CN101829360A - Method for preparing acellular ligament or tendon stent - Google Patents
Method for preparing acellular ligament or tendon stent Download PDFInfo
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- CN101829360A CN101829360A CN201010149804A CN201010149804A CN101829360A CN 101829360 A CN101829360 A CN 101829360A CN 201010149804 A CN201010149804 A CN 201010149804A CN 201010149804 A CN201010149804 A CN 201010149804A CN 101829360 A CN101829360 A CN 101829360A
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Abstract
The invention relates to the technical field of tissue engineering, in particular to a method for preparing an acellular ligament or tendon stent. In the conventional method for preparing the ligament or tendon acellular ligament stent, acellular treatment is performed by jointly applying physical, chemical and enzyme methods, wherein the use of a detergent is an important step in the process of the acellular treatment. Ligaments and tendons are compact connective tissues and the inside and the outside of the fiber bundle are wrapped by an inner membrane and an outer membrane to make cellular constituents difficult to remove, so that the acellular period is long and the acellular process causes large damage to an extracellular matrix. The invention provides the method for preparing the acellular ligament or tendon stent, and in the method, laser treatment is performed on the surfaces of ligaments or tendons by using a dot matrix with suitable density and energy and then few detergent or no detergent can be used according to the conditions in the process of acellular treatment, so that influence on the extracellular matrix is reduced and adhesion and growth of the cells are facilitated.
Description
Technical field
The present invention relates to the tissue engineering technique field, be specifically related to the preparation method of a kind of acellular ligament or tendon scaffold.
Background technology
Repairing back healing ability difference after ligament and the tendon injury is common clinically problem, and this makes multiple substitute be applied to the reconstruction of ligament and tendon.Wherein allosome, xenogenesis ligament and tendon graft stock support have the inherent characteristics of ligament and tendon scaffold, along with the development of taking off cell technology and sterilization technology, the host reduces greatly to the immunologic rejection and the disease propagation of allosome, the tough belt supporting frame of xenogenesis, and making ligament and tendon take off cytoskeleton becomes possibility in clinical practice.Present ligament and tendon acellular ligament support preparation method be the capable cell that takes off of use in conjunction physics, chemistry and enzyme process often, and wherein the use of detergent is cell free important step.To use difference little for report ligament or the cell free physical method of tendon, enzyme process and conventional reagent on the document, but the selecting method report of detergent is differed, and it is also inconsistent to take off the cell result.(referring to document 1.Gratzer PF, Harrison RD, Woods T.Matrix alteration and not residualsodium sulfate cytotoxicity affects the cellular repopulation of a decellularizedmatrix.Tissue-Eng.2006; 12 (10): 2975-83.2.Whitlocka PW, Smitha TL, Poehlinga GG, Shilta JS, Dykeb MV.A naturally derived, cytocompatible, andarchitecturally optimized scaffold for tendon and ligament regeneration.Biomaterials 2007; 28:4321-4329.3.Woods T, Gratzer PF.Effectiveness of threeextraction techniques in the development of a decellularized bone-anteriorcruciate ligament-bone graft.Biomaterials 2005; 26:7339-7349.) ligament and tendon be compact connective, has inner membrance and adventitia to hold inside and outside the fibre bundle and make cell component remove difficulty, so it is long to take off cell cycle, takes off cell processes pair cell epimatrix and destroy bigger.The various cell reagents that take off carry out the cell free while, and therefore also interference cell epimatrix structure reduces the influence that the use of taking off cell reagent can alleviate the pair cell epimatrix, more helps cell adhesion and growth.
Dot matrix laser is between the minimally-invasive treatment technology that has between wound and the non-invasive therapy, is usually used in cosmesis in recent years.Laser can reduce damage [Jih MH, the Kimyai-Asadi A.Fractional photothermolysis:a review and update.Semin Cutan Med Surg.2008Mar of surrounding tissue when swashing saturating organizing; 27 (1): 63-71].Still does not have at present report dot matrix laser and take off application in the cytoskeleton process preparing ligament/tendon.
Summary of the invention
The object of the present invention is to provide a kind of preparation ligament/tendon to take off cytoskeletal method.
The inventor takes off in the cytoskeleton process at preparation ligament/tendon and finds that complete adventitia/aponeurosis (aponeuroses) has the effect with extraneous barrier, and the block cell composition removes equally.Take off in the cell processes, but when adventitia/aponeurosis (aponeuroses) was complete, residual cell component mainly was deposited in adventitia/aponeurosis (aponeuroses) below, the inner residual cell composition of tendon is a small amount of, and the imperfect place of adventitia/aponeurosis (aponeuroses), substrate inner cell composition removes thoroughly.
To take off cell behind the tendon aponeurotomy, find to use hypotonic loss of thick fluid cell 48h after, aponeurotomy section cell removes thoroughly, and aponeurosis (aponeuroses) complete segment cell does not have obviously and removes.
As seen cut the tendon aponeurosis (aponeuroses) and help cell and remove, can obviously reduce the use of taking off cell reagent, thereby reduce the interference of pair cell epimatrix.But the tendon aponeurosis (aponeuroses) has the collagen fiber of keeping structure function, and over-drastic incision tendon aponeurosis (aponeuroses) sees that collagen structure is obviously loose after taking off cell 96h.
Therefore we think that the adventitia/aponeurotomy method of ideal ligament/tendon must possess (1), the degree of depth of cutting aponeurosis (aponeuroses) is suitable, can not destroy collagen structure; (2), aponeurotomy is even, ratio is suitable, incision tendon aponeurosis (aponeuroses) that can not be suitable helps cell and removes, and does not influence biomechanics.
The invention provides the method that a kind of organizational project prepares acellular ligament or tendon scaffold, this method is hit porosity with 5%-20% earlier before taking off cell, and the dot matrix laser of 5-10mJ energy is handled the adventitia of ligament or the aponeurosis (aponeuroses) of tendon.
The invention provides the method that a kind of organizational project prepares acellular ligament or tendon scaffold, 1) get animal ligament or tendon; 2) before taking off cell, with the super pulse dot matrix of JC25C CO2 type laser, 5%-20% hits porosity earlier, and the 5-10mJ energy is handled the adventitia of ligament or the aponeurosis (aponeuroses) of tendon; 3) conventional method for removing cells rinsing continued to take off cell 4-7 days with hypotonic medium, changed liquid once every 24h.Institute all carries out at shaking table (150-200RPM) in steps, all adds 5ml/L penicillin/streptomycin solution (10000U/ml10000mg/ml) solution prevention infection, finishes each step and all uses PBS flushing 3 times.According to the ligament/tendon compact structure degree of different animals, take the circumstances into consideration to use or do not use detergent; 4) take off cell and finish after, normal temperature drying is preserved after the cold drying.
The thickness of the adventitia/aponeurosis (aponeuroses) of ligament and tendon and extracellular matrix structural compactness depend on the factors such as kind, position and size of animal.That therefore, uses dot matrix laser hits porosity and sharp hole energy fibrous root according to different adjusting of drawing materials.
With the super pulse dot matrix of JC25C CO2 type laser, select for use 5%-20% to hit porosity, the 5-10mJ energy can swash the aponeurosis (aponeuroses) of saturating ligament/tendon, and little to collagen influence under the aponeurosis (aponeuroses), the about 160 μ m of lasing beam diameter.Before taking off cell, with dot matrix laser the adventitia/aponeurosis (aponeuroses) of ligament/tendon is handled, re-use hypotonic medium and continue to take off cell 96-168h, discovery can remove the inner most cell nuclears of patellar ligament and remove more thorough, the a small amount of tissue coagulation of dot matrix laser breakdown place, collagen structure is normal on every side, contains the small amounts of cells nuclear composition under the aponeurosis (aponeuroses).Behind dot matrix laser treatment ligament or the tendon adventitia, biomechanics does not have obvious change, and cell compatibility is good.
Existing bibliographical information method for removing cells shows that method for removing cells all uses detergent Triton-X100, dodecylic acid sodium (SDS), triphosphoric acid butyl ester (TBP) to wait one or more use in conjunction.Take off cell cycle 1-2 week and do not wait, the zoopery of this method for removing cells shows that it is short to take off cell cycle, significantly reduces detergent and is suitable for, and does not even use detergent.
Description of drawings
Fig. 1 takes off the groups of cells specimen to organize HE slice map (100 times)
Wherein A is a haematoxylin dyeing), B is Yihong dyeing.
Fig. 2 is that the matched group specimen is organized the HE slice map
The specific embodiment
Describe the present invention below in conjunction with drawings and Examples, but enforcement of the present invention is not limited only to this.
Embodiment: the rabbit ligament takes off the cytoskeleton preparation and estimates
1, draw materials: select new zealand rabbit (about 3kg, The 2nd Army Medical College zoopery center provides, down together) for use, complete excision patellar ligament and tibiofibula stop thereof are removed the outer synovial tissue of ligament, must guarantee that the ligament adventitia is complete, 4 ℃ of preservations in the aseptic PBS liquid.
2, dot matrix laser treatment: patellar ligament surface PBS liquid is drawn dried with sterile gauze, with the super pulse dot matrix of JC25C CO2 type laser (doctor people company of U.S. section), select for use 10% to hit porosity, 5mJ energy, processing patellar ligament aponeurosis (aponeuroses).
3, patellar ligament takes off the cell processing, gets the normal rabbit patellar ligament and compares.
(1) it is as follows to take off the cell step:
(0.05% phenylmethyl sulfonylfluoride alcohol solvent, 35ml/L 50ml ethanol+25mgPMSF), continue 120h changes liquid once every 24h to hypotonic tris buffer+serpin of 10mM.Institute all carries out at room temperature, shaking table (150RPM) in steps, all adds 5ml/L penicillin/streptomycin solution (10000U/ml 10000mg/ml) solution prevention bacterial reproduction, finishes each step and all uses PBS flushing 3 times.
(2) the rabbit ligament takes off cytoskeletal histological examination
Get take off cell after two groups of specimen give paraffin embedding, section (5 μ m), the HE observation of dyeing.Observation of cell nuclear composition (haematoxylin dyeing), collagen arrangement form (Yihong dyeing) is made comparisons with normal tendon.See that cell removes thoroughly (Fig. 1) after taking off cell 96h after the dot matrix laser treatment peridesmium; Patellar ligament adventitia untreated fish group is seen that cell does not have obviously and is removed (Fig. 2)
(3) the rabbit ligament takes off cytoskeletal biomechanics detection
Get 12 acellular ligament central longitudinal to 12mm * 1mm
2Part detects sample as biomechanics, and 12 fresh patellar ligaments getting same area, identical size as a comparison.Use biomechanics instrument (Instron 5544, Needham, and MA) detection of biological mechanical property continues tractive, and fracture is as the criterion in the middle of the test sample book.Wherein 2 acellular ligaments are not included comparison in the fracture of clamp end.Use independent sample t-check analysis to take off the influence of cell to biomechanics.The results are shown in elastic modelling quantity, maximum displacement, maximum load does not all have significant difference (seeing table 1 for details), illustrates that taking off cell front and back patellar ligament biomechanics does not have obvious change.
Table 1DCA method is taken off the influence of cell to the ligament biomechanics
Claims (4)
1. an organizational project prepares the method for acellular ligament or tendon scaffold, it is characterized in that this method before taking off cell, hits porosity with 5%-20% earlier, and the dot matrix laser of 5-10mJ energy is handled the adventitia of ligament or the aponeurosis (aponeuroses) of tendon.
2. a kind of organizational project according to claim 1 prepares the method for acellular ligament or tendon scaffold, it is characterized in that this method comprises:
A) get animal ligament or tendon;
B) before taking off cell, with the super pulse dot matrix of JC25C CO2 type laser, 5%-20% hits porosity earlier, and the 5-10mJ energy is handled the adventitia of ligament or the aponeurosis (aponeuroses) of tendon;
C) take off cell: continue to take off cell 96-168h with hypotonic medium, change liquid once, on the 150-200RPM shaking table, carry out, add 5ml/L penicillin/streptomycin solution (10000U/ml 10000mg/ml), finish each step and all use PBS flushing 3 times every 24h.
3. a kind of organizational project according to claim 2 prepares the method for acellular ligament or tendon scaffold, it is characterized in that, step C) hypotonic medium is hypotonic tris buffer and the serpin of 10mM in, and serpin wherein is 0.05% phenylmethyl sulfonylfluoride alcohol solvent, 35ml/L 50ml ethanol and 25mgPMSF.
4. the method for preparing acellular ligament or tendon scaffold according to claim 1,2 or 3 described a kind of organizational projects, it is characterized in that step B) middle with the super pulse dot matrix of JC25C CO2 type laser, 10% hits porosity, the 5mJ energy is handled the adventitia of ligament or the aponeurosis (aponeuroses) of tendon.
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| CN 201010149804 CN101829360B (en) | 2010-04-16 | 2010-04-16 | Method for preparing acellular ligament or tendon stent |
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| CN 201010149804 CN101829360B (en) | 2010-04-16 | 2010-04-16 | Method for preparing acellular ligament or tendon stent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105879120A (en) * | 2016-06-08 | 2016-08-24 | 浙江大学 | Preparation method of tendon conjunction bone decellularization material of natural tissue source |
| CN116421789A (en) * | 2023-04-23 | 2023-07-14 | 诺一迈尔（苏州）医学科技有限公司 | Preparation method of acellular dermal matrix composite scaffold |
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| CN1343523A (en) * | 2000-09-19 | 2002-04-10 | 中国人民解放军第二军医大学 | Millipore non-cell xanoepidermis substitute |
| US20030039676A1 (en) * | 1999-02-23 | 2003-02-27 | Boyce Todd M. | Shaped load-bearing osteoimplant and methods of making same |
| CN101332315A (en) * | 2007-06-29 | 2008-12-31 | 上海国睿生命科技有限公司 | Tissue engineering tendon and vitro construction method thereof |
| CN101496912A (en) * | 2008-01-30 | 2009-08-05 | 北京大清生物技术有限公司 | Bio-derivative tendon repair material and preparation method thereof |
-
2010
- 2010-04-16 CN CN 201010149804 patent/CN101829360B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030039676A1 (en) * | 1999-02-23 | 2003-02-27 | Boyce Todd M. | Shaped load-bearing osteoimplant and methods of making same |
| CN1343523A (en) * | 2000-09-19 | 2002-04-10 | 中国人民解放军第二军医大学 | Millipore non-cell xanoepidermis substitute |
| CN101332315A (en) * | 2007-06-29 | 2008-12-31 | 上海国睿生命科技有限公司 | Tissue engineering tendon and vitro construction method thereof |
| CN101496912A (en) * | 2008-01-30 | 2009-08-05 | 北京大清生物技术有限公司 | Bio-derivative tendon repair material and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105879120A (en) * | 2016-06-08 | 2016-08-24 | 浙江大学 | Preparation method of tendon conjunction bone decellularization material of natural tissue source |
| CN105879120B (en) * | 2016-06-08 | 2019-01-25 | 浙江大学 | A kind of tendon joint bone in natural tissues source takes off the preparation method of cell material |
| CN116421789A (en) * | 2023-04-23 | 2023-07-14 | 诺一迈尔（苏州）医学科技有限公司 | Preparation method of acellular dermal matrix composite scaffold |
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