CN113667631A - Bracket for culturing in-vitro skin model and preparation method thereof - Google Patents
Bracket for culturing in-vitro skin model and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a stent for culturing an in vitro skin model and a preparation method thereof, the stent comprises a support layer, a nanofiber membrane positioned on the support layer and at least one closed ring-shaped structure positioned on the nanofiber membrane, wherein the support layer has a porous structure. The scaffold has good supporting strength and permeability, is beneficial to the formation and differentiation of skin tissues when used for culturing an in-vitro skin model, particularly can play a role in promoting the differentiation of skin keratinocyte in gas-liquid separation culture, and can be used as a substitute of a traditional Transwell chamber in the preparation of the in-vitro skin model.
Description
Technical Field
The invention belongs to the field of tissue engineering, and particularly relates to a stent for culturing an in-vitro skin model and a preparation method thereof.
Background
The safety evaluation of cosmetics and skin external drugs is usually carried out through animal models, and the defects of using the animal models mainly include large difference among model individuals, large difference between animal skin structures and human skin, long model preparation period, high cost consumption and the like. In addition, cosmetics and raw materials evaluated for safety by animal experiments have been completely banned from sale in the european union since 2009. Therefore, the use of in vitro skin models is becoming an effective and accurate means for evaluating the safety of cosmetics and skin-type drugs.
Currently, in vitro skin models are prepared by inoculating skin epidermal cells in a Transwell chamber and constructing the skin epidermal cells through gas-liquid separation culture. The Transwell cell is in great demand, and the Transwell cell cost needs special consideration when large-scale skin model preparation is carried out. Meanwhile, the size of the Transwell cell is generally a fixed value and cannot be changed as required, so the size of the skin model is limited to a certain extent.
The composite structure porous scaffold is widely used in tissue engineering, and is characterized in that the scaffold can provide a complex three-dimensional structure for simulating human tissues, and can provide powerful support under a microenvironment for activities such as cell adhesion, proliferation, migration, differentiation and the like through a spatial configuration and apertures and pores in the scaffold. Meanwhile, the micron and nano-scale fiber structures in the composite porous support can effectively realize the delivery of oxygen and nutrients, the discharge of cell metabolites and the like. Compared with the traditional two-dimensional cell culture system, the scaffold with the structure provides an environment which is closer to extracellular matrix in body tissues, so that biological tissues prepared in the structure are closer to normal human physiological tissues.
The cell culture scaffold with the composite three-dimensional porous structure can be prepared by using a fused deposition and solution electrospinning method. Fused deposition techniques may be used to prepare scaffolds with a lattice porous structure. The fused deposition technology generally uses a high polymer material, the material is heated and melted, then the fiber with the diameter ranging from 50 to 1000 μm is prepared in an extrusion mode, and a plurality of fiber filaments are arranged according to a certain arrangement to finally form a grid three-dimensional structure with a certain structure. The scaffold has excellent mechanical properties, can play a good role in supporting and structurally supporting skin tissues in an in-vitro skin model, but cannot provide a good microenvironment for cell attachment and gas-liquid exchange in terms of a cell plane.
For example, citation 1(CN109385393A) discloses a support structure for 3D printing in skin model construction, which includes a base layer and a heightening layer, wherein the heightening layer forms a groove around the edge of the base layer, the base layer includes multiple fiber layers fixedly connected together from bottom to top, each fiber layer includes multiple fiber filaments arranged in parallel, the fiber filaments of two adjacent fiber layers are arranged in a staggered manner, and the base layer forms an interwoven three-dimensional space network structure, so as to facilitate arrangement of other layer structures of the skin model. However, it takes a long time to form such a foundation layer by 3D printing, and the construction of the foundation layer is difficult to satisfy both requirements for supporting strength and porosity.
The solution electrostatic spinning technology generally prepares the polymer into nano-fibers with the diameter of 50-1000nm, and a porous structure bracket formed by nano-spinning can effectively provide adhesion support for cells, and simultaneously provides stable culture conditions for tissues in a gas-liquid separation culture stage in the in-vitro skin model culture process, thereby promoting the proliferation and differentiation processes of keratinocytes. However, the solution electrostatic spinning has the defect that the prepared nanofiber structure has weak mechanical property and cannot effectively support the skin tissue structure for a long time in the gas-liquid culture process required by the skin model.
Disclosure of Invention
Problems to be solved by the invention
In the prior art, the bracket prepared by a single method cannot well meet the requirements of the preparation of an in-vitro skin model and the application of the bracket in corresponding skin tissue engineering, and particularly has certain defects when replacing a Transwell chamber to culture the in-vitro skin model. Therefore, the development of a scaffold which can replace a Transwell chamber and is used for culturing an in-vitro skin model and has good supporting strength and permeability is a problem to be solved urgently.
Means for solving the problems
In order to solve the problems, the invention provides a bracket for culturing an in-vitro skin model and a preparation method thereof. Specifically, the present invention solves the problems of the present invention by the following means.
[1] A scaffold for culturing an in vitro skin model, comprising a support layer, a nanofibrous membrane on the support layer and at least one closed loop structure on the nanofibrous membrane, the support layer having a porous structure.
[2] The stent according to [1], wherein the support layer has a lattice structure, and the nanofiber membrane is an electrospun fiber membrane.
[3] The stent according to [1] or [2], characterized in that the height of the support layer is 1 to 10mm, the average pore size of the support layer is 100-1000 μm, and the support layer is formed of a polymer material.
[4] The scaffold according to [2], wherein the lattice-like structure is formed by fibers arranged in a staggered manner, and the diameter of the fibers is 100-1000 μm.
[5] The scaffold according to [1] or [2], wherein the nanofiber membrane has a thickness of 500nm to 100 μm, preferably 1 μm to 50 μm; the nanofiber membrane has a fiber diameter of 50-1000nm and is formed of a polymer material.
[6] The stent according to [1] or [2], wherein the closed loop structure has a height of 0.5 to 4mm, preferably 2 to 3 mm.
[7] The stent according to [1] or [2], comprising a plurality of the closed loop structures.
[8] The method for producing a stent according to any one of [1] to [7], which comprises the steps of:
(1) forming the support layer by fused deposition;
(2) forming the nanofiber membrane on the support layer by electrospinning, and
(3) forming the at least one closed loop structure on the nanofiber membrane by fused deposition.
[9] The production method according to [8], wherein the electrospinning is solution electrospinning or melt electrospinning, preferably solution electrospinning.
[10] The production method according to [9], wherein the solvent used for the solution electrospinning is water, ethanol, isopropanol, hexafluoroisopropanol, tetrahydrofuran, chloroform, dimethylformamide, or a mixture of two or more thereof.
[11] The preparation method according to [9], wherein in the step (3), the polymer is heated to above the melting temperature and extruded through the nozzle, so as to deposit the fiber filaments with the diameter of 100-.
[12] Use of the scaffold according to any one of [1] to [7] for culturing an in vitro skin model.
[13] The use according to [12], wherein the in vitro skin model is an in vitro skin model for skin toxicology testing.
[14] The use according to [13], characterized in that the skin toxicology test is a cosmetic safety monitoring assessment, a test for damage of a chemical agent to the skin or a toxicology test for the skin against harmful chemicals.
ADVANTAGEOUS EFFECTS OF INVENTION
The scaffold has good supporting strength and permeability, is beneficial to the formation and differentiation of skin tissues when used for culturing an in-vitro skin model, particularly can play a role in promoting the differentiation of skin keratinocyte in gas-liquid separation culture, and can be used as a substitute of a traditional Transwell chamber in the preparation of the in-vitro skin model.
Drawings
FIG. 1: a partial structural schematic of a stent according to one embodiment of the present invention.
FIG. 2: a stent according to one embodiment of the invention is schematically illustrated in longitudinal cross-section.
FIG. 3: schematic representation of the support layer in one embodiment of the invention.
FIG. 4: a schematic representation of a closed loop configuration in one embodiment of the invention.
FIG. 5: scanning electron micrographs of electrospun fibrous membranes in one embodiment of the invention.
FIG. 6: schematic illustration of the preparation of an in vitro skin model using the scaffold of the invention.
FIG. 7: photograph of one embodiment of the stent of the present invention.
Detailed Description
< terms and definitions >
In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.
In the present specification, the numerical ranges indicated by "above" or "below" mean the numerical ranges including the numbers.
In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
As used herein, the use of "optionally" or "optional" means that certain materials, components, performance steps, application conditions, and the like are used or not used.
In the present specification, the unit names used are all international standard unit names, and the "%" used means weight or mass% content, if not specifically stated.
Reference throughout this specification to "a preferred embodiment," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.
< Stent >
One object of the present invention is to provide a scaffold for the culture of an in vitro skin model, comprising a support layer, a nanofibrous membrane on the support layer and at least one closed loop structure on the nanofibrous membrane, the support layer having a porous structure.
The respective parts of the stent of the present invention will be described in detail below.
Supporting layer
The specific structure of the support layer in the present invention is not particularly limited as long as it is a porous structure that allows liquid to flow through the upper and lower surfaces of the support layer. The supporting layer has a porous structure, and can provide good supporting strength and good permeability when an in-vitro skin model is cultured, so that nutrients required by cells can smoothly pass through the supporting layer.
The average pore diameter of the support layer is 100-1000 μm, preferably 200-900 μm, more preferably 300-800 μm, and most preferably 400-700 μm. The porosity of the support layer is 40-80%.
In a particular embodiment, the support layer has a grid-like structure. The lattice-like structure is formed by interlacing of filaments. More specifically, the support layer comprises a plurality of fiber layers, each fiber layer comprises a plurality of fiber filaments which are parallel to each other, the fiber filaments of two adjacent fiber layers are staggered, and at least a part of the fiber filaments are bonded at staggered points, so that a grid-shaped structure is formed. The staggered arrangement means that the fiber filaments of two adjacent fiber layers form an angle of more than 0 degrees and less than 180 degrees, preferably form an angle of 30-150 degrees, more preferably form an angle of 45-135 degrees, even more preferably form an angle of 60-120 degrees, and most preferably form an angle of 80-100 degrees. In a particular embodiment, the filaments of two adjacent fibrous layers are at 90 ° to each other.
In this embodiment, the fiber filament has a diameter of 100-1000 μm, preferably 150-800 μm, and more preferably 200-500 μm. In each fiber layer, the distance between adjacent fiber filaments is 100-.
In the present invention, the size of the support layer can be set as desired. In one embodiment, the height of the support layer is from 1 to 10mm, preferably from 2 to 7mm, more preferably from 3 to 6 mm. The volume of the support layer is 5 × 5 × 1 to 30 × 30 × 10, preferably 5 × 5 × 2 to 20 × 20 × 5, in length × width × height (mm).
The support layer is formed of a polymer material, preferably a polymer material having biocompatibility, and specific examples include, but are not limited to, Polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), L-polylactic acid (PLLA), polylactic-glycolic acid copolymer (PLGA), polyethylene glycol (PEG), or a mixture/copolymer of two or more of the above materials.
Nanofiber membranes
The nanofiber membrane is located on a support layer, which is preferably an electrospun fiber membrane. The electrospun fiber membrane is formed by stacking nanometer-scale fiber filaments, can effectively increase the contact area between cells and materials, provides space for cell proliferation and provides good oxygen and nutrient substance transfer and delivery for the cells.
The thickness of the nanofiber membrane is 500nm-100 μm, preferably 1 μm-50 μm, more preferably 2 μm-20 μm, and most preferably 3 μm-10 μm. The nanofiber membrane has a fiber diameter of 50-1000nm, preferably 100-900nm, more preferably 200-800nm, even more preferably 400-600nm, and most preferably 500 nm. The nanofiber membrane has a porosity of 40-95%, preferably 50-70%, and a pore size of 1-50 μm, preferably 1-10 μm. The size of the nanofiber layer may be set according to the size of the support layer.
The nanofiber membrane is formed of a polymeric material, preferably a biocompatible polymeric material, examples of which include, but are not limited to, Polycaprolactone (PCL), polylactic acid (PLA), L-polylactic acid (PLLA), polyglycolic acid (PGA), polylactic-glycolic acid copolymer (PLGA), Polyurethane (PU), polyethylene glycol (PEG), polyethylene glycol-b-poly (L-lactide-co-caprolactone) (PELCL), chitosan, gelatin, collagen, or a mixture of two or more thereof.
Closed ring structure
The stent of the invention comprises at least one closed ring structure positioned on the nanofiber membrane, wherein the closed ring structure forms a groove in the central area of the closed ring structure, for example, the closed ring structure and the nanofiber membrane at the bottom form a groove structure together to provide a space for culturing an in-vitro skin model.
In one embodiment, the stent of the present invention comprises a plurality of closed loop structures, each of which is located on the nanofiber membrane, and the central region of each closed loop structure can independently serve as a space for accommodating an in vitro skin model. The plurality of closed loop structures may be disposed on the nanofiber membrane in any manner. In a particular embodiment, a plurality of closed loop structures are spaced apart. Wherein spaced apart means that there is a distance between the two closed loop structures. In another embodiment, a plurality of closed loop structures are disposed contiguously, wherein contiguously means that at least a portion of a closed loop structure is bonded to at least a portion of another closed loop structure, or two closed loop structures share a portion of a structure, e.g., a plurality of closed loop structures are disposed in a side-by-side rectangular pattern on a nanofiber membrane. In another embodiment, a portion of the plurality of closed loop structures are disposed apart and another portion are disposed adjacent.
The height of the closed loop structure is between 0.5 and 4mm, preferably 2 to 3 mm. The height of the closed loop structure refers to the maximum of the heights of the plurality of closed loop structures. The peripheral dimension (length × width × height) of each closed loop structure may be set as required, and may be, for example, 10mm × 10mm × 0.5mm to 20mm × 20mm × 4 mm. When a plurality of closed loop structures are included, the dimensions of the individual loop structures may be the same or different.
The closed loop structure is formed of a polymeric material, preferably a biocompatible polymeric material, and specific examples include, but are not limited to, Polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), L-polylactic acid (PLLA), polylactic-glycolic acid copolymer (PLGA), polyethylene glycol (PEG), or a mixture/copolymer of two or more thereof.
In one embodiment, the closed loop structure is formed by a stack of fiber filaments of a polymeric material, wherein the fiber filaments have a diameter of 100-. The wall of the closed loop structure may be formed by a single layer of adjacently arranged filaments or a plurality of layers of adjacently arranged filaments, whereby the wall thickness of the closed loop structure is larger than or equal to the diameter of the filaments.
The scaffold has better mechanical strength and proper pore diameter and porosity, can promote the transportation of nutrient substances of a culture solution required in the culture process of an in-vitro skin model, provides a proper microenvironment for cells in an epidermal layer and a dermal layer of the model, promotes cell proliferation, migration, differentiation and the like.
One specific embodiment of the stent of the present invention is described in detail below with reference to the accompanying drawings. It should be noted that the drawings in the present invention are only schematic, and the size and the ratio of the parts should not be considered as limiting the stent of the present invention.
As shown in fig. 1, a stent 1 for culturing an in vitro skin model comprises a support layer 2, an electrospun fiber membrane 4 on the support layer 2, and a closed loop structure 3. As shown in fig. 2, the support layer 2 is positioned at the bottom, the electrospun fiber membrane 4 is positioned on the support layer 2, and the closed loop structure 3 is positioned on the electrospun fiber membrane 4. As shown in fig. 3, the support layer 2 has a lattice-like structure formed by staggered fibres 5. As shown in fig. 4, in one embodiment, the closed loop structure 3 is rectangular.
< preparation method >
It is another object of the present invention to provide a method for preparing the stent of the present invention, comprising the steps of:
(1) forming the support layer by fused deposition;
(2) forming the electrospun fiber membrane on the support layer by electrospinning, and
(3) forming said at least one closed loop structure on said electrospun fibrous membrane by melt deposition.
The individual steps of the process of the invention are described in detail below.
Step (1)
Fused deposition modeling as used in the present invention is one of the 3D printing techniques, and in the method of the present invention, fused deposition is performed in a conventional manner, and for specific process parameters, one skilled in the art can select depending on factors such as the particular equipment used, the polymer material, and the desired size of the support layer.
In a particular embodiment, the polymer is heated above the melt temperature and extruded through a spray head and then deposited on a support plate to form the fibers in the support layer. The fibers in the supporting layer are formed one by one and layer by changing the movement track of the spray head. Wherein the diameter of the fibers and the spacing between the fibers are as described above.
Step (2)
In the invention, the electrospun fiber membrane is formed on the supporting layer by an electrospinning method, wherein the electrospinning can be solution electrospinning or melt electrospinning, and is preferably solution electrospinning. In the process of the present invention, electrospinning is carried out in a conventional manner and, for specific process parameters, one skilled in the art can select depending on factors such as the particular equipment used, the polymeric material and the desired electrospun fibrous membrane.
In a specific embodiment, the electrospun fiber membrane is formed on the support layer using a solution electrospinning process. Specifically, a polymer is dissolved in a solvent to form a polymer solution, the polymer solution is subjected to jet spinning in a strong electric field, and the resulting polymer filaments are deposited on a support layer to form an electrospun fiber membrane.
In this embodiment, the solvent used is water, ethanol, isopropanol, hexafluoroisopropanol, tetrahydrofuran, chloroform, dimethylformamide, or a mixture of two or more thereof.
Step (3)
And (3) forming at least one closed annular structure on the electrospun fiber membrane by using fused deposition forming. For fused deposition, reference is made to the detailed description above.
In a specific embodiment, the polymer is heated to above the melting temperature and extruded through a nozzle, so that the fiber filaments with the diameter of 100-.
< use >
It is another object of the invention to provide the use of the scaffold of the invention in culturing an in vitro skin model. The cultured in vitro skin model can be used for skin toxicology detection, including but not limited to cosmetic safety monitoring evaluation, damage detection of chemical agents to skin, toxicology detection of skin against harmful chemicals, and the like.
An exemplary embodiment of culturing an in vitro skin model using the scaffold of the present invention is described below. This embodiment comprises the steps of:
step 1: preparation: GelMa solution;
step 2: adding a photoinitiator solution into a GelMa solution to obtain a photo-curable GelMa hydrogel precursor solution;
and step 3: uniformly mixing human skin fibroblasts with the GelMa hydrogel precursor solution to obtain GelMa/cell suspension;
and 4, step 4: adding the GelMa/cell suspension into the closed ring structure of the scaffold, namely pouring the GelMa/cell suspension on the electrospun fiber membrane;
and 5: performing photocuring crosslinking on the GelMa/cell suspension structure poured in the step (4) to obtain hydrogel containing fibroblasts, namely the dermis layer structure of the in-vitro skin model;
step 6: immersing the scaffold containing the dermis structure of the in-vitro skin model in the step (5) in a fibroblast culture solution, and then putting the scaffold into an incubator for culturing to proliferate fibroblasts to form a dermis tissue of the in-vitro skin model;
and 7: inoculating human skin keratinocytes to the surface of the dermal tissue layer of the in vitro skin model formed in the step (6), immersing the human skin keratinocytes in a keratinocyte culture medium, and culturing the human skin keratinocytes in a cell culture box to proliferate the inoculated keratinocytes;
and 8: removing the culture medium on the surface of the keratinocyte layer in the step (7) and enabling only the supporting layer of the scaffold to be in contact with the culture medium, exposing the electrospun fiber membrane and the upper part of the electrospun fiber membrane to the air, and carrying out gas-liquid separation culture on the keratinocyte layer in such a way, and finally obtaining the in vitro skin model with the mature cuticle layer.
The above process of culturing the in vitro skin model should be operated in a clean bench to ensure aseptic conditions, and the required raw materials should also ensure sterility.
Fig. 6 is a schematic view of the preparation of an in vitro skin model using the scaffold of the present invention, which corresponds to the state in step 7 described above. Wherein the dermal layer tissue formed by proliferation of the human skin fibroblasts 6 is positioned on the electrospun fibrous membrane 4 and confined in the recess formed in the central region of the closed annular structure 3, the human skin keratinocytes 7 are seeded on the surface of the dermal layer tissue, which is also confined in the recess formed in the central region of the closed annular structure 3, the scaffold of the invention is placed in the keratinocyte medium 8 and the keratinocyte medium 8 is not passed over the human skin keratinocytes 7.
Examples
The stent of the present invention will be specifically described below with reference to examples.
Example 1
(1) Forming a support layer: polycaprolactone (PCL) was melt extruded into 250 μm diameter filaments using a BioMaker4 bio 3D printer (shangpobo source (beijing) biotechnology limited); and extruding the fiber filaments at intervals of horizontal and vertical directions by changing the motion track of the printing nozzle, thereby forming the supporting layer with a latticed structure. The dimensions of the support layer were 20mm by 2.5mm, with a filament pitch of 500 μm.
(2) Forming an electrospun fiber membrane: polycaprolactone (PCL) solution (solvent Hexafluoroisopropanol (hexafluo) was electrospun onto the surface of the support layer by solution electrospinning using a BioMaker4 bio 3D printer (available from tokyo biotechnology limited, jet size 21G). The electric field intensity is 10kV, the thickness of the electro-spun fiber membrane is 3 μm, the pore diameter is 3 μm, and the porosity is 60%.
(3) Forming a closed ring structure: and (3) melting and extruding Polycaprolactone (PCL) into a fiber filament with the diameter of 250 mu m on the electrospun fiber membrane obtained in the step (2) by using a BioMaker4 biological 3D printer (Shangpobo Yuan (Beijing) Biotechnology Co., Ltd.), and constructing a closed annular structure by changing the movement track of a printing nozzle. The closed loop configuration has peripheral dimensions of 15mm by 2mm (length by width by height) and a wall thickness of 250 μm.
It is specifically noted that those skilled in the art and related fields can make various modifications and changes or rich application fields of the present invention within the scope of the present invention based on the description herein, and the modifications and changes or application fields in other fields are also included in the scope of the present invention.
Industrial applicability
The bracket of the invention can be widely used for culturing in-vitro skin models, and the cultured in-vitro skin models can be used for skin toxicology detection.
Claims (14)
1. A scaffold for culturing an in vitro skin model, comprising a support layer, a nanofibrous membrane on the support layer and at least one closed loop structure on the nanofibrous membrane, the support layer having a porous structure.
2. The stent of claim 1 wherein the support layer has a lattice structure and the nanofiber membrane is an electrospun fiber membrane.
3. The stent according to claim 1 or 2, wherein the height of the support layer is 1-10mm, the average pore size of the support layer is 100-1000 μm, and the support layer is formed of a polymer material.
4. The scaffold according to claim 2, wherein the lattice-like structure is formed by staggered fibers having a diameter of 100-1000 μm.
5. A scaffold according to claim 1 or 2, wherein the nanofibrous membrane thickness is 500nm-100 μ ι η, preferably 1 μ ι η -50 μ ι η; the nanofiber membrane has a fiber diameter of 50-1000nm and is formed of a polymer material.
6. A support according to claim 1 or 2, wherein the closed loop configuration has a height of 0.5-4mm, preferably 2-3 mm.
7. A support according to claim 1 or 2, comprising a plurality of said closed loop formations.
8. Method for the preparation of a scaffold according to any of claims 1-7, comprising the steps of:
(1) forming the support layer by fused deposition;
(2) forming the nanofiber membrane on the support layer by electrospinning, and
(3) forming the at least one closed loop structure on the nanofiber membrane by fused deposition.
9. The method of claim 8, wherein the electrospinning is solution electrospinning or melt electrospinning, preferably solution electrospinning.
10. The method according to claim 9, wherein the solvent used for electrospinning of the solution is water, ethanol, isopropanol, hexafluoroisopropanol, tetrahydrofuran, chloroform, dimethylformamide, or a mixture of two or more thereof.
11. The preparation method according to claim 9, wherein in the step (3), the polymer is heated to a temperature above the melting temperature and extruded through the nozzle, so as to deposit the fiber filaments with the diameter of 100-.
12. Use of a scaffold according to any of claims 1-7 in culturing an in vitro skin model.
13. Use according to claim 12, wherein the in vitro skin model is an in vitro skin model for skin toxicology testing.
14. The use according to claim 13, wherein the skin toxicology test is a cosmetic safety monitoring assessment, a test for damage to the skin by a chemical agent, or a toxicology test for the skin against a harmful chemical.
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