CN111035812A - Human-derived cell biological composite patch - Google Patents
Human-derived cell biological composite patch Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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Abstract
The invention discloses a human-derived cell biological composite patch. It has human cell, which is compounded and grown and replicated on the sheet rack, and the extracellular matrix secreted by the cell is cross-linked with the rack structure. This is a living patch tissue. The patch can provide good compliance matched with autologous abdominal wall tissues, meet the mechanical properties required by the hernia patch graft, and facilitate the abdominal wall tissue cells of a host to grow into the hernia patch graft without forming scar tissues. The human-derived cells can be derived from allogenic mesenchymal cells or mesenchymal stem cells derived from various human tissues, and umbilical cord blood mesenchymal stem cells; allogenic endothelial cells, smooth muscle cells may also be included.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a human-derived cell biological composite patch.
Background
Stem cells (stem cells) are a type of pluripotent cells with the ability to self-replicate (self-renew). Under certain conditions, it can differentiate into a variety of functional cells. The stem cells are classified into embryonic stem cells (ES cells) and adult stem cells (stromal cells) according to the developmental stage in which the stem cells are located. The cells are classified into three categories according to their developmental potential: totipotent Stem Cells (TSC), pluripotent stem cells (pluripotent stem cells) and unipotent stem cells (multipotent stem cells). Stem cells (Stem cells) are insufficiently differentiated and immature cells, have the potential function of regenerating various tissues, organs and human bodies, and are called universal human cells in the medical field. In 2013, 12 and 1, scientists at the university of columbia medical research center, usa, successfully transformed human stem cells into functional lung cells and respiratory cells for the first time. In 4 months 2014, the first manufacturing center of stem cells available in ireland for human bodies received the permission of the ireland drug administration, and was established at the university of iriland national golwell.
Collecting umbilical cord blood comprises collecting umbilical cord blood after birth of newborn, ligating two hemostatic forceps at umbilical cord position of 3-8cm, cutting off umbilical cord, carrying away the infant, sterilizing at hemostatic forceps close to mother end, inserting needle into umbilical vein, and collecting umbilical cord blood. The cord blood collection is different from the traditional bone marrow collection, anesthesia is not needed, the placenta and the umbilical cord are thrown away as waste after the fetus is born, and the cord blood collection is carried out after the placenta and the umbilical cord are completely separated from a mother body and the fetus, so that the cord blood collection has no adverse effect on the mother body and the child, belongs to waste utilization and changes waste into valuable. The inside of cord blood contains a large number of stem cells, called cord blood stem cells, which are the main sources of blood and immune cells for fetal production; cord blood stem cells are important stem cells of a human body, are distributed in various organ tissues, and have potential functions of regenerating various tissue organs.
In 1988, Broxmier et al first discovered that cord blood is rich in stem cells and is available for hematopoietic stem cell transplantation. Same year Gluckman et al first treated 1 male with Fanconi anemia at age 5 with umbilical cord blood transplantationThe sex infant, this infant survives more than 7 years so far after receiving HLS identical sibling umbilical cord blood transplantation, present clinical symptom disappear, sex chromosome, ABO antigen analysis, etc. show 100% hematopoietic stem cell is the blood donor source. In 1995, Wagner et al summarized 44 leukemia patients of all types who received umbilical cord blood transplantation of siblings. Median age 4.0 years, weight 18.6(7.5-50.0) kg; 34 cases with perfect match of HLA, 4 cases with 1 antigen mismatch, 1 case with 2 antigen mismatch, 5 cases with 3 antigen mismatch; number of input nucleated cells 1X 107/kg，CFU-GM4×104In terms of/kg. Absolute Neutrophil Count (ANC) of not less than 0.5X 10 after transplantation9The median time of/L is 22(12-46) days, and the platelet is more than or equal to 50 multiplied by 109The L is 48(15-100) days. The success time of transplantation has no obvious relationship with the input nucleated cells/kg or CFU-GM/kg. Approximately 2/3 patients were given hematopoietic growth factor treatment and no significant improvement in hematopoietic recovery was found. The incidence of GVHD at levels II-IV within 100 days was 3%, the incidence of chronic GVHD within 1 year was 6%, and the actual survival rate of receiving HLA-identical and 1 antigen-mismatched antigens after 1.6 years of median follow-up was 72%.
Cord blood has a variety of advantages that make it a potentially effective, economical, and safe source of hematopoietic stem cells. Besides enhancing HLA matching, the method can amplify the number of umbilical cord blood progenitor cells, improve transplantation efficiency, establish a computer-networked umbilical cord blood stem cell bank, and enable umbilical cord blood to show wide application prospects in aspects of hematopoietic stem cell transplantation, gene therapy and the like.
Hernia, which is the movement of an organ or tissue from its normal anatomical location into another location through a congenital or acquired weak point, defect or aperture. Common hernias include umbilical, inguinal direct, indirect, incisional, recurrent surgical, white line, femoral, etc. Abdominal hernia is caused by increased intra-abdominal pressure due to cough, sneezing, excessive exertion, abdominal obesity, exertion, defecation, pregnancy, excessive crying of infants, and degenerative change of the strength of the abdominal wall of the elderly, and forces the free organs in the abdominal cavity such as: the small intestine, the cecum, the omentum majus, the bladder, the ovary, the fallopian tube and other organs enter another part through the normal or abnormal weak points or defects and pores of the human body.
The hernia repair patch is a hernia repair material for short. With the rapid development of materials science in recent years, various hernia repair materials have been widely applied to clinic, so that the treatment of hernia is fundamentally changed. The currently clinically available hernia repair materials mainly have the following disadvantages:
(1) non-degradable material patch: the polyester patch, the polypropylene patch and the expanded polytetrafluoroethylene patch are easy to cause infection, scar, discomfort, local effusion and fiber envelope formation, and need to be taken out if infected;
(2) degradable material patch: degradable synthetic polymer compounds such as PLGA patches and PCL patches, and polymer compounds extracted from natural substances, such as patches made of gelatin, chitosan, hyaluronic acid and the like, have uncertain degradation time and the problem of premature degradation;
(3) animal-derived patches: the hernia patch prepared from the decellularized pig skin and the small intestine of the pig has potential immunogenicity or virus infection risk;
(4) human-derived patch: the source is extremely limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a humanized cell biological composite patch.
The technical scheme of the invention is as follows: a human-derived cell biological composite patch comprises a dense polymer nanofiber layer and a porous scaffold layer, wherein the dense polymer nanofiber layer is tightly attached to the peritoneal side in an operation, the porous scaffold layer covers the dense polymer nanofiber layer, and human-derived cells and extracellular matrixes generated by growth and proliferation of the human-derived cells grow in the porous scaffold layer;
the pores of the porous scaffold layer are sized to allow the human-derived cells to grow in and are open to allow the extracellular matrix to be interconnected;
the compact polymer nanofiber layer is prepared by electrostatic spinning or 3D printing,
the porous support layer is prepared by foaming rapid freezing molding or 3D printing.
In a preferred embodiment of the present invention, the thickness of the dense polymer nanofiber layer is 10 to 2000 μm, the diameter of pores thereof is 1 to 100 μm, and the fiber diameter is 10 to 1000 nm.
In a preferred embodiment of the present invention, the thickness of the porous scaffold layer is 10 to 5000 μm.
In a preferred embodiment of the present invention, the thickness of the dense polymer nanofiber layer is 10 to 2000 μm, the diameter of pores thereof is 1 to 100 μm, and the diameter of fibers is 10 to 1000 nm; the thickness of the porous support layer is 10-5000 μm.
In a preferred embodiment of the present invention, the material of the dense polymer nanofiber layer comprises at least one of polyurethane, polytetrafluoroethylene, expanded polytetrafluoroethylene, silk fibroin, polycaprolactone, polylactic acid, polyethylene terephthalate, polyglycolic acid, polylactic-polyglycolic acid, carboxymethyl cellulose, gelatin, collagen, hyaluronic acid, polyvinyl alcohol, polyacrylamide, polyacrylic acid, polyvinylpyrrolidone, and marine animal and plant derived polymer including chitosan, carboxymethyl chitosan, and alginic acid/alginate.
In a preferred embodiment of the present invention, the material of the porous scaffold layer comprises at least one of polyurethane, polytetrafluoroethylene, expanded polytetrafluoroethylene, silk fibroin, polycaprolactone, polylactic acid, polyethylene terephthalate, polyglycolic acid, polylactic-polyglycolic acid, carboxymethyl starch, starch acetate, carboxymethyl cellulose, gelatin, collagen, hyaluronic acid, polyvinyl alcohol, polyacrylamide, polypropylene, polyacrylic acid, polyvinylpyrrolidone, and marine animal and plant-derived high molecular polymer, including chitosan, carboxymethyl chitosan, and alginic acid/alginate.
In a preferred embodiment of the present invention, the human-derived cells include human tissue-derived allogenic mesenchymal cells or mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, allogenic endothelial cells and smooth muscle cells.
The invention has the beneficial effects that: the invention has human cells, which can be derived from variant mesenchymal cells or mesenchymal stem cells of various human tissue sources, and umbilical cord blood mesenchymal stem cells; allogenic endothelial cells, smooth muscle cells may also be included. The invention is a living patch tissue, avoids immunoreaction, improves the histocompatibility of the patch, simultaneously can provide good compliance matched with abdominal wall tissue, meets the mechanical property required by the hernia patch, and can allow normal tissue cells of a human body to grow into the patch without forming scar tissue.
Drawings
FIG. 1 is a photograph showing the appearance of the human-derived cell bio-composite patch prepared in example 1 of the present invention.
FIG. 2 is a scanning electron micrograph of a dense polymer nanofiber layer obtained by electrospinning with PCL as a main component and gelatin as an auxiliary component in example 1 of the present invention.
FIG. 3 is a SEM image of a porous scaffold layer with PCL as the host in example 1 of the present invention.
FIG. 4 is a graph showing the results of the tensile force test of the biocomposite patch (without cells) prepared in example 1 of the present invention.
Fig. 5 is a view showing the anatomy of the material obtained in step (4) of example 1 of the present invention after 6 months of implantation into the inguinal hernia site.
FIG. 6 is a photograph showing the appearance of the biocomposite patch (without cells) prepared in example 1 of the present invention after being transplanted into rats for 6 months.
FIG. 7 is a scanning electron micrograph of a dense nanofiber layer obtained by electrospinning PVA according to example 2 of the present invention.
FIG. 8 is a SEM image of a porous scaffold layer obtained by foaming a gelatin solution in example 2 of the present invention.
Fig. 9 is a scanning electron micrograph of a dense nanofiber layer obtained by PLA-collagen composite electrospinning in example 3 of the present invention.
FIG. 10 is a scanning electron micrograph of a porous support layer of PVA according to example 3 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1
The appearance of the human umbilical cord blood stem cell biological composite patch prepared in this example is shown in fig. 1, and includes a dense polymer nanofiber layer and a porous scaffold layer, and the preparation method specifically includes:
(1) dissolving PCL and gelatin in hexafluoroisopropanol at a mass ratio of 7: 3 to form 6 wt% solution, thereby obtaining electrostatic spinning solution;
(2) performing electrostatic spinning on the electrostatic spinning stock solution to obtain a dense polymer nanofiber layer (with the thickness of 200 μm, the average pore size of 5-10 μm, and the fiber diameter of 200nm-400nm) shown in figure 2;
electrostatic spinning parameter control
(3) Weighing 7.7g of PCL and 6g of NaCl particles with the size of 300-500 mu m, dissolving in 90mL of dichloromethane, and stirring at 300rpm for dissolution; adding 40mL of deionized water, and continuing stirring for 1h to obtain a foaming solution;
(4) and (3) uniformly coating the foaming solution on the upper surface of the compact polymer nanofiber layer prepared in the step (2), pre-freezing at-80 ℃, and then freezing and vacuum-drying at-4 ℃ for 24 hours. Soaking in deionized water for 72 hr, changing water at appropriate time to remove NaCl, soaking in petroleum ether for 24 hr, and sterilizing with 70% ethanol to obtain porous scaffold layer with thickness of 2mm as shown in FIG. 3.
And (4) respectively carrying out tensile force detection and histocompatibility detection on the material obtained in the step (4). As shown in fig. 4, the material tensile properties were good; as shown in FIG. 5, the drug was well absorbed after being transplanted into rats and completely absorbed after 6 months. As shown in FIG. 6, the biocomposite patch (acellular) prepared in this example showed no scar formation at 6 months after transplantation of the rat (right part of the shaved part in the figure).
(5) Matrigel (BD matrigel (TM) basement membrane matrix, GFR, phenol Red free, cat # 356231) was converted to a solution at room temperature, and 1-2mL of this solution was added to the procedure by means of a pipette gunCoating the porous support layer obtained in the step (4), and then placing the porous support layer in a cell culture box for balancing; centrifuging the human umbilical cord blood stem cells which are quickly thawed, dropwise adding the human umbilical cord blood stem cells into a 15mL centrifuge tube containing a complete culture medium of the human umbilical cord blood stem cells, and centrifuging for 3min at 1000 rpm; centrifuging, removing supernatant, adding 1mL of human cord blood stem cell complete culture medium, blowing, sucking and mixing the sediment uniformly; after the uniform blowing and sucking, discarding matrigel in the balanced porous scaffold layer, and adding stem cell suspension with uniform blowing and sucking into the porous scaffold layer to uniformly distribute cells; then placed at 37 ℃ in 2, 5% CO2Culturing for 4h in the constant-temperature incubator, and adding 1mL of human cord blood stem cell complete culture medium; changing the liquid once every 1-2 days from the time of resuscitation; culturing for 1-3 days to obtain the human cell biological composite patch for transplantation.
The preparation method of the human umbilical cord blood stem cell complete culture medium comprises the following steps: will be provided with
500mL (BM0008) of cord blood stem cell phenol-free medium,
Fetal bovine serum 25mL (BM0001) and
cord blood stem cell culture supplement 1mL, (BM0003) was thawed at 37 ℃ and mixed rapidly, plus 1% double antibody for contamination protection.
Example 2
(1) PVA (MW: 90000-130000) is dissolved in water to form 10 wt% of spinning solution, and electrostatic spinning stock solution is obtained;
(2) performing electrostatic spinning on the electrostatic spinning stock solution to obtain a dense polymer nanofiber layer (with the thickness of 20-500 μm, the average gap of 2-10 μm and the fiber diameter of 200nm-400nm), wherein the scanning electron microscope result is shown in FIG. 7;
electrostatic spinning parameter control
(3) Weighing 10g of gelatin, dissolving in 100mL of deionized water at 50 ℃ in a water bath, foaming at a mechanical rotation speed of 1000rpm until a stable foaming solution is formed, uniformly coating the stable foaming solution on the surface of the compact polymer nanofiber layer, freezing and shaping at-20 ℃, and vacuum drying at room temperature to obtain a porous scaffold layer with the thickness of 1mm, wherein the microstructure of the porous scaffold layer is as shown in a scanning electron microscope result in fig. 8.
(4) Coating 1-2mL laminin solution (20 μ g/mL) in the porous scaffold layer, and placing in a cell culture box for balancing for 4 h; adding 1mL of human mesenchymal stem cell culture medium into the centrifuged human umbilical cord blood mesenchymal stem cells to blow and suck the cell sediment uniformly; after the blowing and sucking are uniform, sucking off the liquid in the balanced porous scaffold layer by using a vacuum pump, and adding 200 mu L-1mL of cell suspension liquid which is uniformly blown and sucked into the porous scaffold layer to uniformly distribute the cells; then placed at 37 ℃ with 5% CO2The constant temperature incubator is used for 4 hours, and 3mL of culture medium is added; changing the liquid once every 1-2 days from the time of resuscitation; culturing for 1-3 days to obtain the human cell biological composite patch for transplantation.
The mesenchymal stem cell culture medium is specially designed for in vitro culture of human mesenchymal stem cells and is the culture medium which is most suitable for growth of the human mesenchymal stem cells. Is a sterilized liquid medium containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals and low-concentration fetal bovine serum (5%). The buffer system of the culture medium is bicarbonate containing 5% CO2The pH value after equilibration in the cell culture box of (1) was 7.4. The formula of the culture medium can selectively promote the proliferation and growth of normal human microvascular endothelial cells in vitro culture, and can reach the optimal nutrient balance state.
Specifically, the mesenchymal stem cell culture medium comprises 500mL of basal medium, 25mL of fetal bovine serum (FBS, cat.no.0025), 5mL of mesenchymal stem cell growth supplement (MSCGS, cat.no.7552) and 5mL of penicillin/streptomycin solution (P/S, cat.no.0503).
Example 3
(1) Dissolving PLA and collagen in hexafluoroisopropanol at a mass ratio of 9: 1 to form a 10 wt% solution, namely obtaining an electrostatic spinning stock solution;
(2) performing electrostatic spinning on the electrostatic spinning stock solution to obtain a dense polymer nanofiber layer (with thickness of 50-100 μm, average void of 2-10 μm, and fiber diameter of 200nm-400nm), wherein the scanning electron microscope result is shown in FIG. 9;
electrostatic spinning parameter control
(3) Weighing 2g of PVA (MW: 90000-130000), dissolving in 100mL of deionized water, pouring into a mold paved with the dense polymer nanofiber layer, freezing and shaping at-20 ℃, and vacuum drying at room temperature to obtain a porous scaffold layer with the thickness of 2mm, wherein the microstructure of the porous scaffold layer is shown as the result of a scanning electron microscope in FIG. 10.
(4) The smooth muscle cells were planted in the porous layer in the same manner as in example 2.
(5) The smooth muscle cell culture medium contained 500mL of basal medium, 10mL of fetal bovine serum (FBS, Cat.No.0010), 5mL of smooth muscle cell growth factor (SMCGS, Cat.No.1152), and 5mL of penicillin/streptomycin solution (P/S, Cat.No.0503).
Example 4
(1) Dissolving PCL in hexafluoroisopropanol to form a 10 wt% solution, and dissolving chitosan in trifluoroacetic acid to form a 1% solution, thereby obtaining an electrostatic spinning stock solution;
(2) performing electrostatic spinning on the electrostatic spinning stock solution by using a double-nozzle to obtain a compact polymer nanofiber layer (with the thickness of 50-100 μm, the average gap of 2-10 μm and the fiber diameter of 200nm-400nm), wherein the scanning electron microscope result is shown in FIG. 9;
electrostatic spinning parameter control
(3) Weighing 2g of PVA (MW: 89000-98000), dissolving in 100mL of deionized water, pouring into a mold paved with the dense polymer nanofiber layer, freezing and shaping at-20 ℃, and vacuum drying at room temperature to obtain a porous scaffold layer with the thickness of 2mm, wherein the microstructure of the porous scaffold layer is shown as the result of a scanning electron microscope in FIG. 10.
(4) The planting method of the human endothelial cells on the porous layer is the same as that of the embodiment 2.
(5) The endothelial cell culture medium is from Shanghai's Xinzhou science and technology Co., Ltd, and is a complete culture medium specially designed for in vitro culture of normal human microvascular endothelial cells and most suitable for growth of the endothelial cells. Is a sterilized liquid medium containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals and low-concentration fetal bovine serum (5%). The buffer system of the culture medium is bicarbonate containing 5% CO2The pH value after equilibration in the cell culture box of (1) was 7.4. The formula of the culture medium can selectively promote the proliferation and growth of the endothelial cells of normal human microvascular in vitro culture and achieve the optimal nutrient balance state for the endothelial cells.
The endothelial cell culture medium contained 500mL of basal medium, 25mL of fetal bovine serum (FBS, Cat.No.0025), 5mL of endothelial cell growth factor (ECGS, Cat.No.1052), 5mL of penicillin/streptomycin solution (P/S, Cat.No.0503).
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (7)
1. A human-derived cell biological composite patch is characterized in that: the surgical operation stent comprises a dense polymer nanofiber layer and a porous scaffold layer, wherein the dense polymer nanofiber layer is tightly attached to the peritoneal side in an operation process, the porous scaffold layer covers the dense polymer nanofiber layer, and human-derived cells and extracellular matrix generated by growth and proliferation of the human-derived cells grow in the porous scaffold layer;
the pores of the porous scaffold layer are sized to allow the human-derived cells to grow in and are open to allow the extracellular matrix to be interconnected;
the compact polymer nanofiber layer is prepared by electrostatic spinning or 3D printing,
the porous support layer is prepared by foaming rapid freezing molding or 3D printing.
2. The biogenic cell biocomposite patch of claim 1, wherein: the thickness of the compact polymer nanofiber layer is 10-2000 mu m, the diameter of pores is 1-100 mu m, and the diameter of fibers is 10-1000 nm.
3. The biogenic cell biocomposite patch of claim 1, wherein: the thickness of the porous support layer is 10-5000 μm.
4. The biogenic cell biocomposite patch of claim 1, wherein: the thickness of the compact polymer nanofiber layer is 10-2000 mu m, the diameter of pores is 1-100 mu m, and the diameter of fibers is 10-1000 nm; the thickness of the porous support layer is 10-5000 μm.
5. The biogenic cell biocomposite patch of claim 1, wherein: the material of compact polymer nanofiber layer includes at least one of polyurethane, polytetrafluoroethylene, expanded polytetrafluoroethylene, silk fibroin, polycaprolactone, polylactic acid, polyethylene glycol terephthalate, polyglycolic acid, polylactic acid-polyglycolic acid, carboxymethyl cellulose, gelatin, collagen, hyaluronic acid, polyvinyl alcohol, polyacrylamide, polyacrylic acid, polyvinylpyrrolidone and marine animal and plant sourced high molecular polymer, and this marine animal and plant sourced high molecular polymer includes chitosan, carboxymethyl chitosan and alginic acid/alginate.
6. The biogenic cell biocomposite patch of claim 1, wherein: the material of porous support layer includes polyurethane, polytetrafluoroethylene, expanded polytetrafluoroethylene, silk fibroin, polycaprolactone, polylactic acid, polyethylene glycol terephthalate, polyglycolic acid, polylactic acid-polyglycolic acid, carboxymethyl starch, starch acetate, carboxymethyl cellulose, gelatin, collagen, hyaluronic acid, polyvinyl alcohol, polyacrylamide, polypropylene, polyacrylic acid, polyvinylpyrrolidone and at least one of the high molecular polymer of marine animal and plant source, and this high molecular polymer of marine animal and plant source includes chitosan, carboxymethyl chitosan and alginic acid/alginate.
7. The biogenic cell biocomposite patch of claim 1, wherein: the human-derived cells comprise human tissue-derived allogeneic mesenchymal cells or mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, allogeneic endothelial cells and smooth muscle cells.
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