction period used medium are as follows: SFM+ β-phosphoglycerol disodium salt (10-2Mol/L)+dexamethasone (10-8mol/ L)+ascorbic acid (10-5mol/L)；
Wherein SFM are as follows: the sub- oil of DMEM culture solution+1%HSA+1%PSG+1%HEPES+ (0.5-2) %ITS+ (0.3-1.2) % Acid.
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