CN117363563A

CN117363563A - Chondrocyte composite culture method based on silk fibroin-hydroxyapatite scaffold and application

Info

Publication number: CN117363563A
Application number: CN202210751718.9A
Authority: CN
Inventors: 陈仲春; 赵霞; 苗变梁; 姚晋荣; 邵正中
Original assignee: Huashan Hospital of Fudan University
Current assignee: Huashan Hospital of Fudan University
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2024-01-09

Abstract

The invention belongs to the field of tissue engineering and medical materials, and relates to a method for compositely culturing a chondrocyte-silk fibroin hydroxyapatite scaffold and an application prospect in reconstruction of tracheal defects. The invention adopts the silk fibroin hydroxyapatite composite as a scaffold material, and the isolated primary human nasal septum chondrocyte is used as a seed cell, and the chondrocyte is subjected to compound culture on the silk fibroin hydroxyapatite composite material to form the cartilage-silk fibroin hydroxyapatite composite. The cartilage-silk fibroin hydroxyapatite compound can be used for preparing trachea defect repair products, and can provide meaningful references for development of tissue engineering trachea.

Description

Chondrocyte composite culture method based on silk fibroin-hydroxyapatite scaffold and application

Technical Field

The invention belongs to the technical field of tissue engineering and medical materials, and relates to a chondrocyte compound culture method. In particular to a method for compositely culturing human chondrocytes based on a silk fibroin hydroxyapatite scaffold and application of the prepared cartilage-silk fibroin hydroxyapatite complex in reconstruction of tracheal defects.

Background

The prior art discloses that long-term tracheal defects or stenosis may be caused by trauma, tumor, congenital disease, tracheotomy, long-term tracheal intubation, etc. There are still several difficulties with tissue repair after airway lesion excision. In clinical practice, the airway reconstruction needs to achieve the following purposes or effects: 1. completely closing the operation cavity to avoid emphysema and the like; 2. has the supporting function similar to the strength of cartilage so as to maintain the shape of a tube cavity and avoid the collapse of the trachea during inspiration; 3. can be shaped conveniently according to the shape and size of the defect in the operation; 4. after repair of organs where the airways are in direct contact with ambient air, it is desirable that mucous membranes can grow on the repair tissue or material to expel sputum and reduce obstruction and infection. The reconstruction method commonly used in clinic at present is difficult to achieve satisfactory effect, and incorrect repair is extremely easy to cause the impairment of the functions of pronunciation, swallowing, breathing, removal of inhaled antigens by mucociliary and immune response, thereby causing the reduction of life quality and even endangering life. Moreover, since the blood vessels of the supply air tube are slim and are distributed in a segmental manner, the blood vessels are difficult to anastomose, so that the allogenic air tube transplantation similar to liver and kidney transplantation is difficult to clinically apply, the allogenic tissue is reconstructed, the source of a donor is tense, and an immunosuppressant needs to be taken for a long time after operation; autologous tissue reconstruction, because of limited materials, cannot repair larger defects and can cause damage to the donor area of the human body, and the like.

To improve the possibility and success rate of repairing the defect of the long-segment trachea, the skilled person pays attention to the development of an artificial trachea repairing material with excellent performance. At present, more tracheal repair materials are researched to be non-degradable synthetic materials such as polypropylene, degradable synthetic materials such as polylactic acid-polyglycolic acid, natural polymer materials such as collagen and chitosan, but the materials have certain limitations. Synthetic materials have several distinct advantages: 1. the source is rich, and no donor is needed. 2. The manufacturing process is simple and quick, and can meet the individual needs of different patients. 3. The mechanical strength is better. However, the disadvantages of synthetic materials (such as polylactic acid-polyglycolic acid and polypropylene scaffolds) are also apparent, and the synthetic materials have poor biocompatibility, are easy to cause inflammatory reaction and are easy to form granulation tissues and infection. Collagen, chitosan, decorin and other degradable natural macromolecular biological materials, and has the advantage of similar structure to extracellular matrix components. However, although their histocompatibility is very good, they are not suitable for use alone in tracheal stents because of their obvious lack of mechanical strength.

The silk fibroin is used as a functional biological material, and in recent years, the application research of the silk fibroin is paid attention to by researchers, and 1. The silk fibroin is rich in source and strong in material preparation controllability. The silk fibroin is fibrous protein obtained by degumming silk cocoons, and the conditions of the silk fibroin manufacturing process are changed, so that brackets with different pore diameters, morphologies, mechanical properties and degradation time can be obtained to adapt to different tissue repair requirements; silk fibroin can be degraded and absorbed, and the absorption rate is related to the implantation site, mechanical environment, host health condition, physiological characteristics, types, silk fibroin morphology, structure and the like; 2. the silk fibroin has good biocompatibility, has cytokine binding modification sites such as growth factors and the like, and can promote cell adhesion growth; silk fibroin has the same amino acid composition as human keratin; silk fibroin is beneficial to cell adhesion because it contains a cell binding domain. The three-dimensional porous structure of silk fibroin scaffolds is similar to extracellular matrix, providing sites for cells to acquire nutrition, gas exchange, excrete waste, and grow metabolism, facilitating cell adhesion, proliferation, and differentiation. The excellent tissue compatibility of silk fibroin has been confirmed by studies, which can promote the growth of retinal pigment epithelium, cornea epithelium, tympanic membrane epithelium, fibroblasts, etc., and can support the adhesion, proliferation, differentiation and cartilage formation of chondrocytes; 3. silk fibroin can induce regulation of calcium salt deposition. Is one of the most commonly used polymers for biomineralization and hybridization with hydroxyapatite. This is because, on the one hand, silk fibroin has a similar structure to collagen; on the other hand, exposed carbonyl groups on the surface of silk fibroin play a very important role in inducing mineralization.

Hydroxyapatite is the main component of human bone tissue, and research shows that the hydroxyapatite has good biocompatibility and bioactivity, no immunogenicity, does not cause organism inflammatory reaction, has good bone conductivity, forms good chemical bonding with the tissue of human body, and is a bone substitute which has been clinically applied. It has been reported that hydroxyapatite nanocrystals can induce endothelial cell activation, adhesion and proliferation in vivo;

so far, no report on the application of the human chondrocyte-silk fibroin hydroxyapatite scaffold complex in the aspect of tracheal defect reconstruction exists at home and abroad.

Based on the current state of the art, the inventors of the present application have intended to provide a chondrocyte composite culture method, in particular a method for composite culturing human chondrocytes based on a silk fibroin hydroxyapatite scaffold, and the application of the prepared cartilage-silk fibroin hydroxyapatite composite in reconstruction of tracheal defects.

Disclosure of Invention

The invention aims to provide a chondrocyte composite culture method based on the current state of the art, and in particular relates to a method for compositely culturing human chondrocytes based on a silk fibroin hydroxyapatite scaffold and application of the prepared cartilage-silk fibroin hydroxyapatite composite in reconstruction of tracheal defects.

The invention provides a method for culturing human primary chondrocytes on a silk fibroin hydroxyapatite composite scaffold and application prospects in clinical tracheal defect reconstruction.

The invention adopts a silk fibroin hydroxyapatite composite material as a scaffold material and isolated human nasal septum chondrocytes as seed cells to form the cartilage-porous silk fibroin hydroxyapatite scaffold composite. The invention provides necessary support for repairing the respiratory tract lumen by utilizing the good biocompatibility of the silk fibroin and the hydroxyapatite and the effect of promoting the cell growth.

The chondrocyte-silk fibroin hydroxyapatite scaffold compound can be used for repairing clinical trachea defects, and can provide a meaningful reference for discussing the effects of the chondrocyte-silk fibroin hydroxyapatite compound on promoting trachea repair and clinically reconstructing trachea defects and reducing postoperative complications of reconstructed trachea.

The culture steps of the chondrocyte on the silk fibroin hydroxyapatite are as follows:

1. preparation of porous silk fibroin hydroxyapatite composite scaffold

1. Preparation of silk protein/hydroxyapatite composite porous scaffold

1.1 preparation of regenerated silk protein aqueous solution

Removing silkworm pupa and impurities from silkworm cocoons, then putting the silkworm cocoons into a boiling water solution of sodium carbonate (0.5 wt%) and boiling for 30 minutes to remove sericin, rinsing the silkworm cocoons with deionized water, and drying the silkworm cocoons in a baking oven at 40 ℃ for standby to obtain degummed silk;

the degummed silk is placed in 9.5M lithium bromide water solution, after being dissolved for one hour at 60 ℃, the solution is filtered by 8 layers of medical gauze to remove solid impurities, and the obtained solution is placed in a dialysis bag with the cut-off molecular weight of 12,000-14,000 Da, and is dialyzed by deionized water for three days to remove salt. Centrifuging the dialyzed silk protein solution at 8000rpm for 10 minutes, filtering with 8 layers of medical gauze, collecting the upper clarified part, storing the solution in a refrigerator at 4 ℃, and calibrating the concentration by a weighing method;

the supernatant was concentrated by dialysis against 12.5wt% aqueous polyethylene glycol to give an aqueous regenerated silk protein solution having a concentration of about 14 wt%. The concentration of the aqueous silk fibroin solution was determined by weighing and kept in a refrigerator at 4℃and the other concentration of silk fibroin solution was obtained by diluting a 14wt% solution with deionized water. During the operation, intense stirring, shaking, contact with organic solvent and the like are avoided as much as possible, so as to prevent the denaturation caused by the change of the silk protein conformation in the solution.

1.2 preparation of Silk fibroin/hydroxyapatite composite suspension

Preparing a 0.54M disodium hydrogen phosphate aqueous solution and a 2M calcium chloride aqueous solution; adding a certain amount of calcium chloride solution into silk protein water solution according to the ratio of hydroxyapatite to silk protein, and mixing in a shaking table at 37 ℃ for about thirty minutes; then, adding a certain amount of disodium hydrogen phosphate solution, and continuously mixing for thirty minutes; then, the mixture was cultured in a constant temperature water bath at 37℃for 24 hours to prepare a silk fibroin/hydroxyapatite composite suspension.

1.3 preparation of n-butanol/Silk protein/hydroxyapatite composite emulsion

And taking silk protein/hydroxyapatite composite suspensions with different concentrations, dropwise adding different amounts of n-butanol, and stirring at 400rpm for 2 minutes at room temperature to form stable emulsion.

1.4 preparation of Silk protein/hydroxyapatite composite porous scaffold by emulsion freezing method

Pouring the prepared n-butanol/silk protein/hydroxyapatite composite emulsion into a mould, freezing for 24 hours in a refrigerator at the temperature of minus 20 ℃, thawing at room temperature, rinsing for a plurality of times with deionized water, removing n-butanol, and then obtaining the silk protein/hydroxyapatite composite porous scaffold, and trimming the porous scaffold to the size of 0.5cm in diameter and 1mm in thickness for later use.

2. Primary culture of human nasal septum chondrocytes

Collecting nasal septal cartilage after nasal septal correction, immediately soaking in DMEM culture solution containing 10% FBS, and adding 1×10 extract ⁵ U/L penicillin and 1X 10 ⁵ The U/L streptomycin is washed three times with normal saline, washed three times with PBS, placed in a 60cm dish, immersed in DMEM medium containing 10% FBS, sheared to about 1mm by 1mm size with an ophthalmic scissors, and digested overnight at 4deg.C with type II collagenase. The culture broth was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, washed once more with DMEM medium containing 10% FBS (centrifugation at 1000 rpm. Times.5 min), and 3ml of DMEM medium containing 10% FBS was added, gently swirled with a disposable sterile pipette, and mixed well and placed 25cm ² In a culture flask, the mixture was cultured at 37℃in a 5% CO2 cell incubator. The culture medium was changed every 2-3 days.

3. Composite culture of human nasal septal chondrocyte and porous silk fibroin-hydroxyapatite scaffold

After the growth of human chondrocytes reached 80% confluence, the culture solution was aspirated, washed 3 times with PBS, 0.25% pancreatin was added, and digested for about 3 minutes, see cell rounding. Immediately, DMEM medium containing 10% fbs was added to terminate digestion, and the cells were blown down by repeated pipetting. Centrifuge 1000 rpm x 5 minutes. The supernatant was discarded, and the supernatant was discarded after washing with DMEM medium containing 10% FBS (centrifugation at 1000 rpm. Times.5 minutes). Adding 10% FBSDMEM broth, disposable sterile pipette was gently mixed. Placing porous silk fibroin hydroxyapatite composite scaffold in 24-well culture plate at 1×10 ⁵ /cm ² Human chondrocytes were seeded onto porous silk fibroin hydroxyapatite composite scaffolds and cultured in a 5% CO2 cell incubator at 37℃for 14 days. And (5) periodically scanning an electron microscope and staining with calcein to observe the growth condition of the cells.

The experimental results show that:

1) Primary cell culture of human nasal septal cartilage:

the cell wall is partially observed after the culture for 24 hours, the cells are gradually changed into a fusiform or a polygonal shape from a round shape, the cells start to divide and proliferate after the culture for 2 to 3 days, the cells proliferate and accelerate after the culture for 7 to 9 days, and the cells are fused for about 10 days.

2) Scanning electron microscope observation of chondrocyte growth on porous silk fibroin hydroxyapatite:

the human chondrocyte can grow and proliferate on the porous silk fibroin hydroxyapatite composite scaffold, and scanning electron microscope observation shows that the chondrocyte adheres well on the porous silk fibroin hydroxyapatite composite scaffold, and the proliferation of the chondrocyte begins within 3 days; after 7 days, the proliferation of the cells is obvious and the matrix is secreted; the cells were confluent and maintained in good morphology for 14 days, and the surface of the material was substantially completely covered by cells and matrix.

3) Observation of chondrocyte growth calcein staining on porous silk fibroin hydroxyapatite scaffold:

calcein staining (Calcein AM) of living cells confirmed that chondrocytes with good cell viability had adhered and distributed throughout the scaffold on day 3, and that these cells proliferated gradually throughout the scaffold, with good cell viability remaining on days 7 and 14.

The experimental result shows that the isolated human chondrocyte can be adhered to the porous silk fibroin hydroxyapatite composite scaffold, and well grows, and the living cell calcein staining shows that the human chondrocyte on the porous silk fibroin hydroxyapatite scaffold has good activity, and the scanning electron microscope can see that the cell secretes a large amount of matrixes, so that the whole scaffold is gradually covered.

The invention cultures the human chondrocyte on the porous silk fibroin hydroxyapatite bracket to form the human chondrocyte-porous silk fibroin hydroxyapatite bracket compound, and the compound can be used as an artificial trachea to repair the defect of the trachea, and the artificial trachea is more similar to the strength of a normal trachea, is beneficial to the growth of the epithelium of the trachea mucosa, and further promotes the clinical application of the artificial trachea.

The present invention has the advantage that,

the macromolecular silk fibroin material and the hydroxyapatite are combined to mineralize the silk fibroin, so that the defects of the macromolecular silk fibroin material and the hydroxyapatite serving as artificial trachea materials can be mutually compensated, and the mechanical strength of the composite scaffold is enhanced; silk fibroin can induce hydroxyapatite to deposit on the surface and form a compound through strong chemical crosslinking; the mechanical strength of the mineralized silk fibroin scaffold is obviously enhanced. Meanwhile, the silk fibroin hydroxyapatite composite scaffold has good biocompatibility, and can effectively induce regeneration of bones and cartilages by adjusting an intracellular signal system to generate an osteoinduction effect.

Drawings

Figure 1, primary culture of human nasal septal cartilage, wherein collagen type a, ii is stained, B, methylene blue is stained.

FIG. 2 shows that the human primary cartilage cells can grow and proliferate on the porous silk fibroin hydroxyapatite scaffold, and the fluorescent staining of the calcein living cells shows that the cartilage cells have good activity and morphology;

fig. 3 shows that the human primary cartilage cells can grow and proliferate on the porous silk fibroin hydroxyapatite scaffold by the scanning electron microscopy observation, and the scanning electron microscopy observation shows that the cartilage cells adhere well with the porous silk fibroin hydroxyapatite scaffold, wherein, the cartilage cells proliferate on the materials visible in A and 3 days, the cartilage cells proliferate obviously and secrete the matrix after B and 7 days, and the human cartilage cells are fully paved in C and 14 days, and the layered growth and secretion of a plurality of matrixes are shown.

Detailed Description

EXAMPLE 1 preparation of porous silk fibroin hydroxyapatite scaffold

The silkworm cocoons are put into boiling water solution of sodium carbonate (0.5 wt%) and boiled for 30 min to remove sericin, the degummed silk is put into 9.5M lithium bromide water solution to be dissolved, silk protein solution obtained by dialysis is centrifugated for 10 min at 8000rpm, and supernatant is dialyzed and concentrated by 12.5wt% polyethylene glycol water solution to regenerate the silk protein water solution. And adding a certain amount of calcium chloride solution and disodium hydrogen phosphate solution into the silk protein aqueous solution to obtain a silk protein/hydroxyapatite composite suspension. And taking silk protein/hydroxyapatite composite suspensions with different concentrations, dropwise adding different amounts of n-butanol, and stirring at 400rpm for 2 minutes at room temperature to form stable emulsion. The n-butanol/silk protein/hydroxyapatite composite emulsion is poured into a mould and frozen in a refrigerator at-20 ℃ for 24 hours. Thawing at room temperature, rinsing with deionized water for multiple times, and removing n-butanol to obtain the silk fibroin/hydroxyapatite composite porous scaffold. Trimming to a size of 0.5cm in diameter and 1mm in thickness for later use.

EXAMPLE 2 Primary culture of human nasal septal chondrocytes

Taking nasal septal cartilage after nasal septal orthoses of human, immediately soaking in DMEM culture solution containing 10% FBS, flushing with physiological saline containing 1×105U/L penicillin and 1×105U/L streptomycin for three times, flushing with PBS for three times, placing in a 60cm culture dish, soaking in DMEM culture solution containing 10% FBS, cutting into pieces of about 0.5mm×0.5mm, adding type II collagenase, and digesting overnight at 4deg.C. The culture broth was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, washed once with DMEM medium containing 10% fbs (centrifugation 1000 rpm×5 min), and 3ml DMEM medium containing 10% fbs was added, and gently swirled with a disposable sterile pipette, and incubated in a 25cm2 flask at 37 ℃ in a 5% co2 cell incubator. The culture medium was changed every 2-3 days.

Example 3 composite culture of human chondrocytes and porous Silk fibroin hydroxyapatite scaffold

When the growth of human nasal septum chondrocyte reaches 80% fusion, pancreatin digestion is carried out to adjust the cell concentration to 1 multiplied by 10 ⁵ /cm ² Is to the human nasal septumChondrocytes were seeded onto 3D-printed silk fibroin scaffolds and cultured at 37 ℃ in a 5% co2 cell incubator for 14 days. And (5) periodically scanning an electron microscope and staining with calcein to observe the growth condition.

The experimental result shows that the isolated human primary chondrocyte can grow and proliferate on the porous silk fibroin hydroxyapatite scaffold, and the calcein living cell fluorescent staining shows that the chondrocyte has good activity and morphology;

the primary chondrocyte of the human body can grow and proliferate on the porous silk fibroin hydroxyapatite scaffold, and the scanning electron microscope observation shows that the chondrocyte and the porous silk fibroin hydroxyapatite scaffold are well adhered.

Claims

1. The chondrocyte-silk fibroin hydroxyapatite composite is characterized in that the chondrocyte-silk fibroin hydroxyapatite composite is prepared by taking the silk fibroin hydroxyapatite composite as a scaffold material and taking human nasal septum chondrocytes as seed cells through composite culture.

2. The chondrocyte-silk fibroin hydroxyapatite composite according to claim 1, prepared according to the following method, comprising: the silk fibroin/hydroxyapatite composite porous scaffold is prepared according to the following steps,

(1) Preparing regenerated silk protein aqueous solution;

(2) Preparing a silk protein/hydroxyapatite composite suspension;

(3) Preparing n-butanol/silk protein/hydroxyapatite composite emulsion;

(4) And preparing the silk protein/hydroxyapatite composite porous scaffold by an emulsion freezing method.

3. The chondrocyte-silk fibroin hydroxyapatite composite according to claim 2, wherein the prepared silk fibroin/hydroxyapatite composite porous scaffold has a diameter of 0.5cm and a thickness of 1mm.

4. Use of the chondrocyte-silk fibroin hydroxyapatite composite according to claim 1 in the preparation of a tissue engineered tracheal defect reconstruction product.