CN104117097A - Integrated epiphyseal cartilage scaffold with bionic interface structure and preparation method thereof - Google Patents
Integrated epiphyseal cartilage scaffold with bionic interface structure and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an integrated epiphyseal cartilage scaffold with a bionic interface structure and a preparation method thereof, belonging to the field of prosthesis materials. The integrated epiphyseal cartilage scaffold disclosed by the invention sequentially consists of a cartilage layer, a calcified cartilage layer and a subchondral bone layer from top to bottom, wherein the cartilage layer is a vertical orientated scaffold prepared from a silk fibroin solution through directional crystallization and freeze-drying; the calcified cartilage layer is a compact region consisting of silk fibroin and hydroxyapatite; the calcified cartilage layer is in organic connection with the cartilage layer and the subchondral bone layer through one-time freeze-drying; the subchondral bone layer is a three-dimensional porous structural scaffold prepared from the silk fibroin and the hydroxyapatite through paraffin microsphere perforation, freeze-drying and deparaffinating. The integrated epiphyseal cartilage scaffold disclosed by the invention has good biocompatibility and biodegradability; the calcified cartilage layer is in organic connection with the cartilage layer and the subchondral bone layer to achieve the effect of isolating bone and cartilage microenvironments, so that differentiation and multiplication of cultured cells under two microenvironments are facilitated; the preparation technology is high in controllability, does not need other cross-linking agents or toxic reagents and has large application potentiality.
Description
Technical field
The invention belongs to prosthetic material field, be specifically related to a kind of integrated bone cartilage frame with bionical interfacial structure and preparation method thereof.
Background technology
The joint part osteocartilaginous and subchondralo bone injury being caused by reasons such as wound, disease, regressions is Orthopedic Clinical common disease, particularly common in China, and is ascendant trend year by year.The injury in treating difficulty that merges at present subchondral bone for this cartilage, effect still can not be satisfactory, needs the desirable permanent reconstruction measure of research badly, and expectant treatment can only respite pain, can not stop advancing of disease; Bone and cartilage autotransplantation art source is limited, is difficult to repair larger area damaged; There is immunological rejection and pathophorous possibility in allograph bone cartilage grafting; Artificial joint replacement somewhat expensive, revision rate is higher.Developing rapidly of tissue engineering technique, for ossa articularia cartilage injury's Regeneration and Repair provides new solution.
Once had two kinds of different stent materials of employing to build respectively tissue engineered bone, cartilaginous tissue, the method being then assembled to together builds tissue engineered bone-cartilage complex.The method of assembling comprises that fibrin gel is bonding, is adhesively fixed etc. without wound suture sewing method or polylactic acid, but the bone-cartilage complex constructing exists bone, cartilage portion in conjunction with not good enough problem.
Tissue engineering relates to a cross discipline of cytobiology, material science, engineering and bioreactor, utilize ultimate principle and the method for life sciences and engineering, building needed by human body will organize, for repairing, substituting because of wound, the non-functional tissue of disease or organ.The main contents of Tissue Engineering Study comprise: seed cell, timbering material, somatomedin.Seed cell comprises the cell that chondrocyte, bone marrow stroma stem cell, fat-derived stem cells and process are genetic engineering modified etc.At present both at home and abroad mainly concentrate on choosing of timbering material and seed cell and skeletonization and the aspect such as connected mode that becomes cartilage portion about the research of organizational project bone-cartilage,
Chinese patent application 201010140115.2 discloses " a kind of method for preparing double-layer bionic cartilage tissue engineering scaffold ".First prepare substrate, make double-layer bionic cartilage frame at substrate surface again, finally by the double-layer bionic cartilage frame of NaOH solution impregnation substrate surface, peel off and obtain double-layer bionic cartilage frame from substrate, double-layer bionic cartilage frame thin thickness prepared by the present invention, good mechanical properties, one-shot forming, between upright opening and circular port, be connected closely, do not have the problem of mutual disengaging; Supporting structure approaches natural cartilage structure, and cartilage frame can directly with containing the substrate of polylactic acid PLA/bone meal be combined and form bone cartilage frame, and for the simultaneously damaged reparation of bone cartilage, cartilage layers part still has double-decker.
Chinese patent application 201210088049.8 discloses a kind of " a kind of tissue engineering bone/cartilage timbering material and preparation method thereof and device ".Cartilage frame material of the present invention is followed successively by top layer, middle level and calcification layer from top to bottom; Top layer is made up of lyophilizing cartilage matrix and II Collagen Type VI, and matrix fiber is parallel distribution in the horizontal direction; Middle level is made up of lyophilizing cartilage matrix, GAG, X-type collagen and II Collagen Type VI and TGF-β, and matrix fiber is longitudinal arrangement; Calcification layer is made up of lyophilizing cartilage matrix, GAG and X-type collagen and calcined bone powder, and its fiber is three-dimensional staggered and distributes.Be conducive to timbering material and implant the fusion of rear and subchondral bone, promote the regeneration of subchondral bone; After compound chondrocyte, can be used in holostrome cartilage defect repair, the cartilaginous tissue of regeneration has the space structure consistent with natural cartilage and protein component; Be suitable for the reparation of the heavy burden position articular cartilage such as knee joint, the larger neocartilage of regeneration comprcssive strength, improves wearability and the anti-pressure ability on cartilage frame surface.
Summary of the invention
The present invention proposes in conjunction with not good enough shortcoming in order to overcome the bone and the cartilage portion that exist in prior art, its objective is a kind of integrated bone cartilage frame with bionical interfacial structure and preparation method thereof is provided.
Technical scheme of the present invention is:
A kind of integrated bone cartilage frame with bionical interfacial structure, described support is made up of cartilage layers 1, calcification layer 2 and subchondral bone layer 3 from top to bottom successively, the vertical orientated property support that described cartilage layers 1 forms for fibroin albumen, described calcification layer 2 is the compact area that fibroin albumen and hydroxyapatite form, and described subchondral bone layer 3 is the three-dimensional porous structure support that fibroin albumen and hydroxyapatite form; Organic connection between calcification layer 2 and cartilage layers 1, subchondral bone layer 3.
Fibroin albumen in described calcification layer 2 and subchondral bone layer 3 and the mass ratio of hydroxyapatite are 1:1.
The pore diameter range of the three-dimensional porous structure of described subchondral bone layer 3 is 250 μ m ~ 425 μ m.
The thickness of described calcification layer 2 is less than 500 μ m.
Described cartilage layers 1 is 1:1 with the thickness proportion of subchondral bone layer 3.
A preparation method with the integrated bone cartilage frame of bionical interfacial structure, comprises the following steps:
A. paraffin microsphere template preparation
Paraffin microsphere is put into polyethylene mould, dry for standby;
B. the preparation of calcification layer 2 and subchondral bone layer 3
Be 10% silk fibroin protein solution and 10% hydroxyapatite solution mixing of 1:1 in mass ratio by mass fraction, add in paraffin microsphere template, aspiration vacuum, mixed liquor is fully filled, and exceeds paraffin microsphere template certain distance, and after cooled with liquid nitrogen, room temperature leaves standstill;
C. the preparation of cartilage layers 1
After calcification layer 2 micro-the melting in surface, the silk fibroin protein solution 2mL that is 6% by mass fraction slowly adds on the calcification layer 2 in mould ,-80 DEG C of lyophilization molding;
D. fibroin allosteric and dewaxing
After molding, from polyethylene mould, take out, soaked in absolute ethyl alcohol, filter wax, dries, and obtains bone cartilage frame.
The preparation of described silk fibroin protein solution comprises the following steps:
1. natural silk degumming: 1:200 adds natural silk the Na of 0.02mol/L in mass ratio
2cO
3in solution, boil, washing repeatedly, changes Na
2cO
3after solution, repeat to boil, wash, then ventilate and dry, obtain fibroin silk;
2. fibroin silk lysate: 1:4 adds fibroin silk in the lithium-bromide solution of 0.8g/mL in mass ratio, in 60 DEG C of water-baths, magnetic agitation obtains;
3. dialysis desalting: pouring fibroin silk lysate into molecular cut off is in 12000 ~ 14000 bag filter, dialyses 3 days, changes water every day, centrifugal, get supernatant, proceed to molecular cut off and be in 3500 bag filter, in the polyglycol solution of mass fraction 15%, dialyse, centrifuging and taking supernatant and get final product;
The preparation of described paraffin microsphere comprises the following steps:
1. prepare mass fraction and be 5% poly-vinyl alcohol solution, heating and melting;
2. take 60g paraffin mass, chopping, adds in 1080ml distilled water, then to add 120ml mass fraction be 5% polyvinyl alcohol, heated and stirred, and cooling, 20 ~ 80 eye mesh screens stack successively, sieve;
3. sieve to such an extent that paraffin microsphere water rinses repeatedly, ethanol dewaters, dries, and gets 40 ~ 60 order paraffin microspheres for subsequent use.
Described filter wax technology application apparatus,Soxhlet's, solvent is normal hexane or cyclohexane extraction.
The invention has the beneficial effects as follows:
In the present invention, build a kind of integrated bone cartilage frame with bionical interfacial structure, make its structure closer to physiological environment, it is the new model in a kind of osteochondro tissue regeneration and the field of reconstruction, provide new approaches for building tissue engineering bone cartilage compound, experimental basis is provided to further clinical practice.The material adopting belongs to natural material, have good biocompatibility and degradability, and material source is extensive.Calcification layer is with cartilage layers and subchondral bone layer is organic is connected, utilize the silk fibroin solution itself that occurred conformation does not change to dissolve each other, bone-cartilage two parts support well can be coupled together, preparation simultaneously connects calcification layer, play isolation bone, the effect of cartilage microenvironment, be beneficial to cell better differentiation and proliferation under two kinds of microenvironments of plantation.This support technology of preparing controllability is strong simultaneously, and without other cross-linking agent or toxic reagent, application potential is huge.The present invention on composition, form, mechanical characteristic close to " hyaline cartilage-interfacial structure-subchondral bone " extracellular matrix structure of human synovial, and in conjunction with closely avoiding layering.
Brief description of the drawings
Fig. 1 is structural representation of the present invention;
Fig. 2 is the structure chart of the present invention under scanning electron microscopic observation;
Fig. 3 is the cartilage layers longitudinal section figure under scanning electron microscopic observation;
Fig. 4 is the cartilage layers cross-sectional view under scanning electron microscopic observation;
Fig. 5 is the subchondral bone layer figure under scanning electron microscopic observation;
Fig. 6 is cartilage portion HE colored graph in the present invention;
Fig. 7 is subchondral bone part HE colored graph in the present invention;
Fig. 8 is cartilage portion Toluidine blue staining figure in the present invention;
Fig. 9 is subchondral bone part calcium tuberosity colored graph in the present invention;
Figure 10 is that under scanning electron microscope, cell adhesion is orientated microcellular structure exterior view at cartilage frame;
Figure 11 is that under scanning electron microscope, cell fills the structure chart in subchondral bone brace aperture;
Figure 12 is that LIVE/DEAD dyeing showed cell fills the structure chart in cartilage frame orientation texture;
Figure 13 is that LIVE/DEAD dyeing showed cell adheres to cartilage frame microcellular structure figure;
Figure 14 is that LIVE/DEAD dyeing showed cell fills structure chart in subchondral bone brace aperture.
Wherein:
1. cartilage layers 2. calcification layer 3. subchondral bone layer.
Detailed description of the invention
Below in conjunction with Figure of description and embodiment, the present invention is described in detail:
As shown in Figure 1, a kind of integrated bone cartilage frame with bionical interfacial structure, described support is made up of cartilage layers 1, calcification layer 2 and subchondral bone layer 3 from top to bottom successively, the vertical orientated property support that described cartilage layers 1 forms for fibroin albumen; Described calcification layer 2 is the compact area that fibroin albumen and hydroxyapatite form, organic connection between calcification layer 2 and cartilage layers 1, subchondral bone layer 3; Described subchondral bone layer 3 is the three-dimensional porous structure support that fibroin albumen and hydroxyapatite form.
The silk fibroin protein solution that described cartilage layers 1 is 6% forms through crystallographic orientation, lyophilizing.
Fibroin albumen in described calcification layer 2 and subchondral bone layer 3 and the mass ratio of hydroxyapatite are 1:1, and calcification layer 2 is shaped with cartilage layers 1,3 lyophilizing of subchondral bone layer.The thickness of described calcification layer 2 is less than 500 μ m.
The pore diameter range of the three-dimensional porous structure of described subchondral bone layer 3 is 250 μ m ~ 425 μ m, and subchondral bone layer 3 adopts the method for paraffin microsphere drilling, through lyophilizing, dewax and get final product.
Described cartilage layers 1 is 1:1 with the thickness proportion of subchondral bone layer 3.
A preparation method with the integrated bone cartilage frame of bionical interfacial structure, comprises the following steps:
A. paraffin microsphere template preparation
0.5g 40 ~ 60 object paraffin microspheres are placed in to polyethylene mould, flatten gently, be placed in 55 DEG C of baking box 70min and melt admittedly, room temperature is cooling;
B. the preparation of calcification layer 2 and subchondral bone layer 3
Get respectively that mass fraction is 10% silk fibroin protein solution and the each 1g of hydroxyapatite solution mixes, be added drop-wise in the polyethylene mould that fills paraffin microsphere, vacuum draw, mixed liquor is filled between paraffin microsphere, suck unnecessary liquid, only stay paraffin microsphere surface thin layer, mixed liquor exceeds paraffin microsphere template 300 μ m, after cooled with liquid nitrogen, room temperature leaves standstill 20min;
C. the preparation of cartilage layers 1
After calcification layer 2 micro-the melting in surface, the silk fibroin protein solution that is 6% by mass fraction drips to gently silk fibroin protein solution and above hydroxyapatite mixed liquor, height is advisable to drip full mould, about 2mL;-80 DEG C of cooling metal derbies are placed on mould, are placed horizontally at-80 DEG C of refrigerator overnight, lyophilizing;
D. fibroin allosteric and dewaxing
The support of lyophilizing is taken out from polyethylene mould, and soaked in absolute ethyl alcohol 2h, is put into after drying in the apparatus,Soxhlet's that fills cyclohexane extraction and filters wax 48h, sloughs paraffin, dries and get final product.
The preparation of wherein said paraffin microsphere comprises the following steps:
1. prepare mass fraction and be 5% poly-vinyl alcohol solution, heating and melting 2h;
2. take 60g paraffin mass, chopping, adds in 1080ml distilled water, then to add 120ml mass fraction be 5% poly-vinyl alcohol solution, heated and stirred, and cooling sieving, screen sizes stacks successively from 20 order ~ 80 orders;
3. the paraffin microsphere in screen cloth is rinsed repeatedly with tap water, ethanol dewaters, dries, and class wrapping is by size got 40 ~ 60 order paraffin microspheres for subsequent use.
The preparation that wherein said mass fraction is 10% silk fibroin protein solution comprises the following steps:
1. natural silk degumming: the Na that 40g natural silk is added to 8kg 0.02mol/L
2cO
3in solution, boil 30min, distillation washing 10 times, changes Na
2cO
3solution, repeats aforesaid operations, and cleaning is placed on ventilation and dries, and obtains fibroin silk;
2. fibroin silk dissolves: take 80g lithium bromide standardize solution distilled water 100ml and dissolve, take the fibroin silk 20g drying, slowly fill in lithium-bromide solution 60 DEG C of water-bath magnetic agitation 4h;
3. dialysis desalting: pouring fibroin silk lysate into molecular cut off is in 13000 bag filter, dialyse 3 days, change distilled water every day 3 times, the centrifugal 15min of 11000rpm, get supernatant, pour molecular cut off into and be in 3500 bag filter, 10h dialyses in the polyglycol solution of mass fraction 15%, the centrifugal 15min of 11000rpm, gets supernatant and is silk fibroin protein solution;
4. silk fibroin protein solution measurement of concetration: accurately measure 5 groups of 1ml silk fibroin protein solutions, be placed in culture dish, 55 DEG C of dried overnight, use the empty culture dish of analytical balance Measurement accuracy, are dried weight front and dry rear culture dish, calculate concentration and the density of silk fibroin protein solution.
5. according to fibroin albumen measurement of concetration result, add appropriate distilled water, be mixed with 10% silk fibroin protein solution, stir;
Wherein the preparation method of 10% silk fibroin protein solution and 10% hydroxyapatite solution mixed liquor is as follows:
Adopt nano-grade hydroxy apatite, take the hydroxyapatite powder of 1kg, making mass fraction according to mass ratio 1:10 interpolation distilled water 10kg is 10% hydroxyapatite solution, is placed in shaking table 1h and mixes; According to the ratio of mass ratio 1:1, getting mass fraction is that 10% silk fibroin protein solution and mass fraction are that the each 1kg of 10% hydroxyapatite solution mixes, utilize ultrasonator that the two is combined, obtain 10% silk fibroin protein solution and 10% hydroxyapatite solution mixed liquor.
Stereo microscope, the scanning electron microscope in the present invention with the integrated bone cartilage frame of bionical interfacial structure detect:
Fig. 2 is the structure chart of the present invention of (× 100) under scanning electron microscopic observation, as shown in Figure 2, under integrated bracket Stereo microscope of the present invention, observe 3 part levels obvious, integrated bracket calcification layer 2 is obvious, between cartilage layers 1 and calcification layer 2, calcification layer 2 and subchondral bone layer 3, is connected closely.
Fig. 3 is the cartilage layers longitudinal section figure of (× 500) under scanning electron microscopic observation, and Fig. 4 is the cartilage layers cross-sectional view of (× 500) under scanning electron microscopic observation.
As shown in Figure 3,4, cartilage layers has obvious bionical orientation microcellular structure, is evenly distributed;
Fig. 5 is the subchondral bone layer figure of (× 250) under scanning electron microscopic observation, and as shown in Figure 5, subchondral bone layer 3 has good three-dimensional porous structure, and porous nickel is connective good.
The present invention has the biocompatibility of the integrated bone cartilage frame of bionical interfacial structure:
By fat stem cell, respectively after cartilage and osteogenic induction, plantation is to cartilage, the subchondral bone part of support respectively, through histopathology dyeing, scanning electron microscope, the LIVE/DEAD observation of dyeing.
Fig. 6 is cartilage portion HE colored graph (× 200) in the present invention, Fig. 7 is subchondral bone part HE colored graph (× 100) in the present invention, Fig. 8 is cartilage portion Toluidine blue staining figure in the present invention, and Fig. 9 is subchondral bone part calcium tuberosity colored graph (× 100) in the present invention.
As shown in Fig. 6 ~ 9, histological stain shows that the fat stem cell through inducing evenly adheres in the hole of support, and chondrocyte is spherical in shape, secretes the substrate such as a large amount of proteoglycans, and osteoblast is fusiformis, has a large amount of calcium tuberositys depositions.
Figure 10 is that under scanning electron microscope, (× 1000) cell adhesion is at cartilage frame orientation microcellular structure exterior view, and Figure 11 is that under scanning electron microscope, (× 500) cell fills the structure chart in subchondral bone brace aperture.
As shown in Figure 10,11, scanning electron microscopic observation, cell evenly sticks on support;
Figure 12 is that LIVE/DEAD dyeing showed cell fills the structure chart in cartilage frame orientation texture, Figure 13 is that LIVE/DEAD dyeing showed cell adheres to cartilage frame microcellular structure figure, and Figure 14 is that LIVE/DEAD dyeing showed cell fills structure chart in subchondral bone brace aperture.
As shown in Figure 12 ~ 14, (× 200) are observed in LIVE/DEAD dyeing, are green fluorescence respectively through the fat stem cell of induction on cartilage frame and subchondral bone support, represent that cell survival is good.
Major advantage of the present invention is as follows:
1. proposed according to " hyaline cartilage-interfacial structure-subchondral bone " natural structure in bionics principle simulation joint, the design concept of development " bone support-interfacial structure-cartilage frame "; And the SF solution that proposes to utilize occurred conformation to change itself dissolves each other, three part supports are closely connected, both isolate bone, cartilage microenvironment, strengthen again the mechanical strength of cartilage, bone parts.
2. cartilage layers 1 is bionical orientation microcellular structure, subchondral bone layer 3 possesses the Macro-micro structure of high connectivity and porosity, interfacial structure composition is similar to natural cartilage calcification layer 2, making its structure function closer to physiology, is the new model in a kind of bone-cartilage complex tissue regeneration and the field of reconstruction.
3. cartilage layers 1, subchondral bone layer 3 have good pore structure and connectedness, are conducive to cell migration and enter the transfer of internal stent and nutrient substance and metabolite.
Claims (9)
1. one kind has the integrated bone cartilage frame of bionical interfacial structure, it is characterized in that: described support is made up of cartilage layers (1), calcification layer (2) and subchondral bone layer (3) from top to bottom successively, the vertical orientated property support that described cartilage layers (1) forms for fibroin albumen, described calcification layer (2) is the compact area that fibroin albumen and hydroxyapatite form, and described subchondral bone layer (3) is the three-dimensional porous structure support that fibroin albumen and hydroxyapatite form; Organic connection between calcification layer (2) and cartilage layers (1), subchondral bone layer (3).
2. a kind of integrated bone cartilage frame with bionical interfacial structure according to claim 1, is characterized in that: the fibroin albumen in described calcification layer (2) and subchondral bone layer (3) and the mass ratio of hydroxyapatite are 1:1.
3. a kind of integrated bone cartilage frame with bionical interfacial structure according to claim 1, is characterized in that: the pore diameter range of the three-dimensional porous structure of described subchondral bone layer (3) is 250 μ m ~ 425 μ m.
4. a kind of integrated bone cartilage frame with bionical interfacial structure according to claim 1, is characterized in that: the thickness of described calcification layer (2) is less than 500 μ m.
5. a kind of integrated bone cartilage frame with bionical interfacial structure according to claim 1, is characterized in that: described cartilage layers (1) is 1:1 with the thickness proportion of subchondral bone layer (3).
6. according to the preparation method of a kind of integrated bone cartilage frame with bionical interfacial structure claimed in claim 1, it is characterized in that: comprise the following steps:
A. paraffin microsphere template preparation
Paraffin microsphere is put into polyethylene mould, dry for standby;
B. the preparation of calcification layer (2) and subchondral bone layer (3)
Be 10% silk fibroin protein solution and 10% hydroxyapatite solution mixing of 1:1 in mass ratio by mass fraction, add in paraffin microsphere template, aspiration vacuum, mixed liquor is fully filled, and exceeds paraffin microsphere template certain distance, and after cooled with liquid nitrogen, room temperature leaves standstill;
C. the preparation of cartilage layers (1)
After micro-the melting in calcification layer (2) surface, the silk fibroin protein solution 2mL that is 6% by mass fraction slowly adds the calcification layer (2) in mould upper ,-80 DEG C of lyophilization molding;
D. fibroin allosteric and dewaxing
After molding, from polyethylene mould, take out, soaked in absolute ethyl alcohol, filter wax, dries, and obtains bone cartilage frame.
7. the preparation method of a kind of integrated bone cartilage frame with bionical interfacial structure according to claim 6, is characterized in that: the preparation of described silk fibroin protein solution comprises the following steps:
1. natural silk degumming: 1:200 adds natural silk the Na of 0.02mol/L in mass ratio
2cO
3in solution, boil, washing repeatedly, changes Na
2cO
3after solution, repeat to boil, wash, then ventilate and dry, obtain fibroin silk;
2. fibroin silk lysate: 1:4 adds fibroin silk in the lithium-bromide solution of 0.8g/mL in mass ratio, in 60 DEG C of water-baths, magnetic agitation obtains;
3. dialysis desalting: pouring fibroin silk lysate into molecular cut off is in 12000 ~ 14000 bag filter, dialyses 3 days, changes water every day, centrifugal, get supernatant, proceed to molecular cut off and be in 3500 bag filter, in the polyglycol solution of mass fraction 15%, dialyse, centrifuging and taking supernatant and get final product.
8. the preparation method of a kind of integrated bone cartilage frame with bionical interfacial structure according to claim 6, is characterized in that: the preparation of described paraffin microsphere comprises the following steps:
1. prepare mass fraction and be 5% poly-vinyl alcohol solution, heating and melting;
2. take 60g paraffin mass, chopping, adds in 1080ml distilled water, then to add 120ml mass fraction be 5% polyvinyl alcohol, heated and stirred, and cooling, 20 ~ 80 eye mesh screens stack successively, sieve;
3. sieve to such an extent that paraffin microsphere water rinses repeatedly, ethanol dewaters, dries, and gets 40 ~ 60 order paraffin microspheres for subsequent use.
9. the preparation method of a kind of integrated bone cartilage frame with bionical interfacial structure according to claim 6, is characterized in that: described filter wax technology application apparatus,Soxhlet's, solvent is normal hexane or cyclohexane extraction.
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