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Artificial intervertebral disc complex tissue and preparation method thereof

A complex, intervertebral disc technology, used in prostheses, medical science, spinal implants, etc.

Inactive Publication Date: 2011-01-26
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention aims at the defects of tissue engineering nucleus pulposus or tissue engineering annulus fibrosus grafts with only a single component in the prior art, and provides an artificial intervertebral disc complex tissue, which combines the nucleus pulposus scaffold material and the annulus fibrosus scaffold material together , and have better biological properties

Method used

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  • Artificial intervertebral disc complex tissue and preparation method thereof
  • Artificial intervertebral disc complex tissue and preparation method thereof
  • Artificial intervertebral disc complex tissue and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Preparation of annulus fibrosus decalcification and decellularized bone matrix annulus

[0032] Materials: The bilateral femoral condyles of New Zealand white rabbits were taken, the periosteum was removed, the bone marrow was washed with PBS, and the bones were trimmed into oval bone blocks.

[0033]Decellularization method: Prepare according to Courtman's neutral detergent-enzyme four-step decellularization method (see Courtman DW, Pereira C, Kashef V, et al.Development of apericardial cellular matrix biomaterial: Biochemical and mechanical effects of cell extraction.J Biomed Materials Res, 1994, 28:655-666). Put the bone pieces into the enzyme inhibitor working solution (purchased from Sigma, USA), and shake at a constant temperature of 4°C for 3 days; put the bone pieces into the working solution of detergent enzyme inhibitors (purchased from Sigma, USA), and shake at a constant temperature of 4°C for 3 days ; After continuous washing with deionized wate...

Embodiment 2

[0035] Embodiment 2: Construction of nucleus pulposus cell scaffold

[0036] Porcine type II collagen CII was dissolved in 0.01mol / l HCl (pH 2.3), stirred at 4°C to prepare a 1.25% (w / v) CII solution, and the pH of the CII solution was adjusted to 1-2. Under the condition of continuing stirring at 4°C, 4°C, 1.25% HyA (hyaluronic acid) solution dissolved in ultrapure water was added dropwise at a ratio of 9:1 (v / v), and the speed was controlled at 0.5ml / min. After mixing, stir at 300r / min at 4°C for 4h, centrifuge at 3000r / min at 4°C for 15min to remove air bubbles, pour the mixed solution into a disposable petri dish, shake gently to make the liquid level, and immediately store at -70°C Refrigerator chilled.

Embodiment 3

[0037] Example 3: Construction of nucleus pulposus annulus fibrosus complex scaffold

[0038] The CII / HyA mixed solution was injected into the hollow fibrous ring scaffold prepared in Example 1, and freeze-dried. The dried complex is soaked in 20ml of 40% ethanol solution containing 50mmol / l 2-morpholineethanesulfonic acid (MES pH=5.5) for 30 minutes (at room temperature) per 50mg dry weight, then soaked in 20ml containing 50mmol / l l MES, 24mmol / l EDC, 5mmol / l succinamide (NHS) and 2wt% 6-CS chondroitin sulfate in 40% ethanol solution, crosslink at room temperature (20-25°C) for 24h. Put this complex scaffold in 0.1mol / l Na 2 HPO 4 Wash twice in 1mol / l NaCl for 1 hour in total; wash twice in 1mol / l NaCl for 2 hours in total; wash in 2mol / l NaCl for 24 hours (replaced 6 times during the period), wash ten times with ultrapure water, and freeze-dry twice in a freeze dryer Obtain the complex scaffold. Sterilized by cobalt 60 irradiation and stored at -20°C for later use.

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Abstract

The invention relates to the field of tissue engineering, and discloses an artificial intervertebral disc complex tissue and a preparation method thereof. The artificial intervertebral disc complex tissue comprises nucleus pulposus and fibrous rings, wherein the fibrous rings consist of bone matrix rings of demineralization cells; the nucleus pulposus is formed by growing nucleus pulposus cells and fibrous ring cells on a nucleus pulposus-fibrous ring complex scaffold consisting of collagen II, hyaluronic acid and chondroitin sulfate. The shape and the size of the artificial intervertebral disc are similar to those of the natural intervertebral disc, the outer fibrous ring tissue is dense, and the inner nucleus pulposus is of semitransparent gel. The detection results of biochemical components show that the contents of DNA, protein polysaccharide and hydroxyproline of the artificial intervertebral disc complex tissue are remarkably increased along with the time and similar to the content of the natural intervertebral disc at the 12th week. The gene expression analysis result of collagen also shows that the complex intervertebral disc of the tissue engineering is similar to the natural intervertebral disc, and shows that the artificial intervertebral disc complex tissue has good biological function and wide application prospect.

Description

technical field [0001] The invention relates to the field of tissue engineering, in particular to an artificial intervertebral disc complex tissue and a preparation method thereof. Background technique [0002] Tissue Engineering (Tissue Engineering) is the application of the principles and technologies of life science and engineering, on the basis of a correct understanding of the relationship between the tissue structure and function of mammals in both normal and pathological states, research and development for repair, maintenance, An emerging discipline that promotes the function and form of biological substitutes for various tissues or organs of the human body after injury. Tissue engineering research mainly includes four aspects: seed cells, biomaterials, methods and techniques for constructing tissues and organs, and clinical application of tissue engineering. [0003] The basic principle and method of tissue engineering are: the normal tissue cells cultured and expa...

Claims

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Application Information

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IPC IPC(8): A61L27/40A61L27/38A61L27/24A61L27/20A61F2/44
CPCA61F2/44A61F2002/30001
Inventor 庄颖李长青周跃黄博
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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