CN110903380A - Solid concentrated growth factor and preparation method and application thereof - Google Patents
Solid concentrated growth factor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a solid concentrated growth factor and a preparation method and application thereof, comprising the following steps: freeze-drying or vacuum-drying the extracted gel-like concentrated growth factor to obtain the solid concentrated growth factor; the solid concentrated growth factor prepared by the invention contains a large amount of fibrin with a three-dimensional matrix reticular structure, is a porous structure, has longer storage period and obviously better biomechanical strength compared with a gel concentrated growth factor, simultaneously has better capability of promoting the proliferation, differentiation and mineralization of BMSCs, can be independently used as an implant material for bone defect repair, and obviously improves the biomechanical strength of a damaged vertebra after repair; the preparation method of the solid concentrated growth factor is simple to operate, venous blood can be used as a raw material, and the prepared material has better performance and wider application, and has wide clinical application prospect and potential economic value.
Description
Technical Field
The invention belongs to the field of medical biology, and particularly relates to a solid concentrated growth factor, and a preparation method and application thereof.
Background
Osteoporotic Vertebral Fracture (OVF) is one of the serious complications of Osteoporosis (OP), and belongs to the clinical treatment difficulty. After OVF occurs, compression insertion changes occur, and bone defects in the vertebral body are left after posture reduction or manual reduction, so that poor fracture healing and poor mechanical strength recovery of the vertebral body easily occur, pain is not relieved, the vertebral body is subjected to progressive collapse, and spinal nerves are subjected to compression injury in severe cases, so that the requirement for treating OVF cannot be completely met in clinic by simple pain relief, bed rest, brace, manual reduction and other conservative treatments. Promoting the early healing of fractured vertebral bodies, maintaining the normal force line of the vertebral column and recovering the normal function of the vertebral column become a new standard for treating OVF.
Polymethylmethacrylate (PMMA) is one of the most commonly used implant materials for the current treatment of OVF, and although it provides sufficient support strength and adhesion, it also has some significant disadvantages: the traditional Chinese medicine composition can not be degraded and absorbed in vivo, has poor biocompatibility, no bioactivity, overlarge elastic modulus, large amount of heat released during polymerization, toxic unpolymerized monomers and the like, and causes serious complications frequently occurring in clinical application. The autologous bone transplantation is taken as the current gold standard for treating bone defects, has quick absorption and good effect, but has limited sources and many complications of bone extraction; allogeneic bone and xenogeneic bone are limited in clinical application due to immune rejection, and have risks of rejection and cross infection. To promote OVF healing, the search for biomaterials with good biocompatibility and capable of providing sufficient support strength is a hot spot of current research.
The Concentrated Growth Factors (CGF) are third-generation plasma extracts, which contain a large amount of Growth Factors, the CGF promotes bone regeneration and soft tissue repair, which is a hotspot and difficulty of the current research, and the CGF extracted from animal experiments has great significance when applied to experimental research. At present, CGF is widely applied to the field of dental implant in oral medicine, and related animal experimental research reports that CGF can promote repair of skull defects of rats.
Compared with PRF and PRP, CGF has more completely separated cellular components in blood, fibrin clot is larger in volume, viscosity and strength, and growth factors such as Platelet-derived growth factor (PDGF), Transforming growth factor- β (Transforming growth factor- β - β), Insulin-like growth factor (Insulin-like growth factor), Vascular Endothelial Growth Factor (VEGF), and Bone Morphogenetic Protein (BMP) are contained in CGF at higher concentration, which can promote regeneration repair and angiogenesis of bone tissue or soft tissue.
At present, experiments are carried out after CGF is prepared by centrifuging blood taken from goat, beagle dog, rabbit vein and Vista rat heart, the cost is high, the blood taking difficulty is high, and the method is not convenient to master; after extraction, the extract can be stored in a refrigerator at the temperature of-4 ℃ for 0-48 hours, so the extract is generally required to be used for extraction. In addition, in orthopedics, the bone is usually prepared into gel-like CGF, then the gel-like CGF is cut into small pieces and then the gel-like CGF is implanted into a bone defect part together with autologous bone or allogeneic bone, and at present, a CGF product suitable for being implanted into a bone defect area alone is not available, so that the effect of the CGF on bone defect repair by being implanted into the bone defect area alone is not clear. In addition, the CGF gel is used alone, but has the problems of poor mechanical properties, incapability of long-term storage and the like, and the clinical application of the CGF gel is severely limited.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a concentrated growth factor which is easy to operate, low in cost, better in mechanical properties, and suitable for clinical application, and can be used as an implant material for treating OVF.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of solid-state concentrated growth factors comprises the following steps: the gel-like concentrated growth factor is subjected to freeze drying or vacuum drying to obtain the solid concentrated growth factor.
In order to improve the biomechanical strength and the storage time of the CGF, the solid concentrated growth factor convenient for transplantation is prepared by a freeze drying method or vacuum drying method, can be placed in a refrigerator at the temperature of between 80 ℃ below zero and 60 ℃ below zero, and can be stored for at least 3 months for a long time, so that the use convenience of the CGF is greatly improved.
As a preferred embodiment of the present invention, the gel-like concentrated growth factor is prepared by centrifuging animal venous blood.
As a preferred embodiment of the present invention, the venous blood is rat abdominal main venous blood.
The invention firstly discloses that the SD rat is used for abdominal main vein blood sampling, the operation is simple and convenient, the grasping and the development are convenient, and the research cost is greatly reduced while the experiment purpose is ensured to be realized by adopting the SD rat to extract the CGF.
In a preferred embodiment of the present invention, the centrifugation step in step (1) is accelerated for 30 seconds to 2700rpm, and after centrifugation for 2min, the speed is decreased to 2400rpm, centrifugation is performed for 4min, then accelerated to 2700rpm and centrifuged for 4min, and centrifugation is performed at 3300rpm for 3min, and finally decelerated for 36 seconds and stopped.
The centrifugation step is performed by using a special Italy centrifuge (Medifuge, Silfradetsrl, Italy), which can separate the concentrated growth factors in the blood sample well, and the concentrated growth factors are derived from self bodies, do not cause infectious diseases, have excellent biological safety, have small wound and are easy to be accepted by patients. (ii) a After centrifugation, the blood in the centrifuge tube was poured into a sterilized 10X 10cm cell culture dish, and the CGF gel fraction was cut with sterilized scissors and the CGF gel was stored in a sterile, enzyme-free centrifuge tube for further lyophilization.
In a preferred embodiment of the present invention, the freeze-drying in step (2) is performed at-80 ℃ to-60 ℃ for 24 to 48 hours.
As a preferred embodiment of the present invention, the conditions of the vacuum oven drying in the step (2) are as follows: drying for 24-48 h at 37 ℃.
The invention also claims the solid concentrated growth factor prepared by the preparation method.
The solid concentrated growth factor contains a large amount of fibrin with a three-dimensional matrix reticular structure, is a porous structure, and has obviously improved biomechanical strength compared with a gel concentrated growth factor; cell experiments show that the solid concentrated growth factor has better capabilities of promoting the proliferation, migration, osteogenic differentiation and mineralization of BMSCs compared with the gel concentrated growth factor.
The invention also claims the application of the solid concentrated growth factor in preparing a product for promoting bone defect repair.
The solid concentrated growth factor prepared by the invention can be directly used as an implant material for promoting bone defect repair, and can also be mixed with other materials or medicines or auxiliary materials to prepare other products for promoting bone defect repair.
The invention also claims a product prepared by utilizing the solid concentrated growth factor and used for promoting bone defect repair.
The implant material prepared by the solid concentrated growth factor has better bone defect repairing effect than gel CGF on a rat OVF model, and the biomechanical strength of the injured vertebra after bone defect repair is obviously improved by adopting the solid concentrated growth factor prepared by the invention.
The solid concentrated growth factor prepared by the invention can promote the proliferation of BMSCs and the repair of bone defects under different concentrations.
The invention relates to a preparation method of a solid-state concentrated growth factor, which is characterized in that the solid-state concentrated growth factor is prepared by further freeze drying or vacuum drying the gelatinous concentrated growth factor; the preparation method is simple and convenient in preparation process, can be prepared by adopting a special instrument, can effectively improve the success rate, prevents the problems of pollution and the like, and provides a good foundation for clinical use; the method can be used for preparing by taking blood from vein, and can effectively reduce cost; the solid concentrated growth factor prepared by the method has better storage property and is further convenient for clinical use; the biological mechanical property of the solid concentrated growth factor prepared by the invention is obviously improved, and the original functions of promoting the proliferation, migration, osteogenic differentiation and mineralization of BMSCs are better than that of a gelatinous concentrated growth factor; in addition, the solid concentrated growth factor prepared by the method can be independently used as an implant material, the fracture repair promotion effect of the solid CGF product is verified on a rat osteoporosis vertebral body bone defect model, a reference can be provided for the preparation and clinical application of the human solid CGF product, and the application of the solid concentrated growth factor is further expanded. Furthermore, the method provides reference and basis for centrifugal extraction of gel-like CGF from human venous blood and preparation of solid CGF products by a freeze-drying method, and has wide clinical application prospect and potential economic value.
Drawings
FIG. 1 is an SEM structure of a solid concentrated growth factor prepared by the method of the present invention.
FIG. 2 is a biomechanical comparison of solid-state and gel-state concentrated growth factors prepared by the method of the present invention; (A) the anti-pressure result; (B) tensile strength results.
FIG. 3 shows the result of the CCK8 method for detecting the BMSCs proliferation promotion effect of the solid-state concentrated growth factor and the gel-state concentrated growth factor prepared by the present invention; (A) the gelatinous concentrated growth factors with different concentrations promote the proliferation of BMSCs; (B) the solid concentrated growth factors with different concentrations promote the proliferation of BMSCs.
FIG. 4 is a scratch test for detecting the BMSCs migration promotion effect of the solid-state concentrated growth factor and the gel-state concentrated growth factor prepared by the present invention; (A) promoting BMSCs to migrate under a microscope after 12 and 24 hours of intervention in each group; (B) results of quantitative migration analysis were performed for each group.
FIG. 5 shows the detection of the proliferation, migration, osteogenic differentiation and mineralization of BMSCs by the solid and gel concentrated growth factors prepared by the present invention; (A) ALP staining results for each group; (B) alizarin red staining for each group was quantified.
FIG. 6 shows the results of testing the therapeutic effects of solid-state concentrated growth factors and gel-state concentrated growth factors prepared according to the present invention for promoting the repair of the bone defects of the OVF model of SD rats; (A) micro-CT results; (B) bone growth volume ratio of each group of bone defect parts; (C) HE staining results; (D) mechanical properties: compressive resistance and tensile strength.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
A method for preparing solid concentrated growth factor comprises the following steps: centrifuging 5-10 mL of rat abdominal main venous blood to obtain a gel-like concentrated growth factor; and drying the extracted gel-like concentrated growth factor for 24-48 h at-80 to-60 ℃ by using a freeze dryer (SCIENTZ-10N, Ningbo new ganoderma) to obtain the solid concentrated growth factor.
Example 2
A method for preparing solid concentrated growth factor comprises the following steps: centrifuging 6-10 mL of rat abdominal main venous blood to obtain a gel-like concentrated growth factor; drying the extracted gel-like concentrated growth factor with a vacuum drying oven (DZF-6051, Shanghai-Hengchang scientific instruments Co., Ltd.) under the following conditions: and drying for 24-48 h at 37 ℃ to obtain the solid concentrated growth factor.
Example 3 structural testing
The structure of the solid concentrated growth factor of the invention is detected by a scanning electron microscope:
the detection method comprises the following steps: detection by SEM (FEI-Q25, FEI corporation, USA); fixing the prepared sample on a copper table by using conductive adhesive, spraying gold, and observing under a scanning electron microscope; the test conditions were: 15kV electron beam.
The detection result is shown in figure 1, and the solid concentrated growth factor prepared by the method contains a large amount of fibrin with a three-dimensional matrix network structure, and is a porous structure.
Example 4 biomechanical testing
The treated gel-like concentrated growth factor and the solid-state concentrated growth factor prepared by the invention are placed on a universal material testing machine platform for testing, and a universal material testing instrument is set to be compressed downwards at the speed of 1 mm/min. Analyzing by using self-contained analysis software Bluehill3 to obtain parameters such as compressive strength, tensile strength and the like of each sample (calculation standard: GB/T1040.3-2006); the results are shown in FIG. 2(CGF gel, i.e., gel-like enriched growth factor; CGF preparation, i.e., solid-state enriched growth factor).
As shown in FIG. 2, the solid-state concentrated growth factor prepared by the method of the present invention has significantly better compressive strength and tensile strength than the gel-like concentrated growth factor, which indicates that the solid-state concentrated growth factor prepared by the method of the present invention can significantly improve the biomechanical properties (P <0.001) of the prepared concentrated growth factor.
Example 5 detection of the proliferation, migration, osteogenic differentiation, mineralization of BMSCs
And (3) anesthetizing a 2-4-week-old SD rat, taking the double femurs and the tibia, flushing bone marrow, extracting BMSCs, and performing operations such as culture, passage, plating, intervention and the like. And changing the solution for 1 time in 2-3 days, and selecting passage time according to the growth condition of the cells. Based on BMSCs, the following cell experiments were performed.
1) Detection of BMSCs proliferation promoting effect by CCK8 method
Collecting BMSCs cultured as above at 1X 106The cell density of/mL is inoculated in a 96-well plate which is respectively placed with a gel-like concentrated growth factor and a solid-state concentrated growth factor in advance; the OD values of the 96-well plate at 1, 3, 5 and 7 days were measured by the CCK8 method, and the proliferation of the cells was calculated and a growth curve was plotted. (CCK8 method refers to CCK8 proliferation kit); the results are shown in FIG. 3(CGF gel, i.e., gel-like enriched growth factor; CGF preparation, i.e., solid-state enriched growth factor).
As shown in FIG. 3, after intervention of BMSCs by gel-like concentrated growth factors or solid-state concentrated growth factor soaking solutions with different concentrations, the OD values are increased with time, the BMSCs can be promoted to proliferate, the effect is equivalent to that of 5 days and 7 days, and the concentration of 10% concentrated growth factors is the optimal concentration. The solid concentrated growth factor prepared by the invention has the function of promoting the proliferation of BMSCs, and the optimal concentration of the function is 10%.
2) Scratch test for detecting BMSCs migration promotion effect
The 6-well plate cells are randomly divided into a blank group, a gel-like concentrated growth factor intervention group and a solid-state concentrated growth factor intervention group. When the cells in the 6-well plate proliferated to about 95%, a fine mark was drawn perpendicular to the cells as much as possible with a sterile tip, and then washed twice with PBS to remove cell debris and floating cells, followed by gel-like concentration of growth factorCulturing in culture medium containing 10% gel-like concentrated growth factor, culturing in culture medium containing 10% solid concentrated growth factor, culturing in blank culture medium, culturing cells at 37 deg.C and 5% CO2Culturing in an incubator. The migration of each group of cells was observed under a microscope at 0, 12 and 24h after the dry scratch, and the results are shown in FIG. 4(CGF gel, i.e. gel-like concentrated growth factor; CGF product, i.e. solid concentrated growth factor).
From fig. 4, after scratch experiments for 12h and 24h, both the gel-like concentrated growth factor intervention group and the solid-state concentrated growth factor intervention group significantly promoted BMSCs migration (P <0.001), and the two groups had no statistical difference in migration promotion effect (P > 0.05). The solid concentrated growth factor prepared by the method has the function of promoting the migration of BMSCs.
3) ALP staining and alizarin red staining for detecting osteogenic differentiation and mineralization promoting effects
Collecting BMSCs cultured as above at 1X 106Cell density in/mL was seeded in 12-well plates. The method comprises a BMSCs group, a BMSCs + osteogenesis induction + gelatinous concentrated growth factor group and a BMSCs + osteogenesis induction + solid concentrated growth factor group. The results of osteogenic differentiation and mineralization test analysis after 7 and 14 days of culture are shown in FIG. 5(BMSCs, i.e., BMSCs group, OI, i.e., BMSCs + osteogenic induction group, OI + 10% CGF gel, i.e., BMSCs + osteogenic induction + gel-like concentrated growth factor group, and OI + 10% CGF product, i.e., BMSCs + osteogenic induction + solid concentrated growth factor group).
And (3) displaying a detection result: as shown in fig. 5A, the quantitative analysis values for ALP staining were significantly higher for BMSCs + osteogenic induction + gel-like concentrated growth factor group and BMSCs + osteogenic induction + solid concentrated growth factor group than for the BMSCs + osteogenic induction group (P <0.001, P <0.05) at 7 days and 14 days of ALP staining. In addition, the growth factor group and BMSCs + osteogenic induction + solid state concentrated growth factor group were higher than BMSCs + osteogenic induction + gel state concentrated growth factor group, but the difference was not statistically significant (P > 0.05). Quantitative analysis of alizarin red staining OD values also showed the same trend (fig. 5B); the solid concentrated growth factor prepared by the method has better osteogenic differentiation and mineralization promoting effects, and the effect is slightly better than that of the gel concentrated growth factor.
Example 6 curative effect test of promoting the repair of bone defect of SD rat OVF model
SD rat model construction and CGF implantation: rat OVF bone defect models were constructed according to published procedures (Oncotarget, 2017Sep 1; 8(43): 73559-: A. model group b.cgf coagulation intervention group c.cgf preparation intervention group. The group B is implanted with the extracted gel-like concentrated growth factor in the defect area, and the group C is implanted with the solid-state concentrated growth factor with the volume equivalent to that of the gel-like concentrated growth factor in the defect area.
Material taking and index detection: the materials are taken after 8 weeks of transplantation, and the damaged vertebra defect repair condition is detected through micro-CT, HE staining and biomechanics; the results are shown in FIG. 6.
According to the detection result, after 2 months of treatment, the gel-like concentrated growth factor and the solid-state concentrated growth factor prepared by the method can promote the regeneration of the bone defect bone tissue of the SD rat (as shown in fig. 6A), but the solid-state concentrated growth factor prepared by the method has better bone tissue generation amount (as shown in fig. 6A-B) and better bone tissue morphology than the gel-like concentrated growth factor, more osteoblasts and bone cell aggregation can be seen in the bone defect (as shown in fig. 6C and the HE staining result), and the solid-state concentrated growth factor prepared by the method has higher vertebral injury mechanical parameters (compressive strength and yield strength) than the gel-like concentrated growth factor (P < 0.05).
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A preparation method of a solid-state concentrated growth factor is characterized by comprising the following steps: the gel-like concentrated growth factor is subjected to freeze drying or vacuum drying to obtain the solid concentrated growth factor.
2. The method of claim 1, wherein the gel-like concentrated growth factor is prepared by centrifuging animal venous blood.
3. The method of claim 2, wherein the venous blood is rat abdominal main venous blood.
4. The method of claim 2, wherein the centrifugation step is: accelerating for 30 seconds, reaching 2700rpm, centrifuging for 2min, then reducing to 2400rpm, centrifuging for 4min, then accelerating to 2700rpm, centrifuging for 4min, centrifuging for 3min at 3300rpm, finally decelerating for 36 seconds and stopping.
5. The preparation method according to claim 1, wherein the freeze-drying condition in the step (2) is-80 ℃ to-60 ℃ and the drying time is 24 to 48 hours.
6. The method according to claim 1, wherein the conditions for drying in the vacuum oven in the step (2) are: drying for 24-48 h at 37 ℃.
7. A solid concentrated growth factor prepared by the process of any one of claims 1 to 6.
8. Use of the solid concentrated growth factor of claim 7 in the preparation of a product for promoting repair of a bone defect.
9. An article of manufacture for promoting repair of a bone defect comprising the solid concentrated growth factor of claim 7.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103272258A (en) * | 2013-05-15 | 2013-09-04 | 中国人民解放军第四军医大学 | Novel freeze-drying platelet-rich fibrin, as well as preparation and application thereof |
| US20150080300A1 (en) * | 2013-09-17 | 2015-03-19 | Bestop Group Holdings Limited | Growth factor concentrate and the use thereof |
| US20150224173A1 (en) * | 2012-08-17 | 2015-08-13 | Kasiak Research Pvt. Ltd. | Method of preparing a growth factor concentrate derived from human platelets |
| CN106693064A (en) * | 2016-12-07 | 2017-05-24 | 同济大学 | Composite material for maxillary sinus floor elevation and preparation method |
| US20170157018A1 (en) * | 2013-09-17 | 2017-06-08 | Bestop Group Holdings Limited | Growth factor concentrate and the use thereof |
| CN109528692A (en) * | 2018-12-03 | 2019-03-29 | 王丽萍 | Growth factor slow-release freeze-drying film and preparation method thereof is concentrated |
-
2019
- 2019-11-21 CN CN201911163078.4A patent/CN110903380A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150224173A1 (en) * | 2012-08-17 | 2015-08-13 | Kasiak Research Pvt. Ltd. | Method of preparing a growth factor concentrate derived from human platelets |
| CN103272258A (en) * | 2013-05-15 | 2013-09-04 | 中国人民解放军第四军医大学 | Novel freeze-drying platelet-rich fibrin, as well as preparation and application thereof |
| US20150080300A1 (en) * | 2013-09-17 | 2015-03-19 | Bestop Group Holdings Limited | Growth factor concentrate and the use thereof |
| US20170157018A1 (en) * | 2013-09-17 | 2017-06-08 | Bestop Group Holdings Limited | Growth factor concentrate and the use thereof |
| CN106693064A (en) * | 2016-12-07 | 2017-05-24 | 同济大学 | Composite material for maxillary sinus floor elevation and preparation method |
| CN109528692A (en) * | 2018-12-03 | 2019-03-29 | 王丽萍 | Growth factor slow-release freeze-drying film and preparation method thereof is concentrated |
Non-Patent Citations (5)
| Title |
|---|
| HAIQIONG YUE: "Promotion of skin fibroblasts collagen synthesis by polydioxanone mats combined with concentrated growth factor extracts", 《JOURNAL BIOMATERIALS APPLICATIONS》 * |
| LIPING WANG: "A comparative study of the effects of concentrated growth factors in two different forms on osteogenesis in vitro", 《MOLECULAR MEDICINE REPORTS》 * |
| 刘如林: "《微生物工程概论》", 31 October 1995, 南开大学出版社 * |
| 宿玉成, 辽宁科学技术出版社 * |
| 王昕: "浓缩生长因子注射改善框周皱纹的临床观察", 《中国美容整形外科杂志》 * |
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