CN102178981B - Method for preparing cartilage repairing scaffold material - Google Patents
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- CN102178981B CN102178981B CN 201110099363 CN201110099363A CN102178981B CN 102178981 B CN102178981 B CN 102178981B CN 201110099363 CN201110099363 CN 201110099363 CN 201110099363 A CN201110099363 A CN 201110099363A CN 102178981 B CN102178981 B CN 102178981B
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Abstract
The invention discloses a method for preparing a cartilage repairing scaffold material. According to the method, the cartilage repairing scaffold material is prepared by performing cell removing, reconstructing, crosslinking, freeze-drying, surface modifying and other treatment processes on a dermal matrix from animal skin. The scaffold material is made from the dermal matrix and has good biocompatibility, surface activity and plasticity and even pore structure, contains chondroitin sulfuric acid and other substances, can promote the attachment and the growth of cartilage cells and the healing of articular cartilage injury, and can repair joint coloboma efficiently, therefore, the scaffold material is a novel scaffold material which can effectively repair articular cartilage coloboma.
Description
Affiliated technical field:
The present invention relates to medical endoprosthesis cartilage injury repair materials technical field, particularly relate to a kind of preparation method of repair of cartilage timbering material.
Background technology:
In the orthopaedics field, articular cartilage defect is very common disease.Articular cartilage is the single connective tissue of no blood vessel, lymphatic vessel, nerve, and it is limited in one's ability that self repairs, and the damage back is difficult to regenerate.Can produce pain, the articular dyskinesia of joint area after the damage.Therefore, the repairing and treating of articular cartilage damage is a difficult problem in orthopaedics field always.From the body chondrocyte cell transplantation, be cartilage tissue engineered content one of, be inchoate a kind of method of repairing cartilage defect in recent years.It is characterized in that: collect cartilaginous tissue from patient's normal position, according to final requirement, through external chondrocyte amplification, with the cell kind to suitable timbering material.Through operation, the timbering material of compound chondrocyte is implanted defect.Because damage field has been filled the chondrocyte that flushes, they directly in the growth of damaged place, propagation, form excellent repairing to defect.But desirable timbering material and good short cell proliferation method are its difficult points in this method.
Desirable repair of cartilage timbering material should have following condition: 1 excellent biological compatibility: nontoxic, no teratogenesis is conducive to attaching, the propagation of cell; 2 suitable biological degradabilities: the degradation rate of material should adapt with the histiocyte rate of growth, and degradation time should adapt with the tissue growth rate; 3 surface activitys: be conducive to the attaching of cell, and provide good microenvironment in its superficial growth, propagation and secretion substrate for cell; 4 certain plasticity: be convenient to the required shape of machine-shaping, and certain mechanical strength is arranged, still can keep its shape in the certain hour that implants; 5 three-dimensional porous structures: have porous and high porosity, porosity preferably reaches 80-90%, and internal surface area is big, both has been conducive to the attaching of cell and grows into, and is conducive to the infiltration of nutritional labeling and the discharge of metabolite again.
Natural collagen is the main constituent of organism connective tissue, has excellent biological compatibility.Stone etc. detect collagen from aspects such as toxicity, aseptic degree, hemolytic, pyrogenicities, prove that it is a kind of transplantation, safety and renovating bracket material (the Stone KR that can support the chondrocyte growth, Steadman JR, Rodkey WG, et al.Regeneration of meniscal cartilage with use of a collagen scaffold.J Bone Joint Surge Am, 1997,79 (12): 1770-1777).As far back as the 1980s, the immunological rejection that allogeneic skin graft takes place of discovering of Heck etc. mainly comes from epidermis cell, (Heck EL Bergstresser PR Baxter CR.Composite skin graft:frozen dermal allografts support the engraftment and expansion of autologousepidermis.J Trauma.1985 such as endotheliocyte and fibroblast, 25 (2): 106-12), therefore, as long as the epidermal tissue of skin can be removed and the cell component in the dermal tissue is removed, just should be able to avoid the generation of immunological rejection.Acellular dermal (accellular dermal matrix ADM) has not only removed cell component, and complete reservation the basement membrane of dermal papilla laminar surface, for new life and the expansion of transplanted cells provides favourable support.Nineteen ninety-five, people such as Livesey have at first reported preparation success (the Livesey SA of ADM, Herndon DN, HollyoakMA, et al.Transplanted acellular allograft dermal matrix.Potential as a template forthe reconstruction of viable dermis.Transplantation.1995,60 (1): 1-9).After this ADM is as a kind of novel repair of cartilage timbering material, in the clinical practice in the fields such as reparation of burning wound and wound surface and oral and maxillofacial surgery skin to have obtained good result.But because cell free animal corium exists biological degradability poor, cell compatibility is not good enough, and the restriction of the undesirable grade of porosity can't be well as the repair of cartilage timbering material.Therefore, seeking the treatment that simple, the from the horse's mouth carrier material of a kind of preparation method is used for articular cartilage defect is the current important topic that faces in this area.
Summary of the invention:
It is good to the purpose of this invention is to provide a kind of biocompatibility and degradability, and surface activity and plasticity are strong, has the preparation method that is used for the repair of cartilage timbering material of pore structure.
In order to achieve the above object, the present invention by the following technical solutions, a kind of preparation method of repair of cartilage timbering material, employing derives from the dermal matrix of new calves/pony skin of back, make by taking off treatment process such as cell, reconstruction, crosslinked, lyophilizing and finishing, concrete processing step is as follows:
A. new calves/pony skin of back, 8%Na
2The S depilation, flowing water cleans, and soaks 2 hours in the acetic acid of 0.5mol/L, and PBS cleans, and dries, and cuts skin corium, obtains dermal matrix;
B. the dermal matrix that disposes that step a is obtained is immersed in 0.01% sodium dodecyl sulfide (SDS) and takes off in the cell agent, and vibration is 4 hours on the horizontal oscillator tube, the skin of calf/pony is taken off cell handle, and lyophilizing obtains acellular dermal (ADM);
C. will be cut into specification through the acellular dermal of step b preparation is 1 * 1cm
2Fritter is used 0.5% trypsinization, and lyophilizing merges ultrasonic method and carries out the reconstruction of collagen fiber;
D. with the acellular dermal lyophilizing after step c reconstruction, carry out crosslinked with 0.2% formaldehyde;
E. with the acellular dermal lyophilizing after steps d is crosslinked, make the disk that diameter is 3.5mm, be immersed in 0.5% the chondroitin sulfate and carry out finishing, obtain the repair of cartilage timbering material.
The present invention is by the comparison of multiple different disposal method, the method of the above-mentioned preferred ADM of preparation carrier is provided, because the prepared engineered vector of the present invention derives from dermal matrix, again through taking off step process such as cell, reconstruction, crosslinked and finishing, make carrier have excellent biological compatibility, suitable biological degradability, surface of good activity, plasticity and pore structure.This carrier contains chondroitin sulfate, can promote chondrocyte to attach and growth, promotes the healing of articular cartilage damage.Use this method and can thoroughly remove the cell component of dermal tissue, avoided the generation of immunological rejection; Carrier surface is smooth, porous nickel; Be fit to cell attaching and the growth of long period, be suitable as the repair process that a kind of novel timbering material is applied to cartilage defect.The present invention has shown good effect aspect the treatment articular cartilage defect.
The present invention obtains biocompatibility by simple preparation technology and degradability is good, surface activity and plasticity are strong, has pore structure, is used for the timbering material of repairing articular cartilage damage, has reached goal of the invention.
The specific embodiment:
Below in conjunction with embodiment in detail the present invention is described in detail: a kind of preparation method of repair of cartilage timbering material, employing derives from the dermal matrix of the skin of back of new calves/pony, make by taking off treatment process such as cell, reconstruction, crosslinked, lyophilizing and finishing, concrete processing step is as follows:
Embodiment
A. the preparation of dermal matrix
Select the skin of back of new calves/pony for use, with back wool hair clipper weak point, cut the Corii Bovis seu Bubali of suitable size, 8%Na
2The S depilation was soaked 2 hours in the acetic acid of immersion 0.5mol/L after cleaning, and PBS dries in the ventilating kitchen after cleaning, and according to actual needs, adjusted dermatome thickness, cut skin corium, and is then that dermal matrix is clean with distilled water, is cut into the size shape of actual needs.
B. dermal matrix takes off cell
The dermal matrix that step a obtains is cleaned, immersed under the room temperature among the 0.01%SDS, vibration was used PBS solution washing 20 times after 4 hours on the horizontal oscillator tube, and the reuse distilled water cleans 20 times, and the low-temperature freeze-drying machine lyophilizing is standby after 24 hours.
C. the reconstruction of collagen fiber
To immerse through the acellular dermal (ADM) that step b obtains in 0.5% trypsin, under 37 ℃ of conditions with 220V, 250W, 40% power ultrasonic 14 hours, again it is immersed and soak after 48 hours in 0.5% trypsin, with PBS solution washing 20 times, flowing water flushing 24 hours, the reuse distilled water cleans 20 times, and the low-temperature freeze-drying machine lyophilizing is standby after 24 hours.
D. collagen fiber is crosslinked
ADM after the step c reconstruction is immersed in 0.2% formalin, soaked 2 hours under the room temperature.After the reuse distilled water cleaned 20 times, the low-temperature freeze-drying machine lyophilizing was standby after 24 hours to remove the removing residue formaldehyde composition in 3 days in the flowing water flushing.
E. the modification of chondroitin sulfate
Steps d is obtained ADM after crosslinked be soaked in 0.5% the chondroitin sulfate cellulose solution, 220V, 25W power jolting 10min fully penetrate in the dermal tissue it, pack after the lyophilizing, with obtaining the repair of cartilage timbering material after the Co60250 ten thousand rads metering irradiation.
Detection to the repair of cartilage timbering material:
1. hole is measured
The corium that will take off after cell, the reconstruction is fixed with Formalin, ethanol gradient dehydration, dimethylbenzene is transparent, paraffin embedding, make 5 μ m serial section after, carry out MASSON dyeing, om observation, and carry out the hole measurement with LICAQwin software.The result shows, treated corium porous nickel afterwards, the about 530 μ m of average pore size.
2. the detection of biocompatibility
5 of SD healthy adult rat, about body weight 250g, male and female are not limit, behind chlore-ammonia ketone (100mg/kg) intraperitoneal injection of anesthesia, under aseptic condition, in the rat spine both sides, the rectangle flap of 3 0.5cm * 1cm of design, the space is 1.0cm, the base of a fruit is positioned in the middle of the back.Start flap, three groups of disinfecting are taken off cell calf dermal matrix, and (0.5cm * 1cm) implant subcutaneous rat respectively covers corium with flap, uses suturing with thread management.Treat at room temperature to feed after rat revives.Postoperative 2,4,6,8,12 weeks are cut flap along former cutting opening, observe the relation of corium implant and surrounding tissue; Specimen is got in biopsy, carries out histological observation with formalin fixed, paraffin embedding, section, HE dyeing back under light microscopic respectively, shows that the repair of cartilage timbering material has excellent biological compatibility.
The concrete data that detect see table 1 for details:
Biocompatibility after table 1 is crosslinked
3. cell attaches the detection of effect
Be born newborn rabbit in 3 days, after the execution, 75% alcohol-pickled sterilization 15-20min.In the aseptic processing room super-clean bench, get newborn rabbit femoral cartilage, with containing 5 * 10
4After the PBS of the U/100ml penicillin flushing, place culture dish to shred, 2%II Collagen Type VI enzyme in cell rotating and culturing system, 37 ℃, 5%CO
2Rotation digestion is spent the night under the saturated humidity condition.The centrifugal supernatant of abandoning adds the DMEM culture medium, and piping and druming evenly changes 25cm over to
2Culture bottle is in 37 ℃, 5%CO
2Cultivate under the saturated humidity condition.Treat cell be paved with bottle at the bottom of during 80-90%, 0.5% trypsinization goes down to posterity, cell counting is with 3 * 10
5Concentration be inoculated on the ADM carrier 37 ℃, 5%CO
2Cultivated 4 days under the saturated humidity condition, scanning electron microscope shows that a large amount of cells are attached on the carrier, and cell surface has microvillus justacrine substrate, and it is respond well to show that cell attaches.
4. interior animal experiment
Get adult new zealand white rabbit, scrape from the left side hind leg under the aseptic condition and get the patellofemoral joint face and be immersed in the phosphate buffer of aseptic 0.1mol/L from body chondrocyte of former generation.The layering sew up wound.The processing method of cartilage sheet in super-clean bench is identical with the detection that cell attaches effect.Behind the cell counting, still with 3 * 10
5Concentration be inoculated on the ADM carrier 37 ℃, 5%CO
2Cultivated 24 hours under the saturated humidity condition.After 24 hours, get out the cartilage defect of the about deeply 2mm of the about 3.5mm of diameter at new zealand white rabbit right side hind leg patellofemoral joint face with the ophthalmology trepan, with kind be implanted with from the ADM of body chondrocyte insert damaged in, drip in carrier surface the layering sew up wound with biogum.Other gets a New Zealand white rabbit right side hind leg patellofemoral joint face is got out the cartilage defect of the about deeply 2mm of the about 3.5mm of diameter with the ophthalmology trepan, is made as blank, the layering sew up wound.Result of the test shows with the blank group to be compared, using the compound chondrocyte of novel carriers of the present invention transplanted after 12 weeks of reparation, Stereo microscope and om observation, implant is combined with autologous tissue closely, the cell growth is arranged in the implant, repairing articular cartilage is damaged efficiently to confirm carrier of the present invention, is a kind of carrier of effective treatment articular cartilage defect.
Claims (1)
1. the preparation method of a repair of cartilage timbering material adopts the dermal matrix that derives from new calves/pony skin of back, makes by taking off cell, reconstruction, crosslinked, lyophilizing and finishing treatment process, and concrete processing step is as follows:
A. new calves/pony skin of back, 8%Na
2The S depilation, flowing water cleans, and soaks 2 hours in the acetic acid of 0.5mol/L, and PBS cleans, and dries, and cuts skin corium, obtains dermal matrix;
B. the dermal matrix that disposes that step a is obtained is immersed in 0.01% sodium dodecyl sulfide and takes off in the cell agent, and vibration is 4 hours on the horizontal oscillator tube, calf/pony skin is taken off cell handle, and lyophilizing obtains acellular dermal;
C. will be through the acellular dermal of step b preparation, being cut into specification is 1 * 1cm
2Fritter is used 0.5% trypsinization, and the method for supersonic vibration postlyophilization is carried out the reconstruction of collagen fiber;
D. with the acellular dermal lyophilizing after step c reconstruction, carry out crosslinked with 0.2% formaldehyde;
E. with the acellular dermal lyophilizing after steps d is crosslinked, make the disk that diameter is 3.5mm, be immersed in 0.5% the chondroitin sulfate and carry out finishing, obtain the repair of cartilage timbering material.
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| CN102743795A (en) * | 2012-05-11 | 2012-10-24 | 天津大学 | Piceid filling collagen scaffold, preparation method and application thereof |
| CN103785065B (en) * | 2014-01-24 | 2015-05-20 | 北京大清生物技术有限公司 | Preparation method of cartilage regeneration scaffold material |
| CN106730011A (en) * | 2017-01-18 | 2017-05-31 | 烟台正海生物科技股份有限公司 | A kind of xenogenesis acellular nerve graft thing with prevention tissue adhesion function and preparation method thereof |
| CN107029298A (en) * | 2017-04-17 | 2017-08-11 | 河南汇博医疗股份有限公司 | A kind of medical acellular dermal matrix and preparation method thereof |
| CN110193096A (en) * | 2019-05-29 | 2019-09-03 | 上海市第六人民医院 | A kind of marine origin biomimetic type cartilage material and preparation method thereof |
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| US20100296969A1 (en) * | 2007-06-12 | 2010-11-25 | Musculoskeletal Transplant Foundation | Process for sterilizing acellular soft tissue under vacuum |
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