Injectable rhBMP-2-containing bone repair hydrogel and preparation method thereof
阅读说明：本技术 一种可注射型含rhBMP-2的骨修复水凝胶及其制备方法 (Injectable rhBMP-2-containing bone repair hydrogel and preparation method thereof ) 是由 孙亚 党瑞杰 朱彪 于 2020-04-17 设计创作，主要内容包括：本发明涉及一种可注射型含rhBMP-2的骨修复水凝胶及其制备方法,属于生物医学工程技术领域。所述水凝胶材料含有重组人骨形态发生蛋白-2(rhBMP-2),纳米羟基磷灰石,透明质酸-己二酸二酰肼衍生物和主链具有醛基的海藻酸衍生物。其制备方法包含纳米羟基磷灰石制备、海藻酸钠透明质酸衍生物制备及复合水凝胶制备等步骤。本发明为骨缺损治疗提供了一种新的修复方法,具有广阔的应用前景和理想的修复效果。(The invention relates to an injectable rhBMP-2-containing bone repair hydrogel and a preparation method thereof, belonging to the technical field of biomedical engineering. The hydrogel material contains recombinant human bone morphogenetic protein-2 (rhBMP-2), nano hydroxyapatite, hyaluronic acid-adipic dihydrazide derivatives and alginic acid derivatives with aldehyde groups on the main chain. The preparation method comprises the steps of nano hydroxyapatite preparation, sodium alginate hyaluronic acid derivative preparation, composite hydrogel preparation and the like. The invention provides a new repairing method for bone defect treatment, and has wide application prospect and ideal repairing effect.)
1. An injectable bone repair hydrogel containing rhBMP-2 is characterized in that the composite hydrogel material contains recombinant human bone morphogenetic protein-2 (rhBMP-2), 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 rhBMP-2-containing bone repair hydrogel according to claim 1, which comprises a final concentration of nHAP of 5% (w/v), a final concentration of HA-ADH of 5% (w/v), a final concentration of ALG-CHO of 5% (w/v), and a concentration of rhBMP-2 of 150 ug/ml.
3. The injectable rhBMP-2-containing bone repair hydrogel according to claim 1, which is prepared by a method comprising the following steps.
(1) Obtaining rhBMP-2: the rhBMP-2 in this application can be active rhBMP-2 protein from various sources. It can be extracted from natural product or obtained by expression from various host strains by genetic engineering method, or can be a finished product purchased from market.
(2) 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.
(3) 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.
(4) 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.
(5) 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 rhBMP-2, dispersing in a PBS solution, and adding ALG-CHO until the solution is completely dissolved to obtain a solution 2; the two solutions were then loaded into a gun and injected into the defect site for use.
4. The composite hydrogel containing rhBMP-2 for use in bone defect repair according to claim 1, wherein the hydrogel is used for treating craniomaxillofacial and other bone defects.
Technical Field
The invention relates to the field of biomedical materials containing active protein and the technical field of biomedical engineering, in particular to injectable bone repair hydrogel containing rhBMP-2 and a preparation method thereof.
Background
With the aggravation of the aging social process, the incidence of bone diseases such as fracture, bone tumor, osteoporosis and the like is rapidly increased, and the bone defect caused by the disease not only seriously threatens the physical and psychological health of patients, but also brings huge economic burden to the society. Autologous bone grafting has been considered as the "gold standard" for treating bone defects, but has disadvantages of limited autologous bone source and additional damage to donor sites during application. Allogeneic bone grafts are also used clinically, but their use is limited by problems such as high failure rates and the development of immune rejection. In recent years, biomaterials are increasingly used in the treatment of bone defects, and become a new trend.
Bone morphogenetic proteins are important regulators of bone formation and are capable of inducing osteogenesis and ectopic ossification. The recombinant human bone morphogenetic protein 2(rhBMP-2) is a member with the strongest action capability in a BMP family, can stimulate related stem cells to the region and form bones, is expressed in endochondral ossification, fracture healing and limb growth, plays an important role in bone growth and regeneration repair, and is widely applied to the repair and treatment of bone defects clinically.
Bone repair is a long-term process, and due to the short half-life of rhBMP-2, it is metabolized and absorbed very quickly upon topical application, making it difficult to maintain effective drug concentrations. Therefore, how to regulate the release rate and the release period of the rhBMP-2 at the bone defect position to ensure that the rhBMP-2 is well matched with the growth period of new bones and improve the bioavailability of the rhBMP-2, thereby achieving the optimal curative effect is a key problem to be solved by utilizing the rhBMP-2 to carry out bone repair.
Disclosure of Invention
In view of the above problems, the present applicant provides an injectable bone repair hydrogel containing rhBMP-2. The invention adopts sodium alginate-hyaluronic acid hydrogel as a carrier to wrap rhBMP-2 and inject the rhBMP-2 to the bone defect part, thereby realizing the controlled release and the slow release of the rhBMP-2, and simultaneously adding the nano hydroxyapatite, thereby further enhancing the mechanical property and the bone conductivity of the material.
The technical scheme of the invention is as follows:
an injectable rhBMP-2-containing bone repair hydrogel is characterized in that the composite hydrogel material comprises rhBMP-2, nano-hydroxyapatite (nHAP), a hyaluronic acid-adipic acid dihydrazide derivative (HA-ADH) and an alginic acid derivative (ALG-CHO) with aldehyde groups on the main chain.
Furthermore, the hydrogel HAs 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 a rhBMP-2 concentration of 150 ug/ml.
Further, the preparation method comprises the following steps:
(1) obtaining rhBMP-2: the rhBMP-2 in this application can be active rhBMP-2 protein from various sources. It can be extracted from natural product or obtained by expression from various host strains by genetic engineering method, or can be a finished product purchased from market.
(2) 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.
(3) 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.
(4) 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.
(5) 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 rhBMP-2, dispersing in a PBS solution, and adding ALG-CHO until the solution is completely dissolved to obtain a solution 2; the two solutions were then loaded into a gun and injected into the defect site for use.
Further, in the step (2), 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 (3), 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 (4), 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 (5), the mass of nHAP, HA-ADH and ALG-CHO is 150mg, the volume of PBS for dissolving nHAP is 1.5ml, and the volume of PBS for dissolving rhBMP-2 is 450 mu g.
Further, the injectable rhBMP-2-containing bone repair hydrogel is used for treating craniomaxillofacial and other bone defects.
The invention has the advantages that:
1. convenient operation can be adapted to the complicated and changeable bone defect environment 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 rhBMP-2 slow release function;
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 is a scanning electron microscope image of the hydrogel prepared by the present invention.
FIG. 2 shows the swelling, degradation and mechanical property test results of the hydrogel prepared by the present invention, wherein A is the swelling result, B is the degradation result, and C is the mechanical property test result.
FIG. 3 is an in vitro sustained release curve of rhBMP-2.
FIG. 4 is the result of Micro-CT 3D reconstruction of hydrogel in vivo bone defect repair experiment, wherein A is a control group, B is a hydrogel group, and C is a hydrogel group containing rhNMP-2.
Detailed Description
The present invention will be specifically described below with reference to examples and detection examples.
EXAMPLE 1 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 rhBMP-2
Weighing nHAP, dispersing in a PBS solution, and adding HA-ADH until the nHAP is completely dissolved to obtain a solution 1; weighing rhBMP-2, dispersing in a PBS solution, and adding ALG-CHO until the solution is completely dissolved to obtain a solution 2; the two solutions were then loaded into a gun and injected into the defect site for use.
Test example 1
The product obtained in example 1 above was subjected to an internal morphology observation.
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. 1. As can be seen from fig. 1: 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 2
The hydrogel obtained in example 1 was subjected to swelling, degradation and mechanical property examination.
The test method and conditions were: the swelling properties of the hydrogels were determined by weighing, placing the samples in 37 ℃ PBS and in a 37 ℃ environment, then removing the samples from the PBS at set time points, weighing after wiping off the surface liquid, and the weight was recorded as WsThe initial weight of the sample after lyophilization was recorded as WdThe swelling ratio of the hydrogel was calculated according to the following formula: swelling ratio ═ Ws-Wd)/Wd×100％。
The degradation properties of the hydrogels were also measured by weighing. The initial mass of the freeze-dried sample was first weighed and recorded as W0The samples were then placed in PBS and placed in a 37 ℃ environment, then the samples were taken at specific time points, respectively, the samples were weighed after freeze-drying, the weight was recorded as Wt, and the degradation rate of the hydrogel was calculated according to the following formula: the degradation rate is (W0-Wt)/W0 multiplied by 100%.
The mechanical properties of the hydrogel were measured at room temperature using an Instron 5943 mechanical tester, the hydrogel was prepared into a 15mm diameter by 15mm high sample and was compressed at a constant speed of 1mm/min until the sample failed, and the stress-strain curve was recorded.
As shown in FIG. 2, it can be seen that the swelling ratio of the hydrogel is 22.17. + -. 3.01%, the degradation ratio is 52.64. + -. 6.13%, and the maximum stress value is 0.64. + -. 0.07 MPa.
Test example 3
The hydrogel obtained in example 1 was subjected to in vitro rhBMP-2 sustained release test.
The test method and conditions were: and detecting the rhBMP-2 protein release condition by using a BCA kit. And loading 250 mu g of rhBMP-2 in a hydrogel material, incubating in physiological saline at 37 ℃, collecting the incubated liquid every 1 day, detecting the rhBMP-2 protein release condition, and drawing a release curve.
As shown in FIG. 3, it can be seen that the rhBMP-2 was released from the material by about 90.1% after 30 days by consecutive examination.
Test example 4
The product obtained in example 1 above was subjected to in vivo osteogenesis effect test.
The test method and conditions were: 15 SD male rats (250-300g) were randomly divided into 3 groups, namely a blank control group, a hydrogel repair group, and a hydrogel repair group containing rhBMP-2 (containing 300. mu.g of rhBMP-2), and 5 rats were each group. 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 implantation of 8w of material, rats were subjected to heart perfusion under anesthesia (10% neutral formalin), the calvaria bone was removed, the specimen was fixed in 10% neutral formalin, and Micro-CT scanning and 3D reconstruction were performed on the specimen.
As shown in FIG. 4, it can be seen that the defect of the control group had a part of new bone formation at the edge, most of the defect was not repaired, the hydrogel group had a small amount of new bone formation at the middle of the defect in addition to the new bone formation at the edge of the defect, and the hydrogel repair group containing rhBMP-2 had a large amount of new bone formation at the middle of the defect, which was significantly greater than that of the other two groups.