CN108452381A - A kind of organization engineering skin and preparation method thereof with layered structure - Google Patents
A kind of organization engineering skin and preparation method thereof with layered structure Download PDFInfo
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- CN108452381A CN108452381A CN201810458262.0A CN201810458262A CN108452381A CN 108452381 A CN108452381 A CN 108452381A CN 201810458262 A CN201810458262 A CN 201810458262A CN 108452381 A CN108452381 A CN 108452381A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/60—Materials for use in artificial skin
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Abstract
The invention discloses a kind of organization engineering skin and preparation method thereof with layered structure, is related to organization engineering skin technical field；Organization engineering skin has the structure of layering, including collagen layer, the collagen layer for being embedded in human skin fibroblasts, the first multilayer collagen layer, the collagen layer for being embedded in keratinocyte, the second multilayer collagen layer；By using this temporary material of gelatin, innovatively portion generates channel to the present invention within the organization, adhere to Human umbilical vein endothelial cells in channel, the blood vessel of formation more coincide with human vas, the blood vessel conveying distribution speed of formation improves, and cell has higher survival ability and faster growth rate；Using the structure printing skin of layering, between the cell in the skin texture of formation and skin histology interacts with epimatrix and more stablizes, and is conducive to the regeneration of skin histology.
Description
Technical field
The present invention relates to organization engineering skin technical fields, and in particular to a kind of organization engineering skin with layered structure
And preparation method thereof.
Background technology
Skin is the maximum organ of human body, is the vital tissue for being covered in body surface, is the screen that human body is contacted with external environment
Barrier not only carries and the functions such as protects body, perspire, perceive, but also maintains the stabilization of organismic internal environment.Skin repair is for controlling
It is critically important to treat burn, lacerated wound and diabetic wound.Under normal conditions, it in order to treat skin injury, needs from other portions of body
Position obtains healthy skin and is transplanted to wound, however, extensive skin injury needs the skin dose that largely can be used for transplanting, it is this
Method can not only generate new wound in skin donor site, low with transplanting skin survival rate etc. there is also itself normal skin limited source
Defect, therefore be not effective or feasible therapy.
In recent years, with the rapid development of tissue engineering technique, different types of artificial skin is successively had developed, including
Artificial epidermis, artificial dermis and the artificial full thickness skin containing epidermis and corium double-layer structure.This kind of artificial skin can be used for difference
The reparation of the defect of skin of degree promotes the healing of skin wound, becomes one of research hotspot of scientific research personnel.But this side
Method is vulnerable to the limitation for repairing position shape, is not easy to produce in the case where needing organotypic skin to cultivate as mesh using wound reparation
The skin shape at position is repaired in raw fitting, is not easily passed through conventional method and is easily generated.
3D biometric prints technology develops rapidly in recent years, and " cell " factor is added in this on the basis of traditional 3D printing
Biometric print technology positions assembly biomaterial and cell list under the driving of digital three-dimensional model according to increasing material manufacturing principle
Member " makes " tissue of oneself needs to measure for patient by layer-by-layer printing.One considerable advantage of this technology is
It in the position accurately controlled while can deposit living cells, growth factor and biomaterial scaffolds to simulate natural tissues knot
Structure has huge potentiality in organizational project.
Skin is printed with 3D biometric print technologies, can not only generate the skin shape of the fitting surface of a wound completely, but also step
Simply, success rate is high.
Invention content
Overcome the deficiencies in the prior art of the present invention, it is therefore an objective to utilize 3D biometric print technologies, prepare a kind of with layering knot
The organization engineering skin of structure.
To achieve the above object, the technical solution adopted in the present invention is.
A kind of organization engineering skin with layered structure, including skin corium and epidermis are propped up by seed cell and biology
Frame material is prepared by biological 3D printing technique, and the seed cell includes for constituting the application on human skin of corium into fiber
Cell, the keratinocyte for constituting epidermis, the Human umbilical vein endothelial cells for constituting blood vessel；The biological support
Material includes layered-scaffold material and vascular stent material, and the layered-scaffold material is collagen, the blood vessel branch
Frame material is gelatin.
Structure of the organization engineering skin with layering, the layered structure are：By skin inner layer to outer layer
Sequence is followed successively by the collagen layer, collagen layer for being embedded in human skin fibroblasts, the first multilayer collagen layer, embedding
Collagen layer, the second multilayer collagen layer of keratinocyte are entered.
Preferably, the first multilayer collagen is five layers of collagen, and the second multilayer collagen is two layers of glue
Former albumen.
Preferably, the collagen layer for being embedded in human skin fibroblasts has blood vessel structure, is embedded in cutin and is formed
The collagen layer of cell has luminal structure.
Preferably, further include Porcine HGF.
A kind of preparation method of the organization engineering skin with layered structure, including the preparation of biomaterial, biology 3D are beaten
Step is printed, the biological 3D printing includes：
a）One layer of collagen layer is printed in substrate surface（1）；
b）In collagen layer（1）It is upper to print the collagen layer for being embedded in human skin fibroblasts（2）, described is embedded in
The collagen layer of human skin fibroblasts（2）It is first to print one layer of collagen layer, then in collagen layer surface
Both sides print two gelatin with form of straight lines, and gelatin cures at room temperature after 1-2 minutes, deposits people after solidification between gelatin
Skin fibroblasts are spraying one layer of mist NaOH by ultrasonic transducer, uncrosslinked collagen are being coagulated automatically above
Gelatinization；
c）In the collagen layer for being embedded in human skin fibroblasts（2）The first multilayer collagen of upper printing（3）；
d）In the first multilayer collagen layer（3）It is upper to print the collagen layer for being embedded in keratinocyte（4）, described is embedding
The collagen layer of keratinocyte is entered（4）It is first to print one layer of collagen layer, then in collagen layer surface
Both sides with form of straight lines print two gelatin, gelatin cures at room temperature after 1-2 minutes, after solidification between gelatin deposition angles
Matter forms cell, one layer of mist NaOH is being sprayed automatically above, by uncrosslinked collagen gel；
e）In the collagen layer for being embedded in keratinocyte（4）The second multilayer collagen of upper printing（5）；
f）Above-mentioned printed skin is heated to 37 DEG C, further makes collagen gel, and make gelatin liquefaction on the contrary, by liquid
The gelatin of change is taken away, and mild medium flow field irrigation channel is used in combination, and HUVEC suspension is then injected in the channel of that layers of FB, fits
When stirring, so that cell is attached to the bottom and top surface in channel, ultimately form the engineering skin with layered structure.
Preferably, after the completion of printing, the engineering skin of printed layered structure is positioned in cell incubator,
It is cultivated in KC culture mediums；Culture medium is every other day replaced once.
Preferably, further include that the data for needing to print skin are obtained by mr imaging technique, to skin to be printed
Establish model.
More preferably, the data of skin that need to print include edge shape, surface area, depth, volume.
Disclosure sets forth 3D biometric print technologies are utilized, built according to skin wound depth, form scan three-dimensional vertical
Body repairing model goes out to be bonded the method for surface of a wound skin shape for patient's " making to measure ".While in order to improve cell in printing group
Interior survival rate is knitted, innovatively portion generates channel within the organization further through a kind of temporary material, to promote intercellular friendship
Stream, while injecting Human umbilical vein endothelial cells in channel and forming blood vessel, delivery rate is improved, therefore cell has higher life
Deposit ability and faster growth rate.
Whole process includes four scanning to skin wound, reconstruction, parsing and printing steps.
Before the printing, it is necessary first to have gained some understanding to skin wound, the first step is also that a step of most critical is exactly to wound
Face carries out Image Acquisition and biomimetic features modeling.Pass through magnetic resonance imaging (magnetic resonance imaging, MRI)
Technology obtains the critical data of the surface of a wound, including edge shape, surface area, depth, volume, is modeled to the surface of a wound with this.
Especially depth is most important, because can all find different Skin Cells in every layer of skin.
3-D view after reconstruction is parsed, the number of plies of printing skin is determined according to the depth of the surface of a wound.If only lacking
Epidermis then prints epidermis；If lacking skin corium and epidermis, the double-layer structure of skin is printed.These data indicate 3D
Biometric print machine needs which type of cell.
The shape information of 3D tissue complex to be printed is successively converted to two dimension（2D）Plane information, for generating printing
Program to build skin layer on demand.Coordinate and motion assignment parameter are converted into suitable control routine, to instruct 3D biological
Printer moves.
The organization bracket of printing is made of chemically crosslinkable collagen, for constituting the rack body of skin.
This natural polymer is similar to human cell's epimatrix, degradable in vivo.It is in acid condition liquid, and in neutrality
Or under mild alkaline conditions be gel, this phase transformation hydrogel is mild enough, be free of pungent, cell is not damaged.
The crosslinking agent this time selected is the NaOH solution of 1M, and the PH for adjusting collagen is allowed to gelation.Blood vessel access is using one kind
For temperature sensitive type material --- gelatin come what is generated, this is a kind of thermal reversion hydrogel.This material cures at room temperature, and works as temperature
Liquefaction becomes liquid when raising.Using this characteristic of gelatin, portion forms specific channel within the organization.When high temperature by its according to
Definite shape prints, and the gelatin after printing cures at room temperature, increases temperature after the completion of printing, and gelatin can liquefy,
Liquefaction gelatin is taken away, channel is formed.
The Skin Cell structure of layering is for cell necessary to normal skin function and cell and cell and extracellular base
The regeneration of matter interaction is vital.The method of the present invention is to use human skin fibroblasts（Fibroblasts, FB）
And keratinocyte（Keratinocyte, KC）The multilayer engineered tissue compound of composition carrys out the multilayered structure of simulated skin,
FB constitutes the skin corium of skin, and KC constitutes the epidermis of skin.In tube chamber in attachment Human umbilical vein endothelial cells（Human
Umbilical Vein Endothelial Cells, HUVEC）To promote the generation of blood vessel.
The hydrogel precursor of one layer of liquid is printed first at room temperature, then by ultrasonic transducer in automatic spray above
One layer of misty NaOH solution is crosslinked, and is formed hydrogel, is provided structural intergrity for subsequent printable layer, and be embedded in wherein
Cell.Heating gelatin solution as fluid channel expendable material is printed between collagen layer.The mistake is repeated in a manner of successively
Journey generates skin.Then the water-setting blob of printing is heated to 37 DEG C, gelatin is made selectively to liquefy and be discharged, in collagen branch
Hollow channel is generated in frame, is then deposited HUVEC in the skin corium channel of printing, is made it in tube chamber growth inside, gradual shape
At blood vessel.The artificial skin that printing is completed is placed in KC culture mediums and is cultivated, area to be repaired is then introduced.
Compared with prior art the invention has the advantages that：
1）The present invention completes the scanning to skin wound and reconstruction with MRI imaging techniques, and surface of a wound shape is more bonded to print
The skin of shape.Scan image is parsed automatically by analysis software, to determine the number of plies of printing skin, is had more accurate
With flexible advantage.
2）By using this temporary material of gelatin, innovatively portion generates blood vessel access to the present invention within the organization, logical
Adhere to Human umbilical vein endothelial cells in road, the blood vessel of formation more coincide with human vas, and the blood vessel of formation conveys distribution speed
It improves, the cell for forming blood vessel has higher viability and faster growth rate.It is molten to collagen using auto spraying technology
Liquid is crosslinked, and is that molding gel is more consistent uniformly.
3）The present invention prints skin using the structure of layering, between the cell in the skin texture of formation and skin histology
More stablize with epimatrix interaction, is conducive to the regeneration of skin histology.
4）Biological 3D printing and culture total time are short, skin can be disposably rebuild in clinical Wound treating, for hand
Rapid build organization engineering skin in the short time of art room.
5）The organization engineering skin of above-mentioned layered structure and natural skin degree of approach higher, have epidermis and skin corium,
Contain blood vessel in skin corium.Shape, size and the thickness of the organization engineering skin of layered structure are easily changed, can be close with wound
Fitting has good biocompatibility.
6）Above-mentioned organization engineering skin can enhance the skin elasticity after wound healing and flexibility, and organizational project shifting can be improved
Plant success rate.
Description of the drawings
Fig. 1 is the structural schematic diagram of the organization engineering skin of the present invention with layered structure.
Fig. 2 is the organization engineering skin preparation method flow chart of the present invention with layered structure.
Wherein 1 is collagen layer, and 2 be the collagen layer for being embedded in human skin fibroblasts, and 3 be the first multilayer glue
Former albumin layer, 4 be the collagen layer for being embedded in keratinocyte, and 5 be the second multilayer collagen layer.
Specific implementation mode
With reference to the embodiment technical solution that the present invention will be described in detail, but protection domain is not limited by this.
As shown in Figure 1, showing for a kind of structure for the example that the organization engineering skin with layered structure provides of the present invention
It is intended to, including skin corium and epidermis, is prepared by biological 3D printing technique by seed cell and biologic bracket material, group
Structure of the engineering skin with layering is knitted, layered structure is：By the sequence of skin inner layer to outer layer be followed successively by collagen layer 1,
It is embedded in the collagen layer 2 of human skin fibroblasts, the first multilayer collagen layer 3, is embedded in keratinocyte
Collagen layer 4, the second multilayer collagen layer 5.First multilayer collagen 3 is five layers of collagen, the second multilayer collagen
Albumen 5 is two layers of collagen.
As shown in Fig. 2, being organization engineering skin preparation method flow chart with layered structure, wherein material and cell
Prepare
The specific steps are：
1）Material prepares：
NaOH solution（1M）, ultra-pure water preparation, high-temperature sterilization.
Chemically crosslinkable collagen（3mg/ml）, being maintained at before printing in ice bath prevents from being crosslinked in advance.
Temperature sensitivity gelatin, for building the channel in hydrogel scaffold.
Human umbilical vein endothelial cells are cultivated in Endothelial Cell Growth Medium, and culture medium is replaced primary for every two days.
Keratinocyte and fibroblast are cultivated under the standard conditions of 2% fetal calf serum respectively, in FB culture mediums
FB growth additives are added, while adding in KC growth additive KC culture mediums.Pen .- Strep is also added into two kinds of trainings
It supports in base.Culture medium changes every other day.
Cell suspending liquid and collagen are placed in disposable syringe, each syringe is independently added using air tank
Pressure.
Four micro-valves are used to distribute collagen, gelatin, FB suspension and KC suspension in total.
2）Structural remodeling process：
Skin wound is scanned by MRI, obtains the critical datas such as surface area, shape, the depth of skin wound, next
Reconstruction is carried out to the surface of a wound.The 3-D view that counterweight is built up is analyzed, if only lacking epidermis, prints epidermis；If true
Cortex and epidermis then print the double-layer structure of skin.These data indicate that 3D biometric print machines need which type of cell.
Printing epidermis needs KC；Printing double-layer structure then needs two kinds of FB and KC.The data information of reconstruction is fed back into 3D biometric prints
Machine starts to print.
3）3D biometric print processes：
For printing 10 × 10 square millimeter zones, the double-layer structure of skin need to be printed.
NaOH neutralizers are atomized with ultrasonic transducer, are sprayed on substrate surface.
Uncrosslinked collagen layer 1 is printed upon on coating surface, the NaOH of atomization is sprayed on to the collagen of printing
Layer 1 on, before and after print every layer of collagen material, will by the mist of crosslinking agent automatically coat on the surface, with
Step will not be described in great detail afterwards.
The second layer prints one layer of collagen, then prints two in collagen layer surface layer both sides above with form of straight lines
Bar gelatin, gelatin cures at room temperature after 1-2 minutes, FB is deposited between two channels again after solidification, after having planted FB, upper
One layer of NaOH is sprayed in face again, by uncrosslinked collagen gel, forms the collagen layer 2 for being embedded in human skin fibroblasts.
Print 5 layers of collagen layer altogether on the collagen layer for be embedded in human skin fibroblasts.
One layer of collagen is printed on the surface of 5 layers of printed collagen layer, then above with form of straight lines
Collagen layer surface layer both sides print two gelatin, gelatin cures at room temperature after 1-2 minute, solidification later again two channels it
Between deposit KC, after having planted KC, spraying one layer of NaOH above, by uncrosslinked collagen gel, formation is embedded in fell
The fibroblastic collagen layer of skin 4.
Two layers of collagen layer is printed on the collagen layer 4 for being embedded in human skin fibroblasts, forms more than second
Layer collagen 5.
Above-mentioned printed skin is heated to 37 DEG C, to complete collagen gel, and makes gelatin liquefaction on the contrary, by liquid
The gelatin of change is taken away, and mild medium flow field irrigation channel is used in combination, and HUVEC suspension is then injected in the channel of that layers of FB, fits
When stirring, so that cell is attached to the bottom and top surface in channel, ultimately form the engineering skin with layered structure.
After the completion of printing, the engineering skin of printed layered structure is positioned in cell incubator, is cultivated in KC
It is cultivated in base；Culture medium is every other day replaced once.
The above content is combine specific preferred embodiment to the further description done of the present invention, and it cannot be said that
The specific implementation mode of the present invention is only limitted to this, for those of ordinary skill in the art to which the present invention belongs, is not taking off
Under the premise of from the present invention, several simple deduction or replace can also be made, all shall be regarded as belonging to the present invention by being submitted
Claims determine scope of patent protection.
Claims (8)
1. a kind of organization engineering skin with layered structure, including skin corium and epidermis, by seed cell and biological support
Material is prepared by biological 3D printing technique, which is characterized in that the seed cell includes the people for constituting corium
Skin fibroblasts, the keratinocyte for constituting epidermis, the Human umbilical vein endothelial cells for constituting blood vessel；It is described
Biologic bracket material include layered-scaffold material and vascular stent material, the layered-scaffold material is collagen, institute
The vascular stent material stated is gelatin；
Structure of the organization engineering skin with layering, the layered structure are：By the sequence of skin inner layer to outer layer
It is followed successively by collagen layer（1）, be embedded in the collagen layers of human skin fibroblasts（2）, the first multilayer collagen layer
（3）, be embedded in the collagen layer of keratinocyte（4）, the second multilayer collagen layer（5）.
2. a kind of organization engineering skin with layered structure according to claim 1, which is characterized in that described first
Multilayer collagen（3）For five layers of collagen, the second multilayer collagen（5）For two layers of collagen.
3. a kind of organization engineering skin with layered structure according to claim 1, which is characterized in that be embedded in fell
The fibroblastic collagen layer of skin（2）With blood vessel structure, it is embedded in the collagen layer of keratinocyte（4）Have
Luminal structure.
4. a kind of organization engineering skin with layered structure according to claim 1, which is characterized in that further include cell
Growth factor.
5. a kind of preparation method of the organization engineering skin with layered structure as described in claim 1, including biomaterial
Preparation, biological 3D printing step, which is characterized in that the biological 3D printing includes：
a）One layer of collagen layer is printed in substrate surface（1）；
b）In collagen layer（1）It is upper to print the collagen layer for being embedded in human skin fibroblasts（2）, described is embedded in
The collagen layer of human skin fibroblasts（2）It is first to print one layer of collagen layer, then in collagen layer surface
Both sides print two gelatin with form of straight lines, and gelatin cures at room temperature after 1-2 minutes, deposits people after solidification between gelatin
Skin fibroblasts are spraying one layer of mist NaOH by ultrasonic transducer, uncrosslinked collagen are being coagulated automatically above
Gelatinization；
c）In the collagen layer for being embedded in human skin fibroblasts（2）The first multilayer collagen of upper printing（3）；
d）In the first multilayer collagen layer（3）It is upper to print the collagen layer for being embedded in keratinocyte（4）, described is embedding
The collagen layer of keratinocyte is entered（4）It is first to print one layer of collagen layer, then in collagen layer surface
Both sides with form of straight lines print two gelatin, gelatin cures at room temperature after 1-2 minutes, after solidification between gelatin deposition angles
Matter forms cell, one layer of mist NaOH is being sprayed automatically above, by uncrosslinked collagen gel；
e）In the collagen layer for being embedded in keratinocyte（4）The second multilayer collagen of upper printing（5）；
f）Above-mentioned printed skin is heated to 37 DEG C, further makes collagen gel, and make gelatin liquefaction on the contrary, by liquid
The gelatin of change is taken away, and mild medium flow field irrigation channel is used in combination, and HUVEC suspension is then injected in the channel of that layers of FB, fits
When stirring, so that cell is attached to the bottom and top surface in channel, ultimately form the engineering skin with layered structure.
6. a kind of preparation method of organization engineering skin with layered structure according to claim 5, which is characterized in that
After the completion of printing, the engineering skin of printed layered structure is positioned in cell incubator, is cultivated in KC culture mediums；
Culture medium is every other day replaced once.
7. a kind of preparation method of organization engineering skin with layered structure according to claim 5, which is characterized in that
Further include that the data for needing to print skin are obtained by mr imaging technique, model is established to skin to be printed.
8. a kind of preparation method of organization engineering skin with layered structure according to claim 7, which is characterized in that
The data of skin that need to print include edge shape, surface area, depth, volume.
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| CN116077737A (en) * | 2023-04-07 | 2023-05-09 | 云南云科特色植物提取实验室有限公司 | Artificial skin containing vascular structure and preparation method thereof |
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