CN103877614A - Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof - Google Patents
Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof Download PDFInfo
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- CN103877614A CN103877614A CN201410066627.7A CN201410066627A CN103877614A CN 103877614 A CN103877614 A CN 103877614A CN 201410066627 A CN201410066627 A CN 201410066627A CN 103877614 A CN103877614 A CN 103877614A
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Abstract
The invention discloses a double-layer composite scaffold for repairing cartilage in a tissue-engineered bone. The scaffold comprises a cartilage layer and a subchondral layer, wherein the cartilage layer and the subchondral layer are combined into a whole through photopolymerisable modified hyaluronic acid gel, wherein the cartilage layer is made from transformed chondrocyte-loaded photopolymerisable modified hyaluronic acid gel, and the subchondral layer is made of a porous bio-glass scaffold. The invention also discloses a preparation method of the double-layer composite scaffold for repairing cartilage in a tissue-engineered bone, which comprises the following steps: uniformly mixing the transformed chondrocyte-loaded photopolymerisable modified hyaluronic acid gel and in-vitro induced transformed chondrocyte cells, and forming mixed gel through ultraviolet polymerization; putting the porous bio-glass scaffold on the mixed gel, adding the photopolymerisable modified hyaluronic acid gel, and performing light-crosslinking to obtain the double-layer composite scaffold for repairing cartilage in the tissue-engineered bone. The double-layer composite scaffold has excellent biocompatibility and can be degraded, and the preparation method is simple.
Description
Technical field
The invention belongs to osteochondral tissue engineering rack technical field, relate in particular to a kind of supporting structure for bone repair of cartilage and preparation method thereof.
Background technology
There is articular cartilage defect in adult's knee of 61%; Wherein, the 19%th, osteochondral defect.Articular cartilage self repair ability is limited, once impaired dyschezia is in recovery.Conventional therapeutic modality comprises autologous cartilage transplantation, autologous or allograph bone cartilage transplantation at present.These treatment meanss have some limitations, and for example cartilage transplantation source is limited, impaired for district's function, immunologic rejection, and implant is loosening etc.Along with the appearance of tissue engineering technique, the research of bone repair of cartilage has obtained very large development.Build osteochondral tissue engineering rack and need cell, cartilage cell epimatrix and subchondral bone.Be widely used in tissue engineering bone/cartilage reparation from chondrocyte autologous or that allosome extracts, but cartilage limited amount only accounts for 5～10% of cartilaginous tissue, before therefore using, need external breeding.But chondrocyte there will be when monolayer culture and dedifferentes in vitro.There is at present the macromolecular material of more synthetic as cartilage cell epimatrix, as Polyethylene Glycol (PEG), poly-alpha-hydroxy ester, poly-NiPAAm, polypropylene-fumaric acid, polypropylene fumaric acid, polyurethane polyureas carbamate etc., their main feature is to have plasticity, have certain intensity, but there have many problems to have on aspect the control of biocompatibility, physicochemical property, degradation rate to be to be solved.The multiplex porous titanium alloy support of subchondral bone layer, it has higher intensity, good biocompatibility, but can not degrade in vivo.
Summary of the invention
In order to overcome, repair of cartilage Biocompatibility in prior art is poor, the defect of hard degradation, the object of this invention is to provide the two-layer compound support that a kind of tissue engineered bone cartilage is repaired; Have good biocompatibility, modification hyaluronic acid wherein has optical polymerism, can degrade in vivo under the effect of hyaluronic acid catabolic enzyme simultaneously, and catabolite does not have bio-toxicity yet.Modification hyaluronic acid gel can evenly mix with cell, after uv photopolymerization, forms the mixed gel of hyaluronic acid and cell; Chondrocyte wide material sources in cartilage layers.Subchondral bone layer material is the high-intensity Porous bioglass support of having of 3 D-printing, the high subchondral bone reparation that can be used for weight bearing area of its intensity.Bio-vitric can degradation in vivo, is converted into osteoid hydroxyapatite, has good bone conductibility, is widely studied in the bone field of repairing; Bio-vitric, in degradation process, discharges the various elements in glass ingredient, can stimulate bone repair of cartilage.
Another object of the present invention is to provide a kind of preparation method of two-layer compound support of above-mentioned tissue engineered bone cartilage reparation, and the method is simple, and support can require make according to concrete dimensional structure.
To achieve these goals, technical scheme of the present invention is as follows:
The invention provides the two-layer compound support that a kind of tissue engineered bone cartilage is repaired, comprise cartilage layers and subchondral bone layer, described cartilage layers and subchondral bone layer are combined as a whole by photopolymerisable modification hyaluronic acid derivatives, the material of described cartilage layers is the photopolymerisable modification hyaluronic acid derivatives that is loaded with transformed chondrocytes, and the material of described subchondral bone layer is Porous bioglass support.
Wherein, the density of described transformed chondrocytes is preferably 5 × 10
6～1 × 10
8/ mL.
Wherein, described transformed chondrocytes is preferably by mesenchymal stem cells MSCs, embryonic stem cell, fat stem cell or fibroblast Induction Transformation and obtains.
Described transformed chondrocytes is more preferably the chondrocyte of the primary autologous or allogenic bone marrow mesenchymal stem Induction Transformation to the 4th generation of In vitro culture.
In a preferred embodiments of the present invention, described cartilage layers also can interpolation or combined with mesenchymal stem cells, embryonic stem cell, fat stem cell, somatomedin or transgene component in one or more, to keep chondrocyte and substrate synthesis capability.
Wherein, described Porous bioglass support preferably comprises porous silicate bio-vitric support, porous boron silicate bio-vitric support or porous boron hydrochlorate bio-vitric support; In described Porous bioglass support, more preferably also contain and add one or more in micro-Cu, Zn, Ba, Sr, Ag, Fe, F or Mn.The intensity of described Porous bioglass support is preferably 50～100MPa, and porosity is preferably 45～75%, and aperture is preferably 100～300 μ m.
The present invention also provides a kind of preparation method of two-layer compound support of above-mentioned tissue engineered bone cartilage reparation, comprise the following steps: after thering is photopolymerisable modification hyaluronic acid derivatives and mixing homogeneously with external evoked transformed chondrocytes, form mixed gel through ultraviolet polymerization; Porous bioglass stentplacement, on mixed gel, is added and has photopolymerisable modification hyaluronic acid derivatives, after illumination is crosslinked, obtain the two-layer compound support that tissue engineered bone cartilage is repaired.
Wherein, described photopolymerisable modification hyaluronic acid derivatives is preferably made by following preparation method: the aqueous solution of sodium hyaluronate that is 1～5% by mass concentration mixes with methacrylic anhydride after freezing point is carried out stirring and dissolving again, the concentration of described methacrylic anhydride is 20 times of hyaluronate sodium molar concentration, regulate pH to make pH value be greater than all the time 8, freezing point stirring reaction 24 hours, put into the deionized water of 50 times of volumes of dialyzer and dialyse 3 days, lyophilization is mixed to get photopolymerisable modification hyaluronic acid derivatives with normal saline and cross-linking agent I2959 after 48 hours again.
Wherein, the concentration of described hyaluronate sodium is preferably 2wt%.
Wherein, the molecular weight of described hyaluronate sodium is preferably 66～90 kilodaltons.
Wherein, described in, having the hyaluronic concentration of modification in photopolymerisable modification hyaluronic acid derivatives is preferably 0.05wt%.
Wherein, the concentration of described cross-linking agent I2959 is preferably 0.05wt%.
Wherein, the number volume ratio of described transformed chondrocytes or described photopolymerisable modification hyaluronic acid derivatives is preferably 40 × 10
6/ mL.
Wherein, the method for described ultraviolet polymerization is preferably ultra violet lamp, and wavelength is preferably 320～400nm, and intensity is preferably 8900 μ W/cm
2～21700 μ W/cm
2, irradiating is highly preferably 5～25cm, the time is preferably 5～15 minutes.
Wherein, the crosslinked method of described illumination is preferably ultra violet lamp, and wavelength is preferably 320～400nm, and intensity is preferably 8900 μ W/cm
2～21700 μ W/cm
2, irradiating is highly preferably 5～25cm, the time is preferably 5～15 minutes.
The present invention compared with the existing technology, has the following advantages and beneficial effect:
The two-layer compound support of tissue engineered bone cartilage reparation of the present invention has good biocompatibility, and modification hyaluronic acid wherein has optical polymerism, can degrade in vivo under the effect of hyaluronic acid catabolic enzyme simultaneously, and catabolite does not have bio-toxicity yet; Modification hyaluronic acid derivatives can evenly mix with cell, after uv photopolymerization, forms the mixed gel of hyaluronic acid and cell; Chondrocyte wide material sources in cartilage layers; Subchondral bone layer material is the high-intensity Porous bioglass support of having of 3 D-printing, the high subchondral bone reparation that can be used for weight bearing area of its intensity; Bio-vitric can degradation in vivo, is converted into osteoid hydroxyapatite, has good bone conductibility, is widely studied in the bone field of repairing; Bio-vitric, in degradation process, discharges the various elements in glass ingredient, can stimulate bone repair of cartilage.
It is simple that the present invention prepares the method for two-layer compound support that tissue engineered bone cartilage repairs, and support can require make according to concrete dimensional structure.
Accompanying drawing explanation
Fig. 1 is the structural representation of the two-layer compound support of the tissue engineered bone cartilage reparation shown in embodiment 1.
Fig. 2 is the life or death cytological map of the two-layer compound support In vitro culture repaired of tissue engineered bone cartilage shown in embodiment 4 after 4 weeks.
Fig. 3 is the Safranin O colored graph of the two-layer compound support In vitro culture repaired of tissue engineered bone cartilage shown in embodiment 4 after 4 weeks.
Fig. 4 is the Picrosirius red colored graph of the two-layer compound support In vitro culture repaired of tissue engineered bone cartilage shown in embodiment 4 after 4 weeks.
Fig. 5 is the two-layer compound support Toluidine blue staining photo after 12 weeks in vivo that the tissue engineered bone cartilage shown in embodiment 7 is repaired.
The specific embodiment
Below in conjunction with accompanying drawing illustrated embodiment, the present invention is further detailed explanation.The experimental technique of unreceipted actual conditions in the following example, according to conventional method and condition, or selects according to catalogue.In following embodiment, cross-linking agent I2959 used is purchased from Ciba, Tarrytown, NY.
In following embodiment, silicate bio-vitric, borosilicate glass bio-vitric, borate biological glass, the silicate bio-vitric that trace element adds, the borate biological glass that the borosilicate bio-vitric that trace element adds and trace element add all can prepare by traditional fusion method.Porous bioglass support can be selected the Porous bioglass support that utilizes existing porous support technology of preparing to prepare, silicate bio-vitric porous support more preferably preparation process can list of references " Fu; Q.; E.Saiz; and A.P.Tomsia; Direct ink writing of highly porous and strong glass scaffolds for load-bearing bone defects repair and regeneration.ACTA BIOMATERIALIA, 2011.7 (10): p.3547-3554. ".Concrete steps are as follows: silicate bioglass particles is worn into the glass powder that particle diameter is 1～3 μ m by wet grinding.By 40%(percent by volume) glass powder be well dispersed in 20%(percentage by weight) Pluronic F-127 colloidal sol in, form glass dust batch mixing.Glass dust batch mixing is packed in 3mL syringe, be fit into (3D Inks, A3200-Npaq, Stillwater, OK) in three-dimensional printer, print design the three-dimensional dimension, pore size of support in computer software (RoboCAD3.0) after.
Borate biological glass and borosilicate bio-vitric support are preferably adopted with the following method and are prepared: by 30～50%(percent by volume) glass powder be well dispersed in containing 5%～6%(percent by volume) ethyl cellulose and 8%～10%(percent by volume) in the alcohol mixeding liquid of PEG300, mix homogeneously forms glass dust batch mixing.Glass dust batch mixing is packed in 3mL syringe, be fit into (3D Inks, A3200-Npaq, Stillwater, OK) in three-dimensional printer, print design the three-dimensional dimension, pore size of support in computer software (RoboCAD3.0) after.
The support of printing, 60 ℃ of dried overnight, carries out pre-burning according to the temperature increasing schedule of table 1 at air furnace, 560～710 ℃ of sintering 1 hour, obtains Porous bioglass support subsequently.
Table 1 temperature increasing schedule
| Step | Programming rate (℃/min) | Temperature (℃) | Temperature retention time (h) |
| 1 | 0.5 | 200 | 2 |
| 2 | 0.5 | 300 | 2 |
| 3 | 1 | 500～600 | 1 |
In following embodiment, external evoked transformed chondrocytes is made by following method: by mesenchymal stem cells MSCs, and embryonic stem cell, fat stem cell is cultivated 2-4 week for subsequent use in chondrify culture fluid.
In following embodiment, the preparation method of photopolymerisable modification hyaluronic acid derivatives comprises the following steps:
The hyaluronate sodium that is 1～5% by mass concentration (molecular weight is 66～90 kilodaltons) aqueous solution mixes with methacrylic anhydride after freezing point is carried out stirring and dissolving again, the concentration of described methacrylic anhydride is 20 times of hyaluronate sodium molar concentration, regulate pH to make pH value be greater than all the time 8, freezing point stirring reaction 24 hours, put into the deionized water of 50 times of volumes of dialyzer and dialyse 3 days, lyophilization is mixed to get photopolymerisable modification hyaluronic acid derivatives with normal saline and cross-linking agent I2959 after 48 hours again; The concentration of cross-linking agent I2959 is 0.05wt%.
Embodiment 1
The preparation method of the two-layer compound support of tissue engineered bone cartilage reparation comprises the following steps: will have photopolymerisable modification hyaluronic acid derivatives and carry out filter-sterilized through 0.02 μ m filter membrane, after mixing homogeneously with by the external evoked transformed chondrocytes of mesenchymal stem cells MSCs, making cell concentration is 40 × 10
6/ ml, modification hyaluronate sodium mass concentration is that 1%, I2959 mass concentration is 0.05% hydrogel composites, putting into diameter is 5mm, be highly in the mould of 5mm, irradiate after 10 minutes apart from 15cm place through the uviol lamp of wavelength 320～400nm, be cross-linked to form mixed gel; By porous silicate bio-vitric stentplacement on mixed gel, add 50 μ L to there is photopolymerisable modification hyaluronic acid derivatives, the crosslinked two-layer compound support that obtains tissue engineered bone cartilage reparation after 10 minutes of distance 15cm place's illumination under the uviol lamp of wavelength 320～400nm; Structure as shown in Figure 1; Fig. 1 is the structural representation of the two-layer compound support of the tissue engineered bone cartilage reparation shown in embodiment 1.
The two-layer compound support of tissue engineered bone cartilage reparation comprises cartilage layers and subchondral bone layer, described cartilage layers and subchondral bone layer are combined as a whole by photopolymerisable modification hyaluronic acid derivatives, the material of described cartilage layers is the photopolymerisable modification hyaluronic acid derivatives that is loaded with transformed chondrocytes, and the material of described subchondral bone layer is porous silicate bio-vitric support.
Embodiment 2
The preparation method of the two-layer compound support of tissue engineered bone cartilage reparation comprises the following steps: will have photopolymerisable modification hyaluronic acid derivatives ultra violet lamp in super-clean bench and sterilize half an hour, then be dissolved in the solution of the aseptic PBS that contains I2959, after mixing homogeneously with the external evoked transformed chondrocytes of fat stem cell, making cell concentration is 5 × 10
6/ ml, hyaluronate sodium mass concentration is that 2%, I2959 mass concentration is 0.05% hydrogel composites, putting into diameter is 5mm, is highly in the mould of 5mm, irradiates after 15 minutes apart from 25cm place through the uviol lamp of wavelength 320～400nm, is cross-linked to form mixed gel; By porous boron silicate bio-vitric stentplacement on mixed gel, add 30 μ L to there is photopolymerisable modification hyaluronic acid derivatives, the crosslinked two-layer compound support that obtains tissue engineered bone cartilage reparation after 5 minutes of distance 25cm place's illumination under the uviol lamp of wavelength 320～400nm.
The two-layer compound support of tissue engineered bone cartilage reparation comprises cartilage layers and subchondral bone layer, described cartilage layers and subchondral bone layer are combined as a whole by photopolymerisable modification hyaluronic acid derivatives, the material of described cartilage layers is the photopolymerisable modification hyaluronic acid derivatives that is loaded with transformed chondrocytes, and the material of described subchondral bone layer is porous boron hydrochlorate bio-vitric support.
Embodiment 3
The preparation method of the two-layer compound support of tissue engineered bone cartilage reparation comprises the following steps: will have photopolymerisable modification hyaluronic acid derivatives and carry out filter-sterilized through 0.02 μ m filter membrane, after mixing homogeneously with Fibroblasts in vitro Induction Transformation chondrocyte, making cell concentration is 1 × 10
8/ ml, hyaluronate sodium mass concentration is that 5%, I2959 mass concentration is 0.05% hydrogel composites, putting into diameter is 5mm, is highly in the mould of 5mm, irradiates after 5 minutes apart from 5cm place through the uviol lamp of wavelength 320～400nm, is cross-linked to form mixed gel; Porous is mixed to Sr borosilicate bio-vitric stentplacement on mixed gel, add 40 μ L to there is photopolymerisable modification hyaluronic acid derivatives, the crosslinked two-layer compound support that obtains tissue engineered bone cartilage reparation after 10 minutes of distance 5cm place's illumination under the uviol lamp of wavelength 320～400nm.
The two-layer compound support of tissue engineered bone cartilage reparation comprises cartilage layers and subchondral bone layer, described cartilage layers and subchondral bone layer are combined as a whole by photopolymerisable modification hyaluronic acid derivatives, the material of described cartilage layers is the photopolymerisable modification hyaluronic acid derivatives that is loaded with transformed chondrocytes, and the material of described subchondral bone layer is that porous is mixed Sr borosilicate bio-vitric support.
Embodiment 4
The outer culture experiment of two-layer compound stake body that tissue engineered bone cartilage is repaired
The two-layer compound support that tissue engineered bone cartilage prepared by embodiment 1 is repaired is at 37 ℃, normal pressure, CO
2in incubator, cultivate and after 4 weeks, carry out Live/Dead cell test and Safranin O and Picrosirius red dyeing.Experimental result is shown in Fig. 2, Fig. 3 and Fig. 4.Fig. 2 is the life or death cytological map of the two-layer compound support In vitro culture repaired of tissue engineered bone cartilage shown in embodiment 4 after 4 weeks; Fig. 3 is the Safranin O colored graph of the two-layer compound support In vitro culture repaired of tissue engineered bone cartilage shown in embodiment 4 after 4 weeks; Fig. 4 is the Picrosirius red colored graph of the two-layer compound support In vitro culture repaired of tissue engineered bone cartilage shown in embodiment 4 after 4 weeks.As can be seen from Figure 2, mesenchymal stem cells MSCs Induction Transformation chondrocyte normal growth in compound rest, this compound rest has excellent biocompatibility.As can be seen from Figure 3, there is chondrify feature in cartilage layers, and GAG forms; From Fig. 4, can find that there is collagen forms.
Embodiment 5
Mechanical property test
Use the comprcssive strength of the subchondral bone layer in Instron strength-testing machine (Model5881, Norwood, MA) test implementation example 1.Sample (n=5) size is 5mm diameter × 5mm height, and loading velocity is 0.5mm/min, and the setting maximum load of testing machine is 10kN.Experimental result: the comprcssive strength of compound rest subchondral bone layer of the present invention is 88 ± 7MPa.Use nanometer mechanics test machine (NanoBionix; Agilent Technologies; Oak Ridge, TN) the cartilage layers elastic modelling quantity described in test implementation example 1, sample (n=5) size is 7mm diameter × 2mm height, loading velocity is 0.02mm/min, experimental result discovery, the elastic modelling quantity of compound rest cartilage layers of the present invention is 15 ± 2kPa.After In vitro culture 6 weeks, elastic modelling quantity is increased to 41 ± 16kPa.
Embodiment 6
Use the comprcssive strength of the subchondral bone layer in Instron strength-testing machine (Model5881, Norwood, MA) test implementation example 3.Sample (n=5) size is 5mm diameter × 5mm height, and loading velocity is 0.5mm/min, and the setting maximum load of testing machine is 10kN.Experimental result: the comprcssive strength of compound rest subchondral bone layer of the present invention is 71 ± 10MPa.Use nanometer mechanics test machine (NanoBionix; Agilent Technologies; Oak Ridge, TN) the cartilage layers elastic modelling quantity described in test implementation example 1, sample (n=5) size is 7mm diameter × 2mm height, loading velocity is 0.02mm/min, experimental result discovery, the elastic modelling quantity of compound rest cartilage layers of the present invention is 15 ± 2kPa.After In vitro culture 6 weeks, elastic modelling quantity is increased to 41 ± 16kPa.
Embodiment 7
The two-layer compound support that tissue engineered bone cartilage in embodiment 2 is repaired is implanted the damaged place of White Rabbit articular cartilage-subchondral bone (4mm diameter × 4mm degree of depth), and 12 weeks after operation, carries out Histological section, Toluidine blue staining.Fig. 5 is the two-layer compound support Toluidine blue staining photo after 12 weeks in vivo that the tissue engineered bone cartilage shown in embodiment 7 is repaired.Result shows in glass supporter, have new bone to grow into, and has hyaline cartilage to form in cartilage layers.
The above-mentioned description to embodiment is can understand and apply the invention for ease of those skilled in the art.Person skilled in the art obviously can easily make various modifications to these embodiment, and General Principle described herein is applied in other embodiment and needn't passes through performing creative labour.Therefore, the invention is not restricted to the embodiment here, those skilled in the art are according to announcement of the present invention, and not departing from the improvement that category of the present invention makes and revise all should be within protection scope of the present invention.
Claims (10)
1. the two-layer compound support that tissue engineered bone cartilage is repaired, it is characterized in that: comprise cartilage layers and subchondral bone layer, described cartilage layers and subchondral bone layer are combined as a whole by photopolymerisable modification hyaluronic acid derivatives, the material of described cartilage layers is the photopolymerisable modification hyaluronic acid derivatives that is loaded with transformed chondrocytes, and the material of described subchondral bone layer is Porous bioglass support.
2. the two-layer compound support that tissue engineered bone cartilage according to claim 1 is repaired, is characterized in that: the density of described transformed chondrocytes is 5 × 10
6～1 × 10
8/ mL.
3. the two-layer compound support that tissue engineered bone cartilage according to claim 1 is repaired, is characterized in that: described transformed chondrocytes is to be obtained by mesenchymal stem cells MSCs, embryonic stem cell, fat stem cell and/or fibroblast Induction Transformation.
4. the two-layer compound support that tissue engineered bone cartilage according to claim 1 is repaired, is characterized in that: described transformed chondrocytes is the chondrocyte of the primary autologous and/or allogenic bone marrow mesenchymal stem Induction Transformation to the 4th generation of In vitro culture.
5. the two-layer compound support that tissue engineered bone cartilage according to claim 1 is repaired, is characterized in that: described cartilage layers can interpolation or combined with mesenchymal stem cells, embryonic stem cell, fat stem cell, somatomedin or transgene component in one or more.
6. the two-layer compound support that tissue engineered bone cartilage according to claim 1 is repaired, is characterized in that: described Porous bioglass support comprises porous silicate bio-vitric support, porous boron silicate bio-vitric support or porous boron hydrochlorate bio-vitric support; In composition, add one or more in micro-Cu, Zn, Ba, Sr, Ag, Fe, F or Mn;
Or described Porous bioglass support employing is water base and/or organic facies is prepared;
Or the intensity of described Porous bioglass support is 10～100MPa, porosity is 45～75%, and aperture is 100～500 μ m.
7. the preparation method of the two-layer compound support of the arbitrary described tissue engineered bone cartilage reparation of claim 1 to 6, it is characterized in that: comprise the following steps: after thering is photopolymerisable modification hyaluronic acid derivatives and mixing homogeneously with external evoked transformed chondrocytes, form mixed gel through ultraviolet polymerization; Porous bioglass stentplacement, on mixed gel, is added and has photopolymerisable modification hyaluronic acid derivatives, after illumination is crosslinked, obtain the two-layer compound support that tissue engineered bone cartilage is repaired.
8. the preparation method of the two-layer compound support that tissue engineered bone cartilage according to claim 7 is repaired, it is characterized in that: the preparation method of described photopolymerisable modification hyaluronic acid derivatives comprises the following steps: the aqueous solution of sodium hyaluronate that is 1～5% by mass concentration mixes with methacrylic anhydride after freezing point is carried out stirring and dissolving again, the concentration of described methacrylic anhydride is 20 times of hyaluronate sodium molar concentration, regulate pH to make pH value be greater than all the time 8, freezing point stirring reaction 24 hours, putting into the deionized water of 50 times of volumes of dialyzer dialyses 3 days, lyophilization is mixed to get photopolymerisable modification hyaluronic acid derivatives with normal saline and cross-linking agent I2959 after 48 hours again.
9. the preparation method of the two-layer compound support that tissue engineered bone cartilage according to claim 8 is repaired, is characterized in that: the concentration of described hyaluronate sodium is 2wt%.
10. the preparation method of the two-layer compound support that tissue engineered bone cartilage according to claim 8 is repaired, is characterized in that: the molecular weight of described hyaluronate sodium is 66～90 kilodaltons;
Or described in to have the hyaluronic concentration of modification in photopolymerisable modification hyaluronic acid derivatives be 0.05wt%;
Or the concentration of described cross-linking agent I2959 is 0.05wt%;
Or the number volume ratio of described transformed chondrocytes or described photopolymerisable modification hyaluronic acid derivatives is 40 × 10
6/ mL;
Or the method for described ultraviolet polymerization is ultra violet lamp, wavelength 320～400nm, intensity is 8900 μ W/cm
2～21700 μ W/cm
2, irradiate height 5～25cm, the time is 5～15 minutes;
Or the crosslinked method of described illumination is ultra violet lamp, wavelength 320～400nm, intensity is 8900 μ W/cm
2～21700 μ W/cm
2, irradiate height 5～25cm, the time is 5～15 minutes.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090076624A1 (en) * | 2007-09-14 | 2009-03-19 | Mohamed Naushad Rahaman | Synthetic osteochondral composite and method of fabrication thereof |
| CN101773685A (en) * | 2010-02-04 | 2010-07-14 | 同济大学 | Method for preparing high-elasticity support promoting cartilage regeneration in situ |
| CN101914225A (en) * | 2010-08-05 | 2010-12-15 | 江南大学 | Method for preparing hyaluronic acid gel by using macromolecule photocrosslinking agent |
| WO2013171736A1 (en) * | 2012-05-17 | 2013-11-21 | Cartiheal(2009) Ltd | Biomatrix hydrogels and methods of use thereof |
-
2014
- 2014-02-26 CN CN201410066627.7A patent/CN103877614A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090076624A1 (en) * | 2007-09-14 | 2009-03-19 | Mohamed Naushad Rahaman | Synthetic osteochondral composite and method of fabrication thereof |
| CN101773685A (en) * | 2010-02-04 | 2010-07-14 | 同济大学 | Method for preparing high-elasticity support promoting cartilage regeneration in situ |
| CN101914225A (en) * | 2010-08-05 | 2010-12-15 | 江南大学 | Method for preparing hyaluronic acid gel by using macromolecule photocrosslinking agent |
| WO2013171736A1 (en) * | 2012-05-17 | 2013-11-21 | Cartiheal(2009) Ltd | Biomatrix hydrogels and methods of use thereof |
Non-Patent Citations (2)
| Title |
|---|
| STEPHANIE K. SEIDLITS等: ""The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation"", 《BIOMATERIALS》, vol. 31, no. 14, 19 February 2010 (2010-02-19) * |
| 邓天政等: ""体外构建组织工程骨-软骨复合组织"", 《中国组织工程研究》, vol. 17, no. 8, 19 February 2013 (2013-02-19), pages 1354 - 1361 * |
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