CN112206356A

CN112206356A - Injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes and preparation method thereof

Info

Publication number: CN112206356A
Application number: CN201910623484.8A
Authority: CN
Inventors: 杨烁; 张建军; 朱彪; 赵立升; 付之光; 温宁
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-07-11
Filing date: 2019-07-11
Publication date: 2021-01-12

Abstract

The invention relates to an injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes and a preparation method thereof, belonging to the technical field of biomedical engineering. The hydrogel material contains human umbilical cord mesenchymal stem cell exosome, nano hydroxyapatite, hyaluronic acid-adipic acid dihydrazide derivatives and alginic acid derivatives with aldehyde groups on the main chain. The preparation method comprises the steps of separating human umbilical cord mesenchymal cells, extracting exosomes, preparing nano hydroxyapatite, preparing sodium alginate hyaluronic acid derivatives, preparing composite hydrogel and the like. The invention provides a new repairing method for bone defect treatment, and has wide application prospect and ideal repairing effect.

Description

Injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes and a preparation method thereof.

Background

Bone defect repair is a hot problem in the research of the orthopedic field. Clinically, even common fractures, approximately 2% -10% of fractures may progress to nonunion due to poor bone regeneration. Orthopedic clinicians also often encounter problems with bone defects due to reasons such as fracture, bone infection, bone tumor removal, etc. In addition, obesity, diabetes, etc. cause an increase in skeletal muscle diseases and a decrease in bone regeneration, and elderly patients with osteoporosis have a high risk of bone fracture and insufficient bone regeneration ability. Thus, how to promote bone regeneration remains a major challenge facing clinicians.

The bone tissue engineering technology based on the mesenchymal stem cells brings new hope for the regeneration and repair of clinical bone defects. Recent research shows that the stem cell derived exosome has similar biological effect with stem cells, and can be used as a substitute of the stem cells for bone tissue repair, so that various risks such as mutant tumorigenesis and immune rejection caused by direct use of the stem cells are avoided.

In the current research of repairing bone defects by using exosomes, exosomes mainly play a role in an injection mode, but the method can lead the exosomes to be eliminated in vivo quickly, and the bone repair is a long-period process. Therefore, how to regulate and control the release rate and the release period of the exosome at the bone defect to ensure that the exosome is well matched with the growth period of a new bone, and improve the bioavailability of the exosome, thereby achieving the optimal curative effect is a key problem to be solved by utilizing the exosome to carry out bone repair.

Disclosure of Invention

In order to solve the problems, the applicant provides an injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes. The invention adopts sodium alginate-hyaluronic acid hydrogel as a carrier, wraps exosome and injects the exosome to a bone defect part to realize the controlled release and slow release of the exosome, and simultaneously adds nano hydroxyapatite, thereby further enhancing the mechanical property and the osteoconductivity of the material.

The technical scheme of the invention is as follows:

an injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes is characterized in that the composite hydrogel material comprises human umbilical cord mesenchymal stem cell exosomes, nano hydroxyapatite (nHAP), hyaluronic acid-adipic acid dihydrazide derivatives (HA-ADH) and alginic acid derivatives (ALG-CHO) with aldehyde groups on the main chain.

Furthermore, the hydrogel had a final nHAP concentration of 5% (w/v), a final HA-ADH concentration of 5% (w/v), a final ALG-CHO concentration of 5% (w/v), and contained exosomes at a concentration of 150 ug/ml.

Further, the preparation method comprises the following steps:

(1) separating and culturing umbilical cord mesenchymal stem cells;

(2) osteogenic induction of umbilical cord mesenchymal stem cells: and (3) continuously culturing the cells cultured in the third generation step (1), and when the cell density reaches 70%, continuously culturing by using an osteogenic induction medium.

(3) Exosome extraction: taking the cell culture supernatant obtained 2 days after the osteogenesis induction in the step (2), centrifuging for 10min at the temperature of 4 ℃ at 300g, centrifuging for 10min at 2000g, centrifuging for 30min at 10000g to remove dead cells and cell debris, filtering the culture medium by using a 0.22um filter membrane, and further centrifuging for 2 hours at the temperature of 4 ℃ at 100000 g. The obtained white precipitate is exosome, and is dissolved in PBS to be stored in a refrigerator at the temperature of minus 80 ℃.

(4) Preparation of nHAP: dissolving calcium nitrate in deionized water, adding sodium citrate, stirring until the calcium nitrate is completely dissolved, and adjusting the pH value of the solution by using ammonia water to obtain a solution A; dissolving diammonium phosphate in deionized water to obtain a solution B; and then starting a supergravity Rotating Packed Bed (RPB), adjusting the rotating speed, and respectively conveying A, B two solutions to a slurry product generated by reaction in the RPB, and obtaining nHAP after centrifugation, washing and vacuum drying.

(5) Preparation of HA-ADH: dissolving hyaluronic acid in deionized water, adding Adipic Dihydrazide (ADH) after dissolving, uniformly stirring, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) into the solution for catalytic reaction, then adjusting the pH of the solution with HCl, reacting at room temperature for 12h, dialyzing with deionized water, and freeze-drying to obtain HA-ADH.

(6) ALG-CHO preparation: dissolving sodium alginate in deionized water, dissolving sodium periodate in deionized water, reacting at room temperature for 24h, adding ethylene glycol to terminate the reaction, dialyzing, and freeze-drying to obtain ALG-CHO.

(7) Preparing the composite hydrogel: weighing nHAP, dispersing in a PBS solution, and adding HA-ADH until the nHAP is completely dissolved to obtain a solution 1; weighing ALG-CHO, and dissolving in a PBS solution containing exosome to obtain a solution 2; the two solutions were then loaded into a gun and injected into the defect site for use.

Further, the step (1) is as follows:

soaking umbilical cord 15cm near fetal segment in PBS containing 10% streptomycin, taking out in a superclean bench, placing in a culture dish, repeatedly washing umbilical cord with PBS, stripping adventitia and umbilical vessel, and shearing tissue to 1mm³The tissue blocks were transferred to centrifuge tubes, washed with PBS and centrifuged at 2500rpm for 5 min. Resuspend the tissue mass in a-MEM complete medium, inoculate in a cell dish, incubate at 37 ℃ in 5% CO2, supplement the a-MEM complete medium the next day, and then change the medium completely every 3 days. When the cell growth reaches 70-80% fusion, 0.25% pancreatin is digested for 5min, the cell is observed under the mirror to shrink and become round, the digestive juice is immediately discarded, complete culture medium is dripped to stop digestion, and subculture is carried out according to the proportion of 1: 3.

Further, the osteogenic induction medium in the step (2) comprises DMEM, fetal bovine serum, dexamethasone, ascorbic acid, beta-glycerol phosphate, L-glutamine and double antibody.

Further, in the step (4), the mass of calcium nitrate is 5g, the volume of deionized water for dissolving calcium nitrate is 100ml, the mass of sodium citrate is 0.4g, the mass of ammonia water is 5mol/L, the pH value is adjusted to 11, the mass of diammonium hydrogen phosphate is 1.675g, and the volume of deionized water for dissolving diammonium hydrogen phosphate is 60 ml.

Further, in the step (5), the mass of the hyaluronic acid is 1g, the volume of the deionized water is 200ml, the ADH is 12.5g, the pH value is 4.75, and the dialysis time is 5 days.

Further, in the step (6), the mass of the sodium alginate is 2g, the volume of the deionized water for dissolving the sodium alginate is 90ml, the mass of the sodium periodate is 1g, the volume of the deionized water for dissolving the sodium periodate is 10ml, and the volume of the ethylene glycol is 2 ml.

Further, in the step (7), the mass of nHAP, HA-ADH and ALG-CHO is 150mg, the volume of PBS for dissolving nHAP is 1.5ml, and the concentration of exosome in PBS for dissolving ALG-CHO is 300ug/ml, and the volume is 1.5 ml.

Further, the injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes is used for treating craniomaxillofacial and other bone defects.

The invention has the advantages that:

1. the operation is convenient, and the device can be suitable for complex and changeable bone defect environments in vivo;

2. can provide a three-dimensional scaffold for cells, and is beneficial to cell colonization and bone matrix deposition;

3. can be slowly degraded at the bone defect part and has good slow release function of exosome;

4. the successful development of the method can bring a more efficient treatment method for patients with bone defects, and simultaneously provides a repair mode with better prospect for the fields of tissue engineering, regenerative medicine and the like.

Drawings

FIG. 1 shows the identification result of flow cytometry of human umbilical cord mesenchymal stem cells.

FIG. 2 shows the exosome identification results, wherein A is a transmission electron microscope image, B is a NanoSight nano-particle size analysis result, and C is a Western Blot result.

FIG. 3 is a scanning electron microscope image of the hydrogel prepared by the present invention.

Figure 4 is an in vitro sustained release profile of exosomes.

FIG. 5 shows the HE staining results of hydrogel in vivo bone defect repair experiments, wherein A is a control group, B is a hydrogel group, and NB represents a new bone.

Detailed Description

The present invention will be specifically described below with reference to examples and detection examples.

Example 1 extraction of exosomes from human umbilical cord mesenchymal stem cells

(1) Isolated culture of umbilical cord mesenchymal stem cells

(2) Exosome extraction

When the cell growth density reaches 70%, the basic culture medium is discarded, the osteogenic induction culture medium is used after PBS is washed twice, the cell is continuously cultured for 2 days, then cell culture supernatant is collected, the cell culture supernatant is centrifuged for 10min at 300g under the condition of 4 ℃, 10min at 2000g and 30min at 10000g to remove dead cells and cell debris, then the culture medium is filtered by a 0.22um filter membrane, and the cell culture supernatant is further centrifuged for 2 hours at 100000g and 4 ℃. The obtained white precipitate is exosome, and is dissolved in PBS to be stored in a refrigerator at the temperature of minus 80 ℃.

EXAMPLE 2 preparation of hydrogel

(1) Preparation of nano-hydroxyapatite

Dissolving 5g of calcium nitrate in 100ml of deionized water, adding 0.4g of sodium citrate, stirring until the calcium nitrate is completely dissolved, and adjusting the pH value of the solution to 11 by using 5mol/L ammonia water to obtain a solution A; dissolving 1.675g of diammonium phosphate in 60ml of deionized water to obtain a solution B; and then starting a super-gravity Rotating Packed Bed (RPB), adjusting the rotating speed to 2500rpm, respectively conveying A, B two solutions to the RPB, washing the slurry product generated by reaction twice by centrifugation, and adding water for ultrasonic dispersion for 10min to obtain the nano HAP dispersion. Vacuum drying to obtain nHAP powder.

(2) Preparation of hyaluronic acid derivatives

Dissolving 1g of hyaluronic acid in 200ml of deionized water, adding 12.5g of Adipic Dihydrazide (ADH) after dissolving, uniformly stirring, adding 2g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) into the solution for catalytic reaction, then adjusting the pH of the solution to 4.75 by using HCl, reacting for 12 hours at room temperature, continuously dialyzing for 5 days by using deionized water, and freeze-drying to obtain HA-ADH.

(3) Preparation of sodium alginate derivatives

Dissolving 2.0g of sodium alginate into 90ml of deionized water, dissolving 1.0g of sodium periodate into 10ml of deionized water, reacting for 24 hours at room temperature, adding 2ml of ethylene glycol to terminate the reaction, dialyzing, and freeze-drying to obtain ALG-CHO.

(4) Preparation of injectable bone repair hydrogel containing exosomes of human umbilical cord mesenchymal stem cells

Weighing 150mg of nano-hydroxyapatite, dispersing the nano-hydroxyapatite in 1.5ml of PBS solution, and adding 150mg of HA-ADH until the nano-hydroxyapatite is completely dissolved to obtain a solution 1; weighing 150mg of ALG-CHO, and dissolving in 1.5ml of PBS solution containing 300ug/ml of exosome to obtain solution 2; the two solutions were then loaded into a gun and injected into the defect site for use.

Test example 1

The umbilical cord mesenchymal stem cells obtained in the above example 1 were identified.

The test method and conditions were:

digesting the P3 umbilical cord mesenchymal stem cells by trypsin to prepare single cell suspension. Washing cells with PBS for 2 times, centrifuging at 1000rpm for 5min, discarding supernatant, resuspending cells with PBS to adjust cell density to 2 × 10⁵One tube per tube. Mu.l of PE-CD73, PE-CD31, FITC-CD34, FITC-CD44, FITC-CD45, FITC-CD90 and APC-CD 105. Adding PBS with the same amount into the isotype control tube, adding 200 mul of flow cytometry stationary liquid into each tube, detecting by using a BD flow cytometer, observing and recording the expression rate of positive cells.

The test results are shown in fig. 1. Cells positively expressing stem cell surface marker antigens CD44, CD90, CD105, CD73 and negatively expressing hematopoietic stem cell surface marker antigens CD31, CD34 and CD45 can be seen. Indicating that the umbilical cord mesenchymal stem cells are successfully extracted.

Test example 2

The exosomes obtained in example 1 above were identified.

The test method and conditions were:

(1) transmission electron microscope identification of stem cell exosome morphology

Taking 10 mu L of exosome purification stock solution, dripping the exosome purification stock solution on a carrier copper net after heavy suspension, standing for 1min, then dripping 30 mu L of phosphotungstic acid solution with the concentration of 20ml/L on the copper net, and carrying out negative dyeing for 5min at room temperature. The copper mesh was then placed under an incandescent lamp and baked dry, and the image was observed and captured by transmission electron microscopy.

(2) Exosome particle size analysis

And diluting the exosomes, adding the exosomes into an Izon qNano nano analysis system, and measuring the number of the exosomes and the distribution of the particle size.

(3) The western-blot identifies the surface markers of the exosomes of the stem cells:

BCA assay for exosome concentration

b. Preparing separating gel and concentrated gel electrophoresis buffer according to the proportion.

c. Protein denaturation: mixing 5 × loading buffer with protein sample with determined concentration, boiling for 5min to denature, cooling on ice, and storing at-20 deg.C.

d. Electrophoresis: carefully remove the comb and hold the gel in place on the electrophoresis apparatus. Adding 1 XTris-glycine into the inner tank and the outer tank respectively, adding 25 mu l of the sample into the sample application hole for electrophoresis, wherein the electrophoresis conditions are as follows: concentrating gel at 60V, and separating gel at 120V. And taking out the gel after electrophoresis, and marking.

e. Film transfer: the film was transferred to the positive electrode and the glue to the negative electrode. The glue and membrane were pressed with 3 sheets/side of filter paper, respectively. The voltage was adjusted to 100 volts at the time of transfer and the transfer time was 1 hour.

f. And (3) sealing: the electrophoresis device is disassembled, and the transfer membrane is sealed in 10 percent skim milk powder sealing liquid for 2 hours.

g. The membrane was washed 5 times with PBST and the corresponding primary antibody (dilution ratio 1: 1000) was added. The reaction time is 1.5 h.

h. HRP-labeled secondary antibody (concentration 1: 4000) was added thereto, and the reaction was carried out for 1 hour. The membrane was then washed every 5 minutes for 25 minutes.

i. And (3) taking an ECL fluorescent substrate, dripping ECL luminescent liquid on the PVDF membrane, reacting for 3-5 minutes, taking an X-ray film in a dark room to cover, exposing, developing and fixing.

The test results are shown in fig. 2. It can be seen from the figure that: the complete membranous structure of the exosome can be found through observation of a transmission electron microscope, Nanosight nano-particles are tracked and analyzed to show that the particle size distribution peak value of the extracted exosome is about 120nm, and Western Blot identification results show that the extracted exosomes positively express CD9, CD81 and Tsg 101. The above results fully demonstrate that we successfully obtained exosomes.

Test example 3

The product obtained in example 2 was observed by scanning electron microscopy and exosome-sustained release.

The test method and conditions were: the internal appearance of the hydrogel is observed by SEM, the prepared hydrogel is frozen and dried, the conductivity is increased by spraying gold on the surface of the hydrogel, and then the internal appearance is observed under the condition that the accelerating voltage is 15 kV.

The test results are shown in fig. 3. As can be seen in fig. 3: the hydrogel is in a porous structure, the aperture size is about 50um, and meanwhile, hydroxyapatite attached to the side wall of the aperture can be seen.

Test example 4

The hydrogel obtained in example 2 above was subjected to an in vitro exosome sustained-release test.

The test method and conditions were: exosome protein release was detected by BCA kit. Dripping 250 mul of exosome at 1 mug/mul into the material, incubating in physiological saline at 37 ℃, collecting the liquid 1-14 days after incubation, detecting the protein release condition of the exosome, and drawing a release curve.

The test results are shown in fig. 4, and it can be seen that the exosomes were released from the material by about 71.1% after 14 days of continuous detection.

Test example 5

The product obtained in example 2 above was subjected to in vivo osteogenesis effect test.

The test method and conditions were: 16 SD male rats (250-300g) were collected and randomly divided into 2 groups, i.e., a blank control group, and 8 rats per group in a hydrogel repair group (containing 300. mu.l of hydrogel). 0.1ml of new injection of 50% fast dormancy anesthetizes the rat, prepares skin, and 75% alcohol is disinfected, makes a sagittal incision at the top of the head, opens the full-layer mucoperiosteum flap, uses the bone drill of the department of plantation to make a circular defect with a diameter of 8mm, and the drill grinds the in-process and constantly washes with normal saline, prevents to rub the themogenesis and lead to the necrosis of local tissue. After implanting material 8w, rat was subjected to heart perfusion under anesthesia (10% neutral formalin), skull cap bone was removed, a specimen was fixed with 10% neutral formalin, EDTA decalcification was performed on the specimen, ethanol-stepwise dehydration and paraffin embedding were performed, a histological section having a thickness of 4 μm was cut, and morphological change of tissue at microscopic defect was analyzed by HE staining

As shown in FIG. 5, it can be seen that the defect of the control group had a part of new bone formation only at the edge, most of the defect was covered with fibrous connective tissue, and the hydrogel group had a large amount of new bone formation at the middle of the defect, in addition to the new bone formation at the edge of the defect.

Claims

1. An injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes is characterized in that the composite hydrogel material comprises human umbilical cord mesenchymal stem cell exosomes, nano hydroxyapatite (nHAP), hyaluronic acid-adipic acid dihydrazide derivatives (HA-ADH) and alginic acid derivatives (ALG-CHO) with aldehyde groups on the main chain.

2. The injectable bone repair hydrogel containing human umbilical cord mesenchymal stem cell exosomes according to claim 1, which comprises the components of 5% (w/v) of nHAP final concentration, 5% (w/v) of HA-ADH final concentration, 5% (w/v) of ALG-CHO final concentration and 150ug/ml of exosomes.

3. The injectable human umbilical cord mesenchymal stem cell-containing exosome bone repair hydrogel according to claim 1, which is prepared by the following steps.

(1) Separating and culturing umbilical cord mesenchymal stem cells;

(5) Preparation of HA-ADH: dissolving hyaluronic acid in deionized water, adding Adipic Dihydrazide (ADH) after dissolving, uniformly stirring, adjusting the pH of the solution by using HCl, reacting at room temperature for 12h, dialyzing by using deionized water, and freeze-drying to obtain the HA-ADH.

(6) ALG-CHO preparation: dissolving sodium alginate in deionized water, dissolving sodium periodate in deionized water, reacting at room temperature for 24h, adding ethylene glycol to terminate the reaction, dialyzing, and freeze-drying to obtain LG-CHO.

4. The composite hydrogel containing exosomes of human umbilical cord mesenchymal stem cells for bone defect repair according to claim 1, wherein the hydrogel is used for treating craniomaxillofacial and other bone defects.