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Multistage micro-nano structure bone repair scaffold for freeze-drying delivery of exosome

A technology of micro-nano structure and exosomes, which is applied in the field of medical biomaterials, can solve the problems of long-term preservation, biological activity, and functional maintenance for research, so as to achieve complete regeneration/repair, improve bone repair ability and new bone Growth ability, effect of improving biological activity

Pending Publication Date: 2021-06-11
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing studies have combined exosomes with scaffolds through solution adsorption or hydrogel embedding, which cannot be stored for a long time. How to ensure the long-term stable existence of exosomes on scaffolds is another problem that must be solved in its clinical application.
Li et al. (Chinese Tissue Engineering Research (29) 4593-4600) have shown that exosomes may be made into freeze-dried powder for long-term storage, but the antifreeze agent trehalose must be used to maintain their structural stability. This document only verifies The biological safety of exosomes before and after freeze-drying has not been studied, but their biological activity and functional maintenance have not been studied

Method used

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  • Multistage micro-nano structure bone repair scaffold for freeze-drying delivery of exosome
  • Multistage micro-nano structure bone repair scaffold for freeze-drying delivery of exosome
  • Multistage micro-nano structure bone repair scaffold for freeze-drying delivery of exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Extraction and identification of exosomes

[0030] (1) Cell extraction and identification

[0031] Rat BMSC extraction: 4-week-old male SD rats weighing 80-100 g were killed by intraperitoneal injection of 1% pentobarbital sodium in three doses, soaked in 75% ethanol for 10 minutes, and placed under sterile conditions Take out the femur and tibia, remove the epiphysis at both ends with surgical scissors, and expose the two ends of the marrow cavity. Use a 10mL syringe to draw α-MEM medium containing 10% FBS and 1% penicillin / streptomycin double antibody to repeatedly wash the bone marrow cavity, and use a pipette gun to blow evenly to obtain a uniform bone marrow cell suspension. The characteristics of wall growth were inoculated in disposable polystyrene cell culture flasks (Corning Corning 75cm2 vented cap right-angle culture flasks), and cultured in a cell culture box with a temperature of 37°C, 5% CO2 / 95% air, and saturated humidity , the medium was chan...

Embodiment 2

[0038] Example 2 Evaluation of the osteoinductive performance of exosomes

[0039] The osteoinductive properties of exosomes in the four groups were evaluated by ALP activity quantification, ALP staining, mineralization staining, and osteogenesis-related gene rtPCR

[0040] (1) Quantification of ALP activity in vitro

[0041] The rBMSC cell suspension was inoculated in a 96-well plate at a density of 5000 cells / well, and after culturing for 24 hours, the medium was replaced with α-MEM medium containing exosomes (containing 2% exosome-free serum, 1 % double antibody), placed in the incubator to continue culturing for 4 days, taken out to detect ALP activity. The experimental steps for detecting ALP activity are as follows: ALP buffer (0.1mol / L glycine, 1mmol / L MgCl2·6H2O) and ALP working solution (1mg / mL PNPP-Na / ALP buffer) were prepared. Aspirate the cell culture medium and wash it twice with PBS, add 1 mL of 1% NP-40 lysate to each well and shake at 37°C for 90 minutes to l...

Embodiment 3

[0049] Embodiment 3 MBG scaffold and exo-MBG scaffold preparation and material characterization

[0050] (1) Preparation of MBG scaffold and exo-MBG scaffold

[0051] In a water bath at 40°C, add 300g of absolute ethanol, 6mL of 1mol / L HCl and 24g of F127 in sequence, stir vigorously until completely dissolved and the solution is clear; add in sequence 4.56g of calcium nitrate tetrahydrate and 1.29mL of triethyl phosphate, dropwise add 31.2 g tetraethyl orthosilicate, kept in a 40°C water bath and vigorously stirred for 24 hours, and the product was rotary evaporated at 60°C to obtain a viscous MBG sol. The PU sponge was cut into discs with a diameter of 5mm as the scaffold macroporous template, 6g of MBG sol and 2.5g of polymer microspheres were evenly mixed, poured into the PU sponge, and squeezed repeatedly until evenly distributed on the sponge. Dry in an oven at 60°C for 72 hours, and then transfer to a muffle furnace for calcination at 550°C for 6 hours to obtain an MBG...

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Abstract

The invention relates to a preparation method of a multistage micro-nano structure bone repair scaffold for freeze-drying delivery of exosomes. The method comprises the following steps: a, carrying out cell culture on supernatant to obtain an exosome concentrated solution; and b, adsorbing the exosome concentrated solution to an MBG stent with a macroporous / microporous / mesoporous multilevel structure, and performing freeze drying. The multistage micro-nano structure bone repair scaffold for freeze-drying delivery of exosomes overcomes the problem of limited osteogenesis induction performance of a pure MBG scaffold, improves the biological activity and osteogenesis promotion performance of the scaffold, and has an exosome slow release rate meeting the requirements of bone repair treatment, so that the bone repair ability and new bone growth ability of a scaffold material are improved, and complete regeneration / repair of bone tissues is realized. The microporous structure of the scaffold in the repair scaffold plays a role in protecting the form and activity of the exosome, and the multistage micro-nano structure bone repair scaffold for freeze-drying delivery of the exosome has a remarkably improved in-vivo bone defect repair effect.

Description

technical field [0001] The invention belongs to the field of medical biomaterials, and in particular relates to a multi-level micro-nano structured bone repair scaffold for freeze-dried delivery of exosomes and a preparation method thereof. Background technique [0002] In recent years, a large number of studies have found that exosomes secreted by cells can promote osteogenesis, angiogenesis and bone mineralization, and are expected to become a new bone regeneration treatment. [0003] Clinical application needs to consider the long-term storage, activity maintenance of exosomes, and how to release them slowly after entering the bone defect site. Biomaterial scaffolds can be used as carriers to play the role of controlled release and activity maintenance, improving the efficiency of the loaded bioactive substances. Exosome solution can be stored stably at 4°C for 24 hours (Protein&Cell 10(4)(2019)295-299), and stored at -20°C for 6 months (International Journal of Molecula...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/12A61L27/56A61L27/54C03B19/06C03B19/12C03C11/00
CPCA61L27/12A61L27/3604A61L27/56A61L27/54C03B19/12C03B19/06C03C11/007A61L2430/02A61L2300/30A61L2300/412A61L2300/602
Inventor 林丹沈国芳王旭东林开利刘安琪
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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