CN111643729B - Drug-loaded bone scaffold material and preparation method thereof, and functional hybrid bone scaffold and preparation method thereof - Google Patents
Drug-loaded bone scaffold material and preparation method thereof, and functional hybrid bone scaffold and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a bone scaffold material for drug loading, and relates to the field of bone repair materials. The invention comprises a bone scaffold material for drug loading, which comprises an organic template and a deposition material, wherein the deposition material is uniformly deposited on the organic template to form aerogel; the aim of loading the medicine through the bone scaffold material, and simultaneously meeting the requirements that the loading material has certain mechanical strength, strong medicine loading capacity and the material structure is beneficial to the bone repair process is fulfilled; the invention also comprises a functional hybrid aerogel bone scaffold, which comprises the prepared bone scaffold material for drug loading, wherein the bone scaffold material is loaded with a functional substance; the problems that the growth factors are expensive and the local bioavailability is low when the existing bone scaffold is loaded with the growth factors are solved; a single growth factor cannot have both osteogenic and angiopoietic effects; the bionic bone scaffold material has poor osteogenic and angiogenetic inductivity, thereby limiting the clinical transformation and application of the bone scaffold material.
Description
Technical Field
The invention relates to the field of bone repair materials, in particular to a bone scaffold material for drug loading.
Background
Bone defects can be caused by various reasons such as tumor, inflammation, trauma and the like. At present, autologous bone and allogeneic bone transplantation are still the main treatment modes for reconstructing bone defects, but the autologous bone and allogeneic bone transplantation have or partially have the risks of limited donors, complications of bone supplying areas, immunogenic reactions, disease transmission and the like. Therefore, there is a need to develop an artificial bone scaffold material having both osteoinductive and vascular properties to improve bone defect repair effects. The research of bone scaffold material loaded with growth factor (bone morphogenetic protein or vascular endothelial growth factor) for promoting bone formation and blood vessel formation makes it possible for artificial bone scaffold material to repair bone defect. However, current bone defect repair remains deficient: growth factors are expensive and have low local bioavailability; a single growth factor cannot have both osteogenic and angiopoietic effects; the bionic bone scaffold material has poor inductivity of osteogenesis and angiogenesis inside, and limits the clinical transformation and application of the bone scaffold material.
Under the idea of loading drugs by using bone scaffold materials, common loading materials cannot meet the requirements. Therefore, it is highly desirable to develop a carrier material with a certain mechanical strength, a strong drug loading capacity, a material structure that is conducive to bone repair processes, and a relatively flat drug release profile. Therefore, the carrier material is used for loading the drug to overcome the defect that the bone scaffold material loads the growth factors.
Disclosure of Invention
The invention aims to provide a bone scaffold material for drug loading, so as to realize the loading of drugs through the bone scaffold material, and simultaneously meet the aims that the loading material has certain mechanical strength, strong drug loading capacity and the material structure is beneficial to the bone repair process.
In order to achieve the purpose, the invention adopts the following technical means:
a bone scaffold material for drug loading comprises an organic template and a deposition material, wherein the deposition material is uniformly deposited on the organic template to form aerogel;
the organic template comprises a nano-cellulose/collagen composite scaffold, and the deposition material comprises hydroxyapatite.
Preferably, the mass ratio of the nano-cellulose to the collagen is 0.5-2: 1, and the hydroxyapatite accounts for 0.5-2% of the total mass of the nano-cellulose and the collagen.
Compared with the common bone scaffold material, the bone scaffold material for drug loading disclosed by the invention has the following beneficial effects:
1. the organic template scaffold prepared by compounding the nano-cellulose and the collagen has large specific surface area, high biological activity and excellent degradability.
2. The nano hydroxyapatite is compounded on the organic template support, so that the degradation rate can be controlled and the mechanical property of the composite material can be enhanced by adjusting the components of the material;
3. degradation products such as calcium ions, phosphate radicals and the like can increase the internal bone conduction and bone induction of the scaffold material and improve the microenvironment of local osteogenesis and vascularization.
4. The organic-inorganic hybrid aerogel, namely the aerogel prepared by the organic template and the deposition material through hybridization has a macroporous-mesoporous hierarchical structure, and the nano hydroxyapatite has a nano rough surface, so that the adhesion, proliferation and migration of osteoblasts, BMSCs and HUVECs are facilitated.
The invention also provides a preparation method of the bone scaffold material for drug loading, which aims to solve the problem of how to prepare the bone scaffold material for drug loading.
And adopts the following technical scheme:
a preparation method of the bone scaffold material for drug loading comprises the following steps:
a. preparing nano hydroxyapatite;
b. preparing a nano-cellulose/collagen mixed aqueous solution: dispersing nano-cellulose and collagen in deionized water, and adjusting the pH of the system to acidity by using inorganic acid;
c. preparing a nano-cellulose/collagen/hydroxyapatite mixed aqueous solution: adding the nano hydroxyapatite into the nano cellulose/collagen mixed aqueous solution, and continuously stirring until no particles exist to prepare a precursor solution;
d. preparing nano-cellulose/collagen/hydroxyapatite aerogel: and dropwise adding GPTMS into the precursor solution, stirring to react to form a cross-linked solution, standing the cross-linked solution to remove bubbles, then placing the cross-linked solution into a tert-butyl alcohol solvent for replacement, and finally performing freeze drying to obtain the aerogel.
By the preparation method disclosed by the invention, the bone scaffold material for drug loading can be directly prepared into the composite hybrid aerogel, the composite hybrid aerogel not only has excellent performances of various basic materials, but also can generate new physicochemical properties through multiple hybridization complementation, the conversion of large-scale materials to nanoscale materials is realized, and the prepared composite hybrid aerogel has the advantages of sufficient mechanical strength, good osteoinductivity, good vascular formation in the scaffold, controllable degradation rate and the like.
And the aerogel supported by the organic-inorganic hybrid has a macroporous-mesoporous hierarchical structure, so that a good drug loading effect can be achieved, and meanwhile, the nanoscale rough surface of the nano-hydroxyapatite is beneficial to adhesion, proliferation and migration of osteoblasts, BMSCs and HUVECs.
The invention also provides a functional hybrid aerogel bone scaffold, which aims to solve the problems that the growth factors are expensive and the local bioavailability is low when the current bone scaffold is loaded with the growth factors; a single growth factor cannot have both osteogenic and angiopoietic effects; the bionic bone scaffold material has poor osteogenic and angiogenetic inductivity, thereby limiting the clinical transformation and application of the bone scaffold material.
In order to solve the problems, the invention adopts the following technical means:
a functional hybrid aerogel bone scaffold comprises the prepared bone scaffold material for drug loading, and functional substances are loaded on the bone scaffold material.
Further, the functional substance comprises an in-situ loaded drug loaded in situ or a dropwise loaded drug loaded dropwise;
the in-situ loaded drug comprises parathyroid hormone-related protein, and the dropwise loaded drug comprises at least one of sclerostin monoclonal antibody and stromal cell derived factor-1.
The invention discloses a functional hybrid aerogel bone scaffold, which has the following beneficial effects:
the functional filler can be stably loaded on the bone scaffold material, has angiogenesis and osteogenesis inducing bioactivity and controllable biodegradability, and is used for repairing bone defects.
The invention also provides a preparation method of the functional hybrid aerogel bone scaffold, and the preparation method is used for solving the problems that how to prepare the functional hybrid aerogel bone scaffold, the medicine can be better and more uniformly loaded, and the degree of the medicine effect of the loaded medicine can be met in the releasing process.
In order to solve the problems, the invention adopts the following technical means:
a preparation method of a functional hybrid aerogel bone scaffold comprises the following steps:
a. preparing nano hydroxyapatite;
b. preparing a nano-cellulose/collagen mixed aqueous solution: dispersing nano-cellulose and collagen in deionized water, and adjusting the pH of the system to acidity by using inorganic acid;
c. preparing a nano-cellulose/collagen/hydroxyapatite mixed aqueous solution: adding nano hydroxyapatite into the mixed aqueous solution of nano cellulose and collagen, and continuously stirring until no particles exist to prepare a precursor solution;
d. preparing functional hybrid aerogel:
when loading the drug in situ: dropwise adding GPTMS into the precursor solution, simultaneously adding the in-situ load drug, stirring to react to form load cross-linking solution, standing the load cross-linking solution for defoaming, then placing the load cross-linking solution into tert-butyl alcohol solvent for replacement, and finally freeze-drying to obtain a load aerogel support;
when the loading drop loads the drug: dropwise adding GPTMS into the precursor solution, stirring to react to form a cross-linked solution, standing the cross-linked solution to remove bubbles, then placing the cross-linked solution into a tert-butyl alcohol solvent for replacement, finally performing freeze drying to prepare a matrix aerogel, dropwise adding the dropwise added load drug onto the collective aerogel in a sterile environment, and performing freeze drying after dropwise adding is completed.
Preferably, in the step b, the final concentration of the nano-cellulose and the final concentration of the collagen are both 1-5% w/v.
Furthermore, in the step c, the nano hydroxyapatite accounts for 0.5 to 2 percent of the total mass fraction of the nano cellulose and the collagen.
That is to say, in the preparation method of the functional hybrid aerogel bone scaffold disclosed by the invention, parathyroid hormone-related protein can be loaded on the bone scaffold material through in-situ loading to form a functional aerogel bone scaffold, and after the parathyroid hormone-related protein is loaded, the release curve can meet the requirement of local burst release combined with temporary slow release, so that after the parathyroid hormone-related protein is loaded as a functional substance, the functional aerogel bone scaffold can play a role in the application after being implanted into a human body, and adverse effects on the bone repair process due to too slow release of a medicament are avoided.
In addition, the invention can also adopt a dropping type loading mode, when loading the sclerostin monoclonal antibody and/or the stromal cell derived factor-1, the functional substance is loaded on the bone scaffold material only by dripping and solidifying, compared with the in-situ loading, the convenience of the bone scaffold material for loading the medicine can be greatly improved by a dropping loading mode, namely, after the carrier material is prepared, the drug is loaded according to the requirement, and because the carrier material is used as aerogel, a large amount of macroporous-mesoporous hierarchical structures can be formed in the porous structure, the drug loading of the porous structure can be stronger, and the porous structure can be used for loading drugs through delivery, the drug release curve is more stable, the release time is longer, the long-time slow release condition can occur, and the drug which needs to be slowly released for a long time can also play a loading role.
Drawings
Fig. 1 is a surface micro-topography of an SEM of a bone scaffolding material for drug loading of the present invention.
Fig. 2 is a plot of EDS energy spectrum surface scan elemental analysis of the bone scaffold material for drug loading of the present invention.
FIG. 3 is the in vitro release curve of different loading amounts when the functional hybrid aerogel bone scaffold of the present invention is loaded with parathyroid hormone-related protein.
FIG. 4 is the in vitro calcium ion release curves of different loading amounts when the functional hybrid aerogel bone scaffold of the present invention is loaded with parathyroid hormone-related protein.
FIG. 5 is a graph showing the in vitro phosphorus ion release curves of different loading amounts when the functional hybrid aerogel bone scaffold of the present invention is loaded with parathyroid hormone-related protein.
FIG. 6 is a graph showing in vitro release curves of different loading amounts of sclerostin monoclonal antibody and/or stromal cell derived factor-1 loaded on the functional hybrid aerogel bone scaffold of the present invention.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.
Example 1
A bone scaffold material for drug loading comprises an organic template and a deposition material, wherein the deposition material is uniformly deposited on the organic template to form aerogel;
the organic template comprises a nano-cellulose/collagen composite scaffold, and the deposition material comprises hydroxyapatite.
Firstly, preparing nano hydroxyapatite:
100ml of 0.3mol/L diammonium phosphate aqueous solution was added dropwise to 100ml/L calcium nitrate solution under the conditions of stirring speed of 300rpm and temperature of 60 ℃ for reaction for 2 hours. Meanwhile, a proper amount of ammonia water is added in the reaction process to adjust the pH of the reaction system to be approximately equal to 10. After the reaction is finished, the mixture is placed at room temperature for 24 hours, then is centrifuged and washed for 5 times at 5000 rpm/speed, and the obtained precipitate is frozen and dried for 48 hours at the temperature of minus 50 ℃ to prepare the nano-hydroxyapatite.
Then preparing a nano-cellulose/collagen/hydroxyapatite mixed solution:
weighing a certain mass of nano-cellulose and collagen, and dispersing in deionized water with proper volume, wherein the final concentration of the nano-cellulose and the collagen is 2.0% (w/v) and 1.0% (w/v), respectively. Then, a hydrochloric acid adjusting system is applied, the PH value is approximately equal to 4, nano hydroxyapatite accounting for 0.5 of the total mass fraction of the nano cellulose/collagen is added into a nano cellulose/collagen mixing system, and finally, stirring is carried out at 1000rpm until no obvious granular nano hydroxyapatite exists in the system.
And finally preparing nano-cellulose/collagen/hydroxyapatite aerogel:
1) under the stirring condition of 300rpm, dropwise adding GPTMS into a mixed solution system of nano-cellulose/collagen/hydroxyapatite, and continuously stirring for reaction for 48 hours until the crosslinking is completed;
2) injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold;
3) placing the mould into tert-butyl alcohol solvent for a certain time to completely replace water in the system by tert-butyl alcohol; 4) and (3) freeze-drying the completely replaced sample at-20 ℃ for 48 hours to obtain the nano-cellulose/collagen/hydroxyapatite composite aerogel.
Example 2
A bone scaffold material for drug loading comprises an organic template and a deposition material, wherein the deposition material is uniformly deposited on the organic template to form aerogel;
the organic template comprises a nano-cellulose/collagen composite scaffold, and the deposition material comprises hydroxyapatite.
Firstly, preparing nano hydroxyapatite:
100ml of 0.3mol/L diammonium phosphate aqueous solution was added dropwise to 100ml/L calcium nitrate solution under the conditions of stirring speed of 300rpm and temperature of 60 ℃ for reaction for 2 hours. Meanwhile, a proper amount of ammonia water is added in the reaction process to adjust the pH of the reaction system to be approximately equal to 10. After the reaction is finished, the mixture is placed at room temperature for 24 hours, then is centrifuged and washed for 5 times at 5000 rpm/speed, and the obtained precipitate is frozen and dried for 48 hours at the temperature of minus 50 ℃ to prepare the nano-hydroxyapatite.
Then preparing a nano-cellulose/collagen/hydroxyapatite mixed solution:
weighing a certain mass of nano-cellulose and collagen, and dispersing in deionized water with proper volume, wherein the final concentration of the nano-cellulose and the collagen is 2.0% (w/v) and 1.0% (w/v), respectively. Then, a hydrochloric acid adjusting system is applied, the PH value is approximately equal to 4, nano hydroxyapatite accounting for 1.0 of the total mass fraction of the nano cellulose/collagen is added into a nano cellulose/collagen mixing system, and finally, the mixture is stirred at 1000rpm until no obvious granular nano hydroxyapatite exists in the system.
And finally preparing nano-cellulose/collagen/hydroxyapatite aerogel:
1) under the stirring condition of 300rpm, dropwise adding GPTMS into a mixed solution system of nano-cellulose/collagen/hydroxyapatite, and continuously stirring for reaction for 48 hours until the crosslinking is completed;
2) injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold;
3) placing the mould into tert-butyl alcohol solvent for a certain time to completely replace water in the system by tert-butyl alcohol; 4) and (3) freeze-drying the completely replaced sample at-20 ℃ for 48 hours to obtain the nano-cellulose/collagen/hydroxyapatite composite aerogel.
Example 3
A bone scaffold material for drug loading comprises an organic template and a deposition material, wherein the deposition material is uniformly deposited on the organic template to form aerogel;
the organic template comprises a nano-cellulose/collagen composite scaffold, and the deposition material comprises hydroxyapatite.
Firstly, preparing nano hydroxyapatite:
100ml of 0.3mol/L diammonium phosphate aqueous solution was added dropwise to 100ml/L calcium nitrate solution under the conditions of stirring speed of 300rpm and temperature of 60 ℃ for reaction for 2 hours. Meanwhile, a proper amount of ammonia water is added in the reaction process to adjust the pH of the reaction system to be approximately equal to 10. After the reaction is finished, the mixture is placed at room temperature for 24 hours, then is centrifuged and washed for 5 times at 5000 rpm/speed, and the obtained precipitate is frozen and dried for 48 hours at the temperature of minus 50 ℃ to prepare the nano-hydroxyapatite.
Then preparing a nano-cellulose/collagen/hydroxyapatite mixed solution:
weighing a certain mass of nano-cellulose and collagen, and dispersing in deionized water with proper volume, wherein the final concentration of the nano-cellulose and the collagen is 2.0% (w/v) and 1.0% (w/v), respectively. Then, a hydrochloric acid adjusting system is applied, the PH value is approximately equal to 4, nano hydroxyapatite accounting for 1.5 of the total mass fraction of the nano cellulose/collagen is added into a nano cellulose/collagen mixing system, and finally, the mixture is stirred at 1000rpm until no obvious granular nano hydroxyapatite exists in the system.
And finally preparing nano-cellulose/collagen/hydroxyapatite aerogel:
1) under the stirring condition of 300rpm, dropwise adding GPTMS into a mixed solution system of nano-cellulose/collagen/hydroxyapatite, and continuously stirring for reaction for 48 hours until the crosslinking is completed;
2) injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold;
3) placing the mould into tert-butyl alcohol solvent for a certain time to completely replace water in the system by tert-butyl alcohol; 4) and (3) freeze-drying the completely replaced sample at-20 ℃ for 48 hours to obtain the nano-cellulose/collagen/hydroxyapatite composite aerogel.
Example 4
A bone scaffold material for drug loading comprises an organic template and a deposition material, wherein the deposition material is uniformly deposited on the organic template to form aerogel;
the organic template comprises a nano-cellulose/collagen composite scaffold, and the deposition material comprises hydroxyapatite.
Firstly, preparing nano hydroxyapatite:
100ml of 0.3mol/L diammonium phosphate aqueous solution was added dropwise to 100ml/L calcium nitrate solution under the conditions of stirring speed of 300rpm and temperature of 60 ℃ for reaction for 2 hours. Meanwhile, a proper amount of ammonia water is added in the reaction process to adjust the pH of the reaction system to be approximately equal to 10. After the reaction is finished, the mixture is placed at room temperature for 24 hours, then is centrifuged and washed for 5 times at 5000 rpm/speed, and the obtained precipitate is frozen and dried for 48 hours at the temperature of minus 50 ℃ to prepare the nano-hydroxyapatite.
Then preparing a nano-cellulose/collagen/hydroxyapatite mixed solution:
weighing a certain mass of nano-cellulose and collagen, and dispersing in deionized water with proper volume, wherein the final concentration of the nano-cellulose and the collagen is 2.0% (w/v) and 1.0% (w/v), respectively. Then, a hydrochloric acid adjusting system is applied, the PH value is approximately equal to 4, nano hydroxyapatite accounting for 2.0 of the total mass fraction of the nano cellulose/collagen is added into a nano cellulose/collagen mixing system, and finally, the mixture is stirred at 1000rpm until no obvious granular nano hydroxyapatite exists in the system.
And finally preparing nano-cellulose/collagen/hydroxyapatite aerogel:
1) under the stirring condition of 300rpm, dropwise adding GPTMS into a mixed solution system of nano-cellulose/collagen/hydroxyapatite, and continuously stirring for reaction for 48 hours until the crosslinking is completed;
2) injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold;
3) placing the mould into tert-butyl alcohol solvent for a certain time to completely replace water in the system by tert-butyl alcohol; 4) and (3) freeze-drying the completely replaced sample at-20 ℃ for 48 hours to obtain the nano-cellulose/collagen/hydroxyapatite composite aerogel.
Aiming at the bone scaffold material for drug loading prepared by the invention, the common aerogel scaffold can be prepared by physically mixing nano-cellulose and collagen, chemically crosslinking and freeze-drying. However, the nanocellulose/collagen aerogel scaffold has the disadvantages of insufficient mechanical strength, poor osteoinductive properties, poor in-scaffold vascular formation, too fast degradation rate and the like. According to the invention, other inorganic substances, namely nano-hydroxyapatite, are added into the nano-cellulose/collagen aerogel support to compound the nano-hydroxyapatite to form a synergistic effect.
The hydroxyapatite is a main component (about 65 percent) of natural bone tissue, has excellent osteoconductivity and biocompatibility, can promote local bone formation by degradation products of calcium ions and phosphate radicals, and is an excellent bone tissue substitute material. However, the hydroxyapatite has the defects of large brittleness, poor biological absorbability, slow degradation rate, poor bone inductivity and the like, and the single hydroxyapatite material can cause the risks of loosening, even infection and the like after being implanted into the body for a long time.
The invention constructs organic-inorganic hybrid aerogel formed by compounding nano-cellulose/collagen organic template with inorganic nano-hydroxyapatite, selects nano-cellulose and collagen to be chemically cross-linked, adds nano-hydroxyapatite after copolymerization reaction, utilizes collagen carboxyl as calcium phosphate mineralization nucleation site, and uniformly deposits the nano-hydroxyapatite on the nano-cellulose/collagen organic template to prepare the nano-cellulose/collagen/hydroxyapatite bone defect filling scaffold material.
The nano-cellulose/collagen organic scaffold has large specific surface area, high bioactivity and excellent degradability, and the composite nano-hydroxyapatite can control the degradation rate and enhance the mechanical property by adjusting the components of the material. Degradation products such as calcium ions, phosphate radicals and the like can increase the internal bone conduction and bone induction of the scaffold material and improve the microenvironment of local osteogenesis and vascularization; the organic-inorganic hybrid aerogel has a nano-scale rough surface formed by a macroporous-mesoporous hierarchical structure and nano-hydroxyapatite, and is beneficial to adhesion, proliferation and migration of osteoblasts, BMSCs and HUVECs. Thereby preparing the safe and effective nano-cellulose/collagen/hydroxyapatite organic-inorganic hybrid aerogel support material with controllable degradation rate to repair bone defects.
The microstructure of the bone scaffold material for drug loading prepared by the invention is observed by a Scanning Electron Microscope (SEM), as shown in figure 1, a large number of gaps with different sizes exist on the cross section of the aerogel of the bone scaffold material, the diameter of the gap is 75-250 nm, the average diameter is about 190nm, and meanwhile, the porosity of the aerogel of the bone scaffold material is 69-81% after measurement. Therefore, the prepared bone scaffold material aerogel material has a large number of micropores in the material, can provide a large number of loading spaces for loading drugs, and has strong loading capacity.
Then, the bone scaffold material for drug loading prepared by the invention adopts EDS energy spectrum to scan the element composition of the detection material, as shown in figure 2, and the result of element analysis shows that the main components of the bone scaffold material comprise elements such as phosphorus (P), calcium (Ca), oxygen (O), carbon (C) and the like.
The bone scaffold materials for drug loading prepared in the foregoing examples 1, 2, 3 and 4 were subjected to mechanical property test together with a blank sample, i.e., a blank control sample from which nano-hydroxyapatite was removed, and the test structures are shown in table 1.
TABLE 1
From table 1, it can be seen that the compressive property and the elastic modulus of the aerogel are improved after the nano hydroxyapatite is added. The aerogel containing different mass fractions of n-HA HAs different compression resistance and elastic modulus, the elastic modulus of the aerogel containing 100% of the mass fraction of n-HA is 12.95 +/-4.77 MPa, the highest of the groups, the compression resistance is 0.4067 +/-0.084 MPa, and the highest of the groups.
Example 5
A functional hybrid aerogel bone scaffold comprises the bone scaffold material for drug loading disclosed by the invention, and drug loading is carried out by using the bone scaffold material.
In this example, the parathyroid hormone-related protein is loaded in an in-situ loading manner, and a part of the preparation steps in any one of examples 1 to 4 are adopted, that is, after the nanocellulose/collagen/hydroxyapatite mixed solution is prepared, GPTMS is added dropwise into the nanocellulose/collagen/hydroxyapatite mixed solution under the stirring condition of 300rpm, and simultaneously 100ug of parathyroid hormone-related protein is added, and the stirring reaction is continued for 48 hours until the crosslinking is completed; injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold; placing the mould into tert-butyl alcohol solvent for replacement to ensure that water in the system is completely replaced by tert-butyl alcohol; and (3) freeze-drying the replaced sample at-20 ℃ for 48 hours to obtain the functional hybrid aerogel bone scaffold loaded with the parathyroid hormone related protein.
After the preparation, the functional hybrid aerogel bone scaffold loaded with parathyroid hormone-related protein prepared in this example was released with drug, and the released drug was quantitatively detected, and a drug in vitro release curve was plotted, as shown in fig. 3, which corresponds to curve a in fig. 3 in this example.
Example 6
A functional hybrid aerogel bone scaffold comprises the bone scaffold material for drug loading disclosed by the invention, and drug loading is carried out by using the bone scaffold material.
In this example, the parathyroid hormone-related protein is loaded in an in-situ loading manner, and a part of the preparation steps in any one of examples 1 to 4 are adopted, that is, after the nanocellulose/collagen/hydroxyapatite mixed solution is prepared, GPTMS is added dropwise into the nanocellulose/collagen/hydroxyapatite mixed solution under the stirring condition of 300rpm, 400ug of parathyroid hormone-related protein is added at the same time, and the stirring reaction is continued for 48 hours until the crosslinking is completed; injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold; placing the mould into tert-butyl alcohol solvent for replacement to ensure that water in the system is completely replaced by tert-butyl alcohol; and (3) freeze-drying the replaced sample at-20 ℃ for 48 hours to obtain the functional hybrid aerogel bone scaffold loaded with the parathyroid hormone related protein.
After the preparation, the functional hybrid aerogel bone scaffold loaded with parathyroid hormone-related protein prepared in this example was released with drug, and the released drug was quantitatively detected, and a drug in vitro release curve was plotted, as shown in fig. 3, which corresponds to curve B in fig. 3 in this example.
Example 7
A functional hybrid aerogel bone scaffold comprises the bone scaffold material for drug loading disclosed by the invention, and drug loading is carried out by using the bone scaffold material.
In this embodiment, the parathyroid hormone-related protein is loaded in an in-situ loading manner, and a part of preparation steps in any one of embodiments 1 to 4 are adopted, that is, after the nanocellulose/collagen/hydroxyapatite mixed solution is prepared, GPTMS is added into the nanocellulose/collagen/hydroxyapatite mixed solution dropwise under the stirring condition of 300rpm, after the stirring reaction is continued for 46 hours, 100ug of parathyroid hormone-related protein is added, and the stirring reaction is continued for 2 hours until the crosslinking is completed; injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold; placing the mould into tert-butyl alcohol solvent for replacement to ensure that water in the system is completely replaced by tert-butyl alcohol; and (3) freeze-drying the replaced sample at-20 ℃ for 48 hours to obtain the functional hybrid aerogel bone scaffold loaded with the parathyroid hormone related protein.
After the preparation, the functional hybrid aerogel bone scaffold loaded with parathyroid hormone-related protein prepared in this example was released with drug, and the released drug was quantitatively detected, and a drug in vitro release curve was plotted, as shown in fig. 3, which corresponds to curve C in fig. 3 in this example.
Example 8
A functional hybrid aerogel bone scaffold comprises the bone scaffold material for drug loading disclosed by the invention, and drug loading is carried out by using the bone scaffold material.
In this embodiment, the parathyroid hormone-related protein is loaded in an in-situ loading manner, and a part of preparation steps in any one of embodiments 1 to 4 are adopted, that is, after the nanocellulose/collagen/hydroxyapatite mixed solution is prepared, GPTMS is added into the nanocellulose/collagen/hydroxyapatite mixed solution dropwise under the stirring condition of 300rpm, after the stirring reaction is continued for 46 hours, 400ug of parathyroid hormone-related protein is added, and the stirring reaction is continued for 2 hours until the crosslinking is completed; injecting the crosslinked mixed dispersion into a mold, and standing the sample at 0-4 ℃ for 4 hours to remove air bubbles in the mold; placing the mould into tert-butyl alcohol solvent for replacement to ensure that water in the system is completely replaced by tert-butyl alcohol; and (3) freeze-drying the replaced sample at-20 ℃ for 48 hours to obtain the functional hybrid aerogel bone scaffold loaded with the parathyroid hormone related protein.
After the preparation, the functional hybrid aerogel bone scaffold loaded with parathyroid hormone-related protein prepared in this example was released with drug, and the released drug was quantitatively detected, and a drug in vitro release curve was plotted, as shown in fig. 3, which corresponds to curve D in fig. 3 in this example.
Specifically, in the preparation of the in vitro release profile, the release of the parathyroid hormone-related protein is first carried out by immersing the samples prepared in examples 5, 6, 7 and 8 in 5ml of PBS (pH 7.4) and placing them together in a 15ml EP tube sealed at 37 ℃ and 120rpm on a shaker. The measurement time points were 4, 12, 18 hours, 1, 2, 3, 4, 6, 8 and 14 days, respectively, and 1ml of the supernatant was taken out at the different time end points while fresh pre-heated PBS at 37 ℃ was supplemented, and the drug concentration of the supernatant was measured by high performance liquid chromatography.
When the measurement is performed by using a high performance liquid chromatograph,
1) a 2996 type ultraviolet detector (DAD) of 150mm x 4.6mm equipped with a 5 μm Agilent Zorbax 300-SB C3 chromatographic column was selected;
2) the mobile phases were pure water (0.1% trifluoroacetic acid) and acetonitrile (0.1% trifluoroacetic acid). The mobile phase consisted of acetonitrile (HPLC grade, fischer in usa) containing 0.1% trifluoroacetic acid (TFA) and pure water containing 0.1% TFA, with a gradient of 0-50% acetonitrile (0.1% TFA) at a flow rate of 1mL min "1 for 25 minutes;
3) the retention time of PTHrP under the above conditions was 13.5 minutes, detection was carried out with ultraviolet rays at 220nm and chromatograms were recorded using Empower software;
4) the PTHrP (between 30ug/mL and 1 mg/mL) with different concentrations prepared by 0.9% NaCl is used as a standard solution, the linearity is good, and the correlation coefficient of a calibration graph is greater than 0.999.
The results are plotted as an in vitro release curve of the drug, as shown in fig. 3, it can be seen from fig. 3 that the curves C and D of the samples corresponding to examples 7 and 8 release about 88% to 92% of parathyroid hormone-related protein within 24 hours, and the remaining 8% to 12% of parathyroid hormone-related protein can be slowly released for more than 14 days, resulting in a short-time burst release phenomenon; while the release amount of the drug in the curve A and curve B of the samples corresponding to examples 5 and 6 is about 65-75% at 24 hours, the remaining 25-35% of parathyroid hormone related protein can be slowly released for more than 14 days, the release curve is flat, that is, the burst release phenomenon can occur within 24 hours, and then the slow release process is carried out.
The strategy for effective local osteogenesis promoting release of parathyroid hormone related protein mainly comprises local burst release combined with transient slow release, long-term slow release instead promotes osteoclast proliferation to increase new bone resorption, although the samples of example 7 and example 8 are the best samples with local burst release combined with transient slow release through an in vitro release curve of parathyroid hormone related protein, when the medicine is released in vivo, the process is more complicated, the degradation of materials is accelerated by chemical bonds inside a local microenvironment enzymolysis material, and the release amount of the medicine is increased, so that in combination with the pharmacokinetics and release curves of parathyroid hormone related protein, the samples prepared in example 5 and example 6 are preferably released to carry out medicine loading, namely, the samples with the local burst release combined with transient slow release trend are selected from the graph, and the curve is more gentle.
After the drug release profiles were prepared, the samples prepared in examples 5 and 6 were selected and tested for in vitro release of calcium and phosphorus ions.
The bone scaffold material not loaded with the drug, that is, the sample prepared in example 2, the sample prepared in example 5, and the sample prepared in example 6 were immersed in a Tris-HCl buffer solution (pH 7.4, solid-to-liquid ratio 1: 100), respectively, and left at a constant temperature of 37 ℃ for 1, 3, 7, and 14 days. At each time point, the samples were taken out, dried, weighed and then re-immersed in fresh buffer solution. At the same time, the concentration of Ca and P ions in the medium was checked by inductively coupled plasma emission spectrometer at each time point.
As shown in fig. 4, the sample prepared in example 2 corresponds to curve a in the graph, the sample prepared in example 5 corresponds to curve B in the graph, and the sample prepared in example 6 corresponds to curve C in the graph, and it is apparent from the graphs that the calcium ion concentration in the material can be effectively increased after the parathyroid hormone-related protein is loaded;
referring to fig. 5, the curve a in the graph corresponding to the sample prepared in example 2, the curve B in the graph corresponding to the sample prepared in example 5, and the curve C in the graph corresponding to the sample prepared in example 6, it is apparent from the graphs that the concentration of phosphorus ions in the material can be effectively increased after the parathyroid hormone-related protein is loaded; therefore, with reference to fig. 4 and 5, after the parathyroid hormone-related protein is loaded, degradation products such as calcium ions, phosphate radicals and the like in the material can be effectively increased, the internal bone conduction and the bone induction of the scaffold material are further promoted, and the local osteogenesis and angiogenisis microenvironment is improved.
Example 9
A functional hybrid aerogel bone scaffold comprises the bone scaffold material for drug loading disclosed by the invention, and drug loading is carried out by using the bone scaffold material.
In this example, the stromal cell derived factor-1 is loaded by dropping loading,
specifically, the bone scaffold material for drug loading prepared in any one of examples 1 to 4 was used for loading.
And (3) sterilizing the bone scaffold material by using ethylene oxide, dripping 40 mu L of stromal cell derived factor-1 solution with the concentration of 1 mu g/mL under aseptic conditions, and freeze-drying to obtain the aerogel loaded with the stromal cell derived factor-1. Subpackaging under aseptic condition, and storing at low temperature.
Then, the aerogel loaded with the stromal cell derived factor-1 prepared in this example was subjected to drug release detection, and a release curve was plotted as curve a in fig. 6.
In the light of the above example 10,
a functional hybrid aerogel bone scaffold comprises the bone scaffold material for drug loading disclosed by the invention, and drug loading is carried out by using the bone scaffold material.
In this example, the sclerostin monoclonal antibody is loaded by dropping loading,
specifically, the bone scaffold material for drug loading prepared in any one of examples 1 to 4 was used for loading.
And (3) sterilizing the bone scaffold material by using ethylene oxide, dripping 40 mu L of sclerostin monoclonal antibody solution with the concentration of 1 mu g/mL under the aseptic condition, and freeze-drying to obtain the aerogel loaded with the stromal cell derived factor-1. Subpackaging under aseptic condition, and storing at low temperature.
Then, the aerogel loaded with the stromal cell derived factor-1 prepared in this example was subjected to drug release detection, and a release curve was plotted as curve B in fig. 6.
Specifically, in testing the degree of drug release of the samples prepared in example 9 and example 10;
the samples prepared in example 9 and example 10 were placed in a centrifuge tube containing 1mL of PBS, and placed in a constant temperature shaking box at 37 ℃ at a shaking frequency of 60 times/min for 10 time points of 3h, 6h, 12h, 24h, 3 days, 5 days, 7 days, 10 days, 14 days and 21 days, and 100. mu.L of the extract was taken into an EP tube and stored at-20 ℃ in a frozen state, and 100. mu.L of PBS was added after each PBS was taken. And (3) after all time points are taken, melting the extract at room temperature, adding 10 mu L of the extract at each time point according to the specification of the ELISA test box of the stromal cell derived factor-1 and the sclerostin monoclonal antibody, making 2 multiple holes for each test point, adding 40 mu L of sample diluent into the holes to be tested, and making standard holes and control holes at the same time. And (3) placing the plate sealing membrane plate on a plate sealing membrane, incubating for 30 minutes at 37 ℃, taking out the enzyme-labeled coated plate, pouring out liquid in the hole, filling the prepared washing liquid in each hole, standing for 30s, pouring out, repeating for 5 times, and finally drying by patting. Adding 50 μ L of color-developing agent A into the holes including blank, adding 50 μ L of color-developing agent B, shaking gently, mixing, wrapping with tinfoil paper, developing at 37 deg.C for 10min, and adding 50 μ L of stop solution into each hole to stop developing. OD was measured on a microplate reader. And drawing a standard curve according to the OD value of the standard hole and the concentration of the standard solution, calculating the concentrations of the sclerostin monoclonal antibody and the stromal cell derived factor-1 corresponding to each test point according to a standard curve formula, calculating the accumulated release degree, and drawing a release curve.
For a specific curve, refer to fig. 6: within 3 hours, about 40-50% of the total amount of the stromal cell derived factor-1 and the osteopontin monoclonal antibody is released from the material, and a stable release rate is maintained for the next 21 days, and the release of the stromal cell derived factor-1 and the osteopontin monoclonal antibody from the material can be detected for 21 days, and 70-80% of the total release is observed for 21 days. Thereby obtaining the bone repair scaffold material with slow drug release. The drug release curve is more stable, the release time is longer, and the situation of long-time slow release can occur, namely for the drug which needs to be slowly released for a long time, the bone scaffold material for drug loading related by the invention can also play a loading role.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (5)
1. A functional hybrid aerogel bone scaffold, comprising: the aerogel thermal insulation material comprises an organic template and a deposition material, wherein the deposition material is uniformly deposited on the organic template to form aerogel; the organic template comprises a nano-cellulose/collagen composite scaffold, and the deposition material comprises hydroxyapatite;
the preparation method comprises the following steps:
a. preparing nano hydroxyapatite;
b. preparing a nano-cellulose/collagen mixed aqueous solution: dispersing nano-cellulose and collagen in deionized water, and adjusting the pH of the system to acidity by using inorganic acid;
c. preparing a nano-cellulose/collagen/hydroxyapatite mixed aqueous solution: adding the nano hydroxyapatite into the nano cellulose/collagen mixed aqueous solution, and continuously stirring until no particles exist to prepare a precursor solution;
d. preparing nano-cellulose/collagen/hydroxyapatite aerogel: dropwise adding GPTMS into the precursor solution, stirring to react to form a cross-linked solution, standing the cross-linked solution to remove bubbles, then placing the cross-linked solution into a tert-butyl alcohol solvent for replacement, and finally performing freeze drying to obtain the aerogel;
the nano-cellulose/collagen/hydroxyapatite aerogel is loaded with functional substances;
the functional substance comprises an in-situ loaded drug loaded in situ or a dropwise loaded drug loaded dropwise; the in-situ loaded drug comprises parathyroid hormone-related protein, and the dropwise loaded drug comprises at least one of sclerostin monoclonal antibody and stromal cell derived factor-1.
2. The functional hybrid aerogel bone scaffold of claim 1, wherein: the mass ratio of the nano-cellulose to the collagen is 0.5-2: 1, and the hydroxyapatite accounts for 0.5-2% of the total mass of the nano-cellulose and the collagen.
3. A method for preparing the functional hybrid aerogel bone scaffold of claim 1, which is characterized by comprising the following steps: the method comprises the following steps:
a. preparing nano hydroxyapatite;
b. preparing a nano-cellulose/collagen mixed aqueous solution: dispersing nano-cellulose and collagen in deionized water, and adjusting the pH of the system to acidity by using inorganic acid;
c. preparing a nano-cellulose/collagen/hydroxyapatite mixed aqueous solution: adding nano hydroxyapatite into the mixed aqueous solution of nano cellulose and collagen, and continuously stirring until no particles exist to prepare a precursor solution;
d. preparing functional hybrid aerogel: when loading the drug in situ: dropwise adding GPTMS into the precursor solution, simultaneously adding the in-situ load drug, stirring to react to form load cross-linking solution, standing the load cross-linking solution for defoaming, then placing the load cross-linking solution into tert-butyl alcohol solvent for replacement, and finally freeze-drying to obtain a load aerogel support;
when the loading drop loads the drug: dropwise adding GPTMS into the precursor solution, stirring to react to form a cross-linked solution, standing the cross-linked solution to remove bubbles, then placing the cross-linked solution into a tert-butyl alcohol solvent for replacement, finally performing freeze drying to prepare a matrix aerogel, dropwise adding the dropwise added load drug onto the matrix aerogel in a sterile environment, and performing freeze drying after dropwise adding is completed.
4. The preparation method of the functional hybrid aerogel bone scaffold according to claim 3, characterized in that: in the step b, the final concentration of the nano-cellulose and the final concentration of the collagen are both 1-5% w/v.
5. The preparation method of the functional hybrid aerogel bone scaffold according to claim 3, characterized in that: in the step c, the nano hydroxyapatite accounts for 0.5-2% of the total mass fraction of the nano cellulose and the collagen.
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