CN112675360A - Preparation and application of hADSCs-loaded double-layer skin bionic hydrogel composite scaffold - Google Patents
Preparation and application of hADSCs-loaded double-layer skin bionic hydrogel composite scaffold Download PDFInfo
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Abstract
The invention discloses preparation and application of a hADSCs-loaded double-layer skin bionic hydrogel composite scaffold. The composite scaffold takes a gelatin electrostatic spinning fibrous membrane as a substrate, fat stem cell suspension is added for adhesion, then gelatin photo-crosslinking solution is added, and cylindrical gelatin hydrogel is prepared through photo-crosslinking, namely the composite scaffold. The composite scaffold has a double-layer structure, wherein the fibrous membrane can block the invasion of foreign matters and pathogens and prevent tissue fluid loss; the cylindrical gelatin not only ensures the loading capacity, the viability and the adhesion of the hADSCs, but also promotes the migration and the proliferation of the hADSCs and synergistically promotes the regeneration and the formation of skin tissues; and the unique shape of the composite scaffold ensures that the composite scaffold has good fit with the wound, has good air permeability, is beneficial to the transportation of oxygen and nutrient substances of the wound, can also promote the generation of blood vessels, new granulation tissues and collagen at the wound, has a degradation period close to the wound healing period, and is beneficial to the healing of the wound.
Description
Technical Field
The invention relates to the technical field of biological tissue scaffolds, and particularly relates to preparation and application of a hADSCs-loaded double-layer skin bionic hydrogel composite scaffold.
Background
As the most important barrier to the human body, the skin is often subjected to acute and chronic injuries, particularly difficult to heal wounds, which severely affect the quality of life of the patient. Stem cell transplantation has become an effective research direction for these intractable wounds that cannot be healed by various surgical methods. As one of the adult stem cells, adipose stem cells play an indispensable role in repairing skin wounds than other stem cells because they have an immune compatibility advantage. Based on a large number of animals and clinical tests, the adipose-derived stem cells have a larger breakthrough in the field of skin wound repair in the future, particularly in the field of chronic refractory wounds.
Direct injection of stem cells into the repair site minimizes the invasiveness of the procedure. However, in this way, the fusion degree of cells and host tissues is low, and the low cell survival rate is still a main obstacle to the success of clinical transplantation. The microenvironment for the survival of cells in vivo is mostly a nano-scaffold structure formed by collagen fibers and the surfaces of other cells, and besides protein is an important factor for regulating the vital activities of cells, the nano-scaffold structure is another important factor. The nanofiber prepared by electrostatic spinning is beneficial to the implantation of cells, the adhesion, the infiltration of nutrient substances and the discharge of metabolic waste, and provides a good microenvironment for the growth and the proliferation of the cells, so that the functions of adhesion, migration, proliferation and differentiation of stem cells can be enhanced. The electrostatic spinning nanofiber has a superior effect in the aspect of promoting cell growth.
After the nanofiber membrane is spun to a certain thickness, the pore diameter inside the membrane is reduced, and infiltration and growth of cells are limited. For most tissue engineering scaffolds, the depth of cellular infiltration is critical. The wound surface which is difficult to heal is larger, the tissue infiltration of the lamellar structure is poor, the survival and the function of cells are influenced, the wound surface repair promotion capability is poor due to the lack of sufficient oxygen and nutrition supply, and the success rate of clinical transplantation is reduced. In addition, limited interfacial interactions between cells and hydrogel materials limit tissue ingrowth. By adjusting parameters of electrostatic spinning, such as spinning voltage, volume and the like, the fiber membrane suitable for cell adhesion and proliferation is prepared. Therefore, the electrostatic spinning fiber membrane is an excellent carrier of the adipose-derived stem cells,
however, in the existing dressing, a larger lifting space still exists in the aspects of the loading capacity and the adhesiveness of adipose-derived stem cells, the air permeability, the fitting degree, the nutrient transportation, the wound healing and the like of the dressing.
Disclosure of Invention
The invention aims to provide a double-layer skin bionic hydrogel composite scaffold loaded with hADSCs (human chorionic gonadotrophin) aiming at the defects and defects of wound dressings in the prior art in the aspects of single function, biocompatibility, degradability, cell adhesion, loading capacity, air permeability, degree of adhesion with wounds, nutrient substance transportation and wound healing. The hydrogel composite scaffold with the double-layer skin structure is prepared by adopting an electrostatic spinning and photo-crosslinking method, wherein a nanofiber membrane prepared by performing electrostatic spinning on gelatin with good biocompatibility has a structure similar to that of an extracellular matrix, can simulate the extracellular structure to the maximum extent, and provides a basis for the adhesion and proliferation of hADSCs; the photo-crosslinking gelatin forms columnar hydrogel with a certain interval on the nanofiber membrane, so that the composite scaffold material has good air permeability and larger porosity and pore diameter, is beneficial to the transportation of oxygen and nutrient substances of wounds and hADSCs, improves the generation of blood vessels of wound surfaces, promotes the generation of new granulation tissues and collagen, and is beneficial to the recovery of the wounds.
The invention also aims to provide the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold.
The invention further aims to provide application of the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold in preparation of dressings.
The invention also aims to provide application of the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold in drug loading.
The above object of the present invention is achieved by the following scheme:
a composite bracket loaded with hADSCs double-layer skin bionic hydrogel is characterized in that a gelatin electrostatic spinning fiber membrane is used as a substrate, fat stem cell suspension is added, after cells are attached to the gelatin electrostatic spinning fiber membrane, gelatin photocrosslinking solution is added, and then the cells are photocrosslinked to prepare cylindrical gelatin hydrogel, namely the hADSCs double-layer skin bionic hydrogel composite bracket.
Gelatin is a derived protein obtained by hydrolyzing collagen by an acid method or an alkali method, and is an amphoteric high-molecular polyelectrolyte consisting of 18 amino acids, which has similar composition and property with collagen. The gelatin has no antigenicity, excellent biocompatibility, degradability and cell adhesion, and may be used widely in blood swelling agent, hemostatic, wound treatment, tissue engineering and other fields. But its direct application as a dressing is too mechanical and degrades too rapidly, resulting in limited application. The invention generates the three-dimensional photo-crosslinking gelatin patterned scaffold (namely gelatin electrospinning and cross-linking cylindrical gelatin hydrogel) with adjustable physical and biological characteristics by using a photo-crosslinking/electrospinning method in combination, and the patterned gelatin scaffold not only can support cell viability and cell adhesion, but also can promote cell migration and proliferation and promote regeneration and formation of skin tissues. The nanofiber structure like ECM (extracellular matrix) provides high surface to volume ratio, not only can achieve maximal cell-material interaction and material-mediated signaling, but also can promote rapid vascularization.
The gelatin electrospun fiber membrane of the composite scaffold is equivalent to the epidermis layer of the skin, can block the invasion of foreign matters and pathogens, and can prevent the loss of interstitial fluid. In the environment of simulating the wounds of diabetes, after the patterned gelatin (namely the cylindrical gelatin) is degraded, the gelatin electrostatic spinning fiber membrane can be maintained for a long time, so that the wounds are further protected, and the defect of rapid degradation of the gelatin is overcome. Meanwhile, the patterned gelatin improves the specific surface area, can be well attached to the skin, and simultaneously avoids secondary injury of wounds. In addition, in the preparation process of the composite support, the physical properties of the hydrogel, including water retention, degradability and the like, can be adjusted by adjusting the photocrosslinking time and the aperture size of the mask, so that the composite support is suitable for the requirements of different patients.
The composite scaffold has excellent loading capacity and adhesiveness of hADSCs, and has cylindrical gelatin columns, so that the composite scaffold has high degree of fit with a wound surface, and has good air permeability, thereby facilitating the transportation of oxygen and adipose-derived stem cells to the wound, and facilitating the healing of the wound surface.
At present, a composite stent with a similar double-layer structure is disclosed, but the fiber membrane in the prior art is completely different from the composite stent in the application, and in order to ensure the mechanical property of the fiber membrane, the fiber membrane is usually obtained by spinning materials such as polycaprolactone or polyvinyl alcohol and the like, although the mechanical property of the whole composite stent is improved, the biocompatibility and the degradability of the fiber membrane are poor, and when the fiber membrane is applied as a dressing, the aspects of the adhesiveness of the fiber membrane to a wound, the air permeability and the like are obviously inferior to those of the composite stent prepared by gelatin electrostatic spinning fibers in the application.
In addition, the method adopts a photo-crosslinking mode to crosslink cylindrical gelatin on the gelatin electrostatic spinning fiber membrane, on one hand, the process is simple, the conditions are not harsh, and the method is suitable for the composite scaffold to load living cells; on the other hand, the cylindrical gelatin hydrogel increases the air permeability of the composite scaffold on the premise of ensuring good fit between the composite scaffold and the wound, and is convenient for the composite scaffold to convey loaded living cells, other medicines or nutrient substances and the like to the organism.
Although the gelatin electrospun fiber membrane is frequently used as a dressing in the prior art, the gelatin electrospun fiber membrane is usually used as a carrier to load other beneficial factors (such as vitamins, nano silver or other antibacterial substances), but the gelatin electrospun fiber membrane is rarely used for preparing the fiber membrane by alone and is less frequently used for preparing the dressing, and the gelatin electrospun fiber membrane is also prepared into a composite scaffold of the double-layer skin structure.
Preferably, the diameter of the cylindrical gelatin hydrogel is 600-800 μm, and the diameter can be adjusted by controlling the size of the aperture of the mask according to the requirement; the thickness is 300-600 μm.
More preferably, the cylindrical gelatin hydrogel has a diameter of 600 μm and a thickness of 300 μm.
Preferably, the gelatin electrostatic spinning fiber membrane is prepared by taking gelatin as a raw material, spinning by using electrostatic spinning equipment to obtain a fiber membrane, and then placing the obtained fiber membrane in a genipin-alcohol solution for crosslinking and curing. In order to ensure that the gelatin fiber membrane is formed and solidified, when the genipin is used for crosslinking reaction, a genipin-alcohol solution is required instead of a genipin-water solution.
Preferably, when the gelatin electrospun fiber membrane is spun, the voltage is 14-18kV, the distance from a needle to a receiving device is 18-22cm, and the advancing speed is 16-18 muL/min.
More preferably, when the gelatin electrospun fiber membrane is spun, the voltage is 16kV, the distance from the needle to the receiving device is 20cm, and the advancing speed is 16.7 muL/min.
Preferably, the genipin-alcohol solution has a genipin mass concentration of 1% -2%. More preferably, the genipin is present at a mass concentration of 1%.
Preferably, the crosslinking and curing process of the fiber membrane is as follows: and (3) placing the glass slide containing the fiber membrane into a genipin-alcohol solution to submerge the fiber membrane, placing the glass slide in an environment with the temperature of 4-8 ℃, standing for 24-48h, then transferring the glass slide to an environment with the temperature of 32-37 ℃, standing for 36-48h, and finally cleaning the glass slide by using an alcohol solution to obtain the glass slide.
More preferably, the fiber membrane crosslinking and curing process is as follows: and (3) placing the glass slide containing the fiber membrane into a genipin-alcohol solution to submerge the fiber membrane, placing the glass slide in an environment with the temperature of 4 ℃ for standing for 24 hours, then transferring the glass slide to an environment with the temperature of 37 ℃ for standing for 36 hours, and finally cleaning the glass slide by using an alcohol solution to obtain the glass slide.
Preferably, the alcohol solution used for cleaning has a concentration of 75-90% by volume; more preferably, the alcohol concentration is 90% by volume.
Preferably, the preparation process of the gelatin electrospun fiber membrane comprises the following steps: dissolving gelatin in hexafluoroisopropanol to prepare an electrostatic spinning solution, then adopting an 8# flat head needle head (length is 20cm) as a positive high-pressure injection head of electrostatic spinning equipment under the conditions of room temperature and humidity lower than 50%, adjusting voltage to be 16kV, enabling the distance from the needle head to a receiving device to be 20cm, enabling the propelling speed to be 16.7 mu L/min, and enabling the volume of the electrostatic spinning solution to be 320 mu L; aluminum foil paper is laid on a self-assembled platform with the size of 10cm multiplied by 10cm, and a plurality of 15mm glass slides are placed for collecting spinning fiber membranes.
Preferably, the mass volume concentration of the gelatin in the electrostatic spinning solution is 0.1-0.15 g/mL; more preferably, the mass volume concentration of gelatin is 0.1 g/mL.
Preferably, in the adipose-derived stem cell suspension, the number of cells is 1 × 104～3×104A plurality of; more preferably, the number of cells is 1.5X 104And (4) respectively.
Preferably, the preparation process of the adipose-derived stem cell suspension comprises the following steps: hADSCs (human adipose-derived stem cells) are expanded in vitro to P3-P4 generation, cells in a culture flask are washed twice by PBS, digested by pancreatin, counted, centrifuged and added with culture medium for resuspension.
Preferably, the gelatin photo-crosslinking solution is: the composition is prepared from a 10-30% mass concentration Type A gelatin PBS solution, a bipyridyl ruthenium PBS solution, a sodium persulfate PBS solution and a 0.05-0.2% mass concentration carmine PBS solution according to a volume ratio of 8:1:1: 6.
Preferably, the mass concentration of the Type A gelatin PBS solution is 30 percent.
Preferably, the concentration of solute in the bipyridyl ruthenium PBS solution is 16-32 mM; the concentration of solute in the sodium persulfate PBS solution is 160-320 mM.
Preferably, the photo-crosslinking is performed in the presence of a PDMS mold and a mask by: placing the gelatin electrostatic spinning fiber membrane in a PDMS (polydimethylsiloxane) mold, adding the gelatin photo-crosslinking solution, covering a mask, performing a crosslinking reaction by illumination, adding PBS (phosphate buffer solution) to dissolve an uncrosslinked part (the gelatin solution which does not generate the photo-crosslinking effect is well dissolved in the PBS solution at 37 ℃, so that the process is performed at 37 ℃), finally soaking in genipin-buffer solution for further curing, and cleaning to obtain the double-layer skin structure hydrogel composite scaffold.
Preferably, the lighting conditions are: the light source is cold light source LED white light, the illumination time is 8-20 min, and the light source distance is 7-20 cm.
Preferably, the genipin-PBS solution has a genipin volume ratio of 0.5%; the PBS solution was at pH 7.4.
The invention also provides a preparation method of the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold, which comprises the following steps:
s1, preparing a gelatin electrostatic spinning fiber membrane: dissolving gelatin in hexafluoroisopropanol to prepare an electrostatic spinning solution, then preparing the electrostatic spinning solution by electrostatic spinning equipment at room temperature and under the condition that the humidity is lower than 50%, collecting a spinning fiber membrane, performing crosslinking curing in a genipin-alcohol solution, and cleaning to obtain a gelatin electrostatic spinning fiber membrane;
s2, preparing a PDMS mold and a mask: preparing an upper layer mold and a lower layer mold in required shapes by adopting a PDMS mold product; the mask is a PET film, patterns of the PET film can be designed according to needs, and then the gelatin electrostatic spinning fiber film prepared by S1 is placed in a PDMS upper layer die;
s3, loading adipose-derived stem cells: after the adipose-derived stem cells are cultured in an adherent manner, adding cell pancreatin for digestion, counting and centrifuging, adding a cell culture medium for heavy suspension, dropwise adding the cell culture medium onto a gelatin electrostatic spinning fibrous membrane, and standing;
s4, photo-crosslinking: preparing a photo-crosslinking solution, adding the photo-crosslinking solution after the adipose-derived stem cells are attached to the gelatin electrostatic spinning fiber membrane, standing, covering a mask, and performing a crosslinking reaction by illumination;
and S5, after the photocrosslinking reaction is finished, adding PBS to dissolve the uncrosslinked part at 37 ℃, finally soaking the part in a genipin-buffer solution for further curing, and cleaning to obtain the hydrogel composite bracket with the double-layer skin structure.
Preferably, in step S1, the gelatin electrospun fiber membrane is spun at a voltage of 14-18kV, a distance from the needle to the receiving device of 18-22cm, and a forwarding speed of 16-18 μ L/min.
More preferably, when the gelatin electrospun fiber membrane is spun, the voltage is 16kV, the distance from the needle to the receiving device is 20cm, and the advancing speed is 16.7 muL/min.
Preferably, in step S3, the number of cells in the adipose stem cell suspension is 1 × 104～3×104A plurality of; more preferably, the number of cells is 1.5X 104And (4) respectively.
Preferably, the preparation process of the adipose-derived stem cell suspension comprises the following steps: hADSCs (human adipose-derived stem cells) are expanded in vitro to P3-P4 generation, cells in a culture flask are washed twice by PBS, digested by pancreatin, counted, centrifuged and added with culture medium for resuspension.
Preferably, in step S4, the photo-crosslinking solution is: the composition is prepared from a 10-30% Type A gelatin PBS solution, a bipyridyl ruthenium PBS solution, a sodium persulfate PBS solution and a 0.05-0.2% carmine PBS solution according to a volume ratio of 8:1:1: 6.
Preferably, the mass concentration of the Type A gelatin PBS solution is 30 percent.
Preferably, the concentration of solute in the bipyridyl ruthenium PBS solution is 16-32 mM; the concentration of solute in the sodium persulfate PBS solution is 160-320 mM.
Preferably, the crosslinking and curing process of the fiber membrane is as follows: and (3) placing the glass slide containing the fiber membrane into a genipin-alcohol solution to submerge the fiber membrane, placing the glass slide in an environment with the temperature of 4-8 ℃, standing for 24-48h, then transferring the glass slide to an environment with the temperature of 32-37 ℃, standing for 36-48h, and finally cleaning the glass slide by using an alcohol solution to obtain the glass slide.
More preferably, the fiber membrane crosslinking and curing process is as follows: and (3) placing the glass slide containing the fiber membrane into a genipin-alcohol solution to submerge the fiber membrane, placing the glass slide in an environment with the temperature of 4 ℃ for standing for 24 hours, then transferring the glass slide to an environment with the temperature of 37 ℃ for standing for 36 hours, and finally cleaning the glass slide by using an alcohol solution to obtain the glass slide.
Preferably, the process of step S4 is: and (2) placing the gelatin electrostatic spinning fiber membrane in a PDMS (polydimethylsiloxane) mould, adding the gelatin photo-crosslinking solution after the adipose-derived stem cells are attached to the gelatin electrostatic spinning fiber membrane, covering a mask, performing crosslinking reaction by illumination, adding PBS (phosphate buffer solution) to dissolve the part which is not crosslinked at 37 ℃, finally soaking in a genipin-buffer solution for further curing, and cleaning to obtain the hydrogel composite scaffold with the double-layer skin structure.
Preferably, in step S4, the lighting conditions are: the light source is cold light source LED white light, the illumination time is 8-20 min, and the light source distance is 7-20 cm.
The application of the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold in preparation of wound dressings is also within the protection scope of the invention.
The hADSCs-loaded double-layer skin bionic hydrogel composite scaffold is used as a carrier, and the application of the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold in loading cells and/or medicines is also within the protection scope of the invention.
More preferably, the cells are adipose stem cells.
Compared with the prior art, the invention has the following beneficial effects:
according to the application, gelatin is used as a raw material, the double-layer skin structure hydrogel composite scaffold is prepared through electrostatic spinning and photocrosslinking, and hADSCs cells are loaded between a gelatin electrostatic spinning fiber membrane and cylindrical patterned gelatin hydrogel in the preparation process. The composite scaffold has a double-layer structure, wherein the fibrous membrane is equivalent to the epidermal layer of the skin, can block the invasion of foreign matters and pathogens, and can prevent the loss of interstitial fluid; the patterned cylindrical gelatin can not only support the viability of hADSCs and the adhesion of hADSCs, but also promote the migration and proliferation of hADSCs and promote the regeneration and formation of skin tissues; the unique shape of the composite scaffold ensures good fit between the composite scaffold and a wound, has good air permeability, large porosity and pore size, is beneficial to transportation of oxygen and nutrient substances of the wound, can promote generation of blood vessels at the wound, has a degradation speed close to the wound healing period, and is beneficial to healing of the wound.
Furthermore, the composite support can adjust the physical properties of the hydrogel, including water retention, degradability and the like, by adjusting the photocrosslinking time and the aperture size of the mask in the preparation process so as to meet the requirements of different patients.
Drawings
FIG. 1 is a pictorial view of a loaded cell composite scaffold.
FIG. 2 shows the cell loading of gelatin electrospun fiber membranes (abbreviated as fiber membranes in the figure) and composite scaffolds after cell loading and culture for 7 d.
FIG. 3 shows the survival of cells after gelatin electrospinning fibrous membrane (abbreviated as fibrous membrane in the figure) and composite scaffold are loaded with cells 8 d.
FIG. 4 shows the results of blood flow analysis of the wound surface of diabetic rats.
FIG. 5 shows the result of CD31 staining of diabetic wound surface new tissue
Fig. 6 shows the result of OCT imaging analysis of diabetic rat wound optical coherence tomography.
FIG. 7 shows the result of H & E staining of diabetic wound neogenetic tissue.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 double-layer skin bionic hydrogel composite scaffold loaded with hADSCs
Preparation of gelatin electrostatic spinning fiber membrane
Preparing an electrostatic spinning solution: 0.5g of gelatin is dissolved in 5mL of Hexafluoroisopropanol (HFIP) by a micro electronic balance, and then the solution is added with a magnetic stirrer and placed in a magnetic stirrer to be magnetically stirred for 24 hours for standby.
The electrostatic spinning method comprises the following steps: the electrospinning experiments were carried out at room temperature with a humidity below 50%. The positive high-voltage spray head adopts an 8# flat-head needle (the length is 20cm), the voltage is adjusted to be 16kV, the distance from the needle to the receiving device is 20cm, the propelling speed is 16.7 muL/min, and the volume of the electrostatic spinning solution is 320 muL. Aluminum foil paper is laid on a self-assembled platform with the size of 10cm multiplied by 10cm, and a plurality of 15mm slides are placed for sample collection.
And observing the appearance of the fiber membrane by using an optical microscope in the experimental process.
Crosslinking the fiber membrane: cutting the glass slide containing the fiber membrane from an aluminum foil, placing the glass slide in a pore plate, adding 1% genipin solution (dissolved in 90% alcohol) into each pore, immersing the glass slide in the fiber membrane, placing the glass slide in a refrigerator at 4 ℃ for 24 hours, transferring the glass slide to an incubator at 37 ℃ for 36 hours, sucking out the original solution, washing the glass slide with 90% alcohol for three times, air-drying the glass slide on a clean bench, and storing the glass slide in the refrigerator at 4 ℃ for later use.
Preparation of PDMS (polydimethylsiloxane) mold
The PDMS mold was prepared using dow corning DC184 silicone rubber. The components and the curing agent are completely mixed according to the weight ratio of 10:1, and a mold is prepared according to the content of the specification.
(1) Upper layer die
2mL of PDMS was added to a 35mm petri dish, evacuated for 30min, placed on a flat bench at room temperature for 12 hours, and finally placed in an oven at 80 ℃ for 30min for curing, and the mold was perforated in the middle with a 15mm perforator.
(2) Lower-layer die
Adding 1.5mL of PDMS into a 60mm culture dish, vacuumizing for 30min, placing on a flat experiment table at room temperature for 12 h, finally placing in an oven at 80 ℃ for 30min for curing, and cutting the mold into a square.
(3) Preparation of masks
Pattern as designed on PET film using nanosecond laser: the circular film with the diameter of 2cm is processed by laser with the distance between the centers of the circles of 400 mu m being 800 mu m, and the marking times are 50. And spraying opaque black paint on the round PET film, and drying.
Preparation of composite scaffold
Preparation of the principal solution
(1) 30% Type a gelatin solution: weighing 6g of gelatin particles, placing in a 50mL centrifuge tube, sterilizing with high temperature and high pressure steam, adding 20mL sterile PBS solution, oven drying at 50 deg.C to dissolve completely, sealing, and storing in a 4 deg.C refrigerator.
(2) 0.5% genipin solution: weighing 5g of genipin, dissolving in 100mL of PBS solution, magnetically stirring at 600rpm for 2h for sufficient dissolution, filtering, sterilizing, and storing at 4 ℃.
The preparation process comprises the following steps:
1. soaking the PDMS mold and the mask in alcohol for 4h for sterilization, and air-drying on a superclean bench;
2. placing the lower layer die into a 60mm culture dish by using a pair of tweezers, and keeping the lower layer die flat; placing the gelatin electrostatic spinning fiber membrane prepared in the step one in the middle of a die (in a punched hole in the die), placing an upper layer of the die, adding a proper amount of cell culture medium or glycine solution until the gelatin electrostatic spinning fiber membrane is submerged, and placing the gelatin electrostatic spinning fiber membrane in an incubator at 37 ℃ for 1.5 hours; the process ensures that the gelatin electrostatic spinning fiber membrane completely stops the crosslinking reaction and simultaneously ensures that the gelatin electrostatic spinning fiber membrane is in a hydrogel shape;
wherein the cell culture medium is a cell culture medium conventionally used in the art, and the cell culture medium used in this example comprises the following components: DMEM low-sugar medium, fetal calf serum and double antibodies, and the specific preparation process comprises the following steps: 89% DMEM, 10% fetal bovine serum, 1% double antibody.
The mass concentration of the glycine solution is 0.015-0.02 g/mL;
3. preparing a photo-crosslinking solution: taking 0.5mL of the prepared 30% Type A gelatin solution, 62.5 muL of 16-32 mM bipyridyl ruthenium, 62.5 muL of 160-320 mM sodium persulfate and 375 muL of carmine solution with the mass concentration of 0.05-0.2% into an EP tube, and blowing and beating to uniformly mix the solution; the solutions are all PBS solutions with pH of 7.4;
4. dripping 70-200 mu L of photo-crosslinking solution onto a gelatin electrostatic spinning fiber membrane in a PDMS mold, standing for 2min in a dark place, adding a mask after the photo-crosslinking solution is solidified, irradiating for 8-20 min by using cold light source LED white light, and enabling the distance between an LED light source and the gelatin electrostatic spinning fiber membrane to be 7-20 cm;
5. and after the illumination is finished, adding PBS to immerse the bracket, then placing the bracket in an incubator at 37 ℃ for standing for 10min, dissolving the part which is not crosslinked, removing the PBS by suction, adding 1mL of 0.5% prepared genipin solution, placing the bracket in the incubator for 30-60 min, removing the genipin solution by suction, adding the PBS for washing twice, adding a culture medium, and stopping crosslinking. And preparing the hydrogel composite scaffold with the double-layer skin structure.
The hydrogel composite scaffold with the double-layer skin structure and the non-loaded adipose-derived stem cells is prepared by the process. Referring to the process, before the photocrosslinking solution is prepared in the step 3, the fat stem cell suspension is prepared and added to the gelatin electrostatic spinning fibrous membrane for attaching, and then the subsequent steps are carried out according to the process, so that the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold can be obtained.
The process of preparing the adipose-derived stem cell suspension and attaching the adipose-derived stem cell suspension to the gelatin electrospun fiber membrane comprises the following steps: culturing adipose-derived stem cells by adherent culture for P3-P4 generations, adding pancreatin for digestion, counting, centrifuging, adding cell culture medium for resuspension, adding dropwise into gelatin electrospun fiber membrane, and adding 1.5 × 10 of gelatin electrospun fiber membrane into 15mm circular fiber membrane4A cell suspension of cell mass is left to stand for cell attachment to the fibrous membrane.
Embodiment 2 hADSCs-loaded double-layer skin bionic hydrogel composite scaffold
1. The appearance of the prepared hydrogel composite scaffold loaded with the hADSCs bilayer skin structure is shown in FIG. 1.
As can be seen from fig. 1, the prepared hydrogel composite scaffold with the hADSCs-supported double-layer skin structure is in a hydrogel shape, wherein the gelatin electrospun fiber membrane is a substrate layer, cylindrical gelatin hydrogels are uniformly arranged on the substrate layer, and a certain interval is maintained between the cylindrical gelatin hydrogels.
The composite scaffold prepared in example 1 had an overall thickness of 350 μm; wherein the diameter of the cylindrical hydrogel is 600 μm; the height was 300. mu.m.
2. Detecting the cell loading condition of the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold
The effect of the gelatin electrospun fiber membrane prepared in example 1 on the loading capacity and stability of cells was compared with that of the composite scaffold.
The loading conditions of the hADSCs are shown in figure 2 after the gelatin electrospun fiber membranes (referred to as fiber membranes in the figure) and the composite scaffold are loaded with the hADSCs and cultured for 7d, wherein the hADSCs are loaded for 7d and then subjected to F-ACTIN (green) and DAPI (blue) to obtain a dyeing pattern shown in figure 2, and the dyeing pattern is observed as shown in figure 2, wherein the infiltration growth of the hADSCs on the fiber membranes is observed, the cell morphology is good, and the gelatin electrospun fiber membranes are suitable for the adhesion and proliferation of the hADSCs, and the cylindrical hydrogel does not influence the adhesion of the hADSCs on the gelatin electrospun fiber membranes.
The survival of hADSCs after gelatin electrospun fiber membranes (abbreviated as fiber membranes in the figure) and composite scaffolds loaded with hADSCs8d is shown in FIG. 3. The staining results of the hADSCs on Calcein AM/PI day eight after loading are shown in FIG. 3. From the staining results, the composite scaffolds loaded with hADSCs had a large number of live cells (green) and a small number of dead cells (red) after eight days of culture. Cell death and live staining further proves the good cell compatibility of the scaffold, and the good microenvironment can promote cell adhesion and proliferation. Meanwhile, the scaffold can convey cells to a wound area, so that the survival rate of the cells is ensured.
3. Testing the influence of the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold on the wound surface
Type I diabetes rat (TIDM) modeling
(1) SD rats (8 weeks old, 200-250 g in body weight, male) 10 were subjected to TIDM induction molding, and were fasted for 24 hours before molding. Before molding, the weight of each rat was weighed, and tail vein blood was taken to measure the fasting blood glucose level of each rat.
(2) The 1% STZ solution was administered at a dose of 50mg/kg intraperitoneally. After molding, tail vein blood sampling is carried out to measure the blood sugar of the rat. If the blood sugar value before induction is less than 8.9mmol/L and the blood sugar value after induction for three consecutive days is more than or equal to 16.7mmol/L, the molding is considered to be successful. If the TIDM model is not induced, feeding the rats with normal diet, monitoring blood sugar value, and injecting 1% STZ solution to induce the TIDM model again after the blood sugar of the rats is recovered to a normal value and no obvious abnormality.
Establishment of full-thickness skin excision wound model
The above-mentioned TIDM rats successfully molded were fed with normal diet for four weeks, and the body weight and blood glucose values were measured every three days. TIDM rats were subjected to topical abdominal sterilization and were anesthetized by intraperitoneal injection using 10% chloral hydrate solution at a dose of 4.5 mL/kg. After the effect is shown, the front limb and the back side hair of the rat are removed, a layer of depilatory cream is coated, and after 10 minutes, the depilatory cream is removed and is wiped and disinfected by using an alcohol cotton ball. Taking rat vertebra as a central line, setting the interval distance of wound surfaces to be 2cm, establishing 4 full-layer skin excision wound models by using a biopsy punch with the diameter of 15mm to reach a rat muscle layer, and finally cutting out fascia connecting the skin layer and muscle tissue by using surgical scissors. The wound was dried with a sterile cotton ball, self-control was used, left blank (no treatment) and hADSS cell suspension (about 50 μ L, cell mass 3X 10) was added drop-wise to the lower left4) The upper part of the right side is coated with the non-loaded hADSCs composite stent, and the lower part of the right side is coated with the loaded hADSCs composite stent; the wound was then covered with a PI transparent dressing (containing a 20mm diameter gasket) and further secured with a self-adhesive bandage.
The angiogenesis condition of the rat wound is detected by adopting an angiograph. The working principle of the blood vessel imaging instrument is that when infrared light irradiates human tissues, hemoglobin in vein blood vessels has a more obvious absorption effect on the infrared light than surrounding tissues, and can form a very obvious vein blood vessel image when being shot by an infrared camera, and the image is displayed on a screen display after being converted by a special software and hardware image processing system to form a visible image of the vein blood vessels, so that the vein positioning display is assisted.
Three days after the operation, the results of the blood flow analysis of the wound surface of the diabetic rat are shown in fig. 4. The cell group in the figure is a group of cell suspension in which hADSCs are dripped, the scaffold group is a cell composite scaffold group without hADSCs, and the cell composite scaffold group with hADSCs is loaded.
As can be seen from fig. 4, compared with the blank group and the cell group, the composite scaffold group (loaded cells and unloaded cells) wounds have more neovascularization, higher blood vessel density, almost the whole wound surface has blood flow signs, and the effect of the loaded cell scaffold group is more obvious compared with the scaffold group, which further proves that the material can promote the angiogenesis of diabetic wounds.
For diabetic wounds, revascularization is extremely important for wound healing. Immunohistochemical staining of CD31 resulted in fig. 5. And 3, after operation, a large number of blood vessels appear around the combined scaffold group (loaded cells and unloaded cells) materials in the superficial granulation tissues of the wound surface, wherein the density of the blood vessels of the loaded cell scaffold group is highest. The paracrine effect of hADSCs can be enhanced by three-dimensional culture in the gelatin composite scaffold, for example, the expression of genes and proteins such as angiogenesis promoting factors VEGF and the like is increased, thereby promoting the regeneration of blood vessels. The generation of new blood vessels is beneficial to the transportation of nutrient substances and oxygen of the wound surface, improves the microenvironment of the wound surface and promotes the wound healing.
Three and eight days after surgery, the results of OCT imaging analysis of diabetic rat wounds are shown in FIG. 6. The optical coherence tomography scanner scans back reflection or several scattering signals of incident weak coherent light by using different depth layers of biological tissues to obtain reconstructed tissue section tomography images. As can be seen from FIG. 6, the hydrogel of the wound surface still presents patterning in the third day, the loaded cells can be protected, the scaffold material can be well attached to the wound, and the scaffold is uniformly distributed on the wound. On the eighth day, the lower cylindrical hydrogel starts to degrade and absorb, and is replaced by the new tissue, and the new tissue grows toward the wound surface (dotted line frame in the figure)
Three, thirteen and twenty days after surgery, the results of H & E staining of diabetic wound neogenetic tissue are shown in fig. 7. As can be seen in fig. 7, on day 3 post-surgery, there was extensive infiltration of new granulation tissue including inflammatory cells and red blood cells in each group. The hADSCs-loaded cell composite scaffold group can see that more cells migrate and proliferate to the hydrogel, and the number of inflammatory cells is small. On the thirteen days, the hADSCs-loaded cell composite scaffold group had new epidermis covering most of the wound surface. On the twentieth day, mature hair follicles and sebaceous glands can be seen in the hADSCs cell-loaded composite scaffold group, the material is completely degraded, and the new granulation tissue is thicker than other groups.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The composite scaffold is characterized in that a gelatin electrostatic spinning fiber membrane is used as a substrate, adipose-derived stem cell suspension is added, after cells are attached to the gelatin electrostatic spinning fiber membrane, gelatin photocrosslinking solution is added, and then the cells are photocrosslinked to prepare cylindrical gelatin hydrogel, namely the hADSCs-loaded double-layer skin bionic hydrogel composite scaffold.
2. The bilayer skin biomimetic hydrogel composite scaffold loaded with hADSCs of claim 1, wherein the diameter of the cylindrical gelatin hydrogel is 600-800 μm and the thickness is 300-600 μm.
3. The double-layer skin bionic hydrogel composite scaffold loaded with hADSCs (magnetic resonance imaging systems) of claim 1, wherein the gelatin electrospun fiber membrane is prepared by using gelatin as a raw material, spinning the gelatin by using an electrospinning device to obtain a fiber membrane, and then placing the obtained fiber membrane in a genipin-alcohol solution for crosslinking and curing.
4. The bilayer skin biomimetic hydrogel composite scaffold loaded with hADSCs according to claim 1, wherein the number of cells in the adipose stem cell suspension is 1 x 104～3×104And (4) respectively.
5. The bilayer skin biomimetic hydrogel composite scaffold loaded with hADSCs according to claim 1, wherein the gelatin photocrosslinking solution is: the composition is prepared from a 10-30% Type A gelatin PBS solution, a bipyridyl ruthenium PBS solution, a sodium persulfate PBS solution and a 0.05-0.2% carmine PBS solution according to a volume ratio of 8:1:1: 6.
6. The bilayer skin biomimetic hydrogel composite scaffold loaded with hADSCs according to claim 5, wherein the photocrosslinking is performed in the presence of a PDMS mold and a mask by the process of: and placing the gelatin electrostatic spinning fiber membrane in a PDMS (polydimethylsiloxane) mold, then adding the gelatin photo-crosslinking solution, covering a mask, performing crosslinking reaction by illumination, adding PBS (phosphate buffer solution) to dissolve the uncrosslinked part, finally soaking in a genipin-PBS solution for further curing, and cleaning to obtain the hydrogel composite bracket with the double-layer skin structure.
7. The bilayer skin biomimetic hydrogel composite scaffold loaded with hADSCs according to claim 6, wherein the illumination conditions are: the light source is cold light source LED white light, the illumination time is 8-20 min, and the light source distance is 7-20 cm.
8. A preparation method of a double-layer skin bionic hydrogel composite scaffold loaded with hADSCs is characterized by comprising the following steps:
s1, preparing a gelatin electrostatic spinning fiber membrane: dissolving gelatin in hexafluoroisopropanol to prepare an electrostatic spinning solution, then preparing the electrostatic spinning solution by electrostatic spinning equipment at room temperature and under the condition that the humidity is lower than 50%, collecting a spinning fiber membrane, performing crosslinking curing in a genipin-alcohol solution, and cleaning to obtain a gelatin electrostatic spinning fiber membrane;
s2, preparing a PDMS mold and a mask: preparing an upper layer mold and a lower layer mold in required shapes by adopting a PDMS mold product; the mask is a PET film, patterns of the PET film can be designed according to needs, and then the gelatin electrostatic spinning fiber film prepared by S1 is placed in a PDMS upper layer die;
s3, loading adipose-derived stem cells: after the adipose-derived stem cells are cultured in an adherent manner, adding cell pancreatin for digestion, counting and centrifuging, adding a cell culture medium for heavy suspension, dropwise adding the cell culture medium onto a gelatin electrostatic spinning fibrous membrane, and standing;
s4, photo-crosslinking: preparing a photo-crosslinking solution, adding the photo-crosslinking solution after the adipose-derived stem cells are attached to the gelatin electrostatic spinning fiber membrane, standing, covering a mask, and performing a crosslinking reaction by illumination;
and S5, after the photocrosslinking reaction is finished, finally soaking the hydrogel composite scaffold in a genipin-PBS solution for further curing, and cleaning to obtain the hydrogel composite scaffold with the double-layer skin structure.
9. Use of the loaded hADSCs bilayer skin biomimetic hydrogel composite scaffold of any one of claims 1 to 7 in the preparation of a wound dressing.
10. The use of the loaded hADSCs bilayer skin biomimetic hydrogel composite scaffold of any one of claims 1 to 7 as a carrier for drug loading.
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| CN114028603A (en) * | 2021-11-02 | 2022-02-11 | 太原理工大学 | Double-layer asymmetric dressing for promoting skin wound repair and preparation method thereof |
| CN114870089A (en) * | 2022-01-17 | 2022-08-09 | 中国人民解放军联勤保障部队第九八八医院 | PCL-collagen/Gelma biradial composite stent loaded with MSCs and application |
| CN114870089B (en) * | 2022-01-17 | 2023-04-28 | 中国人民解放军联勤保障部队第九八八医院 | PCL-collagen/Gelma dual-radial composite stent loaded with MSCs and application |
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