A kind of 3 D-printing combines biological three-dimensional printing technology to prepare tendon methodTechnical field
The invention belongs to biological three-dimensional printing technology fields, and in particular to a kind of biological 3 D-printing skill of 3 D-printing combinationArt prepares tendon method.
Background technique
Tendon injury is one of most common injury gained in sports, and the common type in clinical soft tissue injury, currentTreatment method there is certain limitation or deficiency, bracket and seed cell the regeneration of damaged tissues is guided jointly andThe structure of regenerating tissues is controlled, is the key that determine whether artificial tendon can be used for clinical treatment；
Currently, preparing tendon scaffold mainly includes weaving class bracket with fibre bundle bracket, it is knitted class bracket, electrostatic spinningSeveral classes such as nano fiber scaffold, however the tendon scaffold of above method preparation, seed cell sticks, is colonized efficiency in reparationLowly, it is difficult to achieve the effect that quickly to repair injury tissue, moreover, obtained tendon scaffold is in vivo easily and surrounding tissueIt is adhered, the recovery of the normal function after influencing tendon rehabilitation.
Summary of the invention
To solve the problems mentioned above in the background art.The present invention provides a kind of 3 D-printings to combine biology is three-dimensional to beatPrint technology prepares tendon method, has the characteristics that be conducive to tendon repair and normal function is abundant.
To achieve the above object, the invention provides the following technical scheme: a kind of 3 D-printing combines biological 3 D-printing skillArt prepares tendon method, preparation method are as follows:
The culture of S1, seed cell；
The preparation of S2, tendon scaffold；
S3, cell coated preparation.
Preferably, in the S1 step seed cell culture, tendon stem cell with containing 10% fetal calf serum DMEM trainFeeding base is incubated in incubator, and stem cell and fat stem cell are to contain 10% fetal calf serum, Connective Tissue Growth FactorCTGF25ng/ml, ascorbic acid 25uM α-MEM culture medium be incubated in incubator.
Preferably, contain 5%CO in incubator used in the S1 step2, the temperature in incubator is 37 DEG C.
Preferably, in the S2 step preparation step of tendon scaffold include S21, model foundation: using 3 D-printingSoftware establishes printer model, is single-layer or multi-layer oblong-shaped, and save, and waits next step；
The preparation of S22, material: measuring suitable bioabsorbable polymer material, waits next step；
The preparation of S23, equipment: the print parameters of adjustment melting electrostatic spinning 3D printing equipment, by the biology of step S22High molecular material is placed in inside the barrel of melting electrostatic spinning 3D printing equipment, and it is three-dimensional that starting device carries out melting electrostatic spinningPrinting obtains tendon scaffold, waits next step；
Preferably, the print parameters of printing device are in the S23 step, and print head diameter is 150-400 μm, printing temperatureDegree is 120-200 DEG C, and barrel air pressure is 600-1000KPa in print procedure, and melt spinning negative high voltage module voltage is -2-10kV, print structure are controlled by printing path, and printing path is 0/90 °, 0/60 ° and 0/60/120 °.
Preferably, preparation step cell coated in the S3 step includes the foundation of S31, model: using 3 D-printingSoftware establishes printer model, is single-layer or multi-layer oblong-shaped, and save.
The preparation of S32, material: it measures suitable sterile natural material and is mixed with seed cell, obtained containing Cellular gels, soAfter take part to be mixed with suitable lubrication related substances containing Cellular gels.
S33, cell coated printing: 3D printing equipment is sterilized, and sets print parameters；
S34, by being added containing Cellular gels to the barrel of 3D printing equipment in step S32, with nano fibrous membraneIt receives, starts print routine, obtain nano fibrous membrane cell coated, the Cellular gels containing greasing substance in S32 are addedTo the barrel of 3D printing equipment, same print parameters are printed, and are printed on cell coated other adjacent area, are used appropriate sideMethod is crosslinked gel.
Preferably, the print parameters in the S33 step are that the print head diameter of 3D printing equipment is 150-400 μm,Print temperature is 18-37 DEG C, and barrel air pressure is 600-1000KPa in print procedure, and print structure is controlled by printing path, is printedPath is 0/90 °, 0/60 ° and 0/60/120 °.
Preferably, preparation method further includes the cell coated processing of S4, nano fibrous membrane-.
Preferably, the cell coated processing step of nano fibrous membrane-includes that will obtain in S34 step in the S3 stepHave cell coated nano fibrous membrane to be crimped, and to be located at outer layer with the cell coated of lubrication related substances, obtains peopleWork tendon, length 2-10cm, diameter 2-10mm cylindric in coiled structure.
Compared with prior art, the beneficial effects of the present invention are:
The present invention, the 3 D-printing combine biological three-dimensional printing technology, and easily controllable fibre diameter obtains tendon scaffold,And lubricant cell coating is obtained in its printout surface by biological three-dimensional printing technology, make itself just to contain the kind being largely colonizedDaughter cell is conducive to cell Proliferation, differentiation and secretion matrix, and has favorable lubricating property, and the production method is simple and convenient, more preferablyThe bionical natural tendon tissue form in ground, cell component and greasy property, are conducive to tendon repair and normal function restores.
Detailed description of the invention:
Fig. 1 is the flow diagram of artificial tendon of the present invention；
Fig. 2 is the schematic diagram data of control experiment of the present invention；
Fig. 3 is the result schematic diagram of qRT-PCR of the present invention detection；
Fig. 4 is stress-strain diagram schematic diagram of the present invention；
Fig. 5 is friction coefficient vs lab diagram of the present invention.
Specific embodiment
Below in conjunction with attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that instituteThe embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention,Every other embodiment obtained by those of ordinary skill in the art without making creative efforts, belongs to this hairThe range of bright protection.
The present invention the following technical schemes are provided: a kind of 3 D-printing combines biological three-dimensional printing technology to prepare tendon method,Preparation method are as follows:
The culture of S1, seed cell；
The preparation of S2, tendon scaffold；
S3, cell coated preparation.
Specifically, in the S1 step seed cell culture, tendon stem cell with containing 10% fetal calf serum DMEM trainFeeding base is incubated in incubator, and stem cell and fat stem cell are to contain 10% fetal calf serum, Connective Tissue Growth FactorCTGF25ng/ml, ascorbic acid 25uM α-MEM culture medium be incubated in incubator.
Specifically, containing 5%CO in incubator used in the S1 step2, the temperature in incubator is 37 DEG C.
Specifically, the preparation step of tendon scaffold includes the foundation of S21, model in the S2 step: using 3 D-printingSoftware establishes printer model, is single-layer or multi-layer oblong-shaped, and save, and waits next step；
The preparation of S22, material: measuring suitable bioabsorbable polymer material, waits next step；
The preparation of S23, equipment: the print parameters of adjustment melting electrostatic spinning 3D printing equipment, by the biology of step S22High molecular material is placed in inside the barrel of melting electrostatic spinning 3D printing equipment, and it is three-dimensional that starting device carries out melting electrostatic spinningPrinting obtains tendon scaffold, waits next step；
Specifically, the print parameters of printing device are in the S23 step, print head diameter is 150-400 μm, printing temperatureDegree is 120-200 DEG C, and barrel air pressure is 600-1000KPa in print procedure, and melt spinning negative high voltage module voltage is -2-10kV, print structure are controlled by printing path, and printing path is 0/90 °, 0/60 ° and 0/60/120 °.
Specifically, preparation step cell coated in the S3 step includes the foundation of S31, model: using 3 D-printingSoftware establishes printer model, is single-layer or multi-layer oblong-shaped, and save.
The preparation of S32, material: it measures suitable sterile natural material and is mixed with seed cell, obtained containing Cellular gels, soAfter take part to be mixed with suitable lubrication related substances containing Cellular gels.
S33, cell coated printing: 3D printing equipment is sterilized, and sets print parameters；
S34, by being added containing Cellular gels to the barrel of 3D printing equipment in step S32, with nano fibrous membraneIt receives, starts print routine, obtain nano fibrous membrane cell coated, the Cellular gels containing greasing substance in S32 are addedTo the barrel of 3D printing equipment, same print parameters are printed, and are printed on cell coated other adjacent area, are used appropriate sideMethod is crosslinked gel.
Specifically, the print parameters in the S33 step are, the print head diameter of 3D printing equipment is 150-400 μm,Print temperature is 18-37 DEG C, and barrel air pressure is 600-1000KPa in print procedure, and print structure is controlled by printing path, is printedPath is 0/90 °, 0/60 ° and 0/60/120 °.
Specifically, preparation method further includes the cell coated processing of S4, nano fibrous membrane-.
Specifically, the cell coated processing step of nano fibrous membrane-includes that will obtain in S34 step in the S3 stepHave cell coated nano fibrous membrane to be crimped, and to be located at outer layer with the cell coated of lubrication related substances, obtains peopleWork tendon, length 2-10cm, diameter 2-10mm cylindric in coiled structure.
Embodiment 1
(1) by inside the barrel of high molecular material polycaprolactone merging melting electrostatic spinning 3D printing equipment, adjustment is moltenMelt the technological parameter of electrostatic spinning 3D printing equipment: print head diameter is 200 μm, and print temperature is 180 DEG C, in print procedureBarrel air pressure is 600-1000KPa, and melt spinning negative high voltage module voltage is -3kV, and print structure is controlled by printing path, beatenPrinting path is 0/90 °, and starting device carries out melting electrostatic spinning 3 D-printing and obtains tendon scaffold.
(2) tendon stem cell is mixed with 10g sodium alginate gel, is added in the barrel of three-dimensional printer, printing temperatureDegree is set as 37 DEG C, and print head diameter is 200 μm, and a height of 200 μm of layer, barrel air pressure is 800KPa.Printing path is set as0/90 °, resulting nanofiber film surface is printed, start print routine, print single layer rectangular support frame, length is3cm, width 1cm.
(3) tendon stem cell, cell factor BMP7 are mixed with 10g sodium alginate gel, is added to three-dimensional printerIn barrel, print temperature is set as 18-37 DEG C, and print head diameter is 200 μm, and a height of 200 μm of layer, barrel air pressure is800KPa, printing path are set as 0/90 °, in resulting nanofiber film surface, are printed with print area adjacent area,Start print routine, prints single layer rectangular support frame, length 2cm, width 1cm are finally obtained with greasy property cellThe nano fibrous membrane of coating.
(4) nano fibrous membrane is crimped along broadside, obtains cylindrical artificial tendon, the length is 5cm, diameter is8mm, detailed process such as Fig. 1.
Embodiment 2
It by gained artificial tendon, is put into incubator and cultivates, with the nano fibrous membrane that common electrostatic spinning obtains, surface kindIt plants tendon stem cell to compare for control group, after cultivating 1,3,5,7 and 9 day respectively, utilizes the flesh in mtt assay detection tendon scaffoldTendon cell vigor, result figure as indicated with 2, show the flesh that melting electrostatic spinning 3 D-printing combines biological three-dimensional printing technology to obtainTenocyte cell on tendon bracket has good proliferation behavior, and cell viability is preferable, and more significant compared with control group proliferation behavior.
Embodiment 3
Obtained tendon scaffold is subjected to qRT-PCR detection, the artificial tendon for having greasy property cell coated gained,It is put into incubator and cultivates, made with the common electrostatic spinning film surface of resulting tendon scaffold and single layer plantation tendon stem cellComparison after cultivating 7 days respectively, carries out qRT-PCR detection, as indicated at 3, two groups of display have cell coated tendon scaffold to result figureOn Tenocyte cell tendon associated products (COLI, COLIII, SCX, TNMD) expression it is obvious more, while having greasy property thinIt is obvious less that the tendon scaffold of born of the same parents' coating sticks associated products (Vinculin) expression.
Embodiment 4
Tendon scaffold obtained in above-described embodiment 1 is placed in progress mechanical stretch test, stress on omnipotent test machineFor strain curve as shown in figure 4, the maximum fracture strength of the bracket is 177.433MPa as the result is shown, mechanical property is preferable.
Embodiment 5
By to artificial tendon scaffold be placed on PVvalue testing machine and carry out tribology tester, to be free of greasing substanceArtificial tendon as control, as a result as shown in figure 5, the artificial tendon coefficient of friction is significantly less than control group as the result is shown.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be usedTo modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the inventionWithin protection scope.