CN104307044B

CN104307044B - A kind of preparation method of the de-cell material of total spinal disc in natural tissues source

Info

Publication number: CN104307044B
Application number: CN201410538175.8A
Authority: CN
Inventors: 林贤丰; 范顺武; 胡志军
Original assignee: Affiliated Sir Run Run Shaw Hospital of School of Medicine Zhejiang University
Current assignee: Zhejiang Disai Biotechnology Co., Ltd.
Priority date: 2014-10-13
Filing date: 2014-10-13
Publication date: 2016-06-08
Anticipated expiration: 2034-10-13
Also published as: CN104307044A

Abstract

The invention discloses de-cell material of the total spinal disc in a kind of natural tissues source and preparation method thereof, the present invention takes vertebrates intervertebral disc, extract bone fragment, aseptic PBS rinses 3 times, then, in the PBS containing 10KIU/ml protease inhibitor that concentration is 10%, shake 4 hours at shaking table 150rpm; Again concentration be 4% containing Triton? the PBS of X-100, adds mixing antimicrobial fluid, shakes 48 hours at shaking table 150rpm; Followed by the PBS containing SDS that concentration is 5%, the mixing antimicrobial fluid of addition, shake 48 hours at shaking table 150rpm; Finally with the PBS containing DNA enzymatic that concentration is 0.5mg/ml, adding mixing antimicrobial fluid, shake 12 hours at shaking table 150rpm, then rinse 1 hour with PBS, the present invention draws materials extensively, and safety is high, can realize normal disc reconstruct.

Description

A kind of preparation method of the de-cell material of total spinal disc in natural tissues source

Technical field

The invention mainly relates to for organizing or the biological field of organ reparation and regeneration thereof, specifically the de-cell material extremely preparation method of the total spinal disc in a kind of natural tissues source

Background technology

Intervertebral disc degeneration usually causes back pain and intervertebral disk hernia. According to statistics, there are about the people of 84% in the U.S. had low-back pain medical history, and associated cost is annual especially up to 100,000,000,000 dollars. And intervertebral disc degeneration is considered as then a pathological process that is irreversible and that increase the weight of with the age. And expectant treatment traditional at present and excision-fusion all can not reach desirably to effect a radical cure effect.

The appearance of regenerative medicine and tissue engineering technique makes radical cure intervertebral disc degeneration be possibly realized. In recent years, many researcheres are attempted using the technology treatment intervertebral disc degenerations such as organizational project (metal-plastic composite disc), molecular biology (injection somatomedin or cytokine inhibitor) and stem-cell therapy. But due to natural intervertebral disc (vertebral pulp and fibrous ring), there is complicated three dimensional structure and many particular make-up compositions not yet clear and definite at present make effect always not fully up to expectations. Such as, synthetic material is very limited owing to the problems such as biomechanics, biocompatibility, material character and long term grind make it use; And the effective acting time of somatomedin and cytokine inhibitor of short duration also makes therapeutic effect not good; Same stem cells technology also cannot solve intervertebral disc structure at present and destroy a series of disease process caused. Therefore the material or treatment technology that really meet normal disc biology and mechanical property are found, thus replacing and reverse degeneration intervertebral disc is a difficult medical problem urgently to be resolved hurrily.

Extracellular matrix (ECM) is containing the various Some Circulating Factors needed for normal structure or organ cell, and has natural macroscopic view and the stereochemical structure of ultra micro three-dimensional. A series of tissues or the allelotaxises such as ECM scalable biophysics stimulates, biochemistry and molecular signal and repair required various factors, thus realizing recovery and the regeneration of tissue or organ.Therefore ECM is just applied to reparation and the regeneration of a series of tissues such as cardiac valve, trachea, muscle, tendon, cartilage or organ widely as a novel natural biologic material. And there is no report prepared by the de-cell material of the intact disc (vertebral pulp and fibrous ring) about natural origin at present.

Summary of the invention

Present invention aim at filling up in prior art, the organization work human disc not enough and only single vertebral pulp or fibrous framework defect in material composition, structure, biomechanics, it is provided that the preparation method of a kind of de-cell material of total spinal disc being suitable for intervertebral disc cells growth, reconstruct and treatment irreversibility intervertebral disc degeneration

The de-cell material of total spinal disc in a kind of natural tissues source and preparation method thereof, specifically includes following steps:

(1) taking vertebrates intervertebral disc, extract bone fragment, aseptic PBS rinses 3 times, every time rinsing 20 minutes, removes blood, remaining muscular tissue and ligament etc.;

(2) in the PBS containing 10KIU/ml protease inhibitor that concentration is 10%, 45 DEG C of shaking table 150rpm of constant temperature shake 4 hours; And rinse 1 hour with PBS;

(3) at the PBS containing TritonX-100 that concentration is 4%, adding the mixing antimicrobial fluid of 10KIU/ml, 10g/ml, buffer and mixing antimicrobial fluid volume ratio is 5:1, buffer and mixing antimicrobial fluid ratio 5:1,45 DEG C of shaking table 150rpm of constant temperature shake 48 hours; And rinse 1 hour with PBS; Described mixing antimicrobial fluid is made up of penicillin and streptomycin, and the volume ratio of penicillin and streptomycin is 1:1;

(4) concentration is the PBS containing SDS of 5%, and adding the mixing antimicrobial fluid of 10KIU/ml, 10g/ml, buffer and mixing antimicrobial fluid volume ratio is 5:1, and 45 DEG C of shaking table 150rpm of constant temperature shake 48 hours; Rinse 1 hour with PBS again; Described mixing antimicrobial fluid is made up of penicillin and streptomycin, and the volume ratio of penicillin and streptomycin is 1:1;

(5) concentration is the PBS containing DNA enzymatic of 0.5mg/ml, add 10KIU/ml, the mixing antimicrobial fluid of 10g/ml, buffer and mixing antimicrobial fluid volume ratio are 5:1,37 DEG C of shaking table 150rpm shake 12 hours, rinsing 1 hour with PBS, described mixing antimicrobial fluid is made up of penicillin and streptomycin, and the volume ratio of penicillin and streptomycin is 1:1 again.

The major advantage of the present invention is as follows:

(1) total spinal disc of the present invention is the biomaterial of natural origin, has good biocompatibility and popularity of drawing materials;

(2) adopt the total spinal disc that de-cell technology obtains without antigenic substances such as cells, the immunological rejection of receptor can be made to be preferably minimized limit, can be free of the harmful components biological safeties such as bacterial virus high simultaneously;

(3) de-cell technology is removing heterocellular while, can retain the integrity of original ECM, has good extracellular microenvironment, Some Circulating Factors and bio-mechanical property etc., it is possible to simulation normal disc composition and structure to greatest extent;

(4) material of the present invention can be not only used for planting various stem cell (embryonic stem cell, mesenchymal stem cells MSCs (MSCs), fat mesenchymal stem cell etc.) and intervertebral disc cells to realize normal disc reconstruct, for patient customized have individuation, be available for transplant total spinal disc, can also be ground and be made powder, the Some Circulating Factors contained by normal disc be dissolved and is used for the intervertebral disc treating regression.

Accompanying drawing explanation

Fig. 1 is total spinal disc (fibrous ring and vertebral pulp) the general appearance figure of the present invention;

Fig. 2 is the HE acellular and acellular nuclear composition residual figure of dyeing of fibrous ring support;

Fig. 3 is the HE acellular and acellular nuclear composition residual figure of dyeing of vertebral pulp support;

The alcian blue dyeing that Fig. 4 is fibrous ring support retains a large amount of glycosaminoglycans component-part diagrams;

The alcian blue dyeing that Fig. 5 is vertebral pulp support retains a large amount of glycosaminoglycans component-part diagrams;

Fig. 6 is that total spinal disc DNA detection by quantitative is practically free of DNA component-part diagram;

Fig. 7 is the detection collagen fiber arrangement of fibrous ring scanning electron microscope and stereoeffect completely reservation figure;

Fig. 8 is the detection collagen fiber arrangement of vertebral pulp scanning electron microscope and stereoeffect completely reservation figure;

Fig. 9 is that CCK-8 detects the increment situation map of MSCs under different support lixiviating solution concentration;

Figure 10 is Live/Dead cell dyeing MSCs growing state figure on the de-cytoskeleton of total spinal disc.

Specific embodiments

Describe the present invention below in conjunction with drawings and Examples, but the enforcement of the present invention is not limited only to this.

1, total spinal disc acellular matrix is prepared

(1) draw materials: taking 4 months (male and female) healthy rabbits breasts 5 to the intervertebral disc of waist 3, extract bone fragment, aseptic PBS fully rinses removal blood and other impurity, and intervertebral disc major axis is about 1cm, and short axle 0.8cm, thickness is about 5mm.

De-cell step is as follows:

Step one: at the PBS containing 10KIU/ml protease inhibitor that 100ml concentration is 10%, low temperature (4 DEG C) shaking table 150rpm shakes 4 hours; And rinse 1 hour with PBS;

Step 2: at the PBS containing TritonX-100 that 500ml concentration is 4%, adds 100ml penicillin and streptomycin (10KIU/ml, 10g/ml) mixes antimicrobial fluid, and low temperature (4 DEG C) shaking table 150rpm shakes 48 hours; And rinse 1 hour with PBS;

Step 3: at the PBS containing SDS that 500ml concentration is 5%, adds 100ml penicillin and streptomycin (10KIU/ml, 10g/ml) mixes antimicrobial fluid, and low temperature (4 DEG C) shaking table 150rpm shakes 48 hours; And rinse 1 hour with PBS;

Step 4: with the PBS containing DNA enzymatic that 100ml concentration is 0.5mg/ml, add 20ml penicillin and streptomycin (10KIU/ml, 10g/ml) mix antimicrobial fluid, after 37 DEG C of shaking table 150rpm shake 12 hours, and within 1 hour, obtain the de-cytoskeleton of total spinal disc with PBS flushing, as shown in Figure 1;

Total spinal disc takes off cytoskeletal Histological evaluation

Fig. 2 and Fig. 3 is that 100 times of acellular and acellular nuclear composition residual figure are amplified in de-cytoskeleton (fibrous ring and vertebral pulp) the HE dyeing of total spinal disc of the present invention; Fig. 4 and Fig. 5 is that 100 times of a large amount of glycosaminoglycans component-part diagrams of reservation are amplified in de-cytoskeleton (fibrous ring and vertebral pulp) the alcian blue dyeing of total spinal disc of the present invention.

(4) total spinal disc takes off cytoskeletal antigenic component detection by quantitative

Fig. 6 is that de-cytoskeleton (fibrous ring and vertebral pulp) the DNA detection by quantitative of total spinal disc of the present invention is practically free of DNA component-part diagram.

(5) total spinal disc takes off the observation of cytoskeletal ultra micro stereochemical structure

Fig. 7 and Fig. 8 is that total spinal disc of the present invention de-cytoskeleton (fibrous ring and vertebral pulp) scanning electron microscope detection collagen fiber arrangement completely retains figure with stereoeffect.

(6) total spinal disc takes off cytoskeletal evaluation of its biocompatibility

Fig. 9 is that the de-cytoskeleton CCK-8 of total spinal disc of the present invention detects the increment situation map of MSCs under different support lixiviating solution concentration. Figure 10 is the de-cytoskeleton Live/Dead cell dyeing MSCs of total spinal disc of the present invention growing state figure of 100 times on the de-cytoskeleton of total spinal disc.

Claims

1. the preparation method of the de-cell material of the total spinal disc in a natural tissues source, it is characterised in that the method specifically includes following steps:

(1) taking vertebrates intervertebral disc, extract bone fragment, aseptic PBS rinses 3 times, every time rinsing 20 minutes, removes blood, remaining muscular tissue and ligament;

(3) at the PBS containing TritonX-100 that concentration is 4%, adding the mixing antimicrobial fluid of 10KIU/ml penicillin and 10g/ml streptomycin, buffer and mixing antimicrobial fluid volume ratio is 5:1, and 45 DEG C of shaking table 150rpm of constant temperature shake 48 hours; And rinse 1 hour with PBS; Described mixing antimicrobial fluid is made up of penicillin and streptomycin, and the volume ratio of penicillin and streptomycin is 1:1;