CN108619573A - A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs - Google Patents
A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs Download PDFInfo
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Abstract
The present invention relates to medical preparation technical fields, and in particular to a kind of preparation method of the collagen tussah silk fibroin compound rest of load BMSCs.The preparation method of the collagen tussah silk fibroin compound rest of load BMSCs includes：Step 1: the preparation of collagen solution；Step 2: the preparation of tussah silk fibroin solution；Step 3: the preparation of BMSCs suspensions；Step 4: the preparation of compound rest.The collagen tussah silk fibroin compound rest of load BMSCs obtained by the present invention has good biocompatibility, and no inflammation reaction, toxic reaction, immunological rejection etc., adhesive forces are strong, can promote skin wound reparation.The compound rest can keep the humidity of the surface of a wound simultaneously, prevent the loss of body fluid and moisture；Biological barrier can be provided, trauma surface infestation, bacterium intrusion are prevented；And has certain flexibility and tensile property, its function and form are kept during organization healing.
Description
Technical field
The present invention relates to medical preparation technical fields, and in particular to a kind of collagen-tussah silk fibroin of load BMSCs is multiple
Close the preparation method of holder.
Background technology
The progress of Study Progress of Stem Cells, especially mescenchymal stem cell for wound repair provide new thinking and
Strategy.Recent researches show that mesenchymal stem cell (BMSCs) can break up epidermal cell, corium under skin microenvironment
Cell, vascular endothelial cell and cutaneous appendages cell etc. provide new seed cell source for tissue engineering skin, and
And basis and clinical research prompt mescenchymal stem cell promote skin repair and depend primarily on its secretion as Graftskin
Active material.As hypoxia condition culture people's Amniotic Fluid-derived Mesenchymal Stem Cells culture solution can by TGF-beta/SMAD2 and
PI3K/Akt signal paths promote skin wound healing.In addition, research confirms that mescenchymal stem cell can secrete the thin of anti-fibrosis
Intracellular cytokine and growth factor, immunological regulation and anti-inflammatory, secretion nitric oxide, the EMT for inhibiting vascular endothelial cell and flesh are at fiber
The differentiation of cell promotes the revascularization of ischemic tissue, inhibits skin excess fibrosis, one is provided again for the tissue of damage
Raw microenvironment improves wound healing quality, is the new strategy of wound repair treatment.
It includes fibroin albumen, collagen, chitosan, sea that wound repair, which treats common bio-medical material mainly, at present
Alginates, hyaluronic acid etc..Collagen is a kind of natural hmw protein, abundant in people's in-vivo content, main to be distributed
In the tissues such as the bone of animal, tendon, ligament, skin and cornea.Be conducive to cell adhesion by the holder of raw material of collagen
And cell is risen and supports protective effect, and be conducive to the importing of foreign cell.But collagen scaffold has degradation speed fast, new
The moulding not good enough, deficiencies such as mechanical stress intensity is low of raw tissue, are often used in combination with other materials at present.
Fibroin albumen has good moisturizing hygroscopicity, can prevent the loss of body fluid and moisture, and the surface of a wound is made to keep certain
Humidity, be a kind of common biomaterial.The study found that the growth that fibroin material does not influence embryonic mesenchymal stem cells is special
Property, surface marker and multi-lineage potential, there is good biocompatibility.Tussah silk peptide, which removes, has bombyx mori silk fibroin in biology doctor
Outside the advantages of medicine field, it also contains sour (RGD) the tripeptides repetitive sequence of a large amount of arginine-glycine-aspartic acids, can be used as thin
Born of the same parents' receptor occurs specificity interaction with the integrin of cell surface, promotes the adherency of cell and receptor.Tussah silk peptide
The good biocompatibility of albumen and special sexual compatibility to cell provide a kind of high property for fields such as cellular system engineerings
Energy, low cost, the bioactive materials with China's characteristic.
However, the bio-medical material for being used for wound repair treatment in the prior art exists, the ability broken up is bad to be lacked
It falls into, therefore, there is an urgent need for be improved to the prior art.
Invention content
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of collagen-tussah silk peptide egg of load BMSCs
The preparation method of white compound rest, collagen-tussah silk fibroin compound rest tool of the load BMSCs obtained by the preparation method
There is good biocompatibility, the ability of derivant induction differentiation can be improved, and then the advantages of wound healing.
To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs is provided, it includes following step
Suddenly：
Step 1: the preparation of collagen solution：Collagen is extracted to collagen-rich animal tissue, is obtained
To crude extract, crude extract is then subjected to purification process, obtains pure collagen solution；
Step 2: the preparation of tussah silk fibroin solution：Tussah silk is subjected to degumming process, then will treated tussah silk
It is put into lysate and dissolves by heating, obtain tussah silk fibroin crude extract, then carry out tussah silk fibroin crude extract
Tussah silk fibroin solution is made in filtering, dialysis treatment；
Step 3: the preparation of BMSCs suspensions：It rinses the ossis from animal repeatedly with DMEM culture mediums, obtains bone
Myeloid tissue flushing liquor, then centrifuges myeloid tissue flushing liquor, and cell is resuspended in culture solution after then discarding supernatant liquid,
Inoculation, is subsequently placed in incubator and carries out original cuiture, carries out full dose for the first time after a certain period of time and changes liquid, retains attached cell, it
Liquid is changed every certain time afterwards, when the attached cell of reservation reaches certain percentage fusion, is digested with pancreatin, and after being centrifuged
Cell secondary culture is carried out by a certain percentage, is then chosen BMSCs, after being digested and being centrifuged, is resuspended, is obtained with culture medium
BMSCs suspensions；
Step 4: the preparation of compound rest：By tussah made from pure collagen solution made from step 1 and step 2
Silk fibroin protein solution is diluted with water to certain concentration respectively, is then mixed in proportion, and mixed liquor is freeze-dried, and is made
Then collagen-tussah silk fibroin compound rest carries out disinfection collagen obtained-tussah silk fibroin compound rest, then will
The BMSCs suspensions that step 3 obtains cultivate certain time in collagen-tussah silk fibroin compound rest, obtain the load
The collagen of BMSCs-tussah silk fibroin compound rest.
In above-mentioned technical proposal, in the step 1, collagen is extracted to collagen-rich animal tissue
Method be that acid system, alkaline process, salt method or one kind in enzyme process or arbitrary two kinds are combined；
In the step 1, the purification process includes the centrifugation carried out successively, is saltoutd and hyperfiltration treatment.
In above-mentioned technical proposal, in the step 2, the degumming process is de- will to be boiled in tussah silk input aqueous slkali
Glue 30min~50min, is then washed with water, and repeats degumming twice.
In above-mentioned technical proposal, in the step 2, the lysate is calcium nitrate ethanol solution, wherein the nitric acid
The molar concentration of calcium nitrate is 8mol/L~10mol/L in calcium ethanol solution；
In the step 2, the temperature of the heating for dissolving is 50 DEG C~60 DEG C.
In above-mentioned technical proposal, in the step 3, rinses the ossis from animal repeatedly with DMEM culture mediums, obtain
To myeloid tissue flushing liquor, then myeloid tissue flushing liquor is centrifuged, is resuspended in culture solution after then discarding supernatant liquid
Cell, inoculation, is subsequently placed in incubator and carries out original cuiture, and full dose for the first time is carried out after 45h~50h and changes liquid, retains adherent thin
Born of the same parents change liquid every other day later, are 0.2%~0.3% with mass concentration when the attached cell of reservation reaches 80%~90% fusion
Pancreatin digestion, and by 1 after being centrifuged:2 ratio carries out cell secondary culture, then chooses the 3rd~8 generation BMSCs, carries out
After digestion and centrifugation, it is resuspended with culture medium, obtains BMSCs suspensions.
In above-mentioned technical proposal, in the step 3, the inoculation be that addition 4mL~6mL containing mass concentration is 10% tire
The DMEM/F12 culture solutions of cow's serum are inoculated in after then gently blowing and beating mixing in Tissue Culture Flask.
In above-mentioned technical proposal, in the step 3, the condition of the cell secondary culture is to be placed in 37 DEG C, 5%CO2Training
It supports and is cultivated in case.
In above-mentioned technical proposal, in the step 3, the BMSCs fills between mouse mesenchymal stem cell, pig bone marrow
Any one of matter stem cell or characteristics of goat bone marrow mesenchymal stem cells.
In above-mentioned technical proposal, in the step 4, pure collagen solution made from step 1 is made with step 2
Tussah silk fibroin solution be diluted with water to a concentration of 5mg/mL~10mg/mL respectively；Then the collagen solution is pressed
Mixing quality ratio with the tussah silk fibroin solution is 1~5:1 ratio mixing.
In above-mentioned technical proposal, in the step 4, then by collagen obtained-tussah silk fibroin compound rest in purple
Carry out disinfection 20h~30h under outside line, then the BMSCs suspensions that step 3 is obtained are in collagen-tussah silk fibroin compound rest
70h~75h is cultivated, collagen-tussah silk fibroin compound rest of the load BMSCs is obtained.
Compared with prior art, advantageous effect is the present invention：
(1) preparation method of collagen-tussah silk fibroin compound rest of a kind of load BMSCs provided by the invention, institute
Collagen-tussah silk fibroin compound rest of load BMSCs obtained is using collagen and tussah silk fibroin as biological
Compound rest system is made in material, provides suitable carrier for BMSCs, simulates the structure and function of body skin histology, favorably
In the NEW TYPE OF COMPOSITE holder of wound repair.
(2) preparation method of collagen-tussah silk fibroin compound rest of a kind of load BMSCs provided by the invention, institute
Collagen-tussah silk fibroin compound rest of load BMSCs obtained can keep the humidity of the surface of a wound, prevent the stream of body fluid and moisture
It loses；And biological barrier can be provided, prevent trauma surface infestation, bacterium intrusion；And have certain flexibility and tensile property, in group
Its function and form are kept during knitting healing.
(3) preparation method of collagen-tussah silk fibroin compound rest of a kind of load BMSCs provided by the invention, institute
The BMSCs of collagen-tussah silk fibroin compound rest of load BMSCs obtained, load can be to bone and the two fatty embryos
Layer differentiation, has the advantages that differentiation capability is good, and collagen-tussah silk fibroin compound rest can improve derivant induction differentiation
Ability, and then the advantages of wound healing.
(4) preparation method of collagen-tussah silk fibroin compound rest of a kind of load BMSCs provided by the invention, institute
Collagen-tussah silk fibroin compound rest of load BMSCs obtained is biodegradable, and degradation product energy trophocyte, is
Cell growth and proliferation provide suitable microenvironment.In addition, the collagen of load BMSCs-tussah silk fibroin compound rest tool
There are good biocompatibility, no inflammation reaction, toxic reaction, immunological rejection etc., adhesive forces are strong, are conducive to the surface of a wound
Healing.
Description of the drawings
Fig. 1 is that each group surface of a wound carries out the display diagram after different disposal in wound repair experiment of the present invention, wherein
A.Control groups, b.Col/TSF groups, c.Col-Sponge groups and d.BMSCs-Col/TSF groups.
Fig. 2 is the scanning electricity of collagen-tussah silk fibroin compound rest of load BMSCs in wound repair experiment of the present invention
Mirror figure.
Fig. 3 is skeletonization of the load BMSCs on collagen-tussah silk fibroin compound rest in wound repair experiment of the present invention
Differentiation figure.Wherein, a and b. cultivate BMSCs in culture dish；C and d. is cultivated on collagen-tussah silk fibroin compound rest
BMSCs；A and c. uses ordinary culture medium culture；B and d. uses Osteoinductive differentiation complete medium culture.It is contaminated with alizarin red
Color.
Fig. 4 be in wound repair of the present invention experiment load BMSCs on collagen-tussah silk fibroin compound rest at fat
Differentiation figure.Wherein, a and b. cultivate BMSCs in culture dish；C and d. is cultivated on collagen-tussah silk fibroin compound rest
BMSCs；A and c. uses ordinary culture medium culture；B and d. breaks up complete medium culture using adipogenic induction.It is contaminated with oil red O
Color.
Fig. 5 is the healing state figure of surface of a wound each group wound in the 7th, 14,21 and 28 day in wound repair experiment of the present invention.
Fig. 6 is the Wound Contraction rate of each group in wound repair of the present invention experiment.
Fig. 7 is the HE colored graphs of each group rat skin wound different times in wound repair of the present invention experiment.
Fig. 8 is to use fluorescence microscope on HE dyed paraffins slice by fluorescent traccer technique in wound repair of the present invention experiment
Detect the BMSCs of CM-Dil labels.
Fig. 9 is the Masson colored graphs of each group rat skin wound different times in wound repair of the present invention experiment.
Figure 10 is the anti-smooth muscle actin of each group rat skin wound different times in wound repair of the present invention experiment
(α-SMA) immunohistochemical staining figure.
Specific implementation mode
In order to make the technical problems, technical solutions and beneficial effects solved by the present invention be more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
Embodiment 1
A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs, it includes the following steps：
Step 1: the preparation of collagen solution：Acidity extraction collagen egg is carried out to collagen-rich animal tissue
In vain, crude extract is obtained, crude extract is then subjected to purification process, obtains pure collagen solution；Wherein, purification process packet
It includes the centrifugation carried out successively, saltout and hyperfiltration treatment；
Step 2: the preparation of tussah silk fibroin solution：Tussah silk is subjected to degumming process, then will treated tussah silk
It is put into lysate in 55 DEG C of heating for dissolving, tussah silk fibroin crude extract is obtained, then by tussah silk fibroin coarse extraction
Liquid is filtered, dialysis treatment, and tussah silk fibroin solution is made；In the present embodiment, degumming process is that tussah silk is put into alkali
Degumming 40min is boiled in solution, is then washed with water, and repeats degumming twice；In the present embodiment, lysate is calcium nitrate ethyl alcohol
Solution, wherein the molar concentration of calcium nitrate is 9mol/L in calcium nitrate ethanol solution；
Step 3: the preparation of BMSCs suspensions：It rinses the ossis from animal repeatedly with DMEM culture mediums, obtains bone
Myeloid tissue flushing liquor, then centrifuges myeloid tissue flushing liquor, and cell is resuspended in culture solution after then discarding supernatant liquid,
Inoculation, is subsequently placed in incubator and carries out original cuiture, and full dose for the first time is carried out after 48h and changes liquid, retains attached cell, every other day later
Liquid is changed, when the attached cell of reservation reaches 85% fusion, the pancreatin for being 0.25% with mass concentration digests, and after being centrifuged
By 1:2 ratio carries out cell secondary culture, then chooses the 5th generation BMSCs and is resuspended with culture medium after being digested and being centrifuged,
Obtain BMSCs suspensions；In the present embodiment, it is inoculated with as 5mL is added containing the DMEM/F12 cultures that mass concentration is 10% fetal calf serum
Liquid is inoculated in after then gently blowing and beating mixing in Tissue Culture Flask；Wherein, the condition of cell secondary culture be placed in 37 DEG C, 5%
CO2It is cultivated in incubator；In the present embodiment, BMSCs is mouse mesenchymal stem cell；
Step 4: the preparation of compound rest：By tussah made from pure collagen solution made from step 1 and step 2
Silk fibroin protein solution is diluted with water to a concentration of 8mg/mL respectively, then presses collagen solution and tussah silk fibroin solution
Mixing quality ratio is 3:1 ratio mixing, mixed liquor is freeze-dried, collagen-tussah silk fibroin compound rest is made, then
Collagen obtained-tussah silk fibroin compound rest is carried out disinfection for 24 hours under ultraviolet light, then the BMSCs that step 3 is obtained
Suspension cultivates 72h in collagen-tussah silk fibroin compound rest, obtains collagen-tussah silk peptide egg of the load BMSCs
White compound rest.
Embodiment 2
A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs, it includes the following steps：
Step 1: the preparation of collagen solution：Alkalinity extraction collagen egg is carried out to collagen-rich animal tissue
In vain, crude extract is obtained, crude extract is then subjected to purification process, obtains pure collagen solution；Wherein, purification process packet
It includes the centrifugation carried out successively, saltout and hyperfiltration treatment；
Step 2: the preparation of tussah silk fibroin solution：Tussah silk is subjected to degumming process, then will treated tussah silk
It is put into lysate in 50 DEG C of heating for dissolving, tussah silk fibroin crude extract is obtained, then by tussah silk fibroin coarse extraction
Liquid is filtered, dialysis treatment, and tussah silk fibroin solution is made；In the present embodiment, degumming process is that tussah silk is put into alkali
Degumming 30min is boiled in solution, is then washed with water, and repeats degumming twice；In the present embodiment, lysate is calcium nitrate ethyl alcohol
Solution, wherein the molar concentration of calcium nitrate is 8mol/L in calcium nitrate ethanol solution；
Step 3: the preparation of BMSCs suspensions：It rinses the ossis from animal repeatedly with DMEM culture mediums, obtains bone
Myeloid tissue flushing liquor, then centrifuges myeloid tissue flushing liquor, and cell is resuspended in culture solution after then discarding supernatant liquid,
Inoculation, is subsequently placed in incubator and carries out original cuiture, and full dose for the first time is carried out after 45h and changes liquid, retains attached cell, every other day later
Liquid is changed, when the attached cell of reservation reaches 80% fusion, the pancreatin for being 0.2% with mass concentration digests, and after being centrifuged
By 1:2 ratio carries out cell secondary culture, then chooses the 3rd generation BMSCs and is resuspended with culture medium after being digested and being centrifuged,
Obtain BMSCs suspensions；In the present embodiment, it is inoculated with as 4mL is added containing the DMEM/F12 cultures that mass concentration is 10% fetal calf serum
Liquid is inoculated in after then gently blowing and beating mixing in Tissue Culture Flask；Wherein, the condition of cell secondary culture be placed in 37 DEG C, 5%
CO2It is cultivated in incubator；In the present embodiment, BMSCs is pig bone bone marrow-drived mesenchymal stem；
Step 4: the preparation of compound rest：By tussah made from pure collagen solution made from step 1 and step 2
Silk fibroin protein solution is diluted with water to a concentration of 5mg/mL respectively, then presses collagen solution and tussah silk fibroin solution
Mixing quality ratio is 1:1 ratio mixing, mixed liquor is freeze-dried, collagen-tussah silk fibroin compound rest is made, then
Collagen obtained-tussah silk fibroin compound rest is carried out disinfection 20h under ultraviolet light, then the BMSCs that step 3 is obtained
Suspension cultivates 70h in collagen-tussah silk fibroin compound rest, obtains collagen-tussah silk peptide egg of the load BMSCs
White compound rest.
Embodiment 3
A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs, it includes the following steps：
Step 1: the preparation of collagen solution：Salt method collagen egg is carried out to collagen-rich animal tissue
In vain, crude extract is obtained, crude extract is then subjected to purification process, obtains pure collagen solution；Wherein, purification process packet
It includes the centrifugation carried out successively, saltout and hyperfiltration treatment；
Step 2: the preparation of tussah silk fibroin solution：Tussah silk is subjected to degumming process, then will treated tussah silk
It is put into lysate in 60 DEG C of heating for dissolving, tussah silk fibroin crude extract is obtained, then by tussah silk fibroin coarse extraction
Liquid is filtered, dialysis treatment, and tussah silk fibroin solution is made；In the present embodiment, degumming process is that tussah silk is put into alkali
Degumming 50min is boiled in solution, is then washed with water, and repeats degumming twice；In the present embodiment, lysate is calcium nitrate ethyl alcohol
Solution, wherein the molar concentration of calcium nitrate is 10mol/L in calcium nitrate ethanol solution；
Step 3: the preparation of BMSCs suspensions：It rinses the ossis from animal repeatedly with DMEM culture mediums, obtains bone
Myeloid tissue flushing liquor, then centrifuges myeloid tissue flushing liquor, and cell is resuspended in culture solution after then discarding supernatant liquid,
Inoculation, is subsequently placed in incubator and carries out original cuiture, and full dose for the first time is carried out after 50h and changes liquid, retains attached cell, every other day later
Liquid is changed, when the attached cell of reservation reaches 90% fusion, the pancreatin for being 0.3% with mass concentration digests, and after being centrifuged
By 1:2 ratio carries out cell secondary culture, then chooses the 8th generation BMSCs and is resuspended with culture medium after being digested and being centrifuged,
Obtain BMSCs suspensions；In the present embodiment, it is inoculated with as 6mL is added containing the DMEM/F12 cultures that mass concentration is 10% fetal calf serum
Liquid is inoculated in after then gently blowing and beating mixing in Tissue Culture Flask；Wherein, the condition of cell secondary culture be placed in 37 DEG C, 5%
CO2It is cultivated in incubator；In the present embodiment, BMSCs is characteristics of goat bone marrow mesenchymal stem cells；
Step 4: the preparation of compound rest：By tussah made from pure collagen solution made from step 1 and step 2
Silk fibroin protein solution is diluted with water to a concentration of 10mg/mL respectively, then presses collagen solution and tussah silk fibroin solution
Mixing quality ratio be 5:1 ratio mixing, mixed liquor is freeze-dried, collagen-tussah silk fibroin compound rest is made, so
Collagen obtained-tussah silk fibroin compound rest is carried out disinfection 30h under ultraviolet light afterwards, then step 3 is obtained
BMSCs suspensions cultivate 75h in collagen-tussah silk fibroin compound rest, obtain collagen-tussah of the load BMSCs
Fibroin protein composite bracket.
Embodiment 4
A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs, it includes the following steps：
Step 1: the preparation of collagen solution：Collagen Extraction with Enzyme egg is carried out to collagen-rich animal tissue
In vain, crude extract is obtained, crude extract is then subjected to purification process, obtains pure collagen solution；Wherein, purification process packet
It includes the centrifugation carried out successively, saltout and hyperfiltration treatment；
Step 2: the preparation of tussah silk fibroin solution：Tussah silk is subjected to degumming process, then will treated tussah silk
It is put into lysate in 52 DEG C of heating for dissolving, tussah silk fibroin crude extract is obtained, then by tussah silk fibroin coarse extraction
Liquid is filtered, dialysis treatment, and tussah silk fibroin solution is made；In the present embodiment, degumming process is that tussah silk is put into alkali
Degumming 35min is boiled in solution, is then washed with water, and repeats degumming twice；In the present embodiment, lysate is calcium nitrate ethyl alcohol
Solution, wherein the molar concentration of calcium nitrate is 8.5mol/L in calcium nitrate ethanol solution；
Step 3: the preparation of BMSCs suspensions：It rinses the ossis from animal repeatedly with DMEM culture mediums, obtains bone
Myeloid tissue flushing liquor, then centrifuges myeloid tissue flushing liquor, and cell is resuspended in culture solution after then discarding supernatant liquid,
Inoculation, is subsequently placed in incubator and carries out original cuiture, and full dose for the first time is carried out after 46h and changes liquid, retains attached cell, every other day later
Liquid is changed, when the attached cell of reservation reaches 83% fusion, the pancreatin for being 0.22% with mass concentration digests, and after being centrifuged
By 1:2 ratio carries out cell secondary culture, then chooses the 4th generation BMSCs and is resuspended with culture medium after being digested and being centrifuged,
Obtain BMSCs suspensions；In the present embodiment, it is inoculated with as 4.5mL is added containing the DMEM/F12 trainings that mass concentration is 10% fetal calf serum
Nutrient solution is inoculated in after then gently blowing and beating mixing in Tissue Culture Flask；Wherein, the condition of cell secondary culture be placed in 37 DEG C,
5%CO2It is cultivated in incubator；In the present embodiment, BMSCs is mouse mesenchymal stem cell；
Step 4: the preparation of compound rest：By tussah made from pure collagen solution made from step 1 and step 2
Silk fibroin protein solution is diluted with water to a concentration of 6mg/mL respectively, then presses collagen solution and tussah silk fibroin solution
Mixing quality ratio is 2:1 ratio mixing, mixed liquor is freeze-dried, collagen-tussah silk fibroin compound rest is made, then
Collagen obtained-tussah silk fibroin compound rest is carried out disinfection 22h under ultraviolet light, then the BMSCs that step 3 is obtained
Suspension cultivates 71h in collagen-tussah silk fibroin compound rest, obtains collagen-tussah silk peptide egg of the load BMSCs
White compound rest.
Embodiment 5
A kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs, it includes the following steps：
Step 1: the preparation of collagen solution：Acid system is carried out to collagen-rich animal tissue mutually to tie with enzyme process
Extraction collagen is closed, crude extract is obtained, crude extract is then subjected to purification process, obtain pure collagen solution；Its
In, purification process includes the centrifugation carried out successively, is saltoutd and hyperfiltration treatment；
Step 2: the preparation of tussah silk fibroin solution：Tussah silk is subjected to degumming process, then will treated tussah silk
It is put into lysate in 58 DEG C of heating for dissolving, tussah silk fibroin crude extract is obtained, then by tussah silk fibroin coarse extraction
Liquid is filtered, dialysis treatment, and tussah silk fibroin solution is made；In the present embodiment, degumming process is that tussah silk is put into alkali
Degumming 45min is boiled in solution, is then washed with water, and repeats degumming twice；In the present embodiment, lysate is calcium nitrate ethyl alcohol
Solution, wherein the molar concentration of calcium nitrate is 9.5mol/L in calcium nitrate ethanol solution；
Step 3: the preparation of BMSCs suspensions：It rinses the ossis from animal repeatedly with DMEM culture mediums, obtains bone
Myeloid tissue flushing liquor, then centrifuges myeloid tissue flushing liquor, and cell is resuspended in culture solution after then discarding supernatant liquid,
Inoculation, is subsequently placed in incubator and carries out original cuiture, and full dose for the first time is carried out after 49h and changes liquid, retains attached cell, every other day later
Liquid is changed, when the attached cell of reservation reaches 88% fusion, the pancreatin for being 0.28% with mass concentration digests, and after being centrifuged
By 1:2 ratio carries out cell secondary culture, then chooses the 7th generation BMSCs and is resuspended with culture medium after being digested and being centrifuged,
Obtain BMSCs suspensions；In the present embodiment, it is inoculated with as 5.5mL is added containing the DMEM/F12 trainings that mass concentration is 10% fetal calf serum
Nutrient solution is inoculated in after then gently blowing and beating mixing in Tissue Culture Flask；Wherein, the condition of cell secondary culture be placed in 37 DEG C,
5%CO2It is cultivated in incubator；In the present embodiment, BMSCs is pig bone bone marrow-drived mesenchymal stem；
Step 4: the preparation of compound rest：By tussah made from pure collagen solution made from step 1 and step 2
Silk fibroin protein solution is diluted with water to a concentration of 9mg/mL respectively, then presses collagen solution and tussah silk fibroin solution
Mixing quality ratio is 4:1 ratio mixing, mixed liquor is freeze-dried, collagen-tussah silk fibroin compound rest is made, then
Collagen obtained-tussah silk fibroin compound rest is carried out disinfection 28h under ultraviolet light, then the BMSCs that step 3 is obtained
Suspension cultivates 74h in collagen-tussah silk fibroin compound rest, obtains collagen-tussah silk peptide egg of the load BMSCs
White compound rest.
Wound repair is tested：
1 materials and methods
1.1 main agents and instrument
SD rats (Nanfang Medical Univ's Experimental Animal Center)；Tussah cocoon (Liaoning Province Benxi)；Ox heel string (Zhejiang Province Hangzhoupro
State city)；Pepsin (Zhejiang Song Shan biological products Co., Ltd)；DMEM/F12 fluid nutrient mediums (GIBCO, the U.S.)；Tire ox
Serum (GIBCO, the U.S.)；Trypsase (GIBCO, the U.S.)；Dimethyl sulfoxide (DMSO) DMSO (MP Biomedicals, the U.S.).GL-
20G-H freezes high speed tabletop centrifuge (Anting Scientific Instrument Factory, Shanghai)；(Beijing Bo Kang is real for FD-1-50 vacuum freeze driers
Test Instrument Ltd.)；GZF-6000 electric vacunm dryings case (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.)；It is inverted aobvious
Micro mirror (Nikon, Japan)；Inverted fluorescence microscope (OLYMPUS, Japan)；Cell incubator (Thermo Scientific, it is beautiful
State)；Scanning electron microscope (JSM-6390LV, JEOL, Japan))；Full-automatic microplate reader (OLYMPUS, Japan)；Purify work
Platform (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.).The preparation of 1.2 collagen solutions
The ox heel string removal lipid impurities that will be enriched in collagen, are sliced after being cleaned with pure water, blend.According to ox heel string vinegar
The ratio of acid=1: 60 (g/mL) prepares the acetum of 0.5mol/L, will treated that ox heel string is soaked in acetum,
According still further to ox heel string:Enzyme=1: pepsin is added in the ratio of 0.03 (mass ratio), at low temperature under the conditions of extract 72h, extract
It is slowly stirred always in the process.After extracting 72h, collagen crude extract is obtained.Crude extract is passed through into centrifugation, salt
The methods of analysis, ultrafiltration are purified, and collagen solution is obtained.
The preparation of 1.3 tussah silk fibroin solutions
Tussah silk is in 0.5%Na2CO3Degumming 40min, solid-liquid ratio 1 are boiled in aqueous solution:20, with ion water washing, weight
Multiple operation is twice.Silk tussah fibre after degumming is put into 60 DEG C of drying of baking oven.10g silk tussah fibres are weighed, are dissolved in
In 8.56mol/L calcium nitrate ethanol solutions, 3h is dissolved in 100 DEG C of stirred in water bath.By the silk fibroin protein solution gauze of dissolving
Filtering, is fitted into bag filter (molecular cut off 8000-14000, radius 77mm), then dialyse in distilled water 72h, obtains by filtrate
To tussah silk fibroin solution.The preparation of 1.4BMSCs
Rat cervical dislocation is put to death, and 75% alcohol disinfecting 20min is soaked in.Removed rapidly with aseptic apparatus bilateral tibial and
Femur removes remaining muscle and connective tissue, keeps the complete of periosteum and metaphysis.It is rinsed 3 times with containing dual anti-sterile PBS,
Then bilateral metaphysis is cut off, DMEM culture mediums is extracted with 5ml disposable syringes and rinses ossis repeatedly, myeloid tissue is complete
Portion is gone out, and myeloid tissue flushing liquor is then moved into 15ml centrifuge tubes, and 1000r/min centrifuges 10min, abandons supernatant.5ml is added to contain
The DMEM/F12 culture solutions of 10% fetal calf serum are inoculated in Tissue Culture Flask after gently blowing and beating mixing, are placed in 37 DEG C, 5%CO2
It is cultivated in incubator.Full dose changes liquid for the first time after 48h, retains attached cell, changes liquid every other day later, when attached cell reaches 80%~
It when 90% fusion, is digested with 0.25% pancreatin, 1000r/min presses 1 after centrifuging 5min:2 ratios carry out secondary culture.
1.5 living cells dye：
To the BMSCs stand densities in the 3rd generation up to when 90% fusion, trypsin digestion is adjusted at individual cells suspension for culture
Whole total number of cells are 1x 106It is a, it is resuspended with 1ml phosphate buffers (PBS), the CM-Dil dyes of 2 a concentration of 1mg/ml of μ l is added
Liquid is protected from light, and 37 DEG C of incubation 5min, 4 DEG C are incubated 15min, and PBS is washed 2 times, trained completely with the DMEM/F12 containing 10% fetal calf serum
It supports after base weight is hanged and moves into culture dish culture.(stand density is merged up to 90% after waiting for the cell growing way dyed by CM-Dil to stablize
When), it digested, centrifuged, 1 × 10 is diluted to the DMEM/F12 culture mediums containing 10% fetal calf serum5The cell of/ml is outstanding
Liquid.
The preparation of 1.6 collagens-tussah silk fibroin compound rest
It is 8mg/ml that collagen solution obtained and silk fibroin protein solution, which are diluted to concentration, by collagen solution
It is 3 with tussah silk fibroin solution mass ratio:2 are mixed, and mixed liquor is freeze-dried, and collagen-tussah silk fibroin is made
Compound rest.Holder is sterilized under ultraviolet light for 24 hours, then BMSCs suspensions are cultivated into 72h in gained holder, is loaded
The collagen of BMSCs-tussah silk fibroin compound rest.
1.7 scanning electron microscope detect
Collagen-tussah silk fibroin compound rest of BMSCs will be loaded respectively in 2% glutaraldehyde and 1% osmium tetroxide
It is fixed, after the processing of ethyl alcohol continuous dehydration, sample is placed dry in carbon dioxide critical point drying instrument.Sample through metal spraying at
After reason, the observation of surface and section structure is carried out to it under accelerating potential using scanning electron microscope.
The influence that 1.8 collagens-tussah silk fibroin compound rest breaks up BMSCs
BMSCs is seeded on collagen-tussah silk fibroin compound rest, in 37 DEG C, 5%CO2, under the conditions of saturated humidity
Culture discards original culture medium after cell is adherent, and addition adipogenic induction differentiation complete medium and Osteoinductive differentiation are complete
Culture medium requires to change liquid on time according to each kit, induction is terminated after forming a large amount of fat drips and calcium tubercle, with 4% poly
After formaldehyde is fixed, oil red O and Alizarin red staining liquid is used to dye respectively, analysis tussah silk peptide-collagen compound rest is to BMSCs
At the influence of fat and Osteoblast Differentiation.
1.9 zoopery
1.9.1 grouping：50 SD rats are randomly divided into two groups, every group 25, every rat back left and right sides is made not
With processing.
1.9.2 prepared by animal model：SD rats are anaesthetized with 10% chloraldurate solution 0.3ml/kg intraperitoneal injections.
Rat takes prone position, back preserved skin, rat back median line both sides to draw a diameter of 2cm circular incisions, along cut line that skin is complete
Layer is cut.The surface of a wound of blank control group (Control) is only wrapped up with gauze, does not do other processing, other three groups respectively will be peculiar
Nation's medical collagen sponge (Col-Sponge), collagen-tussah silk fibroin compound rest (Col/TSF) load BMSCs's
Collagen-tussah silk fibroin compound rest (BMSCs-Col/TSF) applies on the rat skin surface of a wound, is used in combination sterile gauze wrapping big
The mouse surface of a wound, is shown in Fig. 1.Postoperative injection of antibiotics, experimental mouse pay attention to warming, the sub-cage rearing after its revival.
1.9.3 animal is drawn materials：It is postoperative to take out 5 rats at random in each group respectively at the 7th, 14,21,28 day, with 10% water
Chloralization is closed, back skin tissues sample is taken, uses cervical dislocation to put to death rat after materials.
1.10 Wound Contraction rates measure
After wrapping up 3d, dressing, surface of a wound exposure, self-healing are removed.It, will in 0d, 7d, 14d and 21d that the surface of a wound is formed
The each group surface of a wound is traced on onionskin and draws engineer's scale respectively.The onionskin for recording the surface of a wound is scanned into computer simultaneously
It is calculated using OPTPro softwares.
Wound Contraction rate (%)=(former wound area-do not heal area)/former wound area × 100.
1.11 histological observation
Skin histology is placed in 4% paraformaldehyde to fix for 24 hours solidifies the protein denaturation of tissue, cell, then into
Row specimens paraffin embedding slices.After paraffin section routinely dewaxing, dyed by HE dyeing and Masson, microscopic observation neoplastic skin is thin
The growing state of born of the same parents.The paraffin section of HE dyeing is subjected to fluoroscopic examination by fluorescence microscope, sees Figure 10.
1.12 immunohistochemistry is identified
Paraffin section routinely dewaxes and aquation, PBS rinsings, 3% hydrogen peroxide closes 10min；It is added an antiantibody, 4 DEG C
Overnight；PBS is rinsed, and corresponding secondary antibody is added, 30 minutes are stood at 37 DEG C；PBS is rinsed, DAB reagent colour developments, under the microscope, is occurred
Stop immediately after brown, fully washes.Haematoxylin lining core is redyed, indigo plant is returned in hydrochloride alcohol differentiation, 75%, 85%, 95%,
100% dehydration of alcohol, neutral gum mounting.PBS is added and sets negative control piece.
2, experimental result
2.1 structural analysis
The configuration of surface of dressing is observed by scanning electron microscope.As shown in Fig. 2, collagen-tussah silk of load BMSCs
There is fibroin compound rest the porous structure of high connectivity, BMSCs can be attached on collagen-tussah silk fibroin compound rest
It is grown on hole wall surface.The smooth proliferation being arranged with conducive to cell of porous structure, micronutrient and gas exchange and wound
The absorption of mouth exudate.
2.2 differentiation influence situation
The ability that BMSCs breaks up to skeletonization and at fat is detected respectively using alizarin red and oil red O.As shown in Figure 3 and Figure 4,
Due to the formation of red calcium scoring and fat drips, BMSCs is possible to bone and the two fatty differentiation of germinal layers.In compound rest
In the presence of, induction group forms more calcium scorings and fat drips so that and the differentiation potential of BMSCs increased, and
Non-induced group does not form calcium scoring and fat drips but, illustrates collagen-tussah silk fibroin compound rest itself without promoting to divide
The ability of change, but it can improve the ability of derivant induction differentiation.It is therefore contemplated that collagen-tussah silk fibroin is multiple
Close holder has synergistic effect for the inducibility of differentiating inducer.
2.3 wound healing situations
As shown in figure 5, the wound of BMSCs-Col/TSF groups can heal substantially in 14d, with other three groups (Col/TSF,
Col-Sponge and Control groups) compare, wound healing effect is best, illustrates the collagen-tussah silk fibroin for loading BMSCs
Compound rest has special efficacy to wound healing.
In 7d, Col-Sponge groups compared to other group wound healings it is slower, Control groups, Col/TSF groups and
The wound healing status difference unobvious of BMSCs-Col/TSF groups.To after 14d, Col/TSF groups and BMSCs-Col/TSF groups are bright
Aobvious better than the wound healing situation of Control groups, Wound Contraction rate has significant difference (P<0.05), see Fig. 6.
2.4 histology
Postoperative 1st, 7,14,21,28d HE dyeing, is shown in Fig. 7.In 14d, neoblastic re-epithelialization is observed, and each group
The regeneration of epithelial tissue is not exactly the same.The re-epithelialization of BMSCs-Col/TSF groups is better than other three groups.In 21d, each group goes out
Existing moderate epithelialization, and there are the prematurity appendages of skin at first in BMSCs-Col/TSF groups.In 28d, each group observes that skin is attached
Part, the BMSCs-Col/TSF groups appendages of skin are more ripe than other three groups of developments.Therefore, the effect of BMSCs-Col/TSF groups, is better than
Other three groups.
The paraffin section dyed using fluorescence microscopy HE is to detect and track the BMSCs of CM-Dil labels.Such as Fig. 8
It is shown, in Control groups, so slice cannot all detect fluorescence, and in BMSCs-Col/TSF groups, in all slices
(the 1,7,14,21st and 28 day) can detect fluorescence.Illustrate in entire agglutination, (CM-Dil is marked the BMSCs of inoculation
Note) sustainably play the role of promoting wound healing.
In order to identify the deposition of collagen in skin, Masson dyeing is carried out to histotomy.Masson decoration methods
Collagen is dyed blue (see Fig. 9).The 14th day after surgery, collagen started ordered arrangement, and the deposition of collagenous fibres is bright
It is aobvious more dense.The 21st day after surgery, collagenous fibres were orientated more preferable, linear array.Col/TSF group collagen deposition amount highests, collagen
Arrangement is the most regular.The collagen of followed by BMSCs-Col/TSF groups and Col-sponge groups, the two arranges than Control group
It is good.Collagenous fibres height deposits the healing for illustrating wound.As a result, it was confirmed that BMSCs-Col/TSF groups and Col/TSF groups can all promote to hinder
Mouth healing.
2.4 immune modularization interpretations of result
In the early stage (7d) of wound healing, all shown in the vascular endothelial cell of each group and fibroblast intensive
Positive immune reactivity.Immuno positive α-the SMA of BMSCs-Col/TSF groups and Col/TSF groups are than Control group and Col-
Sponge groups are stronger.Fibroblast and vascular endothelial cell are more, are more conducive to wound healing.In the later stage rank of wound healing
Section (28d), the fibroblast of the regeneration intradermal of each group and vascular endothelial cell only have slight local immunity reactivity.With it
He three groups compare, BMSCs-Col/TSF group vascularization is more ripe, and wound healing is more perfect.
3 experiments discuss
In summary it tests, the structure and composition for loading collagen-tussah silk fibroin compound rest of BMSCs is conducive to carefully
Intracellular growth, adherency, proliferation and differentiation.The dressing has good efficacy to skin repair, is a kind of promising wound dressing.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than limiting the scope of the invention, although
The present invention is explained in detail with reference to preferred embodiment, it will be understood by those of ordinary skill in the art that, it can be to this hair
Bright technical solution is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention.
Claims (10)
1. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs, it is characterised in that：It include with
Lower step：
Step 1: the preparation of collagen solution：Collagen is extracted to collagen-rich animal tissue, is obtained thick
Then crude extract is carried out purification process by extracting solution, obtain pure collagen solution；
Step 2: the preparation of tussah silk fibroin solution：Tussah silk is subjected to degumming process, then tussah silk is put by treated
It is dissolved by heating in lysate, obtains tussah silk fibroin crude extract, be then filtered tussah silk fibroin crude extract,
Tussah silk fibroin solution is made in dialysis treatment；
Step 3: the preparation of BMSCs suspensions：It rinses the ossis from animal repeatedly with DMEM culture mediums, obtains marrow group
Flushing liquor is knitted, then centrifuges myeloid tissue flushing liquor, cell is resuspended in culture solution after then discarding supernatant liquid, connects
Kind, it is subsequently placed in incubator and carries out original cuiture, carry out full dose for the first time after a certain period of time and change liquid, retain attached cell, later
Change liquid every certain time, when the attached cell of reservation reaches certain percentage fusion, digested with pancreatin, and after being centrifuged by
Certain proportion carries out cell secondary culture, then chooses BMSCs, after being digested and being centrifuged, is resuspended, is obtained with culture medium
BMSCs suspensions；
Step 4: the preparation of compound rest：By tussah silk peptide made from pure collagen solution made from step 1 and step 2
Protein solution is diluted with water to certain concentration respectively, is then mixed in proportion, and mixed liquor is freeze-dried, and glue is made
Then original-tussah silk fibroin compound rest carries out disinfection collagen obtained-tussah silk fibroin compound rest, then will step
Rapid three obtained BMSCs suspensions cultivate certain time in collagen-tussah silk fibroin compound rest, obtain the load
The collagen of BMSCs-tussah silk fibroin compound rest.
2. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 1, to collagen-rich animal tissue extract collagen method be acid system,
Alkaline process, salt method or one kind in enzyme process or arbitrary two kinds are combined；
In the step 1, the purification process includes the centrifugation carried out successively, is saltoutd and hyperfiltration treatment.
3. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 2, the degumming process be will tussah silk put into aqueous slkali in boil degumming 30min~
Then 50min is washed with water, and repeat degumming twice.
4. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 2, the lysate is calcium nitrate ethanol solution, wherein in the calcium nitrate ethanol solution
The molar concentration of calcium nitrate is 8mol/L~10mol/L；
In the step 2, the temperature of the heating for dissolving is 50 DEG C~60 DEG C.
5. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 3, the ossis from animal is rinsed repeatedly with DMEM culture mediums, obtain myeloid tissue punching
Washing lotion, then centrifuges myeloid tissue flushing liquor, and cell is resuspended in culture solution after then discarding supernatant liquid, inoculation, so
It is placed in incubator and carries out original cuiture, full dose for the first time is carried out after 45h~50h and changes liquid, retains attached cell, changes every other day later
Liquid, when the attached cell of reservation reaches 80%~90% fusion, the pancreatin for being 0.2%~0.3% with mass concentration digests, and
By 1 after being centrifuged:2 ratio carries out cell secondary culture, then chooses the 3rd~8 generation BMSCs, after being digested and being centrifuged,
It is resuspended with culture medium, obtains BMSCs suspensions.
6. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 3, the inoculation is that 4mL~6mL is added containing the DMEM/ that mass concentration is 10% fetal calf serum
F12 culture solutions are inoculated in after then gently blowing and beating mixing in Tissue Culture Flask.
7. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 3, the condition of the cell secondary culture is to be placed in 37 DEG C, 5%CO2It is cultivated in incubator.
8. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 3, the BMSCs is mouse mesenchymal stem cell, pig bone bone marrow-drived mesenchymal stem or sheep
Any one of mesenchymal stem cell.
9. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 4, by tussah silk peptide egg made from pure collagen solution made from step 1 and step 2
White solution is diluted with water to a concentration of 5mg/mL~10mg/mL respectively；Then the collagen solution and the tussah silk are pressed
The mixing quality ratio of fibroin solution is 1~5:1 ratio mixing.
10. a kind of preparation method of collagen-tussah silk fibroin compound rest of load BMSCs according to claim 1,
It is characterized in that：In the step 4, then collagen obtained-tussah silk fibroin compound rest is disappeared under ultraviolet light
Malicious 20h~30h, then BMSCs suspensions that step 3 is obtained cultivated in collagen-tussah silk fibroin compound rest 70h~
75h obtains collagen-tussah silk fibroin compound rest of the load BMSCs.
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