CN114949350A - Collagen scaffold loaded with basic fibroblast growth factor - Google Patents
Collagen scaffold loaded with basic fibroblast growth factor Download PDFInfo
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- CN114949350A CN114949350A CN202210608106.4A CN202210608106A CN114949350A CN 114949350 A CN114949350 A CN 114949350A CN 202210608106 A CN202210608106 A CN 202210608106A CN 114949350 A CN114949350 A CN 114949350A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- 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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- 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
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- A—HUMAN NECESSITIES
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The invention discloses a collagen scaffold loaded with basic fibroblast growth factor, which is prepared by taking calf bone or pig bone as a raw material, and the collagen scaffold is loaded with the basic fibroblast growth factor; the collagen scaffold is of a double-type gap structure, and simultaneously comprises large gaps larger than 1 mu m and micro gaps smaller than 1 mu m, and the compressive strength of the collagen scaffold reaches 0.5-2.0 MPa; the load capacity of the basic fibroblast growth factor is 5-20 mu g/cm 3 . The collagen bracket can guide and promote the regeneration of alveolar bone for extracting the dental socket, effectively promote the repair and healing of maxillary sinus perforation, provide a good foundation for later-stage planting repair, and have good medical treatmentValue and application prospect.
Description
Technical Field
The invention relates to the field of biological repair materials, in particular to a collagen scaffold loaded with basic fibroblast growth factors.
Background
In the medical treatment process of oral diseases, when teeth in oral cavities of patients suffer from tooth trauma, malposition teeth, extra teeth, retained deciduous teeth and the like, the patients often need tooth extraction operations. The implant of the filling material in the tooth extraction socket is needed to be transplanted immediately after tooth extraction so as to support and fill the tooth extraction socket, block or slow down the absorption of alveolar bone, prevent gingival epithelium or fibrous tissue from entering the tooth extraction socket, guide and promote the bone regeneration of the tooth extraction socket and realize the storage or increment of the alveolar bone.
Currently, the socket filling material can be largely classified into a synthetic material and a biological bone material. Among them, the synthetic materials have the main problems that: on one hand, the degradation speed of the artificially synthesized material in a human body is not easy to control and is asynchronous with the bone regeneration and repair speed of periodontal tissue repair and tooth extraction; on the other hand, the degradation products of synthetic materials, such as polylactic acid, can cause strong acidity in local environment, but can inhibit the repair growth of repaired tissues, so that the synthetic materials are gradually replaced by biological bone materials. The existing biological bone material is mostly selected from a dental pit filling material prepared from pig bone or cow bone material, the degradation speed of the biological bone material in a human body can be almost synchronous with the tissue repair speed, the degradation product can not cause the change of local environment, the bone regeneration of periodontal tissue repair and dental pit extraction is not influenced, and the bone regeneration of dental pit extraction is facilitated.
Basic fibroblast growth factor (bFGF) is a very trace polypeptide substance present in mammals and humans. bFGF can stimulate the division and proliferation of mesoderm and ectoderm derived cells such as fibroblasts, vascular endothelial cells, smooth muscle cells, corneal epithelial cells, crystalline epithelial cells, muscle cells, bone cells, nerve cells and the like, so that the bFGF has very wide physiological functions, is a multifunctional cell growth factor and has the effects of promoting repair and healing of various tissue injuries clinically.
At present, the basic fibroblast growth factor (bFGF) preparations which are applied to clinical use mainly comprise rb-bFGF spray, eye drops, films applied to various epidermal injuries and oral ulcers and the like, but the basic fibroblast growth factor (bFGF) preparations cannot be applied to oral surgery treatment. The Chinese patent document CN113230460A discloses a porous scaffold for guiding the integrated restoration of soft and hard tissues in the oral cavity and a preparation method thereof, and the integrated scaffold is formed by electrostatic spinning and 3D printing. The integral bracket has gradient composition and aperture, the upper layer is a b-FGF-loaded heparin grafted polycaprolactone/gelatin microporous fibrous membrane which plays a barrier role and is used for guiding soft tissue regeneration; the lower layer is a polycaprolactone/gelatin/hydroxyapatite macroporous scaffold for guiding the regeneration of hard tissues. It can be seen that the product is not only complex in preparation method, but also is artificially synthesized by combining the electrostatic spinning of the stent with the 3D printing, which has the problems of the artificially synthesized material, and the b-FGF is combined with the stent after being combined with the heparin-grafted polycaprolactone/gelatin microporous fiber membrane, so that the repair effect of the b-FGF is greatly limited. Another chinese patent document CN102120033A discloses a composite growth factor collagen sustained release carrier material for promoting repair of various wounds on the frontal and facial areas of the oral cavity and a preparation method thereof, the collagen sustained release carrier material is prepared by using modified collagen such as liposome, chitosan, glycosaminoglycan and the like to form a bioactive collagen sustained release material, and combining one or more of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), Vascular Endothelial Growth Factor (VEGF), and Keratinocyte Growth Factor (KGF), and each gram of the bioactive sustained release material contains not less than 10ng of growth factor, and has very significant curative effects on treating various tissue wounds on the facial areas such as tooth extraction, oral ulcer, facial nerve injury, mandibular bone defect and the like. The product also combines bFGF with a plurality of growth factors, and the collagen sustained-release material is composed of collagen modified by liposome, chitosan, glycosaminoglycan and the like, and is also an artificial synthetic material, so that the degradation speed is difficult to control, the speed is not synchronous with the bone regeneration and repair speed of periodontal tissue repair and tooth extraction, and the problems of inhibiting the repair and growth of repaired tissues and the like exist.
The applicant previously proposed a method for preparing a collagen matrix bone repair material (application No. 2022104518724) with antibacterial and bone guiding properties, which is made of animal bone as a scaffold collagen matrix, retains the three-dimensional structure of natural bone, and is placed in a fresh dental extraction socket to provide proper time and space for the formation of new bone, thereby guiding the jaw bone repair. However, this material is only suitable for replacing hydroxyapatite, and since the root tip of the upper posterior molars of the human mouth and the maxillary sinus floor are closely related, the root tip of the molars of some patients is exposed in the maxillary sinus cavity or only a thin layer of bone material is separated, even only a layer of mucous membrane is separated; therefore, when teeth are extracted, the symptoms of perforation of the maxillary sinus are often accompanied, but the existing biological bone material cannot promote the restoration and healing of the perforation of the maxillary sinus, the alveolar bone is easy to be absorbed by progressive atrophy after the teeth are extracted, the bone quantity of the alveolar bone is reduced, and the dental implant restoration in the later period is not facilitated.
Disclosure of Invention
Based on the existing problems, the invention provides a collagen scaffold loaded with basic fibroblast growth factor (bFGF), which is loaded on a collagen scaffold prepared by taking calf bones or pig bones as raw materials, and effectively promotes the repair and healing of maxillary sinus perforation. The specific technical scheme is as follows:
the collagen scaffold loaded with the basic fibroblast growth factor is prepared from calf leg bones or piglet leg bones which are born for 0-2 months as raw materials, and the porosity and the compressive strength of the prepared collagen scaffold are guaranteed. After a series of treatments, the natural three-dimensional porous structure is kept, the collagen scaffold is similar to the structure of human bones, the void ratio is consistent with that of the human bones, and the collagen scaffold is of a double-type void structure, namely, the collagen scaffold simultaneously contains large voids larger than 1 micrometer and micro voids smaller than 1 micrometer. The microporous structure ensures good capillary action of the collagen scaffold, so that blood can rapidly permeate through the collagen scaffold effectively. The interconnected macropores allow blood cells and proteins to enter, ensuring effective bone fusion. The compressive strength of the collagen scaffold reaches 0.5-2.0 MPa, the collagen scaffold can provide sufficient scaffold supporting capacity, and meanwhile, the collagen scaffold can be used as a carrier of basic fibroblast growth factors, the basic fibroblast growth factors can be continuously and effectively released, the effects of guiding and promoting regeneration of alveolar bone for pulling out a dental socket, effectively promoting repair and healing of maxillary sinus perforation can be achieved, and a good foundation is provided for later-stage planting repair.
The preparation method of the collagen scaffold loaded with the basic fibroblast growth factor specifically comprises the following steps:
s1: removing viruses: soaking the cleaned calf bone or pig bone raw material in NaOH solution, and performing virus removal treatment;
s2: degreasing: soaking the bone material after virus removal treatment in n-hexane or a mixed solution of chloroform and methanol, and performing degreasing treatment;
s3: decalcification: soaking the defatted bone material in one or more of hydrochloric acid, formic acid or glacial acetic acid, and decalcifying;
s4: deproteinization: soaking the bone raw material subjected to decalcification treatment in a trypsin solution to perform deproteinization treatment;
s5: removing internal toxicity: soaking the bone raw material subjected to deproteinization treatment in a hydrogen peroxide solution or a sodium hypochlorite solution, and removing internal toxicity to obtain a collagen scaffold;
s6: loading a growth factor: immersing the collagen scaffold in an alkaline fibroblast growth factor solution, and fishing out after repeated immersion;
s7: freeze-drying: and (3) freeze-drying the collagen scaffold loaded with the basic fibroblast growth factor.
Preferably, in step S1, the calf bone or pig bone is a leg bone with a bone age of 0-2 months.
Preferably, in step S1, the concentration of the NaOH solution used for virus removal is 0.1-3 mol/L, and the soaking time is 1-4 h.
Preferably, in step S2, the volume ratio of the reagent for degreasing, chloroform and methanol is 2-3: 1-2, and the soaking time is 8-48 h.
Preferably, in step S3, the soaking time for decalcification is 4-12 h.
Preferably, in step S4, the concentration of the trypsin solution used for deproteinization is 0.1-2%, and the soaking time is 4-12 h.
Preferably, in step S5, the mass concentration of the hydrogen peroxide solution for detoxification is 1% to 10%, the mass concentration of the sodium chlorate solution for detoxification is 0.5% to 3%, and the soaking time is 2 to 10 hours.
Preferably, in step S6, the concentration of the basic fibroblast growth factor solution is 5-50 μ g/cm 3 And repeatedly soaking for 6-10 times, wherein each soaking time is 0.5-1 h, and stirring is carried out at the rotating speed of 50-100 r/min by using a stirrer, so that the loading effect is ensured.
Preferably, in the step S7, the freeze-drying temperature is-40 ℃ to 25 ℃, and the drying time is 30 to 48 hours.
The invention has the beneficial effects that:
1) the invention loads the basic fibroblast growth factor on the collagen bracket prepared by taking the calf bone or the pig bone as the raw material, can effectively promote the repair and healing of the maxillary sinus perforation while guiding and promoting the regeneration of the alveolar bone for pulling out the dental alveolus, and provides a good foundation for the later-stage planting repair.
2) The product only loads the basic fibroblast growth factor on the collagen bracket prepared by taking the calf bone or the pig bone as a raw material, has simple components, does not have the problems caused by artificial bone materials, and is beneficial to the exertion of the repair function of the basic fibroblast growth factor; the collagen scaffold is prepared from calf shank bones or calf shank bones born for 0-2 months as raw materials, and the porosity and the compressive strength of the prepared collagen scaffold are ensured. After a series of treatments, the natural three-dimensional porous structure is kept, the collagen scaffold is similar to the structure of human bones, the void ratio is consistent with that of the human bones, and the collagen scaffold is of a double-type void structure, namely, the collagen scaffold simultaneously contains large voids larger than 1 micrometer and micro voids smaller than 1 micrometer. The microporous structure ensures good capillary action of the collagen scaffold, so that blood can rapidly permeate through the collagen scaffold effectively. The interconnected macropores allow blood cells and proteins to enter, ensuring effective bone fusion. The compressive strength of the collagen scaffold reaches 0.5-2.0 MPa, the collagen scaffold can provide sufficient scaffold supporting capacity, and meanwhile, the collagen scaffold can be used as a carrier of basic fibroblast growth factors, the basic fibroblast growth factors can be continuously and effectively released, the effects of guiding and promoting regeneration of alveolar bone for pulling out a dental socket, effectively promoting repair and healing of maxillary sinus perforation can be achieved, and a good foundation is provided for later-stage planting repair.
3) The collagen scaffold prepared by the invention has the porosity and the type of the void structure consistent with the structure of human bone, thereby being beneficial to the release of the growth factor of the basic fibroblast, combining with the specific receptor on the surface of the gum cell, regulating the division, the propagation and the growth differentiation of the cell, regulating the synthesis, the secretion and the decomposition of the matrix among the cells, promoting the growth and the propagation of the cell tissue, promoting the cell metabolism, enhancing the oxidation effect and further effectively promoting the repair and the healing of the perforation of the maxillary sinus.
4) When the basic fibroblast growth factor is loaded on the collagen scaffold, the basic fibroblast growth factor is repeatedly soaked for 6-10 times, each soaking time is 0.5-1 h, and the stirring is carried out at the rotating speed of 50-100 r/min by using a stirrer, so that the loading effect is ensured, the amount of the basic fibroblast growth factor loaded on the prepared product can fully meet the requirements of repairing and healing of perforation of maxillary sinus, and the treatment effect is ensured.
Drawings
FIG. 1 is a diagram of a finished product of a basic fibroblast growth factor-loaded collagen scaffold according to the present invention;
FIG. 2 is a CBCT image of a maxillary sinus puncture in a lateral palatal root of a patient in accordance with an embodiment of the present invention;
FIG. 3 is a CBCT image of a collagen scaffold loaded with basic fibroblast growth factor according to the present invention implanted in a patient in an application example of the present invention;
fig. 4 is a CBCT image of the maxillary sinus floor after the operation of a patient in the application example of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail and completely with reference to the embodiments and the accompanying drawings.
Example 1
The true bookIn an example of preparing a collagen scaffold loaded with basic fibroblast growth factor, as shown in fig. 1, the collagen scaffold is prepared from a calf bone or a pig bone as a raw material, is loaded with the basic fibroblast growth factor, and has a loading amount of 5 μ g/cm 3 . The collagen scaffold is prepared by taking calf bones born for two months as raw materials, and performing the procedures of virus removal, degreasing, decalcification, protein removal, endotoxicity removal, basic fibroblast growth factor (bFGF) loading, freeze drying and the like; the embodiment specifically comprises the following steps:
s1: removing viruses: and soaking the cleaned calf bone raw material for 4 hours by using 0.5mol/L NaOH solution.
S2: degreasing: soaking the bone raw material after virus removal in n-hexane for 45 h.
S3: decalcification: the defatted bone material was soaked with hydrochloric acid for 5 h.
S4: deproteinization: soaking the decalcified bone material in 0.5% trypsin solution for 10 hr.
S5: removing internal toxicity: and soaking the deproteinized bone raw material in 2% hydrogen peroxide solution for 8 hours to obtain the collagen scaffold.
S6: loading a growth factor: immersing the collagen scaffold after the detoxification treatment into 5ug/cm 3 The alkaline fibroblast growth factor solution is repeatedly soaked for 10 times, each time soaking for 0.5h, and meanwhile, a stirrer is used for stirring at the rotating speed of 50r/min, so that the loading effect is ensured.
S7: freeze-drying: drying at-40 deg.C for 35 h.
Example 2
This example also prepares a collagen scaffold loaded with basic fibroblast growth factor, which selects the leg bone of a piglet born for one month as the raw material, and the collagen scaffold is loaded with basic fibroblast growth factor and the loading amount is 20 mug/cm 3 The method specifically comprises the following steps:
s1: removing viruses: soaking the cleaned pig bone raw material for 2 hours by using 1mol/L NaOH solution.
S2: degreasing: soaking the bone raw material after virus removal for 40 hours by using a mixed solution of trichloromethane and methanol (the mixing volume ratio is 2: 1).
S3: decalcification: soaking the degreased bone raw material for 8 hours by using a mixed solution of formic acid and glacial acetic acid (the mixing volume ratio is 2: 1).
S4: deproteinization: soaking the decalcified bone raw material with 1% trypsin solution for 8 h.
S5: removing internal toxicity: and soaking the deproteinized bone raw material 5 in a 5% hydrogen peroxide solution to obtain the collagen scaffold.
S6: loading a growth factor: immersing the collagen scaffold after the detoxification treatment into 30ug/cm 3 The alkaline fibroblast growth factor solution is repeatedly soaked for 6 times, each time is 1h, and the stirring is carried out by using a stirrer at the rotating speed of 100r/min, so that the loading effect is ensured.
S7: freeze-drying: freeze-drying at-40 deg.C for 35 h.
Example 3
This example also prepared a basic fibroblast growth factor-loaded collagen scaffold, which was prepared by using 45-day-old calf bone as a raw material, and the amount of basic fibroblast growth factor loaded on the collagen scaffold was 50 μ g/cm 3 . The preparation method comprises the following steps:
s1: removing viruses: and soaking the cleaned calf bone raw material for 3 hours by using a 3mol/L NaOH solution.
S2: degreasing: soaking the bone raw material after virus removal for 20 hours by using a mixed solution of trichloromethane and methanol (the mixing volume ratio is 3: 2).
S3: decalcification: soaking the degreased bone raw material for 5 hours by using a mixed solution of formic acid and glacial acetic acid (the mixing volume ratio is 2: 1).
S4: deproteinization: soaking the decalcified bone raw material with 2% trypsin solution for 8 h.
S5: removing internal toxicity: and (3) soaking the deproteinized bone raw material for 6 hours by using a sodium hypochlorite solution with the concentration of 2% to obtain the collagen scaffold.
S6: loading a growth factor: soaking the collagen scaffold subjected to endotoxin removal treatment into 50ug/cm 3 The alkaline fibroblast growth factor solution is repeatedly soaked for 8 times, each time soaking for 1h, and simultaneously the rotating speed of a stirrer is controlled to beStirring at 100r/min to ensure the loading effect.
S7: freeze-drying: freeze-drying at-40 deg.C for 48 h.
Example 4 application example
In this example, the use effect of the basic fibroblast growth factor-loaded collagen scaffold prepared in examples 1 to 3 was verified. This example is a treatment of maxillary sinus perforation due to severe periodontal disease. On the time axis, the case is as follows:
10/12/2021: the patient is examined by repeated swelling, pain and looseness of the upper right posterior tooth, severe periodontal disease is diagnosed, and removal is recommended at that time, and the patient requires observation first.
11/25/2021: patient review symptoms further worsened, CBCT showed: the lateral palatine root has a maxillary sinus perforation, as shown in figure 2.
12 month 2 in 2021: the patient was reviewed again and CBCT showed: the lower density image around the lateral palatine root has penetrated the maxillary floor, and the thickening range and inflammation range of the maxillary sinus mucosa are further expanded, as shown in fig. 3. On the same day, the #17 dental alveolus is removed and scraped thoroughly, inflammatory granulation tissue is cleaned, and the basic fibroblast growth factor-loaded collagen scaffold is implanted.
12 month 30 in 2021: the patient reviewed and the maxillary sinus floor was intact as shown in fig. 4. After the basic fibroblast growth factor-loaded collagen scaffold prepared by the invention is implanted and the reexamination is only carried out for 28 days, the bottom wall of the maxillary sinus of a patient is repaired, which shows that the basic fibroblast growth factor-loaded collagen scaffold can guide and promote the regeneration of alveolar bone for extracting the dental alveolus, effectively promote the repair and healing of the maxillary sinus perforation and provide a good foundation for the later-stage planting repair.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Furthermore, it should be understood that although the present specification describes embodiments, this does not include only one embodiment, and such description is for clarity only, and those skilled in the art should be able to make the specification as a whole, and the embodiments may be appropriately combined to form other embodiments understood by those skilled in the art.
Claims (10)
1. A basic fibroblast growth factor-loaded collagen scaffold is characterized in that: the collagen scaffold is prepared by taking calf bones or pig bones as raw materials, and basic fibroblast growth factors are loaded on the collagen scaffold;
the collagen scaffold is of a double-type gap structure, and simultaneously comprises large gaps larger than 1 mu m and micro gaps smaller than 1 mu m, and the compressive strength of the collagen scaffold reaches 0.5-2.0 MPa;
the loading capacity of the basic fibroblast growth factor is 5-20 mu g/cm 3 。
2. The basic fibroblast growth factor-loaded collagen scaffold according to claim 1, wherein: the preparation method of the collagen scaffold specifically comprises the following steps:
s1: removing viruses: soaking the cleaned calf bone or pig bone raw material in NaOH solution, and performing virus removal treatment;
s2: degreasing: soaking the bone material after virus removal treatment in n-hexane or a mixed solution of chloroform and methanol, and performing degreasing treatment;
s3: decalcification: soaking the defatted bone material in one or more of hydrochloric acid, formic acid or glacial acetic acid, and decalcifying;
s4: deproteinization: soaking the bone raw material subjected to decalcification treatment in a trypsin solution to perform deproteinization treatment;
s5: removing internal toxicity: soaking the bone raw material subjected to deproteinization treatment in a hydrogen peroxide solution or a sodium hypochlorite solution, and removing internal toxicity to obtain a collagen scaffold;
s6: loading a growth factor: immersing the collagen scaffold in an alkaline fibroblast growth factor solution, and repeatedly immersing for a plurality of times;
s7: freeze-drying: and (3) freeze-drying the collagen scaffold loaded with the basic fibroblast growth factor.
3. The basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: and step S1, the calf bone or the pig bone is a leg bone with the bone age of 0-2 months.
4. The basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: and step S1, the concentration of the NaOH solution used for virus removal is 0.1-3 mol/L, and the soaking time is 1-4 h.
5. The basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: step S2, mixing the reagent for degreasing, trichloromethane and methanol in a volume ratio of 2-3: 1-2, and the soaking time is 8-48 h.
6. The basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: and step S3, the soaking time for decalcification is 4-12 h.
7. The basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: and step S4, the concentration of the trypsin solution for deproteinization is 0.1-2%, and the soaking time is 4-12 h.
8. The basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: and step S5, the mass concentration of the hydrogen peroxide solution for removing the endotoxin is 1-10%, the mass concentration of the sodium chlorate solution is 0.5-3%, and the soaking time is 2-10 h.
9. Root of herbaceous plantThe basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: step S6, the repeated soaking times are 6-10 times, and each soaking time is 0.5-1 h; stirring at a stirring speed of 50-100 r/min; the concentration of the basic fibroblast growth factor solution is 5-50 mu g/cm 3 。
10. The basic fibroblast growth factor-loaded collagen scaffold according to claim 2, wherein: and step S7, wherein the freeze-drying temperature is-40 ℃ to 25 ℃, and the drying time is 30-48 h.
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