CN101628131A - Method for preparing ultra-thin porous lamination gradient composite support of tissue engineering - Google Patents
Method for preparing ultra-thin porous lamination gradient composite support of tissue engineering Download PDFInfo
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Abstract
本发明公开了一种超薄多孔叠层梯度组织工程复合支架的制备方法，属于生物医学材料领域。该制备方法包括以下步骤：1)根据修复缺损情况，确定待制备支架的材料和结构；2)制备各层支架，等待溶剂挥发，浆料逐渐变干成型；3)将成型物置于通风处，等待残留溶剂完全挥发后脱模；4)取出成型物，进行干燥，即得超薄多孔叠层梯度组织工程复合支架。使用该方法可制备厚度适合于关节骨软骨修复的超薄多孔叠层复合支架，制备的支架力学性能良好，生物相容性好，降解速率可控，能适应成骨细胞和软骨细胞共同培养及特异性生长因子的复合，可同时进行关节软骨组织和软骨/骨组织综合缺损的修复。The invention discloses a preparation method of an ultra-thin porous laminated gradient tissue engineering composite support, which belongs to the field of biomedical materials. The preparation method includes the following steps: 1) determining the material and structure of the stent to be prepared according to the condition of the repair defect; 2) preparing the stent of each layer, waiting for the solvent to evaporate, and the slurry gradually becomes dry and formed; 3) placing the formed object in a ventilated place, Waiting for the residual solvent to completely volatilize before demoulding; 4) Take out the molded product and dry it to obtain an ultra-thin porous laminated gradient tissue engineering composite scaffold. Using this method, an ultra-thin porous laminated composite scaffold with a thickness suitable for articular osteochondral repair can be prepared. The prepared scaffold has good mechanical properties, good biocompatibility, and controllable degradation rate, and can adapt to the co-culture of osteoblasts and chondrocytes. The combination of specific growth factors can simultaneously repair articular cartilage tissue and cartilage/bone tissue comprehensive defects.
Description
Technical field
The invention belongs to field of biomedical materials, be specifically related to be used for the preparation method of the porous lamination composite support of tissue engineering that the ossa articularia cartilage defect repairs.
Background technology
Articular cartilage defect is very common disease, because articular cartilage special organization structure and biological characteristics, though the known method that many treatment articular cartilage defects are arranged, all these methods all can not reach satisfied repairing effect.The appearance of tissue engineering technique makes the damaged reparation of cartilaginous tissue become possibility.Its ultimate principle is, the cartilaginous tissue cell inoculation that in-vitro separation is cultivated is to the three-dimensional rack with certain space structure and good biocompatibility, be aided with special somatomedin simultaneously, complex with cell-scaffold implants behind the In vitro culture certain hour more then, propagation along with cell, timbering material is constantly degraded, and is replaced by extracellular matrix, forms the tissue with normal function gradually.Wherein, support is one of key element of organizational project.Because the bone cartilage has multiple structure, comprise cartilage layers, calcified cartilage layer, subchondral bone layer and osseous tissue from outside to inside, the cartilage injury may relate to calcified cartilage, subchondral bone even osseous tissue, therefore to select degradation rate controlled and can repair the porous lamination support of multilayer tissue simultaneously as required, this support has multiple structure, and the performance of each layer should be able to adapt to the reparation of respective layer tissue.This support also should have higher porosity, with the growth and the propagation of suitable cell, and the conveying of nutrient substance and metabolic waste.
Report according to pertinent literature, the thickness of outside articular surface cartilage is 1.5～3 millimeters (D.E.T.Shepherd.B.B.Seedhom.Thickness of human articular cartilage in joints of thelower limb.Ann.Rheum.Dis.1999 in the human body knee joint, 58:27-34), because osteochondral defect may relate to subchondral bone even osseous tissue, the thickness that therefore is used to repair ossa articularia cartilage injury's timbering material should be 2～4 millimeters.At present, because technology is limit, the thickness of the compound cartilage tissue engineering rack of porous lamination of development is all much larger than repairing the needed thickness of osteochondral defect both at home and abroad, usually above 6 millimeters, even reach 9 millimeters (S.Ghosh etc., Bi-layered constructs based onpoly (L-lactic acid) and starch for tissue engineering of osteochondral defects, Materials Science and Engineering C, 2008,28:80-86), be used for defect repair and can cause big wound.In the porous lamination compound rest preparation technology of routine, each of support layer quilt prepares respectively, is superimposed and carries out certain bondingly, forms stack trays.In such preparation technology, bond strength is relatively poor between layer and the layer, pore communication is poor, and porous each layer must possess certain thickness to keep its structure, pattern and certain intensity.Report according to pertinent literature, the thickness of porous monolayer compound rest can't be accomplished less than 1.5 millimeters (Anna Tampieri etc., Design of gradedbiomimetic osteochondral composite scaffolds, Biomaterials, 2008,29:3539-3546), therefore, according to above-mentioned conventional preparation technology, the thickness of porous two-layer compound support can't accomplish less than 3 millimeters, and the integral thickness with porous lamination support of structure more than three layers or three layers more can't reach repairs required 2～4 millimeters of ossa articularia cartilage injury.
Summary of the invention
The objective of the invention is the preparation method that provides a kind of ultra-thin porous lamination gradient composite support of tissue engineering for addressing the above problem.Use this method can prepare the ultra-thin porous lamination compound rest that thickness is suitable for the ossa articularia repair of cartilage.
The technical solution adopted in the present invention is:
A kind of preparation method of ultra-thin porous lamination gradient composite support of tissue engineering, this preparation method may further comprise the steps:
1), determines the material and the structure of support to be prepared according to the repair deficiency situation;
2) organic material that will prepare the ground floor support is dissolved in the solvent, solvent for use is an easy volatile solvent, after treating that organic material dissolves fully, add inorganic material again, stir and form uniform mixed slurry, this mixed slurry is poured in the flat mould again, make slurry evenly be tiled in mold bottom, place the environment that helps solvent evaporates then, wait for solvent evaporates, the volatilization time is 0.5～12 hour, the mixed slurry molding that becomes dry gradually;
3) according to step 2) operation, the mixed slurry of preparation second layer support directly is poured on mixed slurry on the ground floor support that has prepared molding, and make the slurry tiling evenly, place the environment that helps solvent evaporates then, wait for the molding that becomes dry gradually of solvent evaporates, slurry;
4), successively prepare the 3rd to N shell support (N 〉=2) according to the operation of step 3);
5) article shaped is placed the ventilation, the demoulding after the wait residual solvent volatilizees fully;
6) take out article shaped, carry out drying, promptly get ultra-thin porous lamination gradient composite support of tissue engineering.
Preferably, described step 2) to step 4), can add porogen, its concrete steps are: with porogen and inorganic material mix homogeneously, add in the solution of organic material again, stir and form uniform mixed slurry.
Preferably, the concrete steps of described adding porogen are: after inorganic material is added the solution of organic material and stirring, add porogen again, stir and form uniform mixed slurry.
Further, described step 2) add porogen to the step 4) after, the article shaped in the step 5) need be soaked with pure water, removes porogen.
Further, in the preparation process of described certain one deck, the amount of solvent for use can be dissolved preparation this layer used organic material fully, and prepares the used inorganic material of this layer and be insoluble to this solvent, but inorganic material must be dispersed in mixed flow mutually in.
Further, to account for the mass percent of each layer support be 0%～60% to described inorganic material.
Preferably, described organic material is artificial synthesizing polymeric material or natural polymeric material, or the mixing of worker's synthesizing polymeric material and natural polymeric material.
More preferably, described artificial-synthetic copolymer's material is a polylactic acid (polylactide, polylactic acid, PLA), polyglycolic acid (poly-Acetic acid, hydroxy-, bimol. cyclic ester, polyglycolic acid, polyglycolic acid, PGA), polylactic acid-glycolic guanidine-acetic acid copolymer (poly (lactic-co-glycolic acid), PLGA), polycaprolactone (polycaprolactone, PCL), polyamide (polyamide, PA), polyvinyl alcohol (polyvinyl alcohol, PVA).
More preferably, described natural polymeric material be fibroin albumen (fibroin), collagen (collagen), chitosan (chitosan), hyaluronic acid (haluronic acid, HA).
Preferably, described inorganic material is a calcium phosphorus series active biomaterial.
More preferably, described calcium phosphorus series active biomaterial is hydroxyapatite (hydroxyapatite, HA), α tricalcium phosphate (α-tricalcium phosphate, α-TCP), β tricalcium phosphate (β-tricalcium phosphate, β-TCP), tetracalcium phosphate and element doping modified calcium phosphor biological material.
Further, described porogen is a solid particle, and particle diameter is 30～500 microns, the solvent that employed porogen uses when being insoluble to this layer of preparation, but water-soluble.
Preferably, described pure water is distilled water, deionized water or reverse osmosis water.
The present invention has the following advantages:
Utilize the present invention can prepare the ultra-thin porous lamination compound rest that thickness is suitable for the ossa articularia repair of cartilage, the support good mechanical properties of preparation, good biocompatibility, degradation rate is controlled, the compound of osteoblast and chondrocyte co-cultivation and specificity growth factor can be adapted to, the comprehensive damaged reparation of articular cartilage tissue and cartilage/bone tissue can be carried out simultaneously.
The specific embodiment
Embodiment 1
Preparation thickness is 1.5 millimeters zinc hydroxyapatite/polylactic acid two-layer compound support.Mould therefor is that an internal diameter is the cylindrical flat glass container of 60mm.Used organic material is Poly-L-lactic acid (PLLA, a molecular weight 50000); (nano-Zn-HA, elongated piece are grown 80～120nm to used inorganic material, and be wide by 20～50nm) in order to contain the zinc nanometer hydroxyapatite; Solvent for use is acetone (C
3H
6O); Used porogen is sodium chloride (NaCl is divided into two kinds of particle diameter 50～200 μ m and particle diameter 200～300 μ m).The stack trays thickness of desire preparation is 1.5mm, and the ground floor of its medium-height trestle (lower floor) thickness is 0.5mm, and the second layer (upper strata) thickness is 1mm.The ground floor composition of support is nano-Zn-HA and PLLA mixture, and the mass ratio of nano-Zn-HA and PLLA is 30: 70; The second layer composition of support is pure PLLA.Preparation is during support, begins preparation from the ground floor of support, prepares the second layer again on ground floor, and promptly mold bottom is the lower floor of support in preparation process, is the upper strata of support on it.
Take by weighing 0.16g PLLA, be dissolved in 1.4ml C
3H
6Among the O, treat that PLLA dissolves the back fully and adds 0.07g nano-Zn-HA, stir and form uniform sizing material, the NaCl that adds 1.8g particle diameter 200～300 μ m again stirs and forms uniform sizing material, and slurry is poured in the mould, and make slurry evenly be laid on mold bottom, left standstill 4 hours, and treated solvent evaporates, form the ground floor of support.
Take by weighing 0.45g PLLA, be dissolved in 2.8ml C
3H
6Among the O, treat that PLLA dissolves the NaCl that the back adds 3.4g particle diameter 50～200 μ m fully, stir to form uniform sizing material, slurry is poured in the mould, and slurry evenly is laid on the support ground floor of shaping, left standstill 4 hours, treat solvent evaporates, form the second layer of support.
Article shaped is placed fume hood, 30 ℃ of volatilization demouldings after 48 hours down.Article shaped is immersed in the deionized water, changed a deionized water, and in the water that soaks usefulness, can not detect chloride ion (Cl in per 6 hours
-).With article shaped lyophilization 36 hours, make the porous lamination compound rest again.
After testing, this support average thickness is 1.53 millimeters, and porosity is 86.6%, adheres to specification.Experiment showed, that through external and vivo degradation this scaffold degradation product is nontoxic, can not cause aseptic inflammation, support degradation time in vivo is 20 days, meets the requirement of clinical bone repair of cartilage.Repair through animal and experiment showed, that this support can repair the ossa articularia cartilage defect of dark and calcified cartilage layer well.
Embodiment 2:
Preparation thickness is three layers of compound rest of hydroxyapatite/polylactic acid-hydroxyacetic acid of 3 millimeters.Mould therefor is that an internal diameter is the cylindrical flat glass container of 40mm.Used organic material is polylactic acid-glycolic guanidine-acetic acid (PLGA, molecular weight 100000, LA: GA=50: 50); Used inorganic material is that (nano-HA, elongated piece are grown 100～150nm to nanometer hydroxyapatite, and be wide by 20～50nm); Solvent for use is dichloromethane (CH
2Cl
2); Used porogen is sodium chloride (NaCl, particle diameter 200～300 μ m).The stack trays thickness of desire preparation is 3mm, and each layer thickness is 1mm.The ground floor of support (bottom) composition is nano-HA and PLGA mixture, and the mass ratio of nano-HA and PLGA is 40: 60; The second layer of support (intermediate layer) composition is nano-HA and PLGA mixture, and the mass ratio of nano-HA and PLGA is 20: 80; The 3rd layer of (top layer) composition of support is pure PLGA.Preparation is during support, begins preparation from the 3rd layer of support, and until ground floor, promptly mold bottom is the top layer of support in preparation process, is the intermediate layer on it, and last is the bottom of support.
Take by weighing 0.2g PLGA, be dissolved in 1.2ml CH
2Cl
2In, treat that PLGA dissolves the back fully and adds 1.8g NaCl, stir and form uniform sizing material, slurry is poured in the mould, and made slurry evenly be laid on mold bottom, left standstill 2 hours, treat solvent evaporates, form the 3rd layer of support.
Take by weighing 0.16g PLGA, be dissolved in 1.2ml CH
2Cl
2In, treat that PLGA dissolves the back fully and adds 0.04g nano-HA, stir and form uniform sizing material, add 1.8g NaCl again, stir to form uniform sizing material, slurry is poured in the mould, and slurry evenly is laid on the 3rd layer on the support of shaping, left standstill 2 hours, and treated solvent evaporates, form the second layer of support.
Take by weighing 0.12g PLGA, be dissolved in 1.2ml CH
2Cl
2In, treat that PLGA dissolves the back fully and adds 0.08g nano-HA, stir and form uniform sizing material, add 1.8g NaCl again, stir and form uniform sizing material, slurry is poured in the mould, and slurry evenly is laid on the support second layer of shaping, left standstill 2 hours, and treated solvent evaporates, form the ground floor of support.
Article shaped is placed fume hood, 30 ℃ of volatilization demouldings after 48 hours down.Article shaped is immersed in the deionized water, changed a deionized water, and in the water that soaks usefulness, can not detect chloride ion (Cl in per 4 hours
-).With article shaped lyophilization 36 hours, make the porous lamination compound rest again.
After testing, this support average thickness is 3.05 millimeters, and porosity is 91.4%, adheres to specification.Experiment showed, that through external and vivo degradation this scaffold degradation product is nontoxic, can not cause aseptic inflammation, support degradation time in vivo is 28 days, meets the requirement of clinical ossa articularia repair of cartilage.Repair through animal and experiment showed, that this support can repair the ossa articularia cartilage defect of dark and subchondral bone well.
Embodiment 3
Preparation thickness is hydroxyapatite/polylactic acid-hydroxyacetic acid/four layers of compound rest of polycaprolactone of 4.5 millimeters.Mould therefor is that an internal diameter is the cylindrical flat glass container of 40mm.50) and polycaprolactone (PCL, molecular weight 70000) used organic material is polylactic-co-glycolic acid (PLGA, molecular weight 100000, LA: GA=50:; Used inorganic material is that (nano-HA, elongated piece are grown 100～150nm to nanometer hydroxyapatite, and be wide by 20～50nm); Solvent for use is chloroform (chloroform, CHCl
3) and oxolane (tetrahydrofuran, THF); Used porogen is sodium chloride (NaCl is divided into particle diameter and is two kinds of 50～200 μ m and 200～300 μ m).The stack trays thickness of desire preparation is 4.5mm, and the ground floor of its medium-height trestle (bottom) thickness is 1mm, and composition is the mixture of nano-HA and PCL, and the mass ratio of nano-HA and PCL is 40: 60; Second layer thickness is 1mm, and composition is the mixture of nano-HA and PCL, and the mass ratio of nano-HA and PCL is 30: 70; The threeply degree is 0.5mm, and composition is the mixture of nano-HA and PLGA, and the mass ratio of nano-HA and PLGA is 20: 80; The 4th layer of (top layer) thickness is 2mm, and composition is pure PLGA.Preparation is during support, begins preparation from the ground floor of support, and until the 4th layer, promptly mold bottom is the bottom of support in preparation process, and last is the top layer of support.
Take by weighing 0.12g PCL, be dissolved among the 1.2ml THF, treat that PCL dissolves the back fully and adds 0.08g nano-HA, stir and form uniform sizing material, add 1.8g NaCl again, stir and form uniform sizing material, slurry is poured in the mould, and made slurry evenly be laid on mold bottom, put into 90 ℃ of baking ovens and left standstill 6 hours, treat solvent evaporates, form the ground floor of support.
Take by weighing 0.14g PCL, be dissolved among the 1.2ml THF, treat that PCL dissolves the back fully and adds 0.06g nano-HA, stir and form uniform sizing material, add 1.8g NaCl again, stir and form uniform sizing material, slurry is poured in the mould, and slurry evenly is laid on the 3rd layer on the support of shaping, put into 90 ℃ of baking ovens and left standstill 6 hours, treat solvent evaporates, form the second layer of support.
Take by weighing 0.08g PLGA, be dissolved in 0.6ml CHCl
3In, treat that PLGA dissolves the back fully and adds 0.02g nano-HA, stir and form uniform sizing material, add 0.9g NaCl again, stir to form uniform sizing material, slurry is poured in the mould, and slurry evenly is laid on the 4th layer on the support of shaping, left standstill 2 hours, and treated solvent evaporates, form the 3rd layer of support.
Take by weighing 0.4g PLGA, be dissolved in 2.5ml CHCl
3In, treat that PLGA dissolves the back fully and adds 1.5g NaCl, stir and form uniform sizing material, slurry is poured in the mould, and slurry evenly is laid on the 3rd layer on the support of shaping, left standstill 6 hours, treat solvent evaporates, form the 4th layer of support.
Article shaped is placed fume hood, 30 ℃ of volatilization demouldings after 48 hours down.Article shaped is immersed in the deionized water, changed a deionized water, and in the water that soaks usefulness, can not detect chloride ion (Cl in per 6 hours
-).With article shaped lyophilization 48 hours, make the porous lamination compound rest again.
After testing, this support average thickness is 4.50 millimeters, and porosity is 88.6%, adheres to specification.Experiment showed, that through external and vivo degradation this scaffold degradation product is nontoxic, can not cause aseptic inflammation, support degradation time in vivo is 40 days, meets the requirement of clinical bone repair of cartilage.Repair through animal and experiment showed, that this support can repair the ossa articularia cartilage defect of dark and osseous tissue well.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
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