CN115944780B - Allogenic cartilage tissue complex based on immune isolation strategy, and preparation method and application thereof - Google Patents
Allogenic cartilage tissue complex based on immune isolation strategy, and preparation method and application thereof Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the field of biomedical tissue engineering, and relates to an allogenic cartilage tissue compound based on an immune isolation strategy, a preparation method and application thereof. The semipermeable membrane material with immune isolation function is a polymer fiber membrane material prepared by an electrostatic spinning technology, and the polymer fiber membrane material can prevent immune cells or inflammatory cytokines from invading without blocking nutrient exchange. And culturing relatively mature regenerated cartilage tissues by a tissue engineering method, and wrapping the relatively mature regenerated cartilage tissues in a semipermeable membrane material with an immune isolation effect to obtain the allogenic cartilage tissue compound based on the immune isolation strategy. The allogenic cartilage tissue complex can be used for regeneration of allogenic cartilage. The invention can be widely applied to the field of cartilage regeneration and defect repair.
Description
Technical Field
The invention belongs to the field of biomedical tissue engineering, and particularly relates to an allogenic cartilage tissue compound based on an immune isolation strategy, and a preparation method and application thereof.
Background
The cartilage tissue engineering technology is an alternative treatment strategy for clinical plastic reconstruction, such as auricle, nose, trachea, joint and the like, and is hopeful to replace the traditional autologous costal cartilage carving technology. Currently, stentless chondropatch technology has the potential to stably regenerate high quality cartilage tissue in vitro and in vivo. Autologous cartilage regeneration is limited mainly by three technical barriers: 1) Seed cell source is deficient; 2) The wound to the patient is larger; 3) Cannot be applied instantaneously. Although allogeneic cartilage regeneration has the advantages of abundant donor sources, immediate application and the like, immune rejection is unavoidable, particularly in immunocompetent large animals or humans.
In recent years, an immunoisolation strategy based on semi-permeable membrane materials has become a viable strategy for protecting allograft cells from host immune attack, such as fibroblasts, mesenchymal stem cells and islet cells. As an ideal semipermeable membrane material, the electrostatic spinning fiber membrane has the advantages of nanofiber structure, controllable pore diameter and the like, and can be used for preparing the semipermeable membrane material with high-efficiency immune isolation and stable tissue survival. However, it is not clear whether the immunoisolation function of the electrospun semipermeable membrane can achieve regeneration of allogenic cartilage in large animals or humans. Cartilage is used as a blood vessel-lack tissue, and survival of the cartilage mainly depends on nutrition diffusion rather than blood vessel ingrowth, so that the cartilage is particularly suitable for realizing allogeneic cartilage regeneration by an immune isolation strategy. In general, semipermeable membrane materials need to meet the following requirements: 1) Biocompatibility and non-immunogenicity; 2) Membrane parameters that block immune cell infiltration, such as fiber diameter, pore size and uniformity; 3) Hydrophilic to facilitate nutrient exchange; 4) Nondegradability to maintain a long-term stable immune-isolation function.
To date, no major breakthrough has been made in preparing electrospun fibrous membranes that meet all of the above requirements as immunoisolation semipermeable membrane materials for allogenic cartilage regeneration.
Disclosure of Invention
Based on the problem of allograft immune rejection in the existing cartilage regeneration technology, the invention provides an allogenic cartilage tissue compound based on an immune isolation strategy, and a preparation method and application thereof.
The aim of the invention can be achieved by the following technical scheme:
it is a first object of the present invention to provide a semipermeable membrane material having an immunoisolation effect.
The semipermeable membrane material with the immune isolation function is a polymer fiber membrane material prepared by an electrostatic spinning technology, and the electrostatic spinning technology refers to a preparation method for forming a fiber membrane by electrostatic atomization of polymer fluid.
In one embodiment of the present invention, the polymer includes a synthetic polymer and a natural polymer, and the synthetic polymer includes polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-hydroxyglycolic acid (PLGA), polycaprolactone (PCL), thermoplastic Polyurethane (TPU), polyvinyl alcohol (PVA), polyethylene (PE), polymethyl acrylate (PMMA), polyetheretherketone (PEEK), and the like. The natural polymer comprises gelatin, collagen, hyaluronic acid, alginic acid, chitosan, cellulose, chondroitin sulfate, polylysine, polyglutamic acid, etc. Preferably PCL, PLGA, TPU, gelatin, hyaluronic acid.
In one embodiment of the present invention, the polymer may be selected as a synthetic-natural composite polymer material. The synthetic-natural composite polymer material is a composite polymer material formed by mixing synthetic polymers and natural polymers according to a certain proportion. The certain mixing proportion (synthetic polymer: natural polymer) is 1:0.005-1:0.95, preferably 1:0.02-1:0.5.
In the invention, the polymer fiber membrane material is structurally characterized in that: 1) The diameter of the fiber is about 0.1-2 mu m; 2) The pore size is about 0.5-5 μm; 3) The uniformity is more than 80%.
The functional characteristics of the polymer fiber membrane material are that it can block immune cell invasion without blocking nutrient exchange.
The semipermeable membrane material with immune isolation function not only can effectively prevent immune cells or inflammatory cytokines from invading allografts, but also does not obstruct nutrient exchange between the material and the grafts.
Wherein the immune cells comprise macrophages, lymphocytes, neutrophils and the like; the cytokines include TNF-alpha, IL-1 beta, IL-6, IL-8, etc.
By allograft is meant a transplanted cell, tissue or organ from a recipient of a genetically non-identical donor of the same species.
In one embodiment of the invention, the allograft is derived from a human, pig, cow, sheep, dog, rabbit, mouse, or the like, preferably rabbit, pig, sheep.
The second object of the invention is to provide a method for preparing an allogenic cartilage tissue complex based on an immune isolation strategy.
In the invention, the preparation method of the allogenic cartilage tissue complex comprises the following steps:
Culturing relatively mature regenerated cartilage tissue by a tissue engineering method, and wrapping the relatively mature regenerated cartilage tissue in the semipermeable membrane material with the immune isolation function to obtain the allogenic cartilage tissue compound based on the immune isolation strategy.
The invention utilizes the semipermeable membrane material with immune isolation effect to encapsulate the relatively mature regenerated cartilage tissue so as to achieve the aim that the relatively mature regenerated cartilage tissue is completely encapsulated.
In the present invention, the tissue engineering method includes a tissue engineering construction method including a scaffold material and a tissue engineering construction method without a scaffold material.
In one embodiment of the invention, the tissue engineering construction method of the scaffold-containing material is to inoculate seed cells with chondrogenic differentiation potential into a high molecular scaffold material or wrap the seed cells into a hydrogel scaffold material, and obtain relatively mature regenerated cartilage tissue through a conventional culture mode.
In one embodiment of the present invention, the polymer scaffold material includes polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-hydroxyglycolic acid (PLGA), polycaprolactone (PCL), thermoplastic Polyurethane (TPU), polyvinyl alcohol (PVA), polyethylene (PE), polymethyl acrylate (PMMA), polyetheretherketone (PEEK), and the like. Preferably PCL, PLGA, TPU.
In one embodiment of the present invention, the hydrogel scaffold material comprises gelatin, collagen, hyaluronic acid, alginic acid, chitosan, cellulose, chondroitin sulfate, polylysine, polyglutamic acid, and the like. Gelatin and hyaluronic acid are preferable.
In one embodiment of the invention, the tissue engineering construction method of the scaffold-free material is to directly perform a high-density inoculation culture dish culture mode on seed cells with chondrogenic differentiation potential, and obtain relatively mature regenerated cartilage tissues through a conventional culture mode.
In one embodiment of the present invention, the seed cells having chondrogenic differentiation potential are selected from chondrocytes, mesenchymal stem cells, adipose stem cells, embryonic stem cells, etc., preferably chondrocytes or mesenchymal stem cells.
In one embodiment of the present invention, the culture method comprises: inducing differentiation and culturing in vitro; or in vivo subcutaneous implantation culture; or in vitro/in vivo co-culture.
The relatively mature regenerated cartilage tissue is characterized by: 1) Histologically, regenerated cartilage exhibits a typical cartilage dimpled structure, with cartilage-specific staining of safranin and type two collagen; 2) On quantitative detection, the total collagen, glycosaminoglycan and PCR (such as Col2, SOX9, aggrecan) levels of regenerated cartilage reached about 60% -90% of normal cartilage tissue.
A third object of the present invention is to provide an allogeneic cartilage tissue complex based on an immunoisolation strategy, which is obtained by a method for preparing an allogeneic cartilage tissue complex based on an immunoisolation strategy.
A fourth object of the present invention is to provide the use of allogeneic cartilage tissue complexes based on immunoisolation strategies in the field of cartilage tissue engineering.
The allogenic cartilage tissue complex based on the immune isolation strategy is used for preparing the allogenic cartilage.
The application of the allogenic cartilage tissue complex based on the immune isolation strategy provided by the invention in the field of cartilage tissue engineering comprises the following steps: cartilage regeneration and cartilage defect repair application. Cartilage regeneration involves ear reconstruction, nose reconstruction, eyelid reconstruction, etc. Cartilage defect repair relates to tracheal defect repair, articular cartilage defect repair, meniscus defect repair, costal cartilage defect repair and the like.
When in use, the allogeneic cartilage tissue compound based on the immune isolation strategy is implanted into different animals or human bodies of the same species, and after a period of in-vivo culture, the regeneration of the allogeneic cartilage is realized.
In the present invention, the in vivo culture period is 2 weeks to 24 weeks, preferably 4 weeks to 12 weeks.
Compared with the prior art, the invention has the following advantages:
(1) The allogenic cartilage regeneration method provided by the invention can solve the defects that autologous cartilage regeneration donors are lack of sources and cannot be instantly applied, and can realize mass and instant tissue engineering cartilage supply.
(2) The immune isolation strategy based on the electrostatic spinning fiber membrane can effectively prevent immune cells from attacking host tissues, does not obstruct nutrient exchange, and realizes allogeneic cartilage regeneration.
(3) The allogenic cartilage regeneration method can be widely applied to the regeneration of specific three-dimensional cartilage (such as ear, nose, meibomian, and the like) and the repair of cartilage defects (such as trachea, joints, meniscus, costal cartilage, and the like).
Therefore, the allogenic cartilage tissue complex based on the immune isolation strategy, the preparation method and the application thereof can be widely applied to the fields of cartilage regeneration and defect repair.
Drawings
FIG. 1 is a schematic representation of an immunoisolation strategy based on semipermeable membrane materials.
FIG. 2 is a general view and scanning electron microscope image of an electrospun fibrous membrane.
Fig. 3 is a fluorescence microscope and scanning electron microscope image of the immunoisolation effect.
FIG. 4 is a diagram of immunohistochemistry after 8 weeks of subchondral culture of the allograft.
FIG. 5 is a histological map after 8 weeks of subchondral culture of the allograft.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
Embodiment one: preparation of PCL (Poly lactic acid) electrospun fiber membrane
Hexafluoroisopropanol (HFIP) is used as a solvent to prepare PCL electrospinning liquid with the mass-volume ratio of 10%. The electrospinning liquid is installed and fixed on an accurate micro-electronic injection pump, and is connected with an electrospinning instrument and a high-voltage power supply, and the electrospinning liquid is subjected to the following electrospinning parameters: the voltage is 20kV, the pushing speed of the injection pump is 0.5mL/h, the receiving distance is 15cm, the room temperature is 20 ℃, and the ambient humidity is 50+/-5%, so that the electrostatic spinning preparation is carried out. The receiving plate is an aluminum foil of 10cm in size. After spinning, vacuum pumping and repeatedly soaking with pure water, and removing residual organic solvent to prepare the PCL polymer electrostatic spinning fiber membrane. The PCL electrostatic spinning fiber membrane can be obtained by scanning electron microscope shooting and image J software analysis, has an average fiber diameter of 0.5+/-0.05 mu m, an average pore diameter of 2.5+/-0.05 mu m and a uniformity of 82+/-5%, and can be used as an immunoisolation semipermeable membrane material.
Embodiment two: preparation of TPU/GT electrospun fibrous membrane
Hexafluoroisopropanol (HFIP) is used as a solvent to prepare the mass-volume ratio of 5% TPU/0.5% Gelatin (GT) electrospinning liquid. The electrospinning liquid is installed and fixed on an accurate micro-electronic injection pump, and is connected with an electrospinning instrument and a high-voltage power supply, and the electrospinning liquid is subjected to the following electrospinning parameters: the voltage is 15kV, the pushing speed of the injection pump is 0.5mL/h, the receiving distance is 15cm, the room temperature is 20 ℃, and the ambient humidity is 50+/-5%, so that the electrostatic spinning preparation is carried out. The receiving plate is an aluminum foil of 10cm in size. After spinning, vacuum pumping and repeatedly soaking with pure water, and removing residual organic solvent to prepare the TPU/GT composite polymer electrostatic spinning fiber membrane. The average fiber diameter of the TPU/GT electrostatic spinning fiber membrane is 0.3+/-0.05 mu m, the average pore diameter is 1.5+/-0.05 mu m, the uniformity is 85% +/-3% which can be used as an immunoisolation semipermeable membrane material (as shown in fig. 1-2, immunocytes is immune cells in fig. 1, nutrients is a nutrient substance, waste is represented by waste, SPM is represented by high molecular electrostatic spinning fiber membrane, CARTILAGE SHEET is represented by cartilage slices, and Immunoisolation is represented by immunoisolation) through scanning electron microscope shooting and analyzing by adopting imageJ software.
Embodiment III: allogenic cartilage regeneration based on PCL immunoisolation semipermeable membrane
Experiment first, a PCL electrospun fiber film material was prepared as in example one.
Cartilage tissue regeneration culture: taking rabbit ear tissue, separating and culturing until the 2 nd generation chondrocyte is obtained, re-suspending the rabbit ear tissue by using high-sugar DMEM cell culture solution, wrapping the rabbit ear tissue in gelatin hydrogel according to the concentration of 2.0X10 5 cells/cm 2, continuously culturing the rabbit ear tissue for 4 weeks by adopting serum-free cartilage re-differentiation culture solution, drilling the cartilage tissue with the diameter of 8mm by using a sterile annular drill bit, and soaking the cartilage tissue in a common high-sugar culture medium for standby.
Allogenic cartilage regeneration based on immunoisolatory semipermeable membranes: the PCL electrostatic spinning fibrous membrane material is adopted to package the cartilage diaphragm tissue to achieve complete package, so that immune cells cannot invade the cartilage tissue. Furthermore, the pre-constructed cartilage tissue complex is implanted into another rabbit body, and after 8 weeks of in-vivo culture, the regeneration of the allogeneic cartilage can be realized. Experimental results indicate that regenerated cartilage exhibits a typical cartilage dimpled structure, with cartilage-specific staining of safranin and type II collagen; on quantitative detection, total collagen, glycosaminoglycan and PCR (Col 2, SOX9, aggrecan) levels of regenerated cartilage reached about 85% of normal cartilage tissue.
Embodiment four: allogenic cartilage regeneration based on TPU/GT immunoisolation semipermeable membrane
Experiment first, a TPU/GT electrospun fibrous membrane material was prepared as in example two.
Cartilage tissue regeneration culture: taking sheep ear tissue, separating and culturing to obtain 2 nd generation chondrocyte, re-suspending with high-sugar DMEM cell culture solution, inoculating into 6-hole plate according to concentration of 2.0X10 6 cells/cm 2, continuously culturing for 4 weeks with serum-free cartilage re-differentiation culture solution, drilling cartilage diaphragm tissue with diameter of 8mm with sterile annular drill bit, and soaking in common high-sugar culture medium for standby.
Allogenic cartilage regeneration based on immunoisolatory semipermeable membranes: encapsulation of cartilage membrane tissue with TPU/GT electrospun fibrous membrane material achieves complete encapsulation such that immune cells cannot invade the interior of cartilage tissue (as shown in fig. 3). And then, the pre-constructed cartilage tissue compound is implanted into another sheep body, and after 8 weeks of in-vivo culture, the regeneration of the allogeneic cartilage can be realized. Experimental results indicate that regenerated cartilage exhibits a typical cartilage dimpled structure, with cartilage-specific staining of safranin and type II collagen; on quantitative detection, total collagen, glycosaminoglycan and PCR (Col 2, SOX9, aggrecan) levels of regenerated cartilage reached about 82% of normal cartilage tissue (as shown in FIGS. 4-5).
Fifth embodiment: application of allogeneic cartilage regeneration in rabbit nose reconstruction
The allogenic cartilage tissue is constructed according to the third embodiment by adopting New Zealand male white rabbits, and cultured in vitro for 8 weeks to obtain the relatively mature tissue engineering cartilage. Then, the pre-constructed allogeneic tissue engineering cartilage is injected into the rabbit nasal part to carry out the in vivo nasal cartilage regeneration process. 8 weeks after the operation, rabbits in the experiment were sacrificed by intravenous air injection, and the experimental effect was evaluated by extracting regenerated nasal cartilage. Experimental results show that the reconstruction of the allogenic tissue engineering nose can basically realize the physiological function of nasal cartilage.
Example six: application of allogeneic cartilage regeneration in rabbit tracheal reconstruction
New Zealand male white rabbits are adopted to manufacture a segmental tracheal defect model at the tracheal part of the rabbits to evaluate the in vivo reconstruction effect of the allogeneic regenerated cartilage tissue. In the experiment, an allogeneic tissue engineering trachea is constructed according to the third embodiment, and the allogeneic tissue engineering trachea is buried in a rabbit trachea in a subcutaneous mode for 8 weeks to obtain a relatively mature tissue engineering trachea. Next, a segmental tracheal defect model 15mm long was made at the tracheal site of the rabbit, and an end-to-end anastomosis procedure was performed. 8 weeks after the operation, rabbits in the experiment were sacrificed by intravenous air injection, and the repairing section trachea was extracted to evaluate the experimental effect. Experimental results show that the trachea of the allogeneic tissue engineering can effectively realize trachea reconstruction and basically recover the mechanical and physiological functions of the trachea.
Embodiment seven: application of allogeneic cartilage regeneration in sheep joint defect repair
A goat animal model was used to produce a model of joint defect having a diameter of 7mm and a depth of 2mm at the weight bearing part of the knee joint of the goat. In the experiment, we divided two groups to perform sheep articular cartilage defect repair experiments: 1) An allogeneic cartilage repair group constructed as in example four; 2) Blank groups were not processed. In the experiment, the regenerated cartilage tissue can be fully filled in the sheep joint cartilage defect. After 3 months and 6 months of operation, we sacrificed sheep in the experiment by intravenous air injection method, and extracted damaged joints to evaluate the experimental repair effect. Experimental results show that the allogeneic regenerated cartilage can realize the repair of articular cartilage defects, and the allogeneic regenerated cartilage can realize complete repair after 6 months of operation.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (8)
1. The preparation method of the allogenic cartilage tissue compound based on the immune isolation strategy is characterized in that relatively mature regenerated cartilage tissue is cultured by a tissue engineering method, and the relatively mature regenerated cartilage tissue is wrapped in a semipermeable membrane material with the immune isolation effect, so that the allogenic cartilage tissue compound based on the immune isolation strategy is obtained;
The semipermeable membrane material with immune isolation function is a polymer fiber membrane material prepared by an electrostatic spinning technology, the polymer fiber membrane material can prevent immune cells or inflammatory cytokines from invading without blocking nutrient exchange, and the structure of the polymer fiber membrane material is characterized in that: 1) The diameter of the fiber is 0.1-2 mu m; 2) Pore size is 0.5-5 μm; 3) The uniformity is more than 80%.
2. The method for preparing an allogeneic cartilage tissue complex based on immune isolation strategy according to claim 1, wherein the macromolecule is selected from synthetic macromolecule, natural macromolecule or synthetic-natural composite macromolecule material;
The synthetic polymer is selected from polylactic acid, polyglycolic acid, polylactic acid-hydroxy glycolic acid, polycaprolactone, thermoplastic polyurethane, polyvinyl alcohol, polyethylene, polymethyl acrylate or polyether ether ketone;
The natural polymer is selected from gelatin, collagen, hyaluronic acid, alginic acid, chitosan, cellulose, chondroitin sulfate, polylysine or polyglutamic acid;
The synthetic-natural composite polymer material is a composite polymer material formed by mixing synthetic polymers and natural polymers in proportion.
3. The method of claim 1, wherein the immune cells comprise macrophages, lymphocytes, and neutrophils; the cytokines include TNF-alpha, IL-1 beta, IL-6, IL-8.
4. The method for preparing the allogenic cartilage tissue composite based on the immune isolation strategy according to claim 1, wherein the tissue engineering method is a tissue engineering construction method containing a scaffold material or a tissue engineering construction method without a scaffold material;
The tissue engineering construction method of the scaffold-containing material comprises the steps of inoculating seed cells with chondrogenic differentiation potential into a high molecular scaffold material or wrapping the seed cells into a hydrogel scaffold material, and obtaining relatively mature regenerated cartilage tissue through a conventional culture mode;
The tissue engineering construction method of the bracket-free material is to directly carry out a high-density inoculation culture dish culture mode on seed cells with chondrogenic differentiation potential, and obtain relatively mature regenerated cartilage tissues through a conventional culture mode.
5. The method of claim 4, wherein the polymer scaffold material is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-hydroxy glycolic acid, polycaprolactone, thermoplastic polyurethane, polyvinyl alcohol, polyethylene, polymethyl acrylate, and polyether ether ketone;
the hydrogel scaffold material is selected from gelatin, collagen, hyaluronic acid, alginic acid, chitosan, cellulose, chondroitin sulfate, polylysine or polyglutamic acid;
the seed cells having chondrogenic differentiation potential are selected from chondrocytes, mesenchymal stem cells, adipose stem cells or embryonic stem cells.
6. The method of claim 4, wherein the relatively mature regenerated cartilage tissue is characterized by: 1) Histologically, regenerated cartilage exhibits a typical cartilage dimpled structure, with cartilage-specific staining of safranin and type two collagen; 2) On quantitative detection, the total collagen, glycosaminoglycan and PCR level of regenerated cartilage reaches 60% -90% of normal cartilage tissue.
7. An allogenic cartilage tissue complex based on an immunoisolation strategy, characterized in that it is obtained by the method for preparing an allogenic cartilage tissue complex based on an immunoisolation strategy according to any one of claims 1-6.
8. The use of the allogeneic cartilage tissue complex based on immunoisolation strategy of claim 7 for preparing allogeneic cartilage.
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