CN102552981A - Method for preparing tissue-engineered bone/cartilage integrated scaffold - Google Patents
Method for preparing tissue-engineered bone/cartilage integrated scaffold Download PDFInfo
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Abstract
The invention discloses a method for preparing a tissue-engineered bone/cartilage integrated scaffold. An integrated scaffold structure of which internal pores are aligned and pore diameters are changed in a gradient way along a thickness direction and which has good mechanical properties is obtained by simulating the composition and spatial structure characteristics of natural articular cartilage and using extracellular matrixes and bone matrix gelatin of the cartilage as main raw materials by methods such as a different concentration superposition method, a directional crystallization method, a freeze-drying method, a cross-linking reaction method and the like. The high biocompatibility of the scaffold is favorable for the adhesion and proliferation of cells; a special pore structure of the scaffold ensures that the content of matrixes secreted by the cells is also changed in a gradient way; and meanwhile, a bone matrix gelatin substrate provides good mechanical support for the instant fixation of the scaffold and an injured part.
Description
Technical field
The invention belongs to tissue engineering technique and technical field of biological material, relate to and be used to repair the damaged tissue engineering bracket preparing technical field of human articular cartilage, particularly have the bionical tissue engineered bone of gradient function/cartilage integral support and preparation method thereof.
Background technology
The articular cartilage damage that is caused by factors such as wound, regression or inflammation is the clinical common disease of orthopaedics; Because regenerating bone or cartilage and repair ability are very limited; In case be difficult to self-regeneration after the damage; And traditional treatment such as expectant treatment, little fracture, arthroscope lavation art etc. all can't obtain satisfied curative effect, and the sustainable development of damage location can cause limbs disturbance and limbs wounded or disabled, have a strong impact on patient's quality of life.At present the clinical treatment articular cartilage damage adopts from body or allosome cartilage transplantation mostly, but can destroy normal articular cartilage from the body cartilage transplantation, and is bigger to patient trauma, and allosome cartilage source is limited and can produce problem such as immunologic rejection.In addition, articular cartilage damage is often followed the pathological changes of subchondral bone, thereby also need repair subchondral bone when repairing cartilage layers.
Tissue engineering technique has been obtained huge effect in recent years in clinical treatment and experiment, also for cartilage injury's reparation new thinking and solution is provided.The tissue engineering technique core is the complex that makes up by cell and cytoskeleton be combined into, and main contents comprise seed cell, support, cytokine and culture environment.Wherein, making up ideal tissue-engineered bone/cartilage frame is the key of repairing cartilage defect.From cartilage surface to subchondral bone; The composition 26S Proteasome Structure and Function of natural cartilage changes and difference with the degree of depth: hyaline cartilage layer main component is II Collagen Type VI, proteoglycan and moisture; Wherein collagen fiber are the arrangement of arched door shape, are full of the extremely strong proteoglycan of water absorption etc. therebetween, produce thus to confine the property bulbs of pressure; Make cartilage layers high resilience, persistent withstand voltage anti-wear performance, have the infiltration lubrication that can supply the ectendotrophy mass exchange simultaneously; The cartilaginous calcification layer mainly is made up of II Collagen Type VI and hydroxyapatite, compact structure and have certain mechanical strength, and this layer is separated into two microenvironments that chemical factors is different with cartilage layers and subchondral bone, makes the two can carry out metabolism separately; The subchondral bone main component is type i collagen and hydroxyapatite, and this is organized as whole cartilage layers main mechanics supporting role is provided.
Above-mentioned many characteristics derive from the complicacy of cartilage biochemical composition and space structure and integrate; Therefore ideal tissue engineered bone/cartilage frame should possess following characteristics: (1) is aspect the material composition; Be mainly the natural component of biogenic property; The main component that also must mate each layer of cartilage simultaneously is beneficial to cell adhesion, propagation and differentiation so that obtain best biocompatibility; (2) aspect space structure; Support should have gradient and orientation texture along thickness direction; When satisfying mechanical strength, be convenient to the chondrocyte arranged distribution; Make the composition requirement near each layer of cartilage of substrate and the moisture of emiocytosis, porosity and pore size also should satisfy the entering of cell and nutrient substance in addition.
How preparing all highly bionical tissue engineered bone/cartilage frame of above-mentioned constituent and space structure is cartilage tissue engineered in recent years hot research fields.Preparation bone/cartilage frame adopts modes such as a minute layer building, fiber bonding, solvent welding and 3 D-printing to carry out more both at home and abroad at present.Timbering material comprises spongy bone, collagen, chitosan, calcium alginate, Fibrin Glue, hyaluronic acid and high molecular polymer, hydroxyapatite etc.Yet there is following problem in the support of said method and material preparation: (1) internal stent layering is obvious, and anti-shearing effect is poor between layer and the layer; (2) the bionical degree of the pore structure of support cartilage layers is low, and no gradient orientations changes; (3) fixedly there is defective in support with the instant of reparation position, can't satisfy the needs of clinical repair; (4) gel-like mechanics of materials support strength is low, and the polymer-based material degraded produces acidic materials, destroys the microenvironment of repair of cartilage; (5) the part technological operation is complicated, and equipment needed thereby is expensive.
Summary of the invention
In order to overcome the defective that above-mentioned technology of preparing exists; The invention provides a kind of method for preparing of novel tissue engineered bone/cartilage integral support; The constituent of bionical natural joint cartilage and spatial structure characteristic; Selecting bone matrix gelatin and cartilage cell epimatrix for use is primary raw material, adopts crystallographic orientation, lyophilization and chemical crosslinking technology, obtains mechanical strength height, internal void orientations, the aperture integrated bracket structure along the thickness direction graded.
Technical scheme of the present invention is following:
The method for preparing of a kind of tissue engineered bone/cartilage integral support may further comprise the steps:
1) gets cartilage cell epimatrix (ECM) slurry that fresh articular cartilage is prepared into variable concentrations; Comprise and concentration be respectively two kinds of ECM slurries of 1～2% and 3～4% (annotate: the percentage composition unit here is g/mL; For example 1% is 0.01g/mL, refers to contain in the 100mL solution 0.1g solute, and this is a concentration method for expressing general when in the biochemical field solid matter being mixed with solution reagent; Together, repeat no more down);
2), obtain bone matrix gelatin (BMG) sheet with spongy bone section and carry out degrease, decalcification, take off cell and handle;
3) mould of selecting for use a depth-adjustment to save, the top of mould is uncovered, but the bottom piston type moves;
The BMG sheet that 4) will be cut to the mould shape of cross section is placed on mold bottom, pours concentration then into and be 3～4% ECM slurry, discharges BMG sheet air entrapment through vibration, makes the ECM slurry soak into the space of BMG sheet and floods the BMG sheet;
5) adding concentration again in the mould is 1～2% ECM slurry, and vertical vibration makes natural fusion between the two-layer ECM slurry up and down;
6) regulate die depth, ECM slurry upper surface is flushed with opening end, cover opening end and contact fully with slurry with sheet metal, then that mould is freezing with reaching the bottom surface around the insulation material parcel mould, carry out crystallographic orientation;
7) sheet metal and the insulation material of removal opening end move into freezer dryer with mould and carry out lyophilization, obtain shaped support;
8) (end with BMG sheet) smeared the suspension of hyaluronic acid solution and nanometer hydroxyapatite in the support bottom, through capillarity suspension got in the brace aperture;
9) support is carried out ultraviolet irradiation and chemical crosslinking, lyophilization once more after the cleaning obtains tissue engineered bone/cartilage integral support.
Above-mentioned steps 1) process of ECM slurry of preparation variable concentrations is: fresh articular cartilage is cleaned be cut into small pieces, under the condition that protease inhibitor exists, carry out the ultra micro waterproof pulverization, gradient centrifugation; Collect supernatant, handle with cell pyrolysis liquid earlier and remove the residual cell composition, reuse DNase and ribonuclease are handled and are removed nuclear matter; Add ethanol precipitation then, gradient centrifugation, collecting precipitation; Water is mixed with the slurry of concentration range at 1～4% variable concentrations, and 4 ℃ of preservations are subsequent use.
In the step 1) concrete operations, said protease inhibitor can be selected Phenylmethanesulfonyl fluoride for use, the articular cartilage fritter is put into Tris-HCL (pH7.4) buffer that contains the 0.35mM Phenylmethanesulfonyl fluoride carry out the ultra micro waterproof pulverization, pulverizes and under 4 ℃, carries out.Pulverize the back add tri-distilled water fully dilute again centrifugal, centrifugal adopt 4 ℃ of following differential centrifugations (3000～6000r/min) 15～30 minutes, collect supernatant.Get supernatant and carry out lysis, cell pyrolysis liquid can be used 1%TritonX-100, at 4 ℃ of following persistent oscillations de-sludging in 24～36 hours and removal residual cells composition.Remove nuclear matter can be under the condition of DNase 40000～60000U/L and ribonuclease 800～1200U/L 37 ℃ of digestion 12 hours.After the digestion, the alcoholic solution of interpolation 75% precipitates, 4 ℃ of following 10000～12000r/min high speed centrifugations, and collecting precipitation adds the ECM slurry that tri-distilled water is mixed with variable concentrations, and 4 ℃ of preservations are subsequent use
Above-mentioned steps 2) process of preparation BMG sheet is: utilizing the undecalcified microtome that the spongy bone piece is cut into thickness is 0.5～1mm spongiosa osteocomma; Cleaning the back is white in color with hydrogen peroxide dipping to osteocomma; The reuse water rinse ,-20 ℃ of hypotonic multigelations make cell membrane broken, carry out degrease, decalcification then, take off the cell processing; Obtain bone matrix gelatin (BMG), 4 ℃ of preservations are subsequent use after the lyophilization.
Step 2) concrete operations can be: after the section with osteocomma earlier with normal saline soaking flushing 15～30 minutes repeatedly, put into 1% hydrogen peroxide again and soak and be white in color to osteocomma in 1～2 hour.-20 ℃ of hypotonic multigelations 2～4 times each 12～24 hours, add degrease then, take off Cell sap: 2% sodium lauryl sulphate, 6% lipase, 1% ethylenediaminetetraacetic acid, 0.1% sodium azide, room temperature vibration 24～36 hours; Again osteocomma is soaked in 10% ethylenediaminetetraacetic acid, preserves 3～5 days to the osteocomma porous spongy that is white in color for 37 ℃; 3%TritonX-100 rinsing osteocomma 12 hours, tri-distilled water cleaned 15～30 minutes, obtained degrease, decalcification, took off cell bone matrix gelatin (BMG), and 4 ℃ of preservations are subsequent use after the lyophilization.
Above-mentioned steps 3) said mould one end is uncovered, but the other end is the bottom of piston type motion, thereby can regulate die depth, for follow-up crystallographic orientation provides condition, and can be used for making the support of different-thickness.
Above-mentioned steps 4) volume ratio of employed 3～4%ECM slurry and the employed 1～2%ECM slurry of step 5) was generally 1: 1～2: 1, and those skilled in the art can suitably adjust according to the architectural feature that support is repaired the position.
Above-mentioned steps 6) said sheet metal uses stainless steel substrates usually, and in low temperature environment, the slurry that contacts with sheet metal produces crystallographic orientation, and said low temperature environment generally is meant-40 ℃ environment, freezing 3～4 hours or longer time.
Above-mentioned steps 7) cryodesiccated temperature is-50 ℃.
Above-mentioned steps 8) suspension of hyaluronic acid solution and nanometer hydroxyapatite gets into BMG and ECM hole through capillarity; Three's mutual group is integral the bottom of support; Wherein the concentration of hyaluronic acid solution is 1～2%, and nanometer hydroxyapatite and hyaluronic mass ratio are 1: 4～1: 6 in the suspension.
Above-mentioned steps 9) cross-linking agent of chemical crosslinking employing is carbodiimides (EDC) and N-hydroxy-succinamide (NHS); With crosslinked 12 hours of the ethanol solution that is placed on carbodiimides and N-hydroxy-succinamide, wherein the concentration of carbodiimides and N-hydroxy-succinamide was respectively 50mM and 20mM.Prepared integral support is vacuum-packed, Co
60Sterilization, 4 ℃ of preservations are subsequent use.
Tissue engineered bone/cartilage the integral support of the inventive method preparation, in conjunction with from body chondrocyte or mescenchymal stem cell, it is damaged to be used for repairing articular cartilage.
The present invention compared with prior art has following advantage and beneficial effect:
1) from the preparation feedstock analysis: this support raw material is the natural component of biogenic property, and its source can be animals such as allosome people or xenogenesis pig, cattle, sheep, dog.Wherein ECM is a cartilage cell epimatrix, and it has kept most of composition of natural joint cartilage cell epimatrix, like II Collagen Type VI and GAG, can adhere to and breed just make chondrocyte need not external evoked cultivation, has excellent biocompatibility.About extracting the method for cartilage cell epimatrix, the inventor has applied for and the multinomial related patent U.S. Patent No. of obtaining the authorization (referring to one Chinese patent application number/patent No.: 200510073008.1,200810057373.7,200810106133.1), technology of preparing is ripe.And BMG passes through degrease, decalcification, takes off the cell processing, and main component is a type i collagen, and is identical with the subchondral bone composition, and has loose three-dimensional porous structure, and mechanical strength is higher, can fixedly provide competent mechanics to support the instant of defect for support.
2) from the internal stent structural analysis: this internal stent hole is orientations, also is columnar arrangement after cell gets into and distributes (like Fig. 1, shown in 2), with natural cartilage layer structural similarity.Because different along thickness direction ECM concentration, cause pore size also graded can occur along thickness direction, therefore implant the new matrix content of emiocytosis and function also can change in gradient.In addition, the molding that is structure as a whole of this support can be born the effect of shearing force, satisfies the mechanics requirement of implant site.The support average thickness is 3mm, near person joint's cartilage thickness, can reduce the modification of stent size during the clinical treatment cartilage defect.
In a word; The integrated orientation bone/cartilage frame of the present invention's preparation has overcome problems such as bone in the traditional handicraft/the bionical degree of cartilage frame internal structure is low, mechanical strength deficiency, in conjunction with chondrocyte or the damaged satisfactory therapeutic effects that obtains of mescenchymal stem cell repairing articular cartilage.In addition, the technical process that the present invention prepares support is simple, and is easy to operate, good reproducibility, and support stability is high, is a kind of application prospect tissue engineered bone/cartilage frame material widely.
Description of drawings
Fig. 1 is the tissue engineered bone/cartilage integral support structural representation of embodiment of the invention preparation;
Fig. 2 is the indicative of local optical microphotograph of tissue engineered bone/cartilage integral support of preparing of the present invention.
The specific embodiment
Below further describe the present invention through embodiment, should be understood that these embodiment only are used for purpose of illustration, do not limit the scope of the invention extremely.
Embodiment 1: the articular chondrocytes epimatrix (ECM) of preparation variable concentrations
Buy fresh pig articular cartilage tissue piece, after rinsing well repeatedly with normal saline, be cut into fritter, put into Tris-HCL (pH7.4) buffer that contains protease inhibitor Phenylmethanesulfonyl fluoride (0.35mM), under 4 ℃, carry out the ultra micro waterproof pulverization.Add tri-distilled water and fully dilute the back 4 ℃ of following differential centrifugations (3000～6000r/min) 30 minutes; Collect supernatant; With 1%TritonX-100 in 4 ℃ of following persistent oscillations de-sludging in 24 hours and remove the residual cells composition; Add DNase 50000U/L and ribonuclease 1000U/L, digested 12 hours down, remove nuclear matter at 37 ℃.Add alcoholic solution 10～20ml of 75% and precipitate, 4 ℃ of following 12000r/min high speed centrifugations, collecting precipitation, adding tri-distilled water, to be mixed with concentration be two kinds of slurries of 1% and 4%, 4 ℃ of preservations are subsequent use.。
Embodiment 2: preparation bone matrix gelatin (BMG)
Choose Radix Achyranthis Bidentatae joint spongy bone, use undecalcified bone slice machine that the spongy bone piece is cut into the osteocomma of thickness as 1mm, with normal saline repeatedly soaking flushing put into 1% hydrogen peroxide after 15～30 minutes and soak and to be creamy white to the bone piece in 1 hour.Behind the tri-distilled water rinsing bone piece 2 times, put into-20 ℃ together and thawed 2 broken cell films of hypotonic multigelation again in freezing 24 hours.Add degrease, take off Cell sap: 2% sodium lauryl sulphate, 6% lipase, 1% ethylenediaminetetraacetic acid, 0.1% sodium azide, room temperature vibration 24 hours.Then osteocomma is put into 10% edta solution, preserved 4 days, and be porous platinum sponge shape for 37 ℃ until osteocomma.Reuse 3%TritonX-100 rinsing osteocomma 12 hours, tri-distilled water rinsing are after 15 minutes, and frozen section dyes, and mirror is observed fat down, bone marrow is removed.After degrease, decalcification, taking off the lyophilization of cell bone matrix gelatin (BMG) sheet, 4 ℃ of preservations are subsequent use.
Embodiment 3: preparation bone matrix gelatin and extracellular matrix integral support
Select for use an end uncovered, but the other end is the mould of piston type motion bottom.Earlier the BMG sheet of preparation among the embodiment 2 is put into mold bottom, the 4%ECM slurry 20ml that gets preparation among the embodiment 1 pours mould into, vibrates 2 minutes, makes the ECM slurry soak into the BMG hole fully, and unnecessary slurry covers the BMG upper surface, and thickness is 2mm; Getting among the embodiment 1 concentration is that 1%ECM slurry 10ml continues to add in the mould, covers the 4%ECM slurry, and vertical vibration makes natural fusion between the two-layer ECM slurry, is integrative-structure, and integral thickness is 3mm; Regulate die depth, slurry is flushed with opening end, cover opening end and contact fully with slurry with stainless steel substrates, wrap up with insulating foam mould periphery and bottom, guarantees the slurry generation crystallographic orientation that contacts with stainless steel substrates; Entire die was placed-40 ℃ of environment freezing 4 hours, and moved in-50 ℃ of freezer dryers then and carried out lyophilization 48 hours; Behind the rack forming; An end that has BMG at support is evenly smeared the hyaluronic acid suspension that contains nanometer hydroxyapatite, and wherein hyaluronic acid concentration is 1.5%, and hydroxyapatite and hyaluronic acid mass ratio are 1: 5; Because capillarity; Suspension gets into BMG and ECM hole, support process ultraviolet irradiation 8 hours, and (carbodiimides concentration was 50mM to the ethanol solution of carbodiimides and N-hydroxy-succinamide (NHS) in crosslinked 12 hours; NHS concentration is 20mM), tri-distilled water rinsing 15 minutes, lyophilization is 48 hours once more, obtains the bone/cartilage integral support of molding.
The indicative of local optical microphotograph of prepared tissue engineered bone/cartilage integral support is seen Fig. 2, and its structure is as shown in Figure 1, and BMG substrate 2 offers the competent mechanics of support and supports; ECM collagen fiber 1 are orientations, and hole changes in gradient; Nanometer hydroxyapatite 3 enters into BMG and ECM hole from the bottom.
Claims (9)
1. the method for preparing of tissue engineered bone/cartilage integral support may further comprise the steps:
1) gets fresh articular cartilage and be prepared into the cartilage cell epimatrix slurry that concentration is two kinds of variable concentrations of 1～2% and 3～4%;
2), obtain the bone matrix gelatin sheet with spongy bone section and carry out degrease, decalcification, take off cell and handle;
3) mould of selecting for use a depth-adjustment to save, the top of mould is uncovered, but the bottom piston type moves;
The bone matrix gelatin sheet that 4) will be cut to the mould shape of cross section is placed on mold bottom, pours concentration then into and be 3～4% cartilage cell epimatrix slurry, makes slurry soak into the space of bone matrix gelatin sheet and floods the bone matrix gelatin sheet;
5) adding concentration again in the mould is 1～2% cartilage cell epimatrix slurry, and vertical vibration makes natural fusion between the two-layer slurry up and down;
6) regulate die depth, the slurry upper surface is flushed with opening end, cover opening end and contact fully with slurry with sheet metal, then that mould is freezing with reaching the bottom surface around the insulation material parcel mould, carry out crystallographic orientation;
7) remove sheet metal and insulation material, mould is moved into freezer dryer carry out lyophilization, obtain shaped support;
8) end that has a bone matrix gelatin sheet at support is smeared the suspension of hyaluronic acid solution and nanometer hydroxyapatite, through capillarity suspension is got in the brace aperture;
9) support is carried out ultraviolet irradiation and chemical crosslinking, clean postlyophilization, obtain tissue engineered bone/cartilage integral support.
2. method for preparing as claimed in claim 1 is characterized in that, said step 1) adopts following method to prepare the cartilage cell epimatrix slurry: fresh articular cartilage is cleaned be cut into small pieces; Under the condition that protease inhibitor exists, carry out the ultra micro waterproof pulverization, gradient centrifugation is collected supernatant; Handle with cell pyrolysis liquid earlier and remove the residual cell composition; Reuse DNase and ribonuclease are handled and are removed nuclear matter, add ethanol precipitation then, gradient centrifugation; Collecting precipitation, water are mixed with the slurry of variable concentrations.
3. method for preparing as claimed in claim 2 is characterized in that, step 1) is put into the Tris-HCL buffer of the pH7.4 that contains the 0.35mM Phenylmethanesulfonyl fluoride with the articular cartilage fritter, carries out the ultra micro waterproof pulverization under 4 ℃; Add tri-distilled water then and fully dilute, 4 ℃ were descended 3000～6000r/min differential centrifugation 15～30 minutes, collected supernatant; Supernatant with 1%TritonX-100 4 ℃ of following persistent oscillations de-sludging in 24～36 hours with remove the residual cells composition; Then under the condition of 40000～60000U/L DNase and 800～1200U/L ribonuclease 37 ℃ digestion 12 hours; Back 75% alcoholic solution that adds of digestion precipitates, 4 ℃ of following 10000～12000r/min high speed centrifugations, and collecting precipitation, the reuse tri-distilled water is mixed with the slurry of variable concentrations.
4. method for preparing as claimed in claim 1; It is characterized in that said step 2) adopt following method to prepare the bone matrix gelatin sheet: utilizing the undecalcified microtome that the spongy bone piece is cut into thickness is 0.5～1mm spongiosa osteocomma, cleans the back and is white in color with hydrogen peroxide dipping to osteocomma; The reuse water rinse;-20 ℃ of hypotonic multigelations make cell membrane broken, carry out degrease, decalcification then, take off the cell processing, obtain the bone matrix gelatin sheet.
5. method for preparing as claimed in claim 4; It is characterized in that; Said step 2) osteocomma after hydrogen peroxide is handled is in-20 ℃ of hypotonic multigelations 2～4 times; Each 12～24 hours, place the solution that contains 2% sodium lauryl sulphate, 6% lipase, 1% ethylenediaminetetraacetic acid and 0.1% sodium azide then, room temperature vibration 24～36 hours; Again osteocomma is soaked in 10% ethylenediaminetetraacetic acid, preserves 3～5 days to the osteocomma porous spongy that is white in color for 37 ℃; Use 3%TritonX-100 rinsing osteocomma 12 hours then, tri-distilled water cleaned 15～30 minutes, obtained the bone matrix gelatin sheet.
6. method for preparing as claimed in claim 1 is characterized in that, the volume ratio of employed 3～4%ECM slurry of step 4) and the employed 1～2%ECM slurry of step 5) is 1: 1～2: 1.
7. method for preparing as claimed in claim 1 is characterized in that, the said sheet metal of step 6) is a stainless steel substrates, and cryogenic temperature is-40 ℃, cooling time 3～4 hours or longer.
8. method for preparing as claimed in claim 1 is characterized in that the concentration of hyaluronic acid solution described in the step 8) is 1～2%, and nanometer hydroxyapatite and hyaluronic mass ratio are 1: 4～1: 6 in the suspension.
9. method for preparing as claimed in claim 1 is characterized in that, the cross-linking agent that the step 9) chemical crosslinking is adopted is carbodiimides and N-hydroxy-succinamide.
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