CN102580156B - Tissue engineering cartilage framework material, as well as preparation method and device thereof - Google Patents
Tissue engineering cartilage framework material, as well as preparation method and device thereof Download PDFInfo
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Abstract
The invention discloses a tissue engineering cartilage framework material. The cartilage framework material provided by the invention sequentially comprises a surface layer, a middle layer and a calcification layer from the top down; the surface layer is composed of freeze-dry cartilage matrix and II-type collagen, and matrix fiber is in parallel distribution in the horizontal direction; the middle layer is composed of freeze-dry cartilage matrix, GAG, X-type collagen, II-type collagen and TGF-Beta, and matrix fiber is arranged longitudinally; and the calcification layer is composed of freeze-dry cartilage matrix, GAG, X-type collagen and sintered bovine bone power, and fiber is distributed in a three dimensional staggered manner. The prepared cartilage framework material has a cartilage repairing effect, is favorable to the integration of the framework material and subchondral bone after the framework material is planted, and promotes the regeneration of the subchondral bone; after cartilage cell compound, the framework material can be used for full-layer cartilage defect repairing, the regenerated cartilage tissue has the space structure and protein components, which are the same with those of natural cartilage; and the framework material is suitable for repairing cartilages in load bearing joints such as knee joints and the like, cartilage with higher regenerated compressive strength can be regenerated, and the abrasion performance and anti-pressure capability of the surface of the cartilage framework are enhanced.
Description
Technical field
The invention belongs to tissue engineering biomaterial for medical purpose technical field, be specifically related to a kind of tissue engineering bone/cartilage timbering material and preparation method thereof and preparation facilities.
Background technology
Articular cartilage damage is clinical common diseases.Investigation both at home and abroad shows, articular cartilage damage prevalence approximately 15%, 40 years old prevalence is 10%～17%, within 60 years old, reaches 50% above, and 80% suffers from the cartilage defect that osteoarthritis is followed in more than 75 years old crowd.In the U.S., articular cartilage damage is the second reason (being only second to ischemic heart desease) that causes 50 years old above male's disablement, is also the chronic main cause disabling of crowd above middle age.According to Americanism damp disease association (ACR) data show: at present, approximately there are 2,000 ten thousand articular cartilage damage patients in the U.S., estimate to the year two thousand twenty, number of the infected will rise to 4,000 ten thousand, in over-65s old people, 70% has articular cartilage damage symptom, and wherein 46% loses function of joint and work capacity to some extent, annual outpatient 7,000,000, inpatient 3,000,000.The U.S. for the third time national health and nutritional survey shows, 8% adult can clarify a diagnosis as joints of hand cartilage injury, means in every 12 Americans and just has 1 people to suffer from joints of hand cartilage injury.
Conservative estimation, China's osteoarthrosis illness is more than 5,000 ten thousand.Research discovery, cartilage injury's morbidity has the trend becoming younger, and the cartilage injury patient of Symptomatic 30～40 years old is much clinically, and x-ray generaI investigation finds that the osteoarthritis incidence rate of 15～24 years old age group is 10%.
Human articular cartilage is without blood vessel, impassivity, and chondrocyte is difficult to migration by substrate restriction around, so can not spontaneous recovery after cartilage injury.Traditional Therapeutic Method (as arthrocentesis and joint debridement art, micro-fracture operation, periosteum transplanting, autologous/allosome cartilage transplantation etc.) can only relief of symptoms, can not have efficient recovery damaged tissues function.In recent years the chondrocyte cell transplantation technology occurring, can be in the structure function of to a certain degree recovering cartilaginous tissue, but needs second operation, and Hui Duigong district causes damage.
The treatment cartilage defect that is developed as of tissue engineering bone/cartilage provides a kind of better selection.Researcher adopts multiple timbering material and various kinds of cell to construct the good tissue engineering bone/cartilage of character; Because natural cartilage is not a unordered structure, but partly formed by top layer, divide a word with a hyphen at the end of a line layer, radiating layer and calcification layer four-layer structure; In surface structure, II Collagen Type VI content is the highest, and glycosaminoglycans (GAG) content is lower, and its matrix fiber distributes by horizontal direction; In layer and the radiating layer structure of dividing a word with a hyphen at the end of a line, II Collagen Type VI declines gradually along with the degree of depth increases its content, and GAG is just in time contrary, and matrix fiber is by vertical direction arrangement, and this structure has given articular cartilage good anti-compression property just; In calcification layer structure, II Collagen Type VI content is minimum, and GAG content is the highest, and has a large amount of X-type collagen.This orderly structure of cartilaginous tissue and composition distribute, and have finally formed wear-resisting, the measuring body functional characteristics of articular cartilage.Physiological characteristics based on articular cartilage, if construct the tissue engineering bone/cartilage that function is consistent with natural cartilage, first to prepare the tissue engineering bone/cartilage timbering material with natural cartilage architectural feature, compound seed cell, realizes the structure of organizing approaching tissue engineering bone/cartilage with human cartilage thereon.
Through retrieval, component and structure that the constructed cartilage frame of Chinese patent application 200610026004.2,200910058053.8,02113378.6 and 201010539861.9 is homogeneous, do not have natural cartilage organizational structure feature.Though Chinese patent application 200910117158.6 has hierarchy, its structure is only that the content of hydroxyapatite is different, and cartilage frame material does not have to increase the vertical orientation structure of cartilage comprcssive strength.The repair of cartilage material that Chinese patent application 201010297976.1 builds is the longitudinal arrangement mode of natural cartilage substrate, but it does not have the composition feature of horizontal fibre structure and the calcification layer on cartilage top layer.Chinese patent application 200710090962.0 discloses a kind of biological rack material for articular cartilage and preparation method, by the natural cartilage substrate composition and the blend of II Collagen Type VI that take off cell, remove antigen, remove I type and X-type collagen, using sodium chloride crystal granule as porogen, by physics and chemistry, process, make a kind of articular cartilage timbering material of porous; It is raw material that the method adopts natural cartilage substrate, guaranteed that structure material composition is consistent with natural cartilage, but between each layer of cartilaginous tissue, component differs greatly, and calcification layer removed the X-type collagen in cartilage matrix, is different from natural cartilage substrate composition.Chinese patent application 200510012257.X adopts the material construction multilamellar cartilage materials such as polyvinyl alcohol (PVA) and hydroxyapatite, the gradual change rule of simulation natural cartilage component in multiple structure, with compositions such as the collagen in the macromolecular material replacement cartilaginous tissues such as PVA, Dan Baiduotang proteoglycan PG, but macromolecule polymeric material katabolism is in vivo completely different from cartilage matrix, the former degraded can produce acid product, cause non-bacterial inflammatory reaction, directly have influence on the fusion of graft and surrounding tissue.
Summary of the invention
The problem existing for prior art, the object of this invention is to provide a kind of tissue engineering bone/cartilage timbering material and preparation method thereof and device, prepared cartilage frame material is after compound chondrocyte, can be used in holostrome cartilage defect repair, the cartilaginous tissue of regeneration has the structure consistent with natural cartilage and component, the reparation of the position articular cartilage that is applicable to bear a heavy burden.
Tissue engineering bone/cartilage timbering material proposed by the invention, has the similar structures with natural cartilage tissue, is followed successively by top layer, middle level and calcification layer from top to bottom; Its mesexine is comprised of lyophilizing cartilage matrix and II Collagen Type VI, and matrix fiber is parallel distribution in the horizontal direction; Middle level is comprised of lyophilizing cartilage matrix, glycosaminoglycans (GAG), X-type collagen and II Collagen Type VI and transforming growth factor (TGF-β), and its matrix fiber is longitudinal arrangement; Calcification layer is comprised of lyophilizing cartilage matrix, GAG and X-type collagen and calcined bone powder, and the fiber in calcification layer is interspersed at three dimensions.Because the layer of dividing a word with a hyphen at the end of a line of natural cartilage tissue is similar in component and structure function with radiating layer, in the present invention, this double-layer structure be one deck, build the middle level of cartilage frame material, its structure and component and natural cartilage divide a word with a hyphen at the end of a line layer and radiating layer approaching.
The preparation method of tissue engineering bone/cartilage timbering material proposed by the invention, is characterized in that natural cartilage matrix composition to become gel, adds after GAG, X-type collagen and forging bone, and calcification layer is prepared in lyophilizing; Natural cartilage substrate is carried out low temperature crystallographic orientation on calcification layer surface after mixing with GAG, X-type collagen, II Collagen Type VI and TGF-β, forms middle level after lyophilization; By natural cartilage substrate and II Collagen Type VI compound after, adopt spin coating method on preparation top layer, surface, middle level, after drying, obtain tissue engineering bone/cartilage timbering material.Concrete steps comprise:
The preparation of step 1, natural cartilage substrate: under 4 ℃ of conditions, mammal is put into liquid nitrogen freezing after 3 hours without perichondral articular cartilage piece, proceed in the pulverizer of pre-cooling and pulverize, adopt the acetum that volumetric concentration is 1% to dissolve, through vacuum freeze-drying, obtain cartilage matrix;
The preparation of step 2, calcification layer: it is in 1% acetum that lyophilizing cartilage matrix, GAG and X-type collagen are dissolved in to volumetric concentration, make final concentration be respectively 5～12mg/ml, 8～15mg/ml and 5～10mg/ml, with aqua calcis, adjust pH to neutral, obtain cartilage matrix solution; The calcined bone powder of particle diameter 0.05～0.2mm is mixed homogeneously with cartilage matrix solution by the amount of 0.1～0.5g/ml, obtain gel solution, be injected into box at the bottom of culture vessel, making gel layer thickness is 1～4mm, after adding a cover under 37 ℃ of conditions after gel solution solidifies, together with vessel, insert in-80 ℃ of environment freezingly, form calcification layer;
Natural cartilage calcification layer is the transition connecting portion of cartilage and subchondral bone, cell is wherein mainly hypertrophic chondrocyte, can secrete in a large number GAG and X-type collagen, so GAG and X-type collagen concentration are higher in calcification layer, form the special high molecular weight protein network structure of calcification layer, given the comprcssive strength that calcification layer is larger.The inventor is by the content feature of two kinds of key component GAG in research natural cartilage calcification layer and X-type collagen, in lyophilizing cartilage matrix, add GAG and X-type collagen is prepared calcification layer, confirm that constructed calcification layer is after compound chondrocyte, can promote that chondrocyte breaks up to hypertrophic chondrocyte, and there is the comprcssive strength that approaches natural cartilage calcification layer.The present invention adds the bone renovating material of this good biocompatibility of forging bone, skeletonization effect stability in calcification layer, can be when repairing cartilage defect, and regeneration of cartilage sending down the fishbone, is conducive to the fusion of calcification layer and subchondral bone layer; And can change by the adjustment of forging bone and lyophilizing cartilage matrix ratio comprcssive strength and the plasticity of calcification layer.For example, when the mass volume ratio of calcined bone powder and lyophilizing cartilage matrix solution is less than 0.2g/ml, prepared calcification layer, after rehydration, suppleness is high, can, according to defect shape random-shaping, be convenient to clinical manipulation.
The preparation in step 3, middle level: it is in 1% acetum that lyophilizing cartilage matrix, GAG, X-type collagen and II Collagen Type VI are dissolved in to volumetric concentration, make final concentration be respectively 5～12mg/ml, 4～10mg/ml, 2～5mg/ml and 4～10mg/ml, with aqua calcis, adjust pH to neutral, adding final concentration is the TGF-β of 2～15ng/ml again, obtains the gel solution in timbering material middle level; The freezing culture vessel that fills calcification layer of step 2 is placed on the base plate of-80 ℃, in the air ambient of 0～10 ℃, middle level gel solution is injected to the calcification layer surface in culture vessel, this gel layer thickness is between 1～3mm, add a cover after gel solution freezes, proceed under-80 ℃ of environment freezing more than 3 hours; Through low-temperature vacuum drying, obtain middle level and the calcification layer structure of cartilage frame material again;
Matrix components and the structural similarity of the radiating layer of natural cartilage and the layer of dividing a word with a hyphen at the end of a line, for the feature of simulating natural cartilage radiating layer and the stromatin fiber of the layer of dividing a word with a hyphen at the end of a line longitudinally distributes, it is ice crystal " formation template " that the present invention adopts the low temperature calcification layer in pre-freeze, utilize crystallographic orientation principle, in the freezing freezing process of middle level gel, ice crystal be take calcification layer surface as starting point upwards growth gradually, after frozen drying, forms the directed timbering material that stromatin fiber is longitudinal arrangement.In order to keep the differentiation state of the chondrocyte of post incoulation in this timbering material, in the building process in middle level, added the transforming growth factor that can promote Chondrocyte Differentiation.
The preparation on step 4, top layer: it is in 1% acetum that lyophilizing cartilage matrix and II Collagen Type VI are dissolved in to volumetric concentration, make final concentration be respectively 1～3mg/ml and 9～30mg/ml, with sodium hydroxide solution, adjust pH to neutral, adopt vacuum rotating coating method that it is evenly coated to the surface, middle level through low-temperature vacuum drying, its rotary speed 500～3000rpm, time 20～100s, obtains tissue engineering bone/cartilage timbering material after drying.
Cartilaginous tissue surface structure complete most important for keeping the normal physiological metabolism of cartilage, surface structure is relatively fine and close, controlling the discrepancy of moisture in cartilage matrix, water content is for keeping the high elastic modulus lubricated and cartilaginous tissue on cartilaginous tissue surface to have important function.In top layer, II Collagen Type VI content accounts for 90% of cartilaginous tissue extracellular matrix total protein content, and collagen fiber are parallel distribution in horizontal direction on top layer, is conducive to reduce coefficient of friction, keeps articular surface function.In order to make prepared tissue engineering bone/cartilage timbering material top layer closer to protein component and the collagen fiber characteristic distributions on natural joint top layer, by take II Collagen Type VI as main stromatin solution is uniformly coated on the stromatin fibre structure of the longitudinal distribution having built, in the mode of drying, increase the fibriilar compactness extent of surface layer adhesive, reduce friction; Wherein, described vacuum rotating coating method can be adjusted by controlling rotary speed and the time of filming compactness extent and the thickness of surface structure.
Cartilage frame material prepared by the present invention be take natural cartilage periplast as basic ingredient, from top layer to calcification layer, II Collagen Type VI content declines gradually, and GAG content raises gradually, and in calcification layer, has added the distinctive X-type collagen of natural cartilage tissue calcification layer composition.In order to realize the characteristic of spatial distribution of each layer of azelon structure in natural cartilage multiple structure, the present invention adopts crystallographic orientation and spin-coating method, make the azelon longitudinal arrangement in prepared middle level, the azelon on top layer is horizontal, and make calcification layer there is bone repairing effect, be conducive to timbering material and implant the fusion of rear and subchondral bone, promote the regeneration of subchondral bone.Prepared cartilage frame material, after compound chondrocyte, can be used in holostrome cartilage defect repair, and the cartilaginous tissue of regeneration has the space structure consistent with natural cartilage and protein component; Be specially adapted to the reparation of the heavy burden position articular cartilage such as knee joint, the larger neocartilage of regeneration comprcssive strength.The present invention is by preparing fine and close cartilage matrix coating at cartilage frame material surface, can improve the wearability on cartilage frame surface, guarantee the complete of repair of cartilage district surface structure, can prevent again the water loss of cartilage after stressed, there is water retention characteristic, the high elastic modulus that has kept cartilaginous tissue, has further strengthened the anti-pressure ability of regeneration of cartilage.
The present invention prepares the culture vessel that tissue engineering bone/cartilage timbering material adopts, and is followed successively by from the bottom to top box at the bottom of culture vessel, heightening ring and upper cover, and it can seal fastening each other, can increase as required the heightening ring adjustment height at middle part.At the bottom of vessel, in box, for material, cultivate, internal diameter is 5～30mm, and the degree of depth is 1～10mm, and wherein the heightening ring degree of depth is 1～5mm; At the bottom of culture vessel, box is made (as medical stainless steel) by the good metal material of heat conduction, heightening ring and upper cover adopt the good material of obstruct heat-conductive characteristic to make (as polystyrene foam), its mutual sealing is good, can prevent conduction of heat and fluid seepage.The internal diameter of culture vessel, external diameter and the depth are determined according to the size of prepared timbering material.During use, end box under, can, at end box side-walls suit heightening ring, finally fasten upper cover.
In preparing tissue engineering bone/cartilage timbering material process, culture vessel is placed in low temperature environment, because bottom heat conduction is good, temperature reduces rapidly, and heightening ring and upper cover still maintain relatively-high temperature in the short time, make solution in vessel first from bottom, form ice crystal, upwards condense gradually, the growth that so just can guarantee solution ice crystal is carried out along a direction, is conducive to prepare the directive timbering material of tool.
Accompanying drawing explanation
Accompanying drawing 1 is the structural representation of culture vessel that preparation method of the present invention adopts, and in figure, 1 is end box, and 2 is heightening ring (can be 1～3), and 3 is upper cover.
Accompanying drawing 2 is the electron micrograph in the prepared cartilage frame material of the present invention middle level, and wherein stromatin fiber is longitudinal distribution as seen, confirms the structural similarity of structure and the natural cartilage substrate of prepared tissue engineering bone/cartilage timbering material.
Accompanying drawing 3 is the electron micrograph of the prepared cartilage frame material of the present invention middle level structure cross section, visible fiber alignment is different from directional characteristic longitudinally, present mushy feature, confirm that constructed cartilage frame material has different construction featuress in vertical and horizontal direction, consistent with natural cartilage.
Accompanying drawing 4 is the electron micrograph of the prepared cartilage frame material of the present invention calcification layer, forging bone granule in visible calcification layer is combined with cartilage matrix closely, after the calcification layer rehydration that bi-material is compounded to form, can keep certain moisture content, can be moulding according to the practical situation of defect.
The cartilage frame material that accompanying drawing 5 is prepared for the present invention after compound chondrocyte for the result photo of 8 weeks after rabbit articular cartilage prothesis, result shows, the fibre structure of neocartilage tissue has the vertical distribution of natural cartilage, and the distinctive multiple structure of cartilage is complete.
The specific embodiment
Below in conjunction with example, technical solution of the present invention is described in further detail.The mammal articular cartilage adopting in example is purchased from slaughterhouse, peels off surperficial perichondrium; The calcined bone powder adopting is bought in commercially available like product (as the Bai Ameng board hydroxyl apatite bone repairing material of Zhuhai Li Zhu group production), also can prepare with reference to existing document (as Chinese Patent Application No. 200910022842.6).The culture vessel of using in example adopts medical stainless steel (mold pressing) and polystyrene (injection moulding) one-shot forming preparation; Specification 1: end box internal diameter is 15mm, is 4mm deeply, and heightening ring is 1mm deeply, 2 of heightening rings; Specification 2: end box internal diameter is 20mm, is 2mm deeply, and heightening ring is 1.5mm deeply.
Embodiment 1,
The preparation of step 1, natural cartilage substrate: under 4 ℃ of conditions, by 10～200mm
3size without perichondral mammal articular cartilage piece, put into liquid nitrogen freezing after 3 hours, proceed in the pulverizer of pre-cooling and pulverize, it is 1% acetum that ground product is dissolved in to volumetric concentration, through vacuum freeze-drying, obtains cartilage matrix;
The preparation of step 2, calcification layer: it is in 1% acetum that lyophilizing cartilage matrix, GAG and X-type collagen are dissolved in to volumetric concentration, makes final concentration be respectively 5mg/ml, 8mg/ml and 5mg/ml, adjusts pH to neutral cartilage matrix solution with aqua calcis; Calcined bone powder (particle diameter 0.2mm) is fully mixed by the concentration of 0.4g/ml with cartilage matrix solution, box at the bottom of injection culture vessel, make gel layer thickness 4mm, after adding a cover at 37 ℃ of environment after gel solution solidifies, together with vessel, insert in-80 ℃ of environment freezing;
The preparation in step 3, middle level: it is in 1% acetum that lyophilizing cartilage matrix, GAG, X-type collagen and II Collagen Type VI are dissolved in to volumetric concentration, make final concentration be respectively 5mg/ml, 4mg/ml, 2mg/ml and 4mg/ml, with aqua calcis, adjust pH to neutral, adding final concentration is the TGF-β of 5ng/ml again, obtains the middle level gel solution of timbering material; The freezing culture vessel that fills calcification layer of step 2 is placed on the rustless steel base plate of-80 ℃, in the air ambient of 0～10 ℃, put two heightening rings, middle level gel solution is injected to the calcification layer surface in culture vessel, this gel layer thickness is 2mm, after adding a cover, treat that gel solution freezes, and proceeds under-80 ℃ of environment freezing more than 3 hours; Through low-temperature vacuum drying, obtain middle level and the calcification layer structure of cartilage frame material again;
The preparation on step 4, top layer: it is in 1% acetum that lyophilizing cartilage matrix and II Collagen Type VI are dissolved in to volumetric concentration, make concentration be respectively 1mg/ml and 9mg/ml, with sodium hydroxide solution, adjust pH to neutral, adopt vacuum rotating coating method that it is evenly coated to the surface, middle level through low-temperature vacuum drying, rotary speed 500rpm, time is 50s, obtains tissue engineering bone/cartilage timbering material after drying.
The calcification layer of tissue engineering bone/cartilage timbering material prepared by this example is thicker, can be used for subchondral bone and follow the cartilage defect repair of cartilage lesion generation excalation, the calcification layer of thickening can go deep into the reparation that subchondral bone carries out cartilage defect, is particularly suitable for the cartilage defect repair that mechanical damage causes.
Embodiment 2,
The preparation of step 1, natural cartilage substrate: under 4 ℃ of conditions, by 10～200mm
3size without perichondral mammal articular cartilage piece, put into liquid nitrogen freezing after 3 hours, proceed in the pulverizer of pre-cooling and pulverize, it is 1% acetum that ground product is dissolved in to volumetric concentration, through vacuum freeze-drying, obtains cartilage matrix;
The preparation of step 2, calcification layer: it is in 1% acetum that lyophilizing cartilage matrix, GAG and X-type collagen are dissolved in to volumetric concentration, makes final concentration be respectively 10mg/ml, 9mg/ml and 10mg/ml, adjusts pH to neutral cartilage matrix solution with aqua calcis; Calcined bone powder (Bai Ameng board hydroxyl apatite bone powder footpath 0.1mm) and cartilage matrix solution are fully mixed by the concentration of 0.15g/ml, box at the bottom of injection culture vessel, make gel layer thickness 2mm, after adding a cover under 37 ℃ of conditions after gel solution solidifies, together with vessel, insert in-80 ℃ of environment freezing;
The preparation in step 3, middle level: it is in 1% acetum that lyophilizing cartilage matrix, GAG, X-type collagen and II Collagen Type VI are dissolved in to volumetric concentration, make final concentration be respectively 8mg/ml, 6mg/ml, 2mg/ml and 8mg/ml, with aqua calcis, adjust pH to neutral, adding final concentration is the TGF-β of 5ng/ml again, obtains the middle level gel solution of timbering material; Step 2 is placed in through the freezing culture vessel that fills calcification layer on the rustless steel base plate of-80 ℃, in the air ambient of 0～10 ℃, put heightening ring, middle level gel solution is injected to the calcification layer surface in culture vessel, this gel layer thickness is 1.5mm, after adding a cover, treat that gel solution freezes, and proceeds under-80 ℃ of conditions freezing more than 3 hours; Through low-temperature vacuum drying, obtain middle level and the calcification layer structure of cartilage frame material again;
The preparation on step 4, top layer: it is in 1% acetum that lyophilizing cartilage matrix and II Collagen Type VI are dissolved in to volumetric concentration, make concentration be respectively 2mg/ml and 18mg/ml, with sodium hydroxide solution, adjust pH to neutral, adopt again vacuum rotating coating method that it is evenly coated to the surface, middle level through low-temperature vacuum drying, rotary speed 2000rpm, time 20s, obtains tissue engineering bone/cartilage timbering material after drying.
Calcification layer and the intima-media thickness of tissue engineering bone/cartilage timbering material prepared by this example are moderate, and surface structure is fine and close, can be used for the cartilage defect repair that osteoarthritis causes; Osteoarthritis conventionally by top layer gradually down-cutting errosion cause cartilage defect, fine and close surface structure is conducive to the fine and close cartilage of rapid regeneration top layer, can prevent the erosion to cartilage again of local pathology environment, rapid recovery symptom, improves patients ' life quality.
Claims (4)
1. a tissue engineering bone/cartilage timbering material, is characterized in that, is followed successively by top layer, middle level and calcification layer from top to bottom; Its mesexine is comprised of lyophilizing cartilage matrix and II Collagen Type VI, and matrix fiber is parallel distribution in the horizontal direction; Middle level is comprised of lyophilizing cartilage matrix, GAG, X-type collagen and II Collagen Type VI and TGF-β, and its matrix fiber is longitudinal arrangement; Calcification layer is comprised of lyophilizing cartilage matrix, GAG and X-type collagen and calcined bone powder, and the fiber in calcification layer is interspersed at three dimensions.
2. the method for preparation tissue engineering bone/cartilage timbering material claimed in claim 1, is characterized in that, is that natural cartilage matrix composition is become to gel, adds after GAG, X-type collagen and calcined bone powder, and calcification layer is prepared in lyophilizing; After natural cartilage substrate is mixed with GAG, X-type collagen, II Collagen Type VI and TGF-β, on calcification layer surface, carry out low temperature crystallographic orientation, after lyophilization, form middle level; By natural cartilage substrate and II Collagen Type VI compound after, adopt spin coating method on preparation top layer, surface, middle level, after drying, obtain tissue engineering bone/cartilage timbering material.
3. preparation method according to claim 2, is characterized in that, concrete steps comprise:
The preparation of step 1, natural cartilage substrate: under 4 ℃ of conditions, mammal is put into liquid nitrogen freezing after 3 hours without perichondral articular cartilage piece, proceed in the pulverizer of pre-cooling and pulverize, adopt the acetum that volumetric concentration is 1% to dissolve, through vacuum freeze-drying, obtain cartilage matrix;
The preparation of step 2, calcification layer: it is in 1% acetum that lyophilizing cartilage matrix, GAG and X-type collagen are dissolved in to volumetric concentration, make final concentration be respectively 5～12mg/ml, 8～15mg/ml and 5～10mg/ml, with aqua calcis, adjust pH to neutral, obtain cartilage matrix solution; The calcined bone powder of particle diameter 0.05～0.2mm is mixed homogeneously with cartilage matrix solution by the amount of 0.1～0.5g/ml, obtain gel solution, be injected into box at the bottom of culture vessel, making gel layer thickness is 1～4mm, after adding a cover under 37 ℃ of conditions after gel solution solidifies, together with vessel, insert in-80 ℃ of environment freezingly, form calcification layer;
The preparation in step 3, middle level: it is in 1% acetum that lyophilizing cartilage matrix, GAG, X-type collagen and II Collagen Type VI are dissolved in to volumetric concentration, make final concentration be respectively 5～12mg/ml, 4～10mg/ml, 2～5mg/ml and 4～10mg/ml, with aqua calcis, adjust pH to neutral, adding final concentration is the TGF-β of 2～15ng/ml again, obtains the middle level gel solution of timbering material; The freezing culture vessel that fills calcification layer of step 2 is placed on the base plate of-80 ℃, in the air ambient of 0～10 ℃, middle level gel solution is injected to the calcification layer surface in culture vessel, this gel layer thickness is 1～3mm, after adding a cover, treat that gel solution freezes, and proceeds under-80 ℃ of environment freezing more than 3 hours; Through low-temperature vacuum drying, obtain middle level and the calcification layer structure of cartilage frame material again;
The preparation on step 4, top layer: it is in 1% acetum that lyophilizing cartilage matrix and II Collagen Type VI are dissolved in to volumetric concentration, make final concentration be respectively 1～3mg/ml and 9～30mg/ml, with sodium hydroxide solution, adjust pH to neutral, adopt vacuum rotating coating method that it is evenly coated to the surface, middle level of low-temperature vacuum drying, its rotary speed 500～3000rpm, time 20～100s, obtains tissue engineering bone/cartilage timbering material after drying.
4. for the custom-designed culture vessel of tissue engineering bone/cartilage timbering material preparation method described in implementing the claims 3, it is characterized in that, culture vessel is followed successively by end box, heightening ring and upper cover from the bottom to top, and it can seal fastening each other; The internal diameter of end box is 5～30mm, and the degree of depth is 1～10mm; The heightening ring degree of depth is 1～5mm.
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| CN104117097B (en) * | 2014-08-14 | 2016-04-20 | 天津市天津医院 | Integrated bone cartilage frame with bionical interfacial structure and preparation method thereof |
| CN107521021B (en) * | 2017-09-13 | 2023-09-08 | 北京华信佳音医疗科技发展有限责任公司 | Device for extrusion molding of collagen |
| CN108451627B (en) * | 2018-04-04 | 2023-11-17 | 中国人民解放军总医院 | Bone repair material blending device |
| CN110180025B (en) * | 2019-07-02 | 2021-11-02 | 上海国睿生命科技有限公司 | Cartilage-bone integrated porous bionic scaffold and preparation method thereof |
| JP7510651B2 (en) * | 2020-05-14 | 2024-07-04 | アンスティチュート ナショナル デ ラ サンテ エ デ ラ レシェルシュ メディカル（アンセルム） | Composite product for bone and joint regeneration of cartilage lesions |
| CN116277634A (en) * | 2023-03-28 | 2023-06-23 | 海南苏生生物科技有限公司 | Tissue engineering bracket production mould capable of directional solidification |
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