CN101979105A - Tissue engineering scaffold material for repairing cartilage defects and preparation method thereof - Google Patents
Tissue engineering scaffold material for repairing cartilage defects and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tissue engineering scaffold material for repairing cartilage defects and a preparation method thereof. The tissue engineering scaffold material is prepared by the following steps of: cleaning the spongy bone at the shoulder blade of a fresh pig; cutting the spongy bone into small blocks with diameter of between 5 and 10mm and thickness of between 3 and 5mm; degreasing, decalcifying and deproteinizing; immersing with alcohol for 30 minutes; and air-drying, wherein the decalcifying time is 6 hours. The prepared demineralized spongy bone matrix (DSBM) has the characteristics of relatively simple and convenient preparation method, and capacity of being obtained on a large scale, can be stored for a long time at a low temperature and is economic and cheap. The material can be prepared into different shapes and sizes according to the shapes of the defects to be repaired, so the material is convenient to apply. The DSBM remains a natural netlike gap structural system of the pig spongy bone, has high cellular compatibility, biocompatibility and degradability and is a very good cartilage tissue engineering scaffold material.
Description
Technical field
The invention belongs to cartilage defect repair materials technical field, be specifically related to a kind of tissue engineering bracket material that cartilage defect is repaired that is used for.The invention still further relates to the preparation method of this timbering material simultaneously.
Background technology
Very common clinically by wound and the joint caused cartilage defect of regression, behind the articular cartilage damage, self repair ability is limited, the how damage of repairing articular cartilage and subchondral bone, promotes articular cartilage self reparation, rebuilds function of joint and become the major issue that clinicist and research worker need solve.
Joint abrasive forming art, cartilage boring or little fracture art etc. down produce the fibrous cartilage reparation by the various CFU-GM in blood and the bone marrow being migrated to the articular cartilage defect district.Though fibrous cartilage all has very big difference with hyaline cartilage on 26S Proteasome Structure and Function, this Therapeutic Method is reduction of patient pain effectively, improves function of joint, is still the first-line treatment method of present clinical treatment limitation cartilage defect.Periosteum, perichondrium, bone cartilage transplantation are that the another kind for the treatment of cartilage defect is clinically selected, though it can produce hyaline cartilage to a certain degree, improve patient's function of joint, but still lack the clinical evidence of long-term efficacy, and, usually limited its clinical practice because donor is limited.From body chondrocyte cell transplantation (autologous chondrocyte transplantation, ACT) can produce to a certain degree hyaline cartilage, be to be applied to clinical a kind of Therapeutic Method in recent years, American-European existing a large amount of patient accepts this kind treatment, short-term clinical research report shows that it has curative effect preferably, but still have the cell distribution inequality, easily spill, cartilage degeneration, leafing, fibrosis cause problems such as graft failure.In order to address these problems, a lot of scholars have carried out repairing based on the Method of Tissue Engineering of cell and three-dimensional stent material the experimentation of cartilage defect, and obtain good result.
Repairing with Method of Tissue Engineering in the process of cartilage defect, the integration of repair tissue and normal structure is particularly important, it is relevant with multiple factor, comprise the type of cartilage defect, the formation of cartilage and the degraded of graft materials etc., and the selection of timbering material is a key factor that influences repairing quality, and the timbering material that is used for tissue engineering bone/cartilage mainly is divided into synthetic material (as PLA, PGA, PLGA etc.) and biologically-derived timbering material (as hyaluronic acid, collagen, alginic acid, chitosan and acellular matrix etc.).Biologically-derived timbering material not only has excellent biological compatibility and favorable biological degradability, and contains the bioactive molecule that can promote that seed cell adheres to, breeds and break up, and has synthetic timbering material incomparable advantage.
(Demineralized bone matrix DBM) is a kind of in the biologically-derived timbering material to decalcified bone matrix, and it mainly is to utilize of the same race or the xenogenesis organ-/ tissue, through taking off cell, removing antigen and handle and obtain the acellular matrix material.This material has extracellular matrix components, the favorable tissue affinity and the compatibility are arranged, help sticking of cell, propagation and differentiation, and has certain mechanical strength, DBM has 3-D natural pore structure system, contain type i collagen simultaneously, collagen is the good support of cell adhesion and growth, with it as timbering material, it has degradability, be easy to be absorbed by body, thereby decalcified bone matrix (DBM) not only has excellent biological compatibility and biological degradability, and has natural pore structure, helps cell and grows into, contain and to promote seed cell to adhere to, the bioactive molecule of propagation and differentiation has synthetic timbering material incomparable advantage.Kasten etc. did from the aspects such as quantity, propagation and differentiation efficiency of the adhesion rate of seed cell, the internal stent of growing into relatively hydroxyapatite (CDHA), tricalcium phosphate and the decalcified bone matrix (DBM) of synthetic, and the result proves that DBM is optimum among the three.Van Osch etc. discovers that the compound perichondrium of decalcified bone matrix (DBM) has reliable one-tenth cartilage ability on the rabbit animal model.Discovery decalcified bone matrix powder (DBP) such as Zhou can inductor the bone marrow stroma stem cell (hMSCs) of going into of outer dimensional culture break up to chondrocyte.Gao etc. repair the rabbit knee district's cartilage defect of bearing a heavy burden after with DBM and the compound cultivation of bone marrow stroma stem cell (BMSCs), and 95% of the cartilage defect degree of depth as a result obtains repairing.But because decalcification intensity and time of DBM is different, the effect that causes repairing bone and cartilage is inconsistent in the prior art.The present inventor studies the back and finds, bone matrix after an amount of decalcification helps cartilage and forms and excessive its one-tenth cartilage ability reduction of decalcified bone matrix, partly cause may be that an amount of decalcification can make bioactie agent compositions such as the bone morphogenetic protein (BMP) that contains in the bone matrix, TGF-β expose, excessive decalcification has then influenced the activity of decalcified bone matrix, and the ability that makes decalcified bone matrix repair cartilage is affected.In addition, main at present both at home and abroad employing Os Bovis seu Bubali, Os Leporis seu Oryctolagi, Os Caprae seu Ovis etc. are made raw material, and these xenogenesis bone material structures and people differ greatly, and allogeneic people bone material source is limited, and easily propagate hepatitis, AIDS etc.How to overcome above-mentioned deficiency, allow this New-support material of decalcified bone matrix, just become problem demanding prompt solution in the prior art better for patient's service.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of and be used for the cartilage defect reparation, have good biocompatibility, degradability and bioactive tissue engineering bracket material.
The present invention also aims to provide a kind of method for preparing described tissue engineering bracket material.
Purpose of the present invention is achieved by the following technical programs.
A kind of tissue engineering bracket material that is used for the cartilage defect reparation, it is characterized in that: with the spongy bone at fresh pig scapula place, cut into the fritter of diameter 5mm～10mm, thickness 3mm～5mm after the cleaning, after defat, decalcification, deproteinization are handled, again with alcohol-pickled 30 minutes, obtain after air-dry; Wherein, decalcification time is 6 hours.
Prepare the method for this tissue engineering bracket material, may further comprise the steps:
1, preparation bone piece: with the fresh pig scapula is raw material, rejects periosteum, cartilage and soft tissue, gets its spongy bone, flowing water stirs flushing repeatedly, removes bone marrow, blood stains and surperficial oils and fats, cuts into diameter 5mm～10mm, the fritter of thickness 3mm～5mm, flushing with clean water is clean;
2, the bone piece of cleaning is placed 1: 1 chloroform methanol mixed liquor of volume fraction, soaking at room temperature defat 2 times, each 6 hours, air-dry then;
3, it is the aqueous hydrochloric acid solution soaking at room temperature decalcification 6h of 0.6mol/L that the bone piece after air-dry places concentration, the envelope-bulk to weight ratio of aqueous hydrochloric acid solution and bone piece is 20mL/g, after decalcification is finished, again the bone piece is placed phosphate buffered solution concentration percetage by weight 0.1%, pH value 7.4 to soak 12～24 hours, air-dry;
4,1: 1 chloroform alcohol mixed liquid dipping defat of bone piece reuse volume fraction is 6 hours, and is air-dry;
5, the bone piece after air-dry with distilled water repeatedly rinsing until pH value 7.0;
6, the bone piece is placed phosphate buffered solution concentration percetage by weight 0.1%, pH value 7.4, constant temperature soaked 72 hours for 37 ℃, and is air-dry;
7, at last air-dry bone piece is placed the ethanol of concentration percetage by weight 75% to soak 30 minutes, promptly obtain the required tissue engineering bracket material that cartilage defect is repaired that is used for after air-dry once more.
With respect to prior art, the present invention has following beneficial effect
1, to have preparation method easy relatively for decalcification cancellous bone matrix of the present invention (DSBM), and the characteristics that can obtain in a large number, and low temperature down can long preservation, and is relatively cheap; Can make different shapes and size by the shape of repair deficiency, it is convenient to use; DSBM had both kept the natural netted gap structure system of pig spongy bone, had good cell and biocompatibility, degradability again, was a kind of good cartilage tissue engineered rack material.
2, according to China's national situation, selecting wide, the suitable pig spongy bone of transplanting of originating for use is raw material, is raw material and adopt Os Bovis seu Bubali abroad more.Because the anatomical physiology of pig is with human close, have an appointment in the high zone of homology 60% homogeny of pig and people MHC-DR gene, it is more receptible object in the organ transplantation donor animal, discover, the inorganic composition content of fresh Os Sus domestica and normal person's bone is more approaching, therefore adopting Os Sus domestica is that raw material carries out the physics and chemistry processed, the easier material of preparing similar body bone tissue natural structure and performance.
3, the pig spongy bone is after series of physicochemical treatment such as process defat, decalcification, deproteinization, its main component is collagen fiber, outward appearance is porous spongy architecture, the mutual traffic of hole can provide roomy internal surface area and space, also contains multiple bioactive substances such as BMP, the space of sticking, breeding and breaking up, growing that helps seed cell, the secretion of extracellular matrix is arranged, and the exchange of gas and nutrient has excellent biological compatibility simultaneously.
4, this product has extremely low immunogenicity, zoopery confirms, it is low that DSBM transplants the back immunological rejection, and has the stronger bone of inducing, cartilage-derived growth, the effect of differentiation, when DSBM has good cell and histocompatibility, also has the characteristic that the sterilization back keeps its chondrocyte induction ability, it has been preserved has the bone morphogenetic protein BMP that induces into the cartilage ability (BMP is a kind of protein molecular in the osseous tissue, to be converted into irreversible bone be cell for free and undifferentiated mescenchymal stem cell and fibrocyte around the main induction of vascular, also can produce cartilage and osseous tissue at position beyond the skeleton, final skeletonization is a kind of typical replacement bone process.
Description of drawings
Fig. 1 DSBM timbering material of the present invention is seen substantially;
Fig. 2 DSBM scanning electron microscopic observation of the present invention shape characteristic;
Fig. 3 DSBM of the present invention is with Image J software measurement pore size;
Fig. 4 DSBM of the present invention calculates porosity with Adobe Photoshop CS4 software rectangular histogram function;
The growth curve of the external compound cultivation of Fig. 5 DSBM of the present invention and BMSCs;
Fig. 6 DSBM of the present invention and BMSCs unite the scanning electron microscope feature of cultivating the 2nd day;
Fig. 7 DSBM of the present invention and BMSCs unite the scanning electron microscope feature of cultivating the 6th day;
Fig. 8 DSBM material of the present invention is implanted the damaged animal model situation of knee cartilage;
Gross examination of skeletal muscle when Fig. 9 DSBM material of the present invention is implanted damaged 2 weeks of knee cartilage;
Gross examination of skeletal muscle when Figure 10 DSBM material of the present invention is implanted damaged 4 weeks of knee cartilage;
Gross examination of skeletal muscle when Figure 11 DSBM material of the present invention is implanted damaged 8 weeks of knee cartilage;
Gross examination of skeletal muscle when Figure 12 DSBM material of the present invention is implanted damaged 12 weeks of knee cartilage;
Histology's performance when Figure 13 DSBM material of the present invention is implanted damaged 2 weeks of knee cartilage;
Histology's performance when Figure 14 DSBM material of the present invention is implanted damaged 4 weeks of knee cartilage;
Histology's performance when Figure 15 DSBM material of the present invention is implanted damaged 8 weeks of knee cartilage;
Histology's performance when Figure 16 DSBM material of the present invention is implanted damaged 12 weeks of knee cartilage;
Safranin O dyeing performance when Figure 17 DSBM material of the present invention is implanted damaged 8 weeks of knee cartilage;
Safranin O dyeing performance when Figure 18 DSBM material of the present invention is implanted damaged 12 weeks of knee cartilage.
The specific embodiment
The present invention is described in further detail below in conjunction with drawings and Examples, but they are not limitation of the invention.
Embodiment
With the fresh pig scapula is raw material, rejects periosteum, cartilage and soft tissue, gets its spongy bone, and flowing water stirs flushing repeatedly, removes bone marrow, blood stains and surperficial oils and fats, cuts into round pie (diameter 5mm-10mm, 3mm-5mm is thick) on demand, the tap water flushing.Chloroform/methanol (1: 1, volume fraction) soaking at room temperature defat 6h * 2 times, the electric heating constant temperature air dry oven is air-dry.0.6mol/L hydrochloric acid soaking at room temperature decalcification 6h, (hydrochloric acid/spongy bone: 20mL/g), soaked overnight in 0.1%PBS (PH7.4); The electric heating constant temperature air dry oven is air-dry.Reuse chloroform/ethanol defat 6h * 1 time the electric heating constant temperature air dry oven is air-dry.With distilled water repeatedly rinsing until pH value 7.0.0.1%PBS (PH7.4) soaks 72h for 37 ℃, and the electric heating constant temperature air dry oven is air-dry.75% alcohol-pickled 30 minutes, obtain decalcification cancellous bone matrix (DSBM) after the electric heating constant temperature air dry oven is air-dry, it is standby to seal 4 ℃ of preservations.
The naked eyes of experimental example 1:DSBM and SEM observe
Naked eyes are seen DSBM timbering material outward appearance and all are white in color spongy, use tweezers clamping good springiness during suction, can squeeze the water out and also can restore, has certain toughness, keep original form and certain intensity is arranged when dry, the surface is seen loose loose structure, and the surface pore is connected with the deep layer hole, and pore size does not wait (Fig. 1).Get 10 of the DSBM that make, in 4 ℃ down 2% glutaraldehyde fix 24 hours, after the rinsing of 0.1mol/L natrium cacodylicum buffer, fix 2 hours with 1% Osmic acid..With the rinsing of 0.1mol/L natrium cacodylicum buffer, ethanol dewaters step by step again, and isoamyl acetate is preserved and spent the night, the carbon dioxide critical point drying, and orientation attaches on the object stage, and behind the vacuum metal spraying, last machine is observed.Surface texture with scanning electron microscopic observation DSBM.SEM observation DSBM is covered with the hole that differs in size and is natural grid structure, the mutual traffic of each hole (Fig. 2).Each specimen is got 10 photos at random, calculates mean pore size and porosity.Pore size calculates (Fig. 3) by the Measure function of Image J software; Porosity is calculated by rectangular histogram (histogram) function of Adobe Photoshop CS4 software, represents pore area (Fig. 4) with pixel (pixel), the pixel of porosity=(pixel that the pixel-bone trabecula of whole photo is shared) whole photo of ÷.The mean pore size of the DSBM that records is 119.42 ± 6.46 μ m and porosity 60.19 ± 6.10% (seeing Table 1).
Table 1 DSBM scanning electron microscope image is measured mean porosities and mean pore size (n=10)
The external compound cultivation of experimental example 2:DSBM and BMSCs
Get decalcification 6h as stated above respectively, each 24 of the DSBM of 12h and three time periods of 24h, cultivate behind the 24h go culture medium with aseptic rayon balls suction after, place 6 24 well culture plates respectively, 1 block of decalcification 6h DSBM material is put in the A1 of every block of plate, B1, C1, D1 hole, 1 block of decalcification 12h DSBM material is respectively put in A2, B2, C2, D2 hole, and 1 block of decalcification 24h DSBM material is respectively put in A3, B3, C3, D3 hole, is 5 * 10 with density
6Third generation mesenchymal stem cells MSCs (BMSCs) the 40 μ L of/mL are inoculated on the pre-wet stock of 24 well culture plates CO
2Cultivated 4 hours in the incubator, add the L-DMEM culture medium continuation cultivation that 1.2mL contains 15% new-born calf serum more respectively, A4, B4, C4, D4 hole add same medium merely as background, and every 2d changes liquid once.The inverted phase contrast microscope observation of cell around the material, adhere in the hole and growing state.Respectively the 2nd, 4,6,8,10, take out a plate during 12d and add MTT 100 μ L, add DMSO 800 μ L behind the 4h, under the 570nm wavelength, measures each hole optical density (OD) with enzyme mark detector after 12 hours and is worth.Determine the influence of material cell growth according to the growth curve of cell on material.The result shows that the DSBM biological activity of decalcification 6h, 12h, 24h has certain difference, and the DSBM activity of decalcification 6h is best, have the good cell compatibility (table 2, Fig. 5).
The maximum OD value of the compound cultivation of three different decalcification time section DSBM of table 2 and BMSCs relatively
Grouping OD (n=4, X ± S)
A organizes (decalcification 6h group) 1.70 ± 0.03
B organizes (decalcification 12h group) 1.57 ± 0.05
C organizes (decalcification 24h group) 1.53 ± 0.03
Three prescription difference analysis, F=27.46, P＜0.01, three group OD
Value difference is different statistical significance; Compare in twos between group, obtain removing between two groups of B, C and do not have statistical discrepancy (p=0.1), between all the other each groups statistical discrepancy (p＜0.01) is arranged more all in twos.
The biological activity that the DSBM of decalcification 6h, 12h, 24h is described has certain difference, and the DSBM activity of decalcification 6h is best
The SEM that experiment 3:DSBM and BMSCs unite cultivation observes
With 6 of the DSBM of above-mentioned decalcification 6h, cultivate behind the 24h with aseptic rayon balls inhale go culture medium after, place 16 well culture plate and perform labelling, press 5 * 10
6/ mL concentration inoculating cell is put in the culture dish and is cultivated, and respectively takes out 3 of materials in the 2nd day and 6 days respectively cultivating, with the fixing 15min of 2.5% glutaraldehyde, drying afterwards in observation of cell under the scanning electron microscope on material, stick, the propagation situation.Observe visible the cultivation through SEM and saw that having some cells to be family's shape invested the timbering material superficial growth on the 2nd day, most cells are spindle shape (Fig. 6), cultivate cell showed increased on the 6th day the carrier, cell is spindle shape, invest material surface and the length of looking unfamiliar deeply, cell surface has a plurality of elongated protrusion, and the mutual juxtaposition of cell process is net lamellar (Fig. 7)
The damaged research of the experiment compound BMSCs repairing articular cartilage of 4:DSBM
Grow up 8 of the southern regions of the Yunnan Province microtia pigs, male and female are not limit, and the about 12-15kg of body weight is provided by unming Medical College's Experimental Animal Center, the laboratory animal quality certification number: SYXK (Yunnan) 2005-0004, build together and stand 16 holostrome cartilage defects.The zoopery right knee joint femoral lateral condyle loading surface that divides into groups is experimental group A group (DSBM/BMSCs), and the condyle loading surface is an experiment contrast group B group (implant DSBM merely, do not contain BMSCs) in the right knee joint femur.Through auricular vein, give 3% pentobarbital 1ml/kg anesthesia with the southern regions of the Yunnan Province microtia pig, suitably increase anaesthesia dosage according to anesthesia situation in the art.After the Animal Anesthesia, dorsal position is fixed on the animal surgery operating board, in right knee surgery district preserved skin, iodophor disinfection, shop aseptic towel.Be longitudinal incision 5-7cm along the patella inboard, cut inboard joint capsule and part vastus medialis, enter articular cavity, patella is dislocated laterally, expose condyle of femur and femur coaster.Check that knee joint cavity does not have hydrops or adhesion, articular cartilage is smooth, color and luster normal after, with drill bit ectocondyle joint loading surface in femur of diameter 7mm, bore a diameter 7mm, the osteochondral defect of dark about 3-4mm is peeled off cartilage to subchondral bone surface, hemostasis by compression with scalpel.The normal saline flushing articular cavity, according to the grouping situation 6 days complex of external compound cultivation is filled in articular cartilage defect place (Fig. 8), successively closed joint capsule and skin incision, intramuscular injection penicillin 1,600,000 IU when every animal art finishes, postoperative intramuscular injection penicillin, 1,600,000 IU/, every day 1 time, logotype 5 days.Animal limb is unfixing, and it is free movable to let alone.In putting to death 2 the southern regions of the Yunnan Province microtia pigs respectively in 2 weeks of postoperative, 4 weeks, 8 weeks, 12 weeks, expose the double knee joint chamber, the perusal intraarticular has or not adhesion when drawing materials, have or not infection, have or not episome to form, observe the filling of damaged place, repair tissue color and luster, smooth, with the integration situation in normal cartilage boundary line on every side.And specimen fixed with 10% neutral formalin, after the 15%EDTA decalcification, conventional ethanol dewaters step by step, paraffin embedding, and the dyeing of HE and safranin O is adopted in section, and light microscopic is observed down, and carries out histological score,
1. the postoperative ordinary circumstance is observed
Postoperative did not like diet the same day or diet is less, and feed later on increases gradually, and diet is normal substantially after 3 days.Postoperative after 5 days activity increase gradually, about 2 weeks activity freely, voluntary activity in canopy.All art knee joint wound healing is good, does not have and infects.
2. postoperative specimen gross examination of skeletal muscle
The gross examination of skeletal muscle of each group during 2 weeks, the repair tissue color and luster is dark red, and the rough surface injustice is with cartilage boundary clear (Fig. 9) on every side; A group cartilage defect district is by translucent or white newborn class cartilaginous tissue filling during 4 weeks, and B group repair tissue color and luster is dark red, and the rough surface injustice is clear with cartilage boundary on every side, matter softer (Figure 10); A, B group repair tissue is with the cartilage form and aspect are near on every side during 8 weeks, and it is smooth that owe on the surface, and with the cartilage boundary is obvious on every side, A group reparation speed is slightly fast, projecting cartilage plane, and timbering material absorbs (Figure 11) fully; 12 when week A, B group repair tissue with the cartilage form and aspect are near on every side, A group repair tissue merges substantially with cartilage on every side, boundary is smudgy, the B group is fuzzy (Figure 12) slightly.
3. Histological section is observed
Specimen is fixed with 10% neutral formalin, and after the 15%EDTA decalcification, conventional ethanol dewaters step by step, paraffin embedding, and HE is adopted in section, and light microscopic is observed down, and carries out histological score, and to 8 weeks, 12 all row and safranin O dyeing.
Respectively organize HE dyeing during 2 weeks and observe A, B group defective region mainly by the timbering material filling, only partially absorb, be mingled with fibrous tissue therebetween, no cartilaginous tissue is not seen bright lymphocytic infiltration, defective region clear border (Figure 13); Respectively organizing HE dyeing observation A group during 4 weeks has cartilaginous tissue to generate, irregular arrangement, fuzzy with the surrounding tissue boundary, rough, B group defective region is mainly filled by timbering material, partially absorbs, and has fiber and cartilaginous tissue to generate, obvious with normal articular cartilage boundary on every side, rough surface injustice (Figure 14); The time respectively organize HE dyeing 8 weeks and observe the A group a large amount of chondrocytes and tissue growth are arranged, with the cartilaginous tissue boundary is fuzzy on every side, the surface is level and smooth slightly, and the B group has a lot of cartilaginous tissues to generate, and is clear with the surrounding tissue boundary, rough (Figure 15); Respectively organize HE dyeing during 12 weeks and observe the A group based on the hyaline cartilage cell, many and the cell space of cell number is big to be integrated closely with cartilage on every side, thickness is close, repair surface is smooth, be difficult to distinguish with boundary on every side, subchondral bone is repaired complete, and B organizes transparent sample cartilage, and cell number is more, the repair tissue smooth surface is with cartilage border still clear (Figure 16) on every side.
A, B group safranin O stained positive is observed in safranin O dyeing during 8 weeks, cartilage matrix dyeing takes on a red color, timbering material absorbs fully, the dyeing of A group is comparatively even, and based on the class hyaline cartilage, chondrocyte is bred in a large number, with cartilaginous tissue obscurity boundary on every side, a large amount of inmature chondrocytes appear in B group, arrange comparatively mixed and disorderly, with cartilaginous tissue clear border (Figure 17) on every side; Respectively organize safranin O dyeing during 12 weeks and observe A, B group safranin O stained positive A group even dyeing, similar to normal cartilage dyeing, repair surface is smooth, is difficult to distinguish that B group repair surface is smooth with boundary on every side, with boundary still clear (Figure 18) on every side.
Claims (2)
1. one kind is used for the tissue engineering bracket material that cartilage defect is repaired, it is characterized in that: with the spongy bone at fresh pig scapula place, cut into the fritter of diameter 5mm～10mm, thickness 3mm～5mm after the cleaning, after defat, decalcification, deproteinization are handled, again with alcohol-pickled 30 minutes, obtain after air-dry; Wherein, decalcification time is 6 hours.
2. prepare the described method that is used for the tissue engineering bracket material of cartilage defect reparation of claim 1, may further comprise the steps:
(1) preparation bone piece: with the fresh pig scapula is raw material, rejects periosteum, cartilage and soft tissue, gets its spongy bone, flowing water stirs flushing repeatedly, removes bone marrow, blood stains and surperficial oils and fats, cuts into diameter 5mm～10mm, the fritter of thickness 3mm～5mm, flushing with clean water is clean;
(2) the bone piece of cleaning is placed 1: 1 chloroform methanol mixed liquor of volume fraction, soaking at room temperature defat 2 times, each 6 hours, air-dry then;
(3) it is the aqueous hydrochloric acid solution soaking at room temperature decalcification 6h of 0.6mol/L that the bone piece after air-dry places concentration, the envelope-bulk to weight ratio of aqueous hydrochloric acid solution and bone piece is 20mL/g, after decalcification is finished, again the bone piece is placed phosphate buffered solution concentration percetage by weight 0.1%, pH value 7.4 to soak 12～24 hours, air-dry;
(4) 1: 1 chloroform alcohol mixed liquid dipping defat of bone piece reuse volume fraction is 6 hours, and is air-dry;
(5) the bone piece after air-dry with distilled water repeatedly rinsing until pH value 7.0;
(6) the bone piece is placed phosphate buffered solution concentration percetage by weight 0.1%, pH value 7.4, constant temperature soaked 72 hours for 37 ℃, and is air-dry;
(7) at last air-dry bone piece is placed the ethanol of concentration percetage by weight 75% to soak 30 minutes, promptly obtain the required tissue engineering bracket material that cartilage defect is repaired that is used for after air-dry once more.
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| CN111494715A (en) * | 2020-04-17 | 2020-08-07 | 东南大学 | High-molecular bone filling material and preparation method thereof |
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