CN111617318A - Preparation method of injectable epiphyseal plate regeneration hydrogel for children - Google Patents
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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
- A61L27/38—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 containing added animal cells
- A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/38—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 containing added animal cells
- A61L27/3839—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 containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3852—Cartilage, e.g. meniscus
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/624—Nanocapsules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Abstract
The invention discloses a preparation method of injectable epiphyseal plate regeneration hydrogel for children, which mainly comprises the following steps: 1. preparing double-layer drug-loaded Sodium Alginate (SA) microspheres with an outer layer serving as an anti-bone bridge formation factor and an inner layer serving as a cartilage differentiation promoting factor by using a flow focusing method and a microfluidic method; 2. and mixing the SA microspheres and the bone marrow mesenchymal stem cells in the hydrogel to prepare the biological ink. The hydrogel is injected into the damaged part of the epiphyseal plate cartilage, and Enbrel + Bevacizumab and IGF-1 are released locally, regularly and quantitatively, so that the formation of a bone bridge can be inhibited, the regeneration and repair of the epiphyseal plate cartilage can be promoted, and a new thought and a new method are provided for solving the problem of repairing the epiphyseal plate damage.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a preparation method of injectable epiphyseal plate regeneration hydrogel for children.
Background
Epiphyseal plates, also known as growth plates, are areas of cartilage where the terminal immature long bone of a child controls the longitudinal growth of bone. Fracture, infection, tumor or iatrogenic injury can cause epiphyseal injury, and about 30% of children are fractured with epiphyseal injury and unbalanced bone growth. Once the epiphyseal plate is damaged, the epiphyseal cartilage tissue is gradually replaced by bone tissue, a bone bridge is formed, growth retardation, angulation or rotation deformity is caused, the joint function is even further influenced, and the physical and mental health of the children is greatly influenced. The currently adopted clinical treatment method mainly comprises the steps of bone bridge resection combined with corresponding material filling, such as adipose tissues, high polymer materials and the like, so as to prevent the bone bridge from relapse and allow peripheral undamaged epiphyseal plate tissues to recover the longitudinal growth capacity of bones, but the success rate of the treatment method is very low, and operations such as osteotomy correction or limb lengthening and the like are mostly required in the later stage.
After the joint efforts of related personnel in the professional field, the construction of the epiphyseal cartilage with bioactivity by adopting the cartilage tissue engineering technology becomes a new direction for treating the epiphyseal injury of children, and the technology mainly utilizes chondrocytes and/or Bone Marrow Mesenchymal Stem Cells (BMSCs) to realize cartilage regeneration by combining with a scaffold made of a specific material. Although a great deal of research is done previously, different seed cells are utilized, different types of bioactive materials are screened, and regulation and control methods are adopted, the problems that the bracket chondrogenesis efficiency is low, and the fusion of regenerated cartilage and surrounding normal tissues is poor are still faced. More importantly, the regenerated cartilage does not have a layered structure similar to a natural epiphyseal plate and cannot control the longitudinal growth of long bones. One of the key reasons for this is that the current study on the scaffold micro-composition mainly focuses on the direction of promoting chondrogenic differentiation and regeneration of cartilage tissues, neglecting that the early damage prevents local osteogenic differentiation and bone bridge formation after epiphyseal plate damage from the source; inhibition of early inflammatory response and angiogenesis after epiphyseal injury is the key to prevention of bone bridge formation, and due to the important role of TNF-alpha and VEGF in this stage, we can use corresponding inhibitors (Enbrel and Bevacizumab) to block, so that the subsequent reaction of bone bridge formation is interrupted. Of course, after controlling osteogenic differentiation to inhibit bone bridge formation, we still need to induce chondrogenic differentiation of exogenous or endogenous mesenchymal stem cells using Growth factors such as Insulin Like Growth factor 1 (IGF-1) to eventually form epiphyseal cartilage tissue at the site of injury. Therefore, we intend to design a double-layer drug-loaded Sodium Alginate (SA) microsphere to realize early release of the outer layer (Enbrel + Bevacizumab) to prevent osteogenic differentiation and bone bridge formation, and then release the inner layer of IGF-1 to promote chondrogenic differentiation and epiphyseal cartilage regeneration.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of injectable children epiphyseal plate regeneration hydrogel, which is characterized in that the hydrogel is implanted into epiphyseal plate cartilage defect parts, and Enbrel + Bevacizumab and IGF-1 are released locally, regularly and quantitatively, so that the formation of bone bridges can be inhibited, the regeneration and repair of epiphyseal plate cartilage can be promoted, and a new thought and a new method are provided for solving the problem of 'epiphyseal plate damage repair'.
In order to achieve the technical purpose and achieve the technical effect, the invention adopts the following technical scheme: a preparation method of injectable epiphyseal plate regeneration hydrogel for children specifically comprises the following steps:
preparing a double-layer drug-loaded sodium alginate microsphere with an outer layer of an anti-osteopontic formation factor Enbrel + Bevacizumab and an inner layer of a chondrogenic differentiation factor IGF-1 by using a flow focusing method and a microfluidic method;
and (II) mixing the double-layer drug-loaded sodium alginate microspheres and bone marrow mesenchymal stem cells BMSCs in hydrogel to prepare the injectable bio-ink.
Further, the specific preparation process of the double-layer drug-loaded sodium alginate microsphere comprises the following steps:
1) placing the microfluidic chip on an objective table of an optical microscope, and respectively connecting six hoses to extend out of six channel ports;
2) adding a 1% sodium alginate solution added with IGF-1 into a single-channel injection pump through an injector, exhausting air, and connecting with a No. 2 channel port through a hose;
3) sucking 1% sodium alginate solution into a two-tube injector, placing the two-tube injector in a two-channel injection pump, and respectively connecting the two 1 # channel ports through flexible tubes;
4) sucking CaCl-containing solution with two-tube syringe2The paraffin oil is arranged in a double-channel injection pump and is respectively connected with two No. 3 channel ports;
5) a No. 4 channel port is connected into an empty centrifuge tube through a hose;
6) opening the injection pump, washing the microfluidic chip and the connecting hose, and collecting waste liquid at the tail end by using a centrifugal tube;
7) the flow rate of a 1% sodium alginate solution mixed with IGF-1 was adjusted to 50. mu.L/h by a syringe pump, and the mixture was mixed with CaCl-containing solution2The flow rate of the paraffin oil injection pump is adjusted to 400 mu L/h, and the flow rate of the 1% sodium alginate solution injection pump is adjusted to 80 mu L/h; sequentially starting the three injection pumps, and observing the balling condition in the microfluidic chip through a microscope;
8) transferring the hose connected with the No. 4 channel port into a centrifugal tube added with a surfactant, and collecting microspheres;
9) sucking the upper oil layer in the centrifugal tube in a super clean bench, filtering for 2-3 times by using a filter membrane with the pore diameter of 50 mu m, and cleaning microspheres;
10) immersing the microspheres into a solution containing ENBREL and Bevacizumab, adsorbing for 1 hour, and freeze-drying to obtain the sodium alginate double-layer microspheres loaded with ENBREL + Bevacizumab and IGF-1 in layers.
Further, in step 4), CaCl dissolved in paraffin oil2The concentration of (2) is 1%.
Further, in the step 7), the synthesis of the microfluidic chip is observed under an optical microscope, and the microspheres can be judged to be formed stably by observing the uniform flow of the spheres.
The invention has the beneficial effects that:
1. the invention discloses a preparation method of injectable epiphyseal plate regeneration hydrogel for children, which comprises the steps of firstly mixing double-layer SA microspheres prepared by the method disclosed in the application with BMSCs in hydrogel to prepare biological ink; injecting the hydrogel into the damaged part of the epiphyseal plate cartilage, and releasing Enbrel + Bevacizumab and IGF-1 locally, timely and quantitatively to inhibit the formation of a bone bridge and promote the regeneration and repair of the epiphyseal plate cartilage, thereby providing a new idea and method for solving the problem of 'repairing the epiphyseal plate damage';
2. the method has the advantages of simple overall implementation process, strong operability, large market application potential and benefit for popularization and use.
Drawings
FIG. 1 is a schematic structural diagram of a sodium alginate double-layer drug-loaded microsphere;
FIG. 2 is a schematic diagram of a micro-fluidic device for preparing sodium alginate double-layer drug-loaded microspheres;
FIG. 3 is a schematic diagram of GelMA loaded SA microspheres and BMSCs;
figure 4 is a diagram showing the pathological process of bone bridge formation after epiphyseal injury and the process of epiphyseal regeneration after injection of hydrogel.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying fig. 1-4, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Example 1
1) Preparation of sodium alginate double-layer drug-loaded microspheres
In the embodiment, the preparation of the sodium alginate double-layer drug-loaded microsphere is mainly carried out by using a flow focusing method and a microfluidic preparation method, and the preparation method mainly comprises the following steps:
1. as shown in fig. 2, the microfluidic chip is placed on the stage of the optical microscope, and six hoses are connected to the stage to extend out of six channel ports;
2. adding a 1% sodium alginate solution added with IGF-1 into a single-channel injection pump through an injector, exhausting air, and connecting with a No. 2 channel port through a hose;
3. sucking 1% sodium alginate solution into a two-tube injector, placing the two-tube injector in a two-channel injection pump, and connecting the two-tube injector with two No. 1 channel ports through a hose;
4. sucking 1% CaCl with a two-tube syringe2The paraffin oil is arranged in a double-channel injection pump and is respectively connected with two No. 3 channel ports;
a No. 5.4 channel port is connected into an empty centrifugal tube through a hose;
6. opening the injection pump, washing the microfluidic chip and the connecting hose, and collecting waste liquid at the tail end by using a centrifugal tube;
7. the flow rate of a 1% sodium alginate solution mixed with IGF-1 was adjusted to 50. mu.L/h, and the mixture was mixed with 1% CaCl2The flow rate of the paraffin oil injection pump is adjusted to 400 mu L/h, and the flow rate of the 1% sodium alginate solution injection pump is adjusted to 80 mu L/h; sequentially starting the three injection pumps, and observing the balling condition in the microfluidic chip through a microscope;
8. the hose connected with the No. 4 port is moved into a centrifuge tube added with a surfactant, microspheres are collected, the surfactant which has no biological toxicity and does not react with components in the microspheres can be used, and generally, the aseptic treatment is not needed;
9. sucking the upper oil layer in the centrifugal tube in a super clean bench, filtering for 2-3 times by using a filter membrane with the pore diameter of 50 mu m, and cleaning microspheres;
10. immersing the microspheres into an ENBREL + Bevacizumab solution, wherein the concentrations of ENBREL and Bevacizumab can be flexibly adjusted according to the specific conditions of an implanted receptor (animal or human), the effect can be screened by observing the effect under different concentrations through specific experiments, and the sodium alginate double-layer microspheres loaded with ENBREL + Bevacizumab and IGF-1 in a layered manner are obtained by freeze-drying after being adsorbed for 1 hour; sodium alginate microspheres containing different concentrations of IGF-1 can be obtained by adjusting the concentration of IGF-1.
2) Preparation of methacrylic anhydride modified gelatin hydrogel: 10 g of Gelatin (Gelatin) was weighed into 100mL of Phosphate Buffered Saline (PBS), the pH of the reaction system was adjusted to 8.5 with a 1 mol/L sodium hydroxide solution, and the mixture was stirred at a constant temperature of 50 ℃ until the Gelatin was completely dissolved. After the gelatin is completely dissolved, the solution is stabilized at 50 ℃, 10 mL of Methacrylic Anhydride (MA) is measured and slowly dropped into the gelatin solution, and a magnetic stirrer is used for stirring and reacting for 3 hours under the condition of keeping 50 ℃. Then 100mL of 50 ℃ PBS solution with pH =8.5 was added to dilute to terminate the reaction. The above solution was filled into a 3500 Da dialysis bag and dialyzed in deionized water at 37 ℃ for 5 days. And after the end, centrifuging the dialysate for 10 min at 3000 rpm, taking supernatant, freeze-drying in a freeze dryer at-80 ℃ for 6 days to obtain a dry white flocculent solid sample which is methacrylic acid modified gelatin hydrogel (GelMA), and storing in a refrigerator at-20 ℃.
3) Mixing SA microspheres and bone marrow mesenchymal stem cells (BMSCs) in hydrogel to prepare injectable bio-ink;
the hydrogel constructed by taking natural biological macromolecules as raw materials has good biocompatibility, can be used as a carrier of bone marrow mesenchymal stem cells, and provides more growth and proliferation spaces for the stem cells; in addition, the hydrogel can provide certain mechanical strength, and has the effect of promoting osteogenic differentiation of stem cells by mechanical stimulation of the stem cells; the hydrogel is added into the implant, so that fibers in gaps can be prevented from growing in, the osteogenesis inducing capacity is increased, and finally, the integration of the material and host bones is realized.
As shown in fig. 4, a represents epiphyseal cartilage, B represents epiphyseal cartilage damage model, C represents entry bone bridge formation pathology, where I is inflammatory reaction phase, which is inflammatory cell infiltration; II is the fibrosis reaction phase, during which BMSCs migrate to the wound site and begin osteogenesis; III is the osteogenesis phase, where the process of angiogenesis and mineralization of tissue is experienced; IV is the bone bridge maturation and reconstruction stage.
D represents the process of entry into epiphyseal cartilage regeneration using the novel hydrogel, wherein V represents the injection of hydrogel at the defect; stage VI shows that under the action of ENBREL + Bevacizumab, inflammatory response, vascularization and osteopontin formation are all inhibited; stage VII shows that after IGF-1 in the inner layer of the microsphere, chondrogenic differentiation and epiphyseal plate cartilage regeneration are promoted; stage VIII shows that epiphyseal cartilage damage has been repaired.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (4)
1. A preparation method of injectable epiphyseal plate regeneration hydrogel for children is characterized by comprising the following steps:
preparing a double-layer drug-loaded sodium alginate microsphere with an outer layer of an anti-osteopontic formation factor Enbrel + Bevacizumab and an inner layer of a chondrogenic differentiation factor IGF-1 by using a flow focusing method and a microfluidic method;
and (II) mixing the double-layer drug-loaded sodium alginate microspheres and bone marrow mesenchymal stem cells BMSCs in hydrogel to prepare the injectable bio-ink.
2. The preparation method of the injectable epiphyseal plate regenerating hydrogel for children as claimed in claim 1, wherein the specific preparation process of the double-layer drug-loaded sodium alginate microsphere is as follows:
1) placing the microfluidic chip on an objective table of an optical microscope, and respectively connecting six hoses to extend out of six channel ports;
2) adding a 1% sodium alginate solution added with IGF-1 into a single-channel injection pump through an injector, exhausting air, and connecting with a No. 2 channel port through a hose;
3) sucking 1% sodium alginate solution into a two-tube injector, placing the two-tube injector in a two-channel injection pump, and respectively connecting the two 1 # channel ports through flexible tubes;
4) sucking CaCl-containing solution with two-tube syringe2The paraffin oil is arranged in a double-channel injection pump and is respectively connected with two No. 3 channel ports;
5) a No. 4 channel port is connected into an empty centrifuge tube through a hose;
6) opening the injection pump, washing the microfluidic chip and the connecting hose, and collecting waste liquid at the tail end by using a centrifugal tube;
7) the flow rate of a 1% sodium alginate solution mixed with IGF-1 was adjusted to 50. mu.L/h by a syringe pump, and the mixture was mixed with CaCl-containing solution2The flow rate of the paraffin oil injection pump is adjusted to 400 mu L/h, and the flow rate of the 1% sodium alginate solution injection pump is adjusted to 80 mu L/h; three injection pumps are started in sequence to be communicatedObserving the balling condition in the microfluidic chip by a microscope;
8) transferring the hose connected with the No. 4 channel port into a centrifugal tube added with a surfactant, and collecting microspheres;
9) sucking the upper oil layer in the centrifugal tube in a super clean bench, filtering for 2-3 times by using a filter membrane with the pore diameter of 50 mu m, and cleaning microspheres;
10) immersing the microspheres into a solution containing ENBREL and Bevacizumab, adsorbing for 1 hour, and freeze-drying to obtain the sodium alginate double-layer microspheres loaded with ENBREL + Bevacizumab and IGF-1 in layers.
3. The method for preparing injectable childhood epiphyseal plate regenerating hydrogel according to claim 2, characterized in that in step 4), CaCl dissolved in paraffin oil is added2The concentration of (2) is 1%.
4. The method for preparing an injectable rejuvenated hydrogel of childproof epiphysis plates according to claim 2, wherein in step 7), the synthesis of the microfluidic chip is observed under an optical microscope, and the microsphere is judged to have started to form stably by observing that the sphere flows at a constant speed.
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