CN103893824A - Three-dimensional bone tissue engineering scaffold material with different stiffness and preparation method of material - Google Patents
Three-dimensional bone tissue engineering scaffold material with different stiffness and preparation method of material Download PDFInfo
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- CN103893824A CN103893824A CN201410153286.7A CN201410153286A CN103893824A CN 103893824 A CN103893824 A CN 103893824A CN 201410153286 A CN201410153286 A CN 201410153286A CN 103893824 A CN103893824 A CN 103893824A
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Abstract
The invention discloses a three-dimensional bone tissue engineering scaffold material with different stiffness and a preparation method of the material. The method comprises the following steps: firstly, mixing collagen with hydroxyapatite according to different ratios, so as to form different concentration gradients of collagen/hydroxyapatite mixed emulsions; immersing the fabricated acellular bone into different concentrations of mixed solutions; reperfusing and lightly vibrating, so as to form an even collagen/hydroxyapatite coating; finally freezing and drying and carrying out crosslinking treatment to obtain the three-dimensional bone tissue engineering scaffold material with different stiffness. Different stiffness of substrates can be formed on the scaffold surface under the condition that the three-dimensional scaffold microstructure is not changed, the scaffold material has good biocompatibility, proliferation and differentiation of different stem cells are facilitated, and the material is low in cost and easy to fabricate.
Description
Technical field
The present invention relates to a kind of bone tissue engineering stent material and preparation method thereof, particularly a kind of acellular bone-collagen/hydroxyapatite three-dimensional stent material with different-stiffness and preparation method thereof.
Background technology
There is good organization's property in order to obtain, functional tissue, tissue engineering bracket material should be simulated structure and the chemical feature of n cell epimatrix as much as possible.Successfully timbering material should analogue body in natural mechanical characteristics stand the ambient stress in body.The mechanical characteristics of timbering material is delivered to and in cell, has important effect affecting cell behavior and controlling outside mechanical signal.Cell can perception and substrate that they are touched make response; different substrate rigidity can inducing mesenchymal stem cell under two-dimensional condition be broken up respectively becomes different cell types; comprise (the Engler AJ such as neurocyte, myogenous cell and osteoblast; Sen S; Sweeney HL, Discher DE.Matrix elasticity directs stem cell lineage specification.Cell.2006; 126:677-689.).Dimensional culture is analog cell true microenvironment in vivo better, but at present about substrate rigidity mainly concentrates under two-dimensional condition the research of differentiation of stem cells.There is bibliographical information to utilize collagen (Hadjipanayi E; Mudera V, Brown RA.Brown.Guiding cell migration in3D:a collagen matrix with graded directional stiffness.Cell Motil Cytoskeleton.2009; 66:121-128.), polylactic acid (poly-lactic acid; PLLA)/Poly(D,L-lactide-co-glycolide (poly-lactic co glycolic acid; PLGA) (Levy-Mishali M; Zoldan J, Levenberg S.Effect of scaffold stiffness on myoblast differentiation.Tissue Eng Part is A.2009; 15:935-944.) etc. study the timbering material with different-stiffness to the impact of differentiation of stem cells.In bone tissue engineering stent material field; existing bibliographical information utilizes hydrogel to form the accurate three-dimensional rack of proper stiffness; can break up (Naito H towards skeletonization direction by inducing mesenchymal stem cell; Dohi Y; Zimmermann WH; Tojo T; Takasawa S; Eschenhagen T, Taniguchi S.The effect of mesenchymal stem cell osteoblastic differentiation on the mechanical properties of engineered bone-like tissue.Tissue Eng Part is A.2011; 17:2321-2329.); The tissue that the accurate three-dimensional rack with different-stiffness and geometry that utilizes poly(ether-urethane) (polyether urethanes) to form can affect MC3T3-E1 cell forms area (Kommareddy KP; Lange C; Rumpler M; Dunlop JW; Manjubala I; Cui J; Kratz K; Lendlein A, Fratzl P.Two stages in three-dimensional in vitro growth of tissue generated by osteoblastlike cells.Biointerphases.2010; 5:45-52.).(the Huebsch N such as Huebsch; Arany PR; Mao AS; Shvartsman D; Ali OA; Bencherif SA, Rivera-Feliciano J, Mooney DJ.Harnessing traction-mediated manipulation of cell/matrix interface to control stem-cell fate.Nat Mater.2010; Research 9:518-26.) shows, different three-dimensional environment rigidity can change the pedigree differentiation of mescenchymal stem cell, and mescenchymal stem cell can be divided into osteoblast under the three-dimensional stiffness of 11～30kPa, especially best toward skeletonization direction differentiation effect in the time of 22kPa.But, yet there are no three-dimensional bone tissue engineering stent material and can in keeping micro structure and porosity substantially constant, form different-stiffness substrate.
Summary of the invention
The technical problem to be solved in the present invention is under three-dimensional condition, to build the bone tissue engineering stent material with different-stiffness, and object is the substrate that forms different-stiffness in the situation that not changing three-dimensional rack micro structure at rack surface.
For solving the problems of the technologies described above, of the present invention have the three-dimensional bone tissue engineering stent material of different-stiffness and be made up of acellular bone and collagen/hydroxyapatite; It is characterized in that, utilize acellular bone to keep the three-dimensional microstructures of timbering material, utilize the coated acellular bone of processing of different proportioning collagen/hydroxyapatites, form the three-dimensional bone tissue engineering stent material with different substrates rigidity, be beneficial to the particular lineage differentiation of induced dry-cell.
In the present invention, acellular bone can adopt conventional method preparation, be in brief, get the capital spongy bone of fresh pig, deliver under clean conditions with 0.9% saline soak of 4 ℃, remove remaining muscle and tissue on sample, making diameter according to the damaged situation of clinical bone is the shape sample such as cylinder or the cube that length × wide × height is 2～20mm × 2～20mm × 2～20mm that 2～20mm, height are 2～20mm, rinse, be then immersed in distilled water 4 ℃ and spend the night.With 1% Triton X-100 processing sample 48h.Sample is carried out to defat 24h with methanol.After 37 ℃ of incubation sample 2h of DNA enzyme/RNA enzyme, clean and continue with PBS and rock.Support is immersed to 4h in dehydrated alcohol, to remove the cell rests thing on support.With a large amount of washed with de-ionized water support 2h, drying bracket, obtains acellular bone, is kept under 4 ℃ of environment.
Acellular bone has three-dimensional micropore structure, and bore dia can be controlled in 300～800 μ m, and porosity can reach 80%～95%, and has good connectivity.The collagen of variable concentrations proportioning and hydroxyapatite mixture are processed above-mentioned acellular bone inside by the mode of perfusion, obtained having the three-dimensional bone tissue engineering stent material of different gradient rigidity.
The preparation method of the three-dimensional bone tissue engineering stent material with different-stiffness described in the present invention also provides, concrete steps are as follows:
1) prepare Acellular bone three-dimensional rack;
2) the collagen/hydroxyapatite solution of preparation variable concentrations ratio, by step 1) timbering material that obtains immerses in institute's obtain solution and carries out perfusion, and vibration gently, guarantees that solution forms uniform coating at material surface;
3) freezing, drying steps 2) obtain through the coated Acellular bone three-dimensional rack of processing of different proportioning collagen/hydroxyapatites;
4) by step 3) obtain through the coated Acellular bone three-dimensional rack of processing of different proportioning collagen/hydroxyapatites by 1-ethyl-3-3-dimethylaminopropyl-carbonization two imido (N-(3-dimethylaminopropyl)-N '-ethylcarbodiimide hydrochloride, and N-maloyl imines (N-hydroxysuccinimide EDC), NHS) after being cross-linked, by 0.1mol/L Na
2hPO
412H
2o, by its soaking and washing 1h, then cleans it with deionized water, afterwards its lyophilization is obtained having the three-dimensional bone tissue engineering stent material of different-stiffness;
5) by step 4) the three-dimensional bone tissue engineering stent material with different-stiffness that obtains passes through
60after Co (2M rad) radiosterilization, by kept dry after its sterile sealing.
Preferably, step 1) described Acellular bone three-dimensional rack preparation method, to get the capital spongy bone of fresh pig, deliver under clean conditions with 0.9% saline soak of 4 ℃, remove remaining muscle and tissue on sample, make required form sample according to the damaged situation of clinical bone, rinse, be then immersed in distilled water 4 ℃ and spend the night.With 1% Triton X-100 processing sample 48h.Sample is carried out to defat 24h with methanol.After 37 ℃ of incubation sample 2h of DNA enzyme/RNA enzyme, clean and continue with PBS and rock.Support is immersed to 4h in dehydrated alcohol, to remove the cell rests thing on support.With a large amount of washed with de-ionized water support 2h, drying bracket, obtains acellular bone, is kept under 4 ℃ of environment.
Preferably, step 1) to be prepared into diameter be the shape sample such as cylinder or the cube that length × wide × height is 2～20mm × 2～20mm × 2～20mm that 2～20mm, height are 2～20mm to described Acellular bone three-dimensional rack.
Preferably, step 2) mass fraction of hydroxyapatite is 5wt%～70wt% in described collagen/hydroxyapatite solution, collagen quality fractional root changes between 0.1wt%～2.0wt% according to required rigidity.
Preferably, step 2) described collagen/hydroxyapatite solution mixed method is: the above-mentioned hydroxyapatite powder that calculates mass fraction slowly added in the consoluet collagen solution that calculates mass fraction, constantly stir, collagen is mixed homogeneously with hydroxyapatite powder.
In sum, of the present invention have the three-dimensional bone tissue engineering stent material of different-stiffness and guarantee that by Acellular bone support the three dimensional structure of material is constant, utilizes the collagen/hydroxyapatite of different proportionings to form different-stiffness three-dimensional stent material at rack surface.Compared with existing three-dimensional bone tissue engineering stent material, three-dimensional bone tissue engineering stent material of the present invention has overcome most of bone tissue engineering stent materials and has also changed in changing substrate rigidity the defect of micro structure, and this is also the key that the present invention proposes.Three-dimensional bone tissue engineering stent material provided by the invention has good biocompatibility, can guarantee natural micro structure and the composition of bone support, and there is good connectivity, and be conducive to adhesion and the growth of cell, be beneficial to entering and the timely discharge of cellular metabolism refuse of nutrient substance.The different-stiffness that utilizes collagen/hydroxyapatite to form, is beneficial to propagation and the differentiation of different dry cell, can promote the generation of new bone.Can select the bone of different animals different piece as material for the preparation of the Acellular bone of three-dimensional bone tissue engineering stent material, as long as meet the requirement that will repair bone.During succinct, the present invention is mainly set forth as an example of the capital spongy bone of pig example, and other animals and part bone also can adopt identical principle.
Accompanying drawing explanation
Fig. 1 shows respectively the young's modulus in compression by a kind of collagen with the variable concentrations proportioning using in the three-dimensional bone tissue engineering stent material preparation method of different-stiffness provided by the invention and the prepared support of hydroxyapatite.
Fig. 2 provided by the inventionly a kind ofly has in the three-dimensional bone tissue engineering stent material preparation method of different-stiffness acellular bone support by the graph of pore diameter distribution before and after 0.7wt% collagen/22wt% hydroxyapatite mixed solution table lining.
Fig. 3 provided by the inventionly a kind ofly has in the three-dimensional bone tissue engineering stent material preparation method of different-stiffness acellular bone support by the Thickness Distribution figure before and after 0.7wt% collagen/22wt% hydroxyapatite mixed solution table lining.
The specific embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Deliver to clean conditions by taking from the capital spongy bone of fresh pig 0.9% saline soak of 4 ℃, remove remaining muscle and tissue on sample, making diameter according to the damaged situation of clinical bone is the shape sample such as cylinder or the cube of 2～20mm × 2～20mm × 2～20mm that 2～20mm, height are 2～20mm, rinse, be then immersed in distilled water 4 ℃ and spend the night.With 1%Triton X-100 processing sample 48h.Sample is carried out to defat 24h with methanol.After 37 ℃ of incubation sample 2h of DNA enzyme/RNA enzyme, with PBS to its cleaning and continue to rock.Support is immersed to 4h in dehydrated alcohol, remove the cell rests thing on support.With a large amount of washed with de-ionized water support 2h, drying bracket, obtains acellular bone, is kept under 4 ℃ of environment.Acellular bone bore dia can be controlled in 300～800 μ m, and controlled porosity is 80%～95%.
The hydroxyapatite powder that calculates mass fraction is slowly added in the consoluet collagen solution that calculates mass fraction, constantly stir, collagen is mixed homogeneously with hydroxyapatite powder.In collagen/hydroxyapatite solution, the mass fraction of hydroxyapatite is 5wt%～70wt%, and collagen quality fractional root changes between 0.1wt%～2.0wt% according to required rigidity.For example collagen content is 0.5wt%, hydroxyapatite is a group of 22wt%, first take collagen 50mg, dissolve completely and dissolve with the HCl of 10mL0.01mol/L, then take hydroxyapatite powder 2.2g, slowly add in dissolved gum original solution, continue that stirring spends the night makes collagen solution mix homogeneously with hydroxyapatite at 4 ℃.
The collagen of variable concentrations proportioning and hydroxyapatite mixture are processed above-mentioned three-dimensional acellular bone inside by the mode of perfusion, made collagen and hydroxyapatite mixture at rack surface uniform fold.The first freezing 24h under-20 ℃ of conditions of timbering material that will coated handle well then carries out lyophilization 24h under-55 ℃, 15Pa condition.Then collagen/hydroxyapatite three-dimensional stent material good lyophilization is cross-linked to 24h with the EDC and the NHS that are dissolved in dehydrated alcohol, then uses the Na of 0.1mol/L
2hPO
412H
2o solution soaking is cleaned support 1h, finally with washed with de-ionized water repeatedly after, under-55 ℃, 15Pa condition, carry out lyophilization 24h, obtain having the three-dimensional bone tissue engineering stent material of different-stiffness, then pass through
60co (2M rad) radiosterilization, by kept dry after its sterile sealing, for subsequent use afterwards.
For the feasibility of checking institute using method, prepare collagen content for being respectively 0.35wt%, 0.5wt% and 0.7wt%, many groups collagen/hydroxyapatite support that hydroxyapatite is 22wt% etc.Below will be with collagen content for being respectively 0.35wt%, 0.5wt% and 0.7wt%, hydroxyapatite is that three groups of collagen/hydroxyapatite supports of 22wt% are that example describes, as shown in Figure 1, the average young's modulus in compression that utilizes the U.S.'s ELF3330 of Bose company mechanical testing instrument to obtain them is 6.74 ± 1.16kPa, 8.82 ± 2.12kPa and 23.61 ± 8.06kPa.Experiment records acellular bone support and is utilizing aperture, 0.7wt% collagen/22wt% hydroxyapatite mixed solution table lining front and back, porosity, pore-size distribution and Thickness Distribution all there is no significant difference.Fig. 2 and Fig. 3 are respectively aperture and the Thickness Distribution figure of acellular bone support before and after 0.7wt% collagen/22wt% hydroxyapatite mixed solution table lining.
Three-dimensional bone tissue engineering stent material provided by the invention is realized nature bone micro structure and composition by Acellular bone, utilize the coated Acellular bone of processing of different proportioning collagen/hydroxyapatites, in the situation that not changing support micro structure, form the three-dimensional porous rack material of different-stiffness.Just because of these composite factors, make the good biocompatibility of three-dimensional bone tissue engineering stent material provided by the invention, be beneficial to propagation and the differentiation of different dry cell, can promote the generation of new bone.Preparation method is simple and cost is low, and the three-dimensional bone tissue engineering stent material of comparing in the past has obvious advantage.
Claims (4)
1. one kind has the three-dimensional bone tissue engineering stent material of different-stiffness, formed by acellular bone and collagen/hydroxyapatite, it is characterized in that: described acellular bone is that the spongy bone that 300～800 μ m, porosity are 80%～95% is made by pig femoral head median pore diameter, this acellular bone is inner by the collagen/hydroxyapatite solution perfusion processing of variable concentrations proportioning, and the collagen that the hydroxyapatite that described collagen/hydroxyapatite solution is 5wt%～70wt% by mass fraction and mass fraction are 0.1wt%～2.0wt% mixes.
2. a kind of three-dimensional bone tissue engineering stent material with different-stiffness as claimed in claim 1, is characterized in that: it is 2～20mm, the height cylinder that is 2～20mm or the cube that length × wide × height is 2～20mm × 2～20mm × 2～20mm that described acellular bone can be made diameter according to the damaged situation of clinical bone.
3. a method of preparing the three-dimensional bone tissue engineering stent material with different-stiffness claimed in claim 1, comprises the steps:
1) prepare Acellular bone three-dimensional rack;
2) the collagen/hydroxyapatite solution of preparation variable concentrations ratio, by step 1) timbering material that obtains immerses in institute's obtain solution and carries out perfusion, and vibration gently, guarantees that solution forms uniform coating at material surface;
3) freezing, drying steps 2) obtain through the coated Acellular bone three-dimensional rack of processing of different proportioning collagen/hydroxyapatites;
4) by step 3) obtain after the coated Acellular bone three-dimensional rack of processing of different proportioning collagen/hydroxyapatites is cross-linked by 1-ethyl-3-3-dimethylaminopropyl-carbonization two imido and N-maloyl imines, by 0.1mol/L Na
2hPO
412H
2o, by its soaking and washing 1h, then cleans it with deionized water, afterwards its lyophilization is obtained having the three-dimensional bone tissue engineering stent material of different-stiffness;
5) by step 4) the three-dimensional bone tissue engineering stent material with different-stiffness that obtains passes through
60co radiosterilization, by kept dry after its sterile sealing.
4. preparation method as claimed in claim 3, it is characterized in that: step 2) the collagen/hydroxyapatite solution allocation method of described variable concentrations ratio is, the hydroxyapatite powder that calculates mass fraction is slowly added in the consoluet collagen solution that calculates mass fraction, constantly stir, collagen is mixed homogeneously with hydroxyapatite powder.
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| CN112957522A (en) * | 2021-02-22 | 2021-06-15 | 重庆大学 | Rigidity-adjustable porous liquid metal bone tissue engineering scaffold and preparation method thereof |
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