CN116942915A - Bone cell gel culture method and application thereof in bone echinococcosis bone defect - Google Patents
Bone cell gel culture method and application thereof in bone echinococcosis bone defect Download PDFInfo
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Abstract
The invention belongs to the technical field of cell culture, and particularly relates to an osteocyte gel culture method and application thereof in bone echinococcosis bone defect. The bone cells are co-cultured in the polydopamine gel film to obtain cell bundle viscous gel, the cell bundle viscous gel is applied to a bone repair functional module, hydroxyapatite is used as a filling main body, the cell bundle viscous gel is adhered and loaded on the surface, and dihydroartemisinin is added into the cell bundle viscous gel as a therapeutic drug, so that the bone echinococcosis is prevented from recrudescence in a slow-release manner, and good repair and prognosis improvement effects are realized.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to an osteocyte gel culture method and application thereof in bone echinococcosis bone defect.
Background
The bone echinococcosis is a common parasitic disease commonly suffered by people and livestock and seriously endangered to human health, and is well developed in the metaphysis of cancellous bone or long bone with rich blood circulation, unlike the growth mode of soft tissue echinococcosis. Echinococcosis granulosa develops from hexagona and parasitizes in bone tissue to form multiple small capsules, and the inner capsule ruptures and the capsule fluid overflows to cause bone destruction. The bag grows slowly and gradually swells, resulting in atrophy and thinning of bone. The bone bag worm non-fibrous coating has the characteristic of exogenous property, so the bone bag worm can expand and spread in the direction of small resistance, firstly grows along the halos tube and the marrow cavity towards the epiphyseal plate and the joint cartilage, and can form secondary bag worm cyst in surrounding soft tissues if the bone bag worm or the joint cartilage is penetrated and broken into the bone bag worm or the joint cartilage and is subjected to pathological fracture or dislocation.
At present, the bone echinococcosis is mainly treated by surgical excision and is assisted by combined chemical medicine treatment, parasitic small bags in bone tissues are different in shape and not concentrated in distribution, a bone defect area caused in the excision process contains more small-angle gaps and miniature cavities, the phenomenon is especially close to articular cartilage, the bone defect repair of the disease has great technical difficulty,
in addition, in the aspect of chemical drug treatment, dihydroartemisinin is one of the first-choice drugs for resisting echinococcosis, preoperative and postoperative application of dihydroartemisinin to kill the echinococcosis can prevent postoperative sowing and postoperative recurrence, but the average cure rate of echinococcosis is only 30% when using dihydroartemisinin tablets, the situation has a great relationship with the factors that the drugs are difficult to enter into the bone echinococcosis, the concentration of the drugs in the bag is extremely low, and the like, and the disease recurrence rate after the surgical operation is used for treating bone echinococcosis is still higher.
Therefore, from two directions of surgery and drug treatment, how to achieve good repair of bone defects of echinococcosis and solve recurrence of the bone defects is a problem to be solved urgently.
(Shao Jun, wang Zhixin, li Yanfei, et al, 1975-2015, medical review of the conditions of diagnosis and treatment of bone echinococcosis in China [ J ]. Hua Xi, 2018, 33 (9): 4.DOI: 10.7507/1002-0179.201611044.)
Disclosure of Invention
In order to solve the technical problems, the invention provides a bone cell gel culture method, which optimizes the culture step of BMSC in gel, obtains cell bundle viscous gel and coats the cell bundle viscous gel to a hydroxyapatite filling main body; the dihydroartemisinin is added into cell bundle viscous gel formed by polydopamine-silk fibroin with bone marrow mesenchymal cells adhered on the surface to serve as a therapeutic drug, so that the recurrence of the bone echinococcosis is prevented by slow release, and good repairing and prognosis improving effects are realized.
The technical scheme of the invention is as follows:
a bone cell gel culture method, comprising the steps of:
(1) Taking bone marrow liquid, centrifuging, removing fat and supernatant to obtain bone marrow mesenchymal stem cell precipitate
(2) Placing the bone marrow mesenchymal stem cell sediment in a non-gel state culture medium, and culturing to obtain a cell suspension;
(3) Placing the cell suspension in a polydopamine gel membrane for co-culture until the confluence of bone marrow mesenchymal stem cells in the polydopamine gel is more than 80%, obtaining a gel cell bundle, and adding 5wt% of silk fibroin and dihydroartemisinin (the use concentration is 0.8-1.2 mg/L) to enable the gel cell bundle to be in a viscous state, thus obtaining a cell bundle viscous gel;
further, the non-gel state culture medium is a DMEM culture medium.
Further, in (3), the conditions under which the cell suspension is co-cultured in the polydopamine gel membrane are: DMEM+50. Mu.g/mL gentamicin medium, 37 ℃, humidity 100%, 5% CO 2 Is cultured in the incubator for 7 days, and the liquid is changed every 2 days.
Further, in the step (3), the viscous state is that the adhesive energy of the cell bundle viscous gel is 45-65J/m when the relative humidity of the cell bundle viscous gel is 40% -60% 2 。
Further, the molecular weight of the silk fibroin is 10000-16000.
Further, the method comprises3) Wherein the cell bundle viscous gel contains bone marrow mesenchymal stem cells 1×10 5 ～1×10 7 And each ml.
The invention also provides application of the cell bundle viscous gel in bone echinococcosis bone defect.
The application comprises the steps of constructing a defect model according to a bone defect area, using hydroxyapatite and polycaprolactone as printing materials, using 3D printing to obtain a hydroxyapatite filling body with a shape suitable for the defect model, adhering cell bundle viscous gel to the hydroxyapatite filling body to obtain a bone repair function module, filling the bone repair function module into the bone defect area of the bone echinococcosis, and having dual metabolic functions of bone formation and bone absorption, and vascular and nerve generation promotion functions.
Further, the hydroxyapatite filling body comprises at least one of autologous/allogeneic skeleton, nano hydroxyapatite skeleton and mineralized bone organ.
Further, the 3D printing is driven by air pressure, the 3D printing temperature is 5-88 ℃, and the 3D printing speed is 3-8 mm/s.
The invention has the beneficial effects that:
the invention provides a bone cell gel culture method, which is used for successfully culturing bone marrow mesenchymal stem cells under a polydopamine gel system, taking polydopamine and silk fibroin as gel matrixes, constructing cell bundle viscous gel with excellent adhesion performance, integrating the bone marrow mesenchymal stem cells with gel materials to form a preliminary osteogenic differentiation bone repair material, overcoming the technical difficulty that high-density gel is difficult to load bone marrow mesenchymal stem cell suspension, and realizing good cell culture effect.
Aiming at the echinococcosis, the dihydroartemisinin is used as a slow-release medicine of the gel, and is gradually released through gradual degradation of the gel to play a role in bones, so that the recurrence probability of the echinococcosis is obviously reduced, and the prognosis effect is improved.
The bone repair functional module has the dual functions of a bone repair filling main body and a gap filling agent, reduces the early stress shielding effect existing after the bone defect repair material is implanted, further promotes early osseointegration compared with the direct implantation of a bone-like hard tissue module (autologous/allogenic bone), and brings the effect into play along with the decomposition of viscous gel on the surface of the bone repair functional module, and the bone marrow mesenchymal stem cells loaded are subjected to osteogenic differentiation, so that bone healing is effectively promoted.
In addition, the polydopamine and silk fibroin provided by the invention have good mechanical properties, can be used for repairing bone defects in a large range, have excellent biocompatibility, can be degraded and absorbed by human bodies, can be degraded into amino acids, is nontoxic to the body, and can be helpful for accelerating the repair of bone defect positions; the catechol group of polydopamine remarkably improves the fusion degree of homosilk fibroin in a gel matrix, can realize instant viscose viscosity, is well fixed on the surface of a hydroxyapatite filling main body, has certain osteoinductive property, can promote proliferation and differentiation of bone marrow mesenchymal stem cells, and is beneficial to regeneration and repair of bones.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a SEM morphological characterization of a cell bundle viscous gel (500 μm);
FIG. 2 is a SEM morphological characterization of a cell bundle viscous gel (1 μm);
FIG. 3 is a CT image of rabbit bone of example 2, wherein a is a 3D model of bone defect obtained by Micro-CT scanning, b is a CT image of BRF group, c is a CT image of Control group, and D is a CT image of bracket group;
FIG. 4 is a HE staining microscope image of bone tissue sections of each group of example 2;
FIG. 5 is a HE staining microscope image of bone tissue sections of each group of example 2;
FIG. 6 is BMSC culture effect comparative data of example 1 and comparative example 1;
FIG. 7 is a comparative drug release profile of example 3.
Detailed Description
The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
The invention aims to successfully culture BMSC in a gel system and construct gel which can adhere to the surface of a filling skeleton, and is applied to bone repair of bone defect micro-cavities, bone cells (including osteoprogenitor cells, preosteoblast cells, osteoblast cells, bone lining cells or bone marrow mesenchymal stem cells) are loaded in the gel, bone differentiation and regeneration repair are carried out between the filling skeleton and the defective bone, in the process, bone defect areas caused by the bone echinococcosis contain more small-angle gaps and micro-cavities, the inventor sequentially tries to construct finer bone filling materials, including self-filled semi-fluid gel, but the material is not beneficial to bone healing and is greatly influenced by gravity, the gel loaded with bone cells is used as biological ink under the demonstration of 3D printing bone repair materials, and one of the feasible methods is provided for printing and preparing the bone filling materials.
Example 1
Preparation of polydopamine gel film (PDA): 200ml of Tris buffer solution with pH=8.5 is added with 0.5g of dopamine monomer powder, 2ml of glycerol is added, and the mixture is stirred for 12 hours in a dark place to form microcosmic polydopamine, and the microcosmic polydopamine gel film is obtained through filtration.
Preparation of Silk Fibroin (SF) solution: placing soluble lyophilized silk fibroin (sterile) in water solution, slowly dissolving, precipitating trace amountFloc, collecting supernatant, adding 0.1mol/L Ca (NO) 3 ) 2 Stirring for 60min, degrading silk fibroin molecular weight, loading into dialysis bag with cut-off molecular weight of 10000, hanging, oven drying at low temperature for 12 hr, and diluting concentrated silk fibroin to obtain 5wt% silk fibroin solution.
The culture method of the bone marrow mesenchymal stem cells comprises the following steps: 3 weeks SD rat, neck-broken femur, PBS to remove redundant muscle tissue, 10% FBS,1% P/S culture medium to blow bone marrow, centrifuging to separate fat and supernatant to obtain BMSC precipitate, culturing in DMEM culture medium, transferring into polydopamine gel membrane (0.1 g/mL polydopamine adhered DMEM+50 μg/mL gentamicin culture medium side wall, placing into BMSC cell suspension), co-culturing at 37deg.C under 100% humidity and 5% CO for 48 hr 2 The culture is carried out for 7 days, the liquid is changed every 2 days, and the culture is repeatedly carried out until the cell confluence reaches 80% -90%, and the culture is carried out until BMSC is third-generation for standby.
The preparation method of the cell bundle viscous gel comprises the following steps: adding 5wt% of silk fibroin solution into the poly-dopamine gel membrane co-cultured with BMSC, adding dihydroartemisinin according to the liquid amount to the final concentration of 1.2mg/L, and stirring at 50r/min until a crosslinked gel state is formed, thus obtaining the cell bundle viscous gel.
Constructing an animal model and repairing bone defects:
(1) Constructing a critical bone defect model (fretsaw cutting) from the femur to the radius diameter of 8mm of a 3 month old New Zealand white rabbit (2 kg plus or minus 0.3 kg) on the right side,
(2) Scanning rabbit bone defect areas by using Micro-CT, and processing CT scanning results by using Mimics software to establish a 3D model of the bone defect areas (shown in figure 3 a);
(3) Weighing 9g of polycaprolactone and 1g of nano hydroxyapatite, uniformly mixing, heating to 90 ℃, extruding by a screw rod to change the mixture into a mobile phase, and 3D printing according to a 3D model of a bone defect area to obtain a hydroxyapatite filling main body;
(4) And uniformly adhering the cell bundle viscous gel to hydroxyapatite to fill 1 mm+/-0.2 mm of the surface layer of the main body, and standing to ensure that the thickness of the cell bundle viscous gel is uniform everywhere by self-leveling, thereby obtaining the bone repair functional module.
(5) The bone repair function module is implanted into a bone defect area, cell bundle viscous gel is injected at a visible pore for refilling, the gel saturation of the defect area is ensured, and the effect of the implant in promoting segmental bone defect repair in a mouse body is evaluated.
Comparative example 1
This comparative example investigated the effect of polydopamine gel on BMSC culture passage.
The culture method of the bone marrow mesenchymal stem cells comprises the following steps: 3 weeks SD rat, femur cut, PBS remove redundant muscle tissue, 10% FBS,1% P/S culture medium blow bone marrow, centrifugate fat and supernatant to obtain BMSC precipitate, culturing in DMEM+50 μg/mL gentamycin culture medium at 37deg.C, humidity 100%, 5% CO 2 The culture is carried out for 7 days, the liquid is changed every 2 days, and the culture is repeatedly carried out until the cell confluence reaches 80% -90%, and the culture is carried out until BMSC is third-generation for standby.
The cell numbers of example 1 and comparative example 1 were separated by 24h, the culture dishes of five groups of parallel controls were air-dried, and the cell colony numbers were counted, and the result is shown in fig. 2, the culture effect of example 1 was similar to that of comparative example 1, and the cell numbers after 5 days were slightly more than that of comparative example 1, which indicates that co-culture of polydopamine gel film and BMSC did not affect proliferation and passage of cells, and probably because the adhesive effect of polydopamine gel film increased the viscosity of the culture medium, sedimentation of blood cell components such as erythrocytes and the like, which had a density heavier than that of mesenchymal stem cells, contained in bone marrow fluid was avoided, thereby being more favorable for adherence of mesenchymal stem cells.
Example 2
This example demonstrates the bone defect repair effect of the bone repair function module.
A blank group (Control group), a cell-free bone repair function module group (NC group), a bone repair function module group (BRF group) and a scaffold group (SF group) were prepared in the same manner as in example 1.
Blank defect group (Control group): the bone defect is not implanted with any framework material;
bone repair function module group (BRF group): filling the bone repair functional module prepared according to the method of example 1 into a bone defect area;
bracket group: filling the bone defect area with a mixture of hydroxyapatite and polycaprolactone;
cell-free bone repair function module group (NC group): the same procedure as in example 1, except for the cell culture step, the obtained cell bundle viscous gel without carrying cells is coated with a bone repair function module, and the bone repair function module is filled into a bone defect area;
after the model is built, the model is fed for 12 weeks in the same environment, 8mm end callus bridging of the BRF group can be observed through X rays (figure 3 b), the density of new bone is higher, a similar marrow cavity-like structure appears, bone healing is achieved, no continuous callus passes through the broken end of the bone defect of the Control group, the bone defect is still filled by fibrous scar tissue (figure 3 c), and BV/TV values of the rest groups are increased by 6 weeks except the Control group after 12 weeks. The Control group bone defects remained largely unhealed, while more new bone was created at the bone defect sites of the BRF and scaffold groups (fig. 3 d) and NC groups.
Further, it can be seen from the conventional HE-stained tissue section that the bone defect area is periostally adhered long and thin fibrous connective tissue, which contains erythrocytes and inflammatory cells, and the bone fracture end has no osteoblast phenomenon and no obvious osteoblast and neobone cell at 12 weeks after the Control group operation (FIGS. 4a and 4d, HE×20).
More inflammatory cells and erythrocytes are visible in the bone marrow cavities of the inner and outer bone plates of the scaffold group (fig. 5b and 5e, he x 20), fibrous connective tissue on both sides separates the bone scaffold from peripheral bone tissue, the intermediate clearance remains, new and old bone bonding lines in the section are obviously visible, and new bone edges are visible as yet uncalcified osteoids (fig. 4b and 4e, he x 40).
The bone repair function modules of the BRF group disappear from the boundary of peripheral bone tissue (fig. 5c and 5f, HE×20), the fusion degree is better, more unequal-sized cavities are formed in new bone, a large number of similar bone marrow cells are contained, a new lamellar structure is arranged in parallel with the defect surface, and the cell bundle viscous gel layer is fused with fibrous connective tissue, and is not limited (fig. 4c and 4f, HE×40).
The boundary between the implantation material of NC group and peripheral bone tissue is obvious, the fusion degree is poor, the bone formation at the boundary is less, the fibrous scab-like tissue is more, and the new blood vessel can be observed (fig. 5a and 5d, HE×40).
The bone repair function module is proved to be an excellent bone material, and is favorable for complete healing of bone defects of the bone echinococcosis.
Example 3
Characterization experiments of cell bundle viscous gels.
The same cell bundle viscous gel as in example 1 was studied for the degree of fusion of polydopamine with silk fibroin using the silk fibroin content as a single factor variable, and four groups of 1wt%, 3wt%, 5wt%, 7wt% silk fibroin and a control group were constructed around the gel density, viscous time, and degree of crosslinking.
| Group of | Gel matrix | Density/g/cm 2 | Viscous time/s | Degree of crosslinking/% |
| 1 | PDA | 0.32 | / | / |
| 2 | PDA+1wt%SF | 0.53 | 50s | 25.6% |
| 3 | PDA+3wt%SF | 1.3 | 35s | 28.1% |
| 4 | PDA+5wt%SF | 1.52 | 8s | 37.5% |
| 5 | PDA+7wt%SF | 1.67 | 7s | 39.4% |
It can be seen that when 5wt% of silk fibroin is added into polydopamine, the crosslinking time is shortest, a three-dimensional network of gel can be quickly constructed, damage to cultured cells caused by excessive stirring is prevented, and the preparation of cell bundle viscous gel is facilitated.
Freeze-drying cell bundle viscous gel, and characterizing the cross section through a scanning electron microscope, the fusion degree of polydopamine and silk fibroin is higher, the gel presents a uniform three-dimensional pore network structure, BMSC cells are maintained to be cultured in pore network gaps (figure 1), and the silk fibroin and polydopamine form a similar communicated tube wall structure from the view of a more microstructure, so that the cell proliferation and the slow release of medicines are facilitated (figure 2).
Cell bundle viscous gel adhesion performance characterization, namely, printing a nano hydroxyapatite plane through 3D, spherically forming cell bundle viscous gel with the diameter of 2mm, and bonding nano hydroxyapatiteThe plane of the limestone is fixed with a pulling wire rope in the ball, and the pulling force required by breaking the cell bundle viscous gel is tested by the pulling force, F= - (3/2) pi E A R，E A Is adhesive energy.
| Group of | Gel matrix | Adhesion energy | Relative humidity of |
| 1 | PDA | 25J/m 2 | 50% |
| 2 | PDA+1wt%SF | 28J/m 2 | 50% |
| 3 | PDA+3wt%SF | 31J/m 2 | 50% |
| 4 | PDA+5wt%SF | 65J/m 2 | 50% |
| 5 | PDA+7wt%SF | 58J/m 2 | 50% |
Therefore, the cell bundle viscous gel has excellent adhesion performance, is tightly combined with nano hydroxyapatite, can avoid gel sedimentation influenced by gravity when being implanted into a bone defect area, and can exert the effect to the greatest extent.
Drug slow release effect: the cell bundle viscous gel has better slow release effect on dihydroartemisinin, and provides good drug carrying effect in an in-vivo environment due to the multiple hydrogen bonding effect of catechol groups and dihydroartemisinin, and the cell bundle viscous gel is soaked in SBF solution, the drug content in the solution is detected for 12h, 48h, 4d and 7d, the release content ratio is calculated, and the silk fibroin content is used as a single factor to be compared with three groups (1 wt%, 5wt% and 7 wt%) according to the preparation method of the embodiment 1 respectively, as shown in fig. 7, the slow release effect of the cell bundle viscous gel formed by 5wt% of silk fibroin is optimal, and the drug effect can be expected to be exerted after 4 weeks.
Claims (10)
1. A method for culturing bone cell gel, comprising the steps of:
(1) Taking bone marrow liquid for centrifugal separation, removing fat and supernatant to obtain bone marrow mesenchymal stem cell sediment;
(2) Placing the bone marrow mesenchymal stem cell sediment in a non-gel state culture medium, and culturing to obtain a cell suspension;
(3) And (3) placing the cell suspension in a polydopamine gel membrane for co-culture until the confluence degree of bone marrow mesenchymal stem cells in the polydopamine gel is more than 80%, obtaining a gel cell bundle, and adding 5wt% of silk fibroin and dihydroartemisinin to enable the gel cell bundle to be in a viscous state, thus obtaining the cell bundle viscous gel.
2. Use of a cell bundle viscous gel in bone defects of echinococcosis.
3. The method of claim 1, wherein the non-gel medium is DMEM medium.
4. The method of claim 1, wherein in (3), the conditions for co-culturing the cell suspension with the polydopamine gel membrane are as follows: DMEM+50. Mu.g/mL gentamicin medium, 37 ℃, humidity 100%, 5% CO 2 Is cultured in the incubator for 7 days, and the liquid is changed every 2 days.
5. The method according to claim 1, wherein in (3), the viscous state is such that the adhesive energy of the cell bundle viscous gel is 25 to 65J/m when the relative humidity of the cell bundle viscous gel is 40 to 60% 2 。
6. The method for culturing bone cell gel according to claim 1, wherein the molecular weight of the silk fibroin is 10000-16000.
7. The method of claim 1, wherein in (3), the cell bundle viscous gel contains bone marrow mesenchymal stem cells 1X 10 5 ～1×10 7 And each ml.
8. The application of claim 2, wherein the application comprises: and constructing a defect model according to the bone defect area, preparing a hydroxyapatite filling body, adhering cell bundle viscous gel to the hydroxyapatite filling body to obtain a bone repair function module, and filling the bone repair function module into the bone defect area of the bone echinococcosis.
9. The use of claim 8, wherein the hydroxyapatite filler comprises at least one of autologous/allogeneic bone, a nano-hydroxyapatite skeleton, a mineralized bone organoid, the components of the nano-hydroxyapatite skeleton comprising polycaprolactone and nano-hydroxyapatite, the method of preparing the hydroxyapatite filler comprising: including one of 3D printing or autologous/allogeneic bone grinding shape adaptation.
10. The use according to claim 9, wherein the 3D printing is driven with air pressure, the 3D printing temperature is 5 ℃ to 88 ℃, and the 3D printing speed is 3 to 8mm/s.
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