CN1314460C - Method for compounding aquo-gel to porous tissue engineering rack - Google Patents
Method for compounding aquo-gel to porous tissue engineering rack Download PDFInfo
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Abstract
The present invention discloses a method for compounding hydrogel to a porous tissue engineering rack, which comprises the following steps: under the state of sol, hydrogel is led into a porous rack, and then an external condition is altered to convert the sol into gel to obtain the porous rack with compounded hydrogel. As required, cell growth factors for cell growth and differentiation can be added into the hydrogel sol. The rack prepared by the method has the excellent mechanical performance of the porous rack and the excellent biocompatible performance of a hydrogel rack simultaneously. The present invention has a wide application range and is suitable for various structures, various porous racks composed of various structures, hydrogel and various cells. The present invention has the advantages of simple preparation process and good repeatability. The prepared rack can be widely applied to the field of tissue engineering.
Description
Technical field
The present invention relates to hydrogel is compound to method in the porous tissue engineering scaffold.
Background technology
Utilize natural or synthetic material analog cell epimatrix, it is combined with cell, so promotion and inducing cell growth formative tissue or organ, repair or the damaged purpose of reconstruction human organ to reach, become the method for widely admitting in the Tissue Engineering Study.Cytoskeletal preparation and combine with intercellular is the major issue that organizational project need solve.
According to the morphosis of tissue regeneration support, a large amount of at present supports that use are divided into two classes: porous support and hydrogel support.Porous support is made up of the gap of material and storeroom, and under the dry state situation, this gap is generally air; In cell culture environment, this gap is generally culture medium.At present, developed the porous support that several different methods prepares various structures, as porogen method, thermally induced phase separation, fibrage method, emulsion lyophilization, microsphere method of piling, 3 dimension-print processes etc.Porous support has the degradation rate of mechanical strength and easy-regulating preferably; Easy macroscopical plastotype also can keep good macroshape during tissue regeneration.But also there is significant disadvantages in this class support.Under condition of in vitro culture, most somatic cells have the adherent growth characteristic, but the growth of being surrounded by substrate under the adherent growth of cell and its interior state has tangible difference, easier the dedifferenting of cell and lose biological function.Cell owing to the restriction that is subjected to the growing space comes in contact inhibition, can't continue division after long-time growth division on the hole wall, cause the cell number that can adhere on the porous support little than the tissue of native state, and the inequality that distributes.In addition, because the biologically inert of most synthetic materials also must carry out the improvement of biocompatibility to support, increased preparation technology's complexity.The hydrogel support also is to be made of material and gap, and this material is generally natural or synthetic hydrophilic macromolecule.Polymeric material forms very thin gel network, contains a large amount of water therebetween.But these water are not freely, are in highly bonded state because of being subjected to the high molecular constraint of aquation on every side.Therefore, hydrogel class support and extracellular matrix have similar structure and performance, and especially the natural macromolecular hydrogel material has good biocompatibility, can more approach intravital growing environment for the growth of cell and differentiation provide.Simultaneously, cell is dispersed in the hydrogel, has bigger growing space.There is significant disadvantages equally in this class support, and promptly mechanical strength is generally relatively poor, and macroscopical plastotype difficulty also can't keep good macroshape during tissue regeneration, therefore can not effectively control the shape of regeneration organ; Simultaneously, operability is relatively poor relatively.
Summary of the invention
The purpose of this invention is to provide a kind of operating process method in the porous tissue engineering scaffold that hydrogel is compound to simple, applied widely, to obtain to have simultaneously the tissue engineering bracket of good mechanical properties and cell compatibility.
The method that hydrogel is compound in the porous tissue engineering scaffold of the present invention is under collosol state hydrogel to be imported in the porous support, changes external condition then and makes collosol and gelization, obtains the porous support of composite aquogel.
Of the present invention hydrogel is compound to method in the porous tissue engineering scaffold, three kinds of technical solutions are arranged.
Scheme one, hydrogel is compound to method in the porous tissue engineering scaffold, specifically may further comprise the steps:
1) under 4 ℃～120 ℃ agar is dissolved in the water and is prepared into the colloidal sol of hydrogel, mix with isopyknic celliferous culture medium solution, the colloidal sol bulking value concentration that obtains is 0.5～5%;
2) porous support is carried out sterilization treatment, the moistening in advance of reuse phosphate buffer;
3) colloidal sol is added drop-wise to the surface of porous support, under gravity and capillarity, colloidal sol is imported internal stent; Or support is immersed in the hydrogel colloidal sol, hydrogel is adsorbed onto porous support inside;
4) support that will be compounded with colloidal sol was placed 10 minutes down at 15 ℃～40 ℃ at least, the porous support of hydrogel that promptly obtained load.
Usually in order to improve the biology performance of this compound rest, can in agar, sneak into gelatin or/and cell growth factor, the ratio of gelatin consumption and agar consumption is 1: 1～1: 100, the ratio of cell growth factor consumption and agar consumption is 1: 10,000～1: 1,000,000.Said cell growth factor can be that alkalescence facilitates fibroblast growth factor, acidity to facilitate fibroblast growth factor, bone morphogenetic protein(BMP) or transforming factor β.
Scheme two, hydrogel is compound to method in the porous tissue engineering scaffold, specifically may further comprise the steps:
1) under 4 ℃～120 ℃ sodium alginate is dissolved in the water and is prepared into the colloidal sol of hydrogel, mix with isopyknic celliferous culture medium solution, the colloidal sol bulking value concentration that obtains is 0.5～5%;
2) porous support is carried out sterilization treatment, the moistening in advance of reuse phosphate buffer;
3) colloidal sol is added drop-wise to the surface of porous support, under gravity and capillarity, colloidal sol is imported internal stent; Or support is immersed in the hydrogel colloidal sol, hydrogel is adsorbed onto porous support inside;
4) will be compounded be placed in 0.1%～2% calcium lactate solution at least 5 minutes of colloidal sol, the porous support of hydrogel that promptly obtained load.
Usually in order to improve the biology performance of this compound rest, can in sodium alginate, sneak into gelatin or/and cell growth factor, the ratio of gelatin consumption and sodium alginate consumption is 1: 1～1: 100, the ratio of cell growth factor consumption and sodium alginate consumption is 1: 10,000～1: 1,000,000.Said cell growth factor can be that alkalescence facilitates fibroblast growth factor, acidity to facilitate fibroblast growth factor, bone morphogenetic protein(BMP) or transforming factor β.
Scheme three, hydrogel is compound to method in the porous tissue engineering scaffold, specifically may further comprise the steps:
1) under 4 ℃～120 ℃ with the phosphoglyceride disodium salt mixture of chitosan and 1～5 times of weight, or polyoxyethylene-polyoxypropylene copolymer, or PNIPAM is dissolved in the water and is prepared into the colloidal sol of hydrogel, mix with isopyknic celliferous culture medium solution, the bulking value concentration that obtains colloidal sol is 0.5～5% again;
2) porous support is carried out sterilization treatment, the moistening in advance of reuse phosphate buffer;
3) colloidal sol is added drop-wise to the surface of porous support, under gravity and capillarity, colloidal sol is imported internal stent; Or support is immersed in the hydrogel colloidal sol, hydrogel is adsorbed onto porous support inside;
4) will be compounded be placed under 30 ℃～40 ℃ conditions at least 5 minutes of colloidal sol, the porous support of hydrogel that promptly obtained load.
Said porous support can be the polymer class porous support in above-mentioned three kinds of technical solutions of the present invention, comprise polylactic acid, collagen, lactic acid-ethanol copolymer, polycaprolactone, polyurethane or polystyrene holder, can be the ceramic-like porous support also, comprise porous hydroxyapatite, tricalcium phosphate or bio-vitric support.
Said cell includes but not limited to chondrocyte, osteoblast, hepatocyte, fibroblast or smooth muscle cell in above-mentioned three kinds of technical solutions of the present invention.
The present invention combines the advantage of porous support and hydrogel support, both keep the favorable mechanical performance of porous support, macroscopical plastotype performance and macroshape hold facility, had the good imitative extracellular matrix structure and the bio-compatible performance of hydrogel support again.By changing the component of colloidal sol, for different cells, can add various promoting growth of cell and differentiation factor targetedly, thereby further improve the biology performance of support, method is simply effective.The present invention is applied widely, is applicable to the porous support and the hydrogel of multiple structure and composition, and various kinds of cell; Preparation technology is simple, good reproducibility, and prepared support can be widely used in field of tissue engineering technology.The present invention provides a kind of practicable method for the best combination of cell in the Tissue Engineering Study and support.Porous support by the present invention preparation can be widely used in the reconstruction and the regeneration of multiple tissue such as skin, cartilage, bone, liver, blood vessel, trachea, esophagus, tendon, cardiac valve or organ.
Description of drawings
Fig. 1 is the distribution situation of chondrocyte in the compound polylactic acid bracket of sodium alginate gel;
Fig. 2 is the distribution situation of chondrocyte in the compound polylactic acid bracket of agar gel;
Fig. 3 is the distribution situation of chondrocyte in agar and the compound polylactic acid bracket of gelatin gel;
Fig. 4 is the distribution situation of chondrocyte in basic fibroblast growth factor and the compound polylactic acid bracket of agar gel;
Fig. 5 is the distribution situation of osteoblast in acid fibroblast growth factor and sodium alginate gel composite collagen support;
Fig. 6 a is the laser confocal microscope photo that 100 microns polylactic acid porous scaffolds (black part) are filled sodium alginate hydrogel (white portion);
Fig. 6 b is the laser confocal microscope photo that 300 microns polylactic acid porous scaffolds (black part) are filled sodium alginate hydrogel (white portion);
Fig. 7 a is the laser confocal microscope photo (rhodamine dyeing) of collagen porous support;
Fig. 7 b is the laser confocal microscope photo (fluorescein sodium dyeing) that is distributed in the sodium alginate in the collagen porous support;
Fig. 7 c is the composite diagram of Fig. 6 a and Fig. 6 b.
The specific embodiment
Following example further specifies the present invention, but these examples are not used for limiting the present invention.
Configuration bulking value concentration is 2% sodium alginate colloidal sol, puts into 120 ℃ of high temperature sterilizes of autoclave 30 minutes, be cooled to room temperature after, sodium alginate is mixed with the chondrocyte suspension, the concentration that makes final sodium alginate is 1%.With average pore size is 300 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.The polylactic acid bracket surface after processing with colloidal sol and cell mixture dropping.Under gravity and capillarity, colloidal sol enters internal stent.To prop up then and be placed in 0.5% the calcium lactate solution 15 minutes, by the crosslinked sodium alginate gelization that makes of calcium ion.At last, compound rest is placed 5%CO
2, hatched 7 days in 37 ℃ of homothermic incubators, the survival of test cell and extracellular matrix secretion situation, and compare with the blank polylactic acid bracket that does not import the sodium alginate hydrogel see Table 1.The form of cell and distribution situation are seen Fig. 1 in the support.As seen chondrocyte can be evenly distributed in the polylactic acid bracket that is compounded with the sodium alginate hydrogel, and cell activity is higher.
Import the sodium alginate hydrogel in table 1 polylactic acid porous scaffold to the chondrocyte Effect on Performance
| Support | The MTT activity | Hydroxyproline (mg/g) | GAG(mg/g) |
| Blank polylactic acid bracket | 100 | 6.22 | 11.47 |
| The compound polylactic acid bracket of sodium alginate | 146.9 | 6.60 | 11.11 |
MTT refers to that (cytoactive of measurement is 100 to carry out normalized with blank polylactic acid bracket for 3-(4, the 5-dimethylthiazole)-2,5-diphenyl tetrazole bromine salt (tetrazolium bromide).GAG represents chondroitin sulfate.The unit of hydroxyproline and GAG is the milligram number of emiocytosis in every gram support.
Embodiment 2 polylactic acid porous scaffolds import the agar hydrogel that contains chondrocyte
Configuration bulking value concentration is 2% agar colloidal sol, puts into 120 ℃ of high temperature sterilizes of autoclave 30 minutes, be cooled to about 45 ℃ after, agar colloidal sol is mixed with the chondrocyte suspension, the concentration that makes final agar is 1%.With average pore size is 300 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.The polylactic acid bracket surface of mixture dropping after processing with colloidal sol and cell.Under gravity and capillarity, colloidal sol enters internal stent.Then support is placed down at 25 ℃ and made agar gelization in 15 minutes.At last, compound rest is placed 5%CO
2, hatched 7 days in 37 ℃ of homothermic incubators, the survival of test cell and extracellular matrix secretion situation, and compare with the blank polylactic acid bracket that does not import the agar hydrogel see Table 2.The form of cell and distribution situation are seen Fig. 2 in the support.As seen chondrocyte can be evenly distributed in the polylactic acid bracket that is compounded with the agar hydrogel, and cell activity is higher, and the secretory volume of hydroxyproline and GAG is bigger.
Import the agar hydrogel in table 2 polylactic acid porous scaffold to the chondrocyte Effect on Performance
| Support | The MTT activity | Hydroxyproline (mg/g) | GAG(mg/g) |
| Blank polylactic acid bracket | 100 | 6.22 | 11.47 |
| The compound polylactic acid bracket of agar | 201.5 | 9.79 | 67.25 |
Embodiment 3 polylactic acid porous scaffolds import agar and the gelatin hydrogel that contains chondrocyte
Configuration bulking value concentration respectively is 2% agar, gelatin mixed sols, put into 120 ℃ of high temperature sterilizes of autoclave 30 minutes, after being cooled to about 45 ℃, agar colloidal sol, gelatin colloidal sol are mixed with the chondrocyte suspension, make that the concentration of final agar and gelatin respectively is 1%.With average pore size is 300 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.The polylactic acid bracket surface of mixture dropping after processing with colloidal sol and cell.Under gravity and capillarity, colloidal sol enters internal stent.Then support is placed down at 25 ℃ and made agar gelization in 15 minutes.At last, compound rest is placed 5%CO
2, hatched 7 days in 37 ℃ of homothermic incubators, the survival of test cell and extracellular matrix secretion situation, and compare with blank polylactic acid bracket that does not import the agar hydrogel and the simple polylactic acid bracket that imports agar see Table 3.The form of cell and distribution situation are seen Fig. 3 in the support.As seen chondrocyte can be evenly distributed in the polylactic acid bracket that is compounded with agar and gelatin hydrogel, and cell activity is higher, and the secretory volume of hydroxyproline and GAG is bigger.
Import agar and gelatin hydrogel in table 3 polylactic acid porous scaffold to the chondrocyte Effect on Performance
| Support | The MTT activity | Hydroxyproline (mg/g) | GAG(mg/g) |
| Blank polylactic acid bracket | 100 | 6.22 | 11.47 |
| The compound polylactic acid bracket of agar | 201.5 | 9.79 | 67.25 |
| Agar and gelatin-compounded polylactic acid bracket | 264.5 | -- | 77.62 |
Because gelatin molecule contains a large amount of hydroxyprolines, thus add can't distinguish behind the gelatin hydroxyproline be emiocytosis or gelatin in.
Embodiment 4 polylactic acid porous scaffolds import and contain chondrocyte and the alkaline agar hydrogel of facilitating fibroblast growth factor
Configuration bulking value concentration is 2% agar colloidal sol, put into 120 ℃ of high temperature sterilizes of autoclave 30 minutes, after being cooled to about 45 ℃, agar colloidal sol and basic fibroblast growth factor (bFGF) are mixed with the chondrocyte suspension, the concentration that makes final agar is 1%, and bFGF concentration is 100 μ g/mL.With average pore size is 300 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.The polylactic acid bracket surface of mixture dropping after processing with colloidal sol and cell.Under gravity and capillarity, colloidal sol enters internal stent.Then support is placed down at 25 ℃ and made agar gelization in 15 minutes.At last, compound rest is placed 5%CO
2, hatched 7 days in 37 ℃ of homothermic incubators, the survival of test cell and extracellular matrix secretion situation, and compare with blank polylactic acid bracket and the simple polylactic acid bracket that imports the agar hydrogel see Table 4.The form of cell and distribution situation are seen Fig. 4 in the support.As seen chondrocyte can be evenly distributed in the polylactic acid bracket that is compounded with the agar hydrogel, and cell activity has the raising of highly significant, and the secretory volume of hydroxyproline and GAG is filled with simple agar does not have significantly difference.
Import bFGF and agar water-setting chamber in table 4 polylactic acid porous scaffold to the chondrocyte Effect on Performance
| Support | The MTT activity | Hydroxyproline (mg/g) | GAG(mg/g) |
| Blank polylactic acid bracket | 100 | 6.22 | 11.47 |
| The compound polylactic acid bracket of agar | 201.5 | 9.79 | 67.25 |
| The compound polylactic acid bracket of agar and bFGF | 370.1 | 9.53 | 55.90 |
Embodiment 5 collagen porous supports import and contain osteoblast and the acid agar hydrogel of facilitating fibroblast growth factor
Configuration bulking value concentration is 2% sodium alginate colloidal sol, put into 120 ℃ of high temperature sterilizes of autoclave 30 minutes, after being cooled to room temperature, facilitate fibroblast growth factor mixing (aFGF) to mix sodium alginate and acidity with the osteoblast suspension, the concentration that makes final sodium alginate is 1%, and aFGF concentration is 30 μ g/mL.Average pore size is about 100 microns, and porosity is 99.9%, and thick about 2 millimeters collagen thin slice support places tri-distilled water to soak, and makes the support swelling.Afterwards support is immersed in the mixed liquor of colloidal sol and cell and shake half an hour, make sodium alginate and chondrocyte enter collagen scaffold inside.To prop up then and be placed in 0.1% the calcium lactate solution 10 minutes, by the crosslinked sodium alginate gelization that makes of calcium ion.At last, compound rest is placed 5%CO
2, hatched 7 days in 37 ℃ of homothermic incubators, the form of cell and distribution situation are seen Fig. 5 in the support.
Embodiment 6 polylactic acid porous scaffolds import the sodium alginate hydrogel
At room temperature pre-configured bulking value concentration is 1%, 2% sodium alginate colloidal sol.With average pore size is 300 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.With the polylactic acid bracket surface of sodium alginate colloidal sol dropping after processing.Under gravity and capillarity, colloidal sol enters internal stent.To prop up then and be placed in 0.5% the calcium lactate solution 60 minutes, by the crosslinked sodium alginate gelization that makes of calcium ion.After rack surface cleaned with tri-distilled water, drying was weighed.Utilize following formula to calculate the importing rate:
Importing rate=actual bracket weightening finish/Theoretical Calculation support weightening finish
Found that in sodium alginate hydrogel concentration to be respectively 1% and at 2% o'clock, the importing rate is respectively 92% and 94%.
Embodiment 7 polylactic acid porous scaffolds import the agar hydrogel
At room temperature pre-configured bulking value concentration is 1%, 2% agar colloidal sol.With average pore size is 300 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.With concentration is that 1% or 2% agar colloidal sol drips the polylactic acid bracket surface after processing.Under gravity and capillarity, colloidal sol enters internal stent.Then support is placed down at 10 ℃ and made agar gelization in 60 minutes.After the rack surface of importing hydrogel cleaned with tri-distilled water, drying was weighed, and calculates the importing rate.Found that in agar hydrogel concentration to be respectively 1% and at 2% o'clock, the importing rate is respectively 100% and 83%.
Embodiment 8 polylactic acid porous scaffolds import aquagel
200mg chitosan (removing the acetyl degree is 91%) is dissolved in the 9ml 0.1M hydrochloric acid solution.560mg phosphoglyceride disodium is dissolved in the 1ml deionized water, and solution dropwise adds in the chitosan solution, forms homogeneous solution.With average pore size is 300 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.The support of handling is dipped in the aquagel, handled 5 minutes at 37 ℃ after 20 minutes, make chitosan gel rubberization.After the rack surface of importing hydrogel cleaned with tri-distilled water, drying was weighed, and recording the importing rate is 84.6%.
Implement of the influence of 9 polylactic acid porous scaffold apertures to hydrogel importing rate
At room temperature pre-configured bulking value concentration is 2% sodium alginate colloidal sol.With average pore size is 300 microns and 100 microns, and thickness is the polylactic acid bracket thin slice of 2-4 millimeter, places 75% alcoholic solution to soak, and by the way of evacuation, the air in the support is removed, and makes alcoholic solution soak into support fully.Soak in alcoholic solution after one day, it is that 7.4 phosphate buffer soaked 1 day that support is put into pH, during change liquid 3 times, to displace the ethanol in the support fully.Sodium alginate colloidal sol is mixed with fluorescein sodium, and the concentration that makes final sodium alginate is 1%, and the concentration of fluorescein sodium is 0.01%, and drips the polylactic acid bracket surface after processing.Under gravity and capillarity, colloidal sol enters internal stent.To prop up then and be placed in 2% the calcium lactate solution 5 minutes, by the crosslinked sodium alginate gelization that makes of calcium ion.Adopt laser confocal microscope to observe hydrogel distribution situation therein, see Fig. 6 a, Fig. 6 b.After rack surface cleaned with tri-distilled water, drying was weighed.Found that in the aperture of polylactic acid porous scaffold the importing rate is respectively 82% and 92% when being 100 microns and 300 microns.Illustrate that the large aperture helps the importing of hydrogel.
Embodiment 10 collagen porous supports import the sodium alginate hydrogel
Average pore size is about 100 microns, and porosity is 99.9%, and thick about 2 millimeters rhodamine labelling collagen thin slice support places tri-distilled water to soak, and makes the support swelling.Again support is immersed the concentration be dissolved with 0.01% fluorescein sodium and be in 1% or 2% the sodium alginate colloidal sol and shake half an hour, make sodium alginate colloidal sol enter collagen scaffold inside.To prop up then and be placed in 0.1% the calcium lactate solution 50 minutes, by the crosslinked sodium alginate gelization that makes of calcium ion.The employing laser confocal microscope is observed, and sees Fig. 7 a, Fig. 7 b, Fig. 7 c.After rack surface cleaned with tri-distilled water, drying was weighed.Found that in sodium alginate hydrogel concentration to be respectively 1% and at 2% o'clock, the importing rate is respectively 100% and 84%.
Claims (10)
1. hydrogel is compound to the method in the porous tissue engineering scaffold, this method may further comprise the steps:
1) under 4 ℃～120 ℃ agar is dissolved in the water and is prepared into the colloidal sol of hydrogel, mix with isopyknic celliferous culture medium solution, the colloidal sol bulking value concentration that obtains is 0.5～5%;
2) porous support is carried out sterilization treatment, the moistening in advance of reuse phosphate buffer;
3) colloidal sol is added drop-wise to the surface of porous support, under gravity and capillarity, colloidal sol is imported internal stent; Or support is immersed in the hydrogel colloidal sol, hydrogel is adsorbed onto porous support inside;
4) support that will be compounded with colloidal sol was placed 10 minutes under 15C～40C at least, the porous support of hydrogel that promptly obtained load.
2. according to claim 1 hydrogel is compound to method in the porous tissue engineering scaffold, it is characterized in that in step 1), in agar, sneak into gelatin or/and cell growth factor, the ratio of gelatin weight and agar weight is 1: 1～1: 100, the ratio of cell growth factor weight and agar weight is 1: 10,000～1: 1,000,000.
3 according to claim 2ly are compound to method in the porous tissue engineering scaffold with hydrogel, it is characterized in that said cell growth factor is that alkalescence facilitates fibroblast growth factor, acidity to facilitate fibroblast growth factor, bone morphogenetic protein(BMP) or transforming factor β.
4. according to claim 1 hydrogel is compound to method in the porous tissue engineering scaffold, it is characterized in that said porous support is the polymer class porous support, comprise polylactic acid, collagen, lactic acid-ethanol copolymer, polycaprolactone, polyurethane or polystyrene holder, or be the ceramic-like porous support, comprise porous hydroxyapatite, tricalcium phosphate or bio-vitric support; Said cell is chondrocyte, osteoblast, hepatocyte, fibroblast or smooth muscle cell.
5. hydrogel is compound to the method in the porous tissue engineering scaffold, this method may further comprise the steps:
1) under 4 ℃～120 ℃ sodium alginate is dissolved in the water and is prepared into the colloidal sol of hydrogel, mix with isopyknic celliferous culture medium solution, the colloidal sol bulking value concentration that obtains is 0.5～5%;
2) porous support is carried out sterilization treatment, the moistening in advance of reuse phosphate buffer;
3) colloidal sol is added drop-wise to the surface of porous support, under gravity and capillarity, colloidal sol is imported internal stent; Or support is immersed in the hydrogel colloidal sol, hydrogel is adsorbed onto porous support inside;
4) will be compounded be placed in 0.1%～2% calcium lactate solution at least 5 minutes of colloidal sol, the porous support of hydrogel that promptly obtained load.
6. according to claim 5 hydrogel is compound to method in the porous tissue engineering scaffold, it is characterized in that in step 1), in sodium alginate, sneak into gelatin or/and cell growth factor, the ratio of gelatin weight and sodium alginate weight is 1: 1～1: 100, the ratio of cell growth factor weight and sodium alginate weight is 1: 10,000～1: 1,000,000.
7. according to claim 6 hydrogel is compound to method in the porous tissue engineering scaffold, it is characterized in that described cell growth factor is that alkalescence facilitates fibroblast growth factor, acidity to facilitate fibroblast growth factor, bone morphogenetic protein(BMP) or transforming factor β.
8. according to claim 5 hydrogel is compound to method in the porous tissue engineering scaffold, it is characterized in that said porous support is the polymer class porous support, comprise polylactic acid, collagen, lactic acid-ethanol copolymer, polycaprolactone, polyurethane or polystyrene holder, or be the ceramic-like porous support, comprise porous hydroxyapatite, tricalcium phosphate or bio-vitric support; Said cell is chondrocyte, osteoblast, hepatocyte, fibroblast or smooth muscle cell.
9. hydrogel is compound to the method in the porous tissue engineering scaffold, this method may further comprise the steps:
1) under 4 ℃～120 ℃ with the phosphoglyceride disodium salt mixture of chitosan and 1～5 times of weight, or polyoxyethylene-polyoxypropylene copolymer, or PNIPAM is dissolved in the water and is prepared into the colloidal sol of hydrogel, mix with isopyknic celliferous culture medium solution, the bulking value concentration that obtains colloidal sol is 0.5～5% again;
2) porous support is carried out sterilization treatment, the moistening in advance of reuse phosphate buffer;
3) colloidal sol is added drop-wise to the surface of porous support, under gravity and capillarity, colloidal sol is imported internal stent; Or support is immersed in the colloidal sol of hydrogel, hydrogel is adsorbed onto porous support inside;
4) will be compounded be placed under 30 ℃～40 ℃ conditions at least 5 minutes of colloidal sol, the porous support of hydrogel that promptly obtained load.
10. according to claim 9 hydrogel is compound to method in the porous tissue engineering scaffold, it is characterized in that said porous support is the polymer class porous support, comprise polylactic acid, collagen, lactic acid-ethanol copolymer, polycaprolactone, polyurethane or polystyrene holder, or be the ceramic-like porous support, comprise porous hydroxyapatite, tricalcium phosphate or bio-vitric support; Said cell is chondrocyte, osteoblast, hepatocyte, fibroblast or smooth muscle cell.
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