CN115845142A - Preparation method of cartilage-like micro tissue for rapidly repairing jaw defects - Google Patents
Preparation method of cartilage-like micro tissue for rapidly repairing jaw defects Download PDFInfo
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Abstract
The invention provides a novel preparation method of cartilage-like micro-tissues for rapidly repairing defects of jawbones. The method sequentially comprises the following steps: s1) separating and culturing jaw bone membrane cells; s2) carrying out large-scale culture of periosteum cell micro-tissues, chondrogenesis induction and cell function identification by using the micropore array; and S3) transplanting the cartilage-like micro tissue into the jaw bone defect to start endochondral osteogenesis to repair the bone defect. The method is suitable for large-scale preparation of the micro-tissue which has the capability of starting bone formation in cartilage to repair the jaw defects after being transplanted into a body, can effectively promote the repair of large-area jaw defects, is simple, convenient and efficient, and has good development and application prospects.
Description
Technical Field
The invention relates to the fields of tissue engineering technology and cell biology, in particular to a preparation method of a cartilage-like micro tissue for rapidly repairing jaw defects.
Background
The jaw bone defect caused by trauma, tumor excision, infection and the like has high morbidity, and the important physiological functions of chewing, swallowing, maintaining the beauty and the like of a patient are influenced, so that the life quality of the patient is reduced. Although bone tissue has a certain regeneration ability, complete regeneration is difficult to achieve in the case of the above-mentioned critical bone defects. The current clinical gold standard is autologous bone graft, but its therapeutic effect is limited by the amount of donor tissue.
The unstable bone defect of jaw bone can be healed by means of endochondral osteogenesis, i.e. firstly, cartilage callus is formed at the bone defect part, and then, bone tissue is formed by remineralization. The large-area bone defect repair often faces the problems of tissue necrosis caused by lack of blood supply and oxygen in the defect center, and the cartilage is used as a non-vascular tissue and can resist a hypoxic environment. Thus, initiating endochondral osteogenesis at a bone defect site is an effective method for promoting repair of a bone tissue defect.
Currently, tissue engineering technology has shown more satisfactory efficacy as an emerging alternative therapy in the clinical transformation process. Tissue engineering technology integrates donor cells (seed cells) for tissue regeneration with cytokines for promoting the process and biomaterial scaffolds providing three-dimensional space, thereby promoting effective regeneration of tissues with poor regeneration capacity. The periosteum is a thin layer of connective tissue covering the surface of bone tissue, contains bone stem cells, is a main tissue source for forming cartilage callus at a bone defect part, and has good in vitro chondrogenesis capability. Thus, the jaw periosteal cells can be used as seed cells for initiating a tissue engineering strategy for the endochondral osteogenesis repair of jaw defects.
However, the step of transplanting seed cells into the body often faces the problem that the cells cultured in vitro cannot perform the function of directional differentiation after being transplanted into the body. Therefore, how to maintain the phenotype of in vitro cultured cells to realize stable clinical transformation application is a technical difficulty to be solved urgently.
Disclosure of Invention
The invention aims to provide a simple and efficient method for repairing jaw defects, and particularly relates to a method for preparing cartilage-like micro tissues for rapidly repairing jaw defects.
The method adopts an enzyme digestion method to separate the jaw bone membrane cells, uses a micropore culture plate to promote the bone membrane cells to aggregate to form a micro tissue, and starts cartilage differentiation. The cartilage-like microtissue is transplanted to the jaw defect part, and the endochondral osteogenesis way is started to repair the jaw defect. In one embodiment of the present invention, it was observed that the periosteal cell micro-tissue transplanted into the body is effective in forming cartilage callus and accelerating the healing of the jaw bone defect.
In order to achieve the above object, the present invention provides a method for preparing cartilage-like micro-tissue for rapidly repairing a jaw defect, which is characterized in that: the method comprises the following steps:
s1: separating and culturing jaw periosteum cells by enzyme digestion;
s2: culturing periosteum cell micro-tissues in a micro-pore array scale, inducing chondrogenesis and identifying cell functions;
s3: the transplantation of cartilage-like micro-tissue into the jaw defects initiates endochondral osteogenesis to repair the bone defects.
Preferably, the jaw periosteum cell separation culture method is a method for inducing cartilage-like microtissue formation by using an agarose micropore array culture plate, a jaw cavity defect construction operation method and a preparation method of a microtissue-containing graft.
Furthermore, in the step S1, the jaw bone periosteum cells are separated and cultured, and the digestion solution components, the digestion step and time and the primary cell inoculation density are combined.
Further, the agarose micro-well array plate induces cartilage-like micro-tissues in the step S2, and the plate with the micro-well array is copied by using agarose on the basis of the PDMS micro-column array template prepared by using a soft lithography method, and is embedded into a common cell culture well plate for use.
Furthermore, in the step S3, the jaw bone hole-shaped defect is constructed by an operation approach for stripping masseter muscle and a drilling operation method of a slow-speed mobile phone connected split drill.
Further, the microtissue graft is contained in the step S3, and prepared by collecting the cartilage-like microtissue by a blow-and-punch centrifugation method, a collagen gel coating method, and a fixation method of the graft to the defect site.
Specifically, in the scheme, the steps of separating the jaw bone periosteum cells by using an enzyme digestion method are as follows:
the neck skin of the mouse is cut open, the neck and face skin is stripped bluntly, and the muscle around the jaw bone of the mouse is continuously separated to free the jaw bone. The mandible was immersed in PBS buffer containing 100units/ml penicillin + 100. Mu.g/ml streptomycin and the surrounding muscles were removed by fine trimming. The remaining muscle was removed by wrapping the condyles with 5% low melting agarose and incubating in digest 1 (α MEM +3mg/ml collagenase type I +4mg/ml separating enzyme) for 20min at 37 ℃. The digestive juice 1 was discarded, and the jaw bone tissue was incubated in fresh digestive juice 2 (. Alpha.MEM +3mg/ml collagenase type I +4mg/ml separating enzyme) at 37 ℃ for 2 hours to digest the periosteal cells, thereby obtaining a periosteal cell suspension. Pass through a 70 μm cell sieve, centrifuge at 1000rpm for 5min and discard the digest, leave the cell pellet, resuspend in proliferation medium (α MEM +10% FBS +100units/ml penicillin +100 μ g/ml streptomycin +100 μ g/ml sodium pyruvate), inoculate in a T25 flask. Standing for 2 days, and changing the liquid every other day. The cells were passaged using 0.25% trypsin digestion at a cell density of about 80%. The third generation of periosteal cells was used for subsequent microtissue culture.
Specifically, the preparation method of the periosteum cell micro-tissue in the scheme is as follows:
the method firstly utilizes the soft lithography technology to prepare the PDSM negative film with the micro-column array, and the silicon wafer is firstly coated with SU-82075 photoresist by a spin coating method and is subjected to soft baking at the temperature of 95 ℃. Fixing a circular hole array mask with a certain diameter and a certain circle center interval on the surface of a silicon wafer coated with photoresist. And (3) carrying out vertical exposure crosslinking under 365nm ultraviolet light, then continuing to carry out exposure baking at 95 ℃, naturally cooling, and then soaking and dissolving the uncrosslinked photoresist by using ethyl acetate to form the micropore array template with a certain diameter and depth. After cleaning, the mixture is subjected to hard baking processing at 200 ℃ and then is naturally cooled. Then, uniformly mixing PDMS A glue and B glue, pouring the mixture on a micropore template, defoaming in vacuum, and solidifying at room temperature. And demolding to form the PDMS micro-column array template with certain diameter and height. After cleaning, 3% -5% agarose is heated and dissolved, poured into a PDMS template, cooled and demoulded, and the agarose micropore array culture plate can be obtained. The agarose block can be intercepted by a biological sample puncher and plugged into a cell culture pore plate (the diameter can be selected, and the agarose block corresponds to cell culture books with different sizes). The periosteum cells are inoculated in the culture medium according to the density of 50-250 cells/micropore, and each pore forms 1 microtissue with the diameter of 50-100 mu m.
The agarose cell culture micropore array plate can further use a puncher to intercept the micropore plate into the required cell culture pore plate size (such as a 24 pore plate, a 12 pore plate and the like) for subsequent cell culture.
Specifically, in the scheme, the method for inducing the periosteum cell micro-tissue into the cartilage in vitro comprises the following steps:
periosteum cell micro-tissue in chondrogenesis induction medium (alpha MEM +10% FBS +100units/ml penicillin + 100. Mu.g/ml streptomycin + 100. Mu.g/ml sodium pyruvate +50ug/ml ascorbic acid +50ug/ml L-proline +1/100ITS + 10) -7 M dexamethasone +10ng/ml TGF beta 1) for 48h. In one embodiment of the invention, the microtissue highly expresses Sox9, acan, etc. chondrogenic marker genes. The cartilage-like microtissue induced for 48h can be used for the repair of jaw defects.
Specifically, in the above scheme, the method for constructing the model for repairing the jaw defect by transplanting the cartilage-like micro-tissue comprises the following steps:
the skin is cut along the inferior mandibular edge, the masseter is bluntly peeled off and the masseter tissue is bluntly separated from the jaw surface, exposing the jaw facets. And drilling a hole with a split drill with the diameter of 1.5mm at the position 2mm above the most sunken part of the mandibular edge. After the cartilage-like microtissue was collected, it was wrapped in 5mg/ml type I collagen gel and transplanted into the hole-shaped defect. In one example of the invention, significant cartilage callus formation at the site of the bone defect was observed after 1 week and significant bone healing was observed after 2 weeks.
The invention also provides a cartilage-like micro-tissue collection method and a function identification method.
The technical principle of the invention is as follows:
in the process of endochondral osteogenesis, mesenchymal stem cells firstly aggregate and then start a subsequent differentiation process, which is a key step for starting endochondral osteogenesis. Therefore, the invention adopts a micropore culture plate, utilizes a bionic tissue engineering strategy to simulate the mesenchymal stem cell condensation process, forms a cartilage-like micro-tissue which can start osteogenesis in cartilage, and promotes defect repair.
The micro-tissue culture method utilizes the self-assembly performance of cells to enable the cells to spontaneously aggregate, effectively simulates the three-dimensional physiological state of the cells in vivo, and has good effects on promoting stem cell differentiation or maintaining the functions of adult cells. And the method does not need additional biological material assistance, and can realize high-throughput and large-scale application. Compared with two-dimensional culture, the three-dimensional culture of the micro-tissues can promote the expression of cell adhesion related molecules and the secretion of extracellular matrixes, and form a stable structure for tightly combining cells and the extracellular matrixes. In addition, the autocrine of cell factors (such as VEGF, TGF, BMP and the like) can be promoted, and the biological function of cells can be maintained. Because of the distance limitation of the infiltration of nutrients (such as oxygen) during in vitro culture, it is important to strictly control the number and diameter of cells contained in the microtissue. In order to facilitate large-scale transformation application, the micro-tissue culture technology requires a culture method with high assembly speed, stable form and stable biological effect, and needs to have the advantages of economy, convenience in operation and the like.
The invention fully utilizes the principle of bionic tissue engineering, introduces agarose which is a material with low price and high biocompatibility, combines with a conventional cell culture plate to manufacture a cell micropore array culture plate, and establishes a method for preparing the cartilage-like microstructure with high flux. The method is suitable for various cell types with self-assembly capability, is safe and efficient, and is economical and practical. The micro-tissue can simulate the condensation process of the mesenchymal stem cells, has good in vivo and in vitro chondrogenic differentiation capacity, can be transplanted into the jaw bone defect to start an in-cartilage osteogenesis mode to repair the bone defect, and can overcome the defect of unstable repair efficiency. The method has the advantages of good repairing effect, high healing speed and good development and application prospects.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention is an innovative technology, the diameter of the formed cartilage-like micro-tissue is smaller, and the problem of cell apoptosis caused by insufficient infiltration of nutrients such as oxygen and the like of central cells due to the larger diameter of the micro-tissue (such as RU 2019134905) can be avoided; meanwhile, the invention can be used for simultaneously producing a large amount of micro-tissues with controllable diameters, and compared with the method for enabling the cells to be self-clustered (such as CN 202220393104.3), the invention has more controllable micro-tissue size and biological effect; in addition, compared with the method for forming the cartilage-like micro tissue by centrifugation, the method has the advantage of high flux, can quickly induce a large amount of micro tissue with the chondrogenic capacity, and is favorable for realizing large-scale application. The invention creatively repairs the jaw bone defect by starting the endochondral osteogenesis mode, and has the characteristics of high repair speed and stable repair effect compared with the modes of directly injecting cells, using osteoconductive biomaterial scaffold to carry cells and the like. The method has the following specific advantages:
1. the invention utilizes the self-made agarose micropore culture plate, can be directly matched with the traditional cell culture plate for use, can realize the high-flux preparation of a large amount of micro-tissues which accord with the target size, and has the advantages of convenience and high efficiency.
2. The invention has stable and controllable size of the micro-tissue, is beneficial to oxygen infiltration, and has good cell activity and stable biological effect.
3. The invention can separate and culture the jaw bone periosteum cells, induce cartilage differentiation through a micro tissue culture mode, and then transplant the cartilage into jaw defects, can start the osteogenesis process in the cartilage to repair large-area bone defects of the jaw, and has the advantages of stable repair effect and high repair speed.
Drawings
FIG. 1 shows the lower view of a jaw periosteum cell mirror in isolated culture.
FIG. 2 is a flow cytometry detection of jaw bone periosteum cell common mesenchymal stem cell surface marker expression;
wherein: CD105 is (A), (CD 90 is (B), and CD29 is (C).
FIG. 3 is a schematic diagram of the operation of the soft lithography method for preparing the agarose micropore array culture plate.
FIG. 4 is a schematic diagram of a method of embedding a microwell array module containing agarose into a conventional cell culture well plate.
Wherein: (A) A schematic punching diagram of an agarose micropore array module which is manufactured by copying from a PDMS micropore array mould; (B) Schematic representation of the embedding of punch-punched agarose into cell culture well plates.
FIG. 5 is a schematic diagram of a culture plate of agarose micropore array for culturing jaw bone membrane cells;
wherein: (A) is the microscopic view of the agarose micropore culture plate; (B) microscopic observation 48 hours after the formation of the microtissue; (C) And (D) the expression of the chondrogenesis-associated marker gene 48 hours after chondrogenesis induction.
FIG. 6 is an intraoperative photograph of a jaw cavity defect build and a general view after the build is successful;
wherein: (A) is an intraoperative photograph; and (B) is a general view.
FIG. 7 is a process of healing a jaw defect after transplantation of cartilage-like micro-tissue;
wherein: (A) And (B) are micro CT three-dimensional reconstruction pictures after 7d and 14d of healing respectively; (C) staining the section specimen with Alcian Blue at 7d after healing; (D) Masson Trichrome staining was done on sections at 14d post-healing.
Detailed Description
The following embodiments are incorporated herein. The invention is further illustrated and it is understood that these examples are intended only to illustrate the invention and are not intended to limit the scope of the invention as claimed, and that the particular experimental conditions and methods not specified in the following examples are generally in accordance with conventional conditions such as: lan Freshney, science press, seventh edition, animal cell culture; osbo et al, scientific press, 5 th edition, fine compiled molecular biology laboratory guidelines; qinchuan et al, national institutes of health, 3 rd edition, books on experimental zoology in medicine, etc., or according to the manufacturer's recommendations.
Example 1 isolation, culture and characterization of mouse jaw periosteal cells
1. Isolation of mouse jaw bone tissue
Taking 4C 57/BL6 mice, killing the mice after neck breaking, soaking the mice in 75% alcohol for 2min, and taking out the mice. The neck skin was cut from the midline of the neck and free facial skin was bluntly isolated upward. The mandible is cut off from the median joint, and the muscles of the mouth bottom and the tongue are cut off from the inner side and the lower edge of the mandible. The masseter muscle is cut from the outside of the mandible, and the other connected muscle tissues are cut to free the mandible. The mandible was immersed in PBS buffer containing 100units/ml penicillin + 100. Mu.g/ml streptomycin and placed on ice.
The remaining muscle in the mandible was trimmed finely with micro-scissors (to avoid tearing the bony matrix) until only a thin layer of muscle tissue remained in the mandible. A total of 8 mandibles were obtained.
2. Digesting to obtain periosteum cell suspension
The mandibular condyle obtained in step 1 was dipped in a small amount of agarose gel by heating 5% low melting agarose (Biofrox corporation) to dissolve, and formed a thin layer of coating on the mandibular condyle after cooling.
To 15ml of α MEM were added 3mg/ml collagenase type I (Biofrox Co.) and 4mg/ml separatory enzyme (Roche Co.) to prepare a digestion solution. 5ml of digestive juice is taken, the mandible wrapped with the condyles is placed in the digestive juice, the digestive juice is discarded after incubation for 20min at 37 ℃, and the residual muscle tissue is removed. 10ml of fresh digestive juice is added, the mixture is incubated for 2 hours at 37 ℃, and the mandible bone surface can be observed to be smooth without residual soft tissue after slight shaking. The mandible tissue was discarded and the digestive juice was retained.
The digest was filtered through a 70 μm cell sieve to remove tissue debris.
3. Periosteal cell culture
The periosteal cell suspension obtained in step 2 is centrifuged at 1000rpm for 5min, the supernatant is removed and resuspended by adding proliferation medium (. Alpha.MEM +10% FBS +100units/ml penicillin + 100. Mu.g/ml streptomycin + 100. Mu.g/ml sodium pyruvate). Inoculating into 2T 25 flasks, shaking, and then, 5% CO at 37 ℃ 2 Culturing in an incubator.
After standing for 2 days, the solution is changed, periosteum cell adherence can be observed under a microscope, and the cell shape is in a polygonal shape of the mesenchymal stem cell (figure 1). After 2 days, the cell density reached 80%. Adherent cells were digested with 0.25% trypsin (Gibco) for passage.
4. Identification of periosteal cell MSC marker by flow cytometry
The periosteal cells passaged to the third generation in step 3 were digested into cell suspension with trypsin at 2 × 10 6 The concentration of individual cells/ml was resuspended using PBS containing 10% FBS, and blocked for 10 minutes.
Using PBS containing 2% FBS, staining buffer containing fluorescent group-conjugated antibody (Biolegend) was prepared as follows.
50ul of cell suspension was added to each tube of staining buffer, mixed well and incubated on ice for 20min in the dark. 800ul of PBS containing 2% FBS was added, mixed, centrifuged at 200g for 5 minutes, the supernatant was discarded, and excess antibody was removed. The cell pellet was resuspended in 2% FBS in PBS, sieved through a 70 μm cell sieve, and loaded onto a machine for flow cytometry analysis.
Compensation adjustment was performed using a single positive tube, fluorescence intensity was gated using FMO control, and the proportion of cell populations expressing common mouse MSC surface markers CD105, CD29, CD90 in periosteal cells was examined, which showed that periosteal cells highly expressed the above markers (fig. 2), which contained a higher proportion of MSC populations.
Example 2 preparation of cartilage-like micro-tissue and chondrogenic Induction
1. Preparing PDMS micro-column array template
As shown in fig. 3, a silicon wafer is first cleaned with Piranha wet etching agent, then coated with SU-82075 photoresist with a thickness of about 150 μm by spin coating, and the photoresist-coated silicon wafer is baked at 95 ℃ for 20-30 minutes. A mask containing a hexagonal array of circular holes with a diameter of 200 μm and a circle center distance of 300 μm is fixed on the surface of a silicon wafer coated with photoresist. After the photoresist is vertically exposed and crosslinked under 365nm ultraviolet light, the photoresist is continuously baked for 10 to 12 minutes at 95 ℃ and then naturally cooled, and then ethyl acetate is used for soaking for 10 to 15 minutes to dissolve the uncrosslinked photoresist, so that the micropore array template with the diameter of 200 mu m and the depth of 150 mu m is formed. After being cleaned, the mixture is baked for 10 to 20 minutes at 200 ℃ and then naturally cooled. Then, mixing PDMS A glue and B glue according to the weight ratio of 10:1, uniformly mixing, pouring on a micropore template, defoaming in vacuum, and solidifying after 24 hours at room temperature. After demolding, a PDMS micro-column array template with a diameter of 200 μm and a height of 150 μm is formed.
2. Preparation of agarose micropore array culture plate
And (2) ultrasonically cleaning the PDMS micro-column array template obtained in the step (1), heating and dissolving 4% agarose in a super clean bench, pouring the agarose into the PDMS template, cooling, and demolding to obtain the agarose micro-pore array culture plate (A in the picture 4). A biological sample puncher can be used cooperatively to cut out agarose blocks with the diameter of 16mm, and the agarose blocks are plugged into a 24-hole cell culture plate (the diameter can be selected, and the cell culture plate can correspond to cell culture plates with different sizes) (B in figure 4). The prepared agarose micropore array culture plate can be soaked in PBS sterile buffer solution containing 100units/ml penicillin and 100 mu g/ml streptomycin for storage.
3. Inoculating periosteal cells
Adherent periosteal cells were digested into single cell suspension using 0.25% trypsin, and after counting, the periosteal cells were seeded at a density of 200 cells/microwell in the agarose microwell array plate prepared in step 2 (a in fig. 5). Each pore in the micropores forms 1 microstructure (B in FIG. 5) having a diameter of about 80 μm.
4. Chondrogenic induction
Chondrogenic induction medium (α MEM +10% FBS +100units/ml penicillin +100 μ g/ml streptomycin +100 μ g/ml sodium pyruvate +50ug/ml ascorbic acid +50ug/ml L-proline +1/100ITS +10 + -7 M dexamethasone +10ng/ml TGF β 1) chondrogenic induction of periosteal cell microtissue for 48h and normal adherent culture of periosteal cells was used as control.
5. Collecting osteochondral-like micro-tissue
And after 48h, repeatedly and gently blowing and beating the cartilage-like micro tissue in the pore plate in the step 4 by using a pipette gun, sucking out and collecting the cartilage-like micro tissue into a centrifuge tube, adding PBS into the pore plate, blowing and beating for a plurality of times again, and repeating for 2 times or more until no micro tissue is observed in all the micro holes under a light microscope. Centrifugation was carried out at 1000rpm for 5 minutes, the supernatant was discarded to remove the medium, resuspended in PBS and centrifuged again, and the microtissue washing was repeated 2 times.
The microtissue pellets were added with 500ul of a GTC buffer containing β -mercaptoethanol, total RNA of the microtissue was extracted using a total RNA extraction kit (Omega Co.), and the expression of cartilage marker genes Sox9 and Acan was detected using qPCR using adherent cells as a control. The results show that microtissue has stronger expression of Sox9 and Acan compared to two-dimensional adherent cells (C in fig. 5, D in fig. 5).
EXAMPLE 3 transplantation of cartilage-like microtissues to repair jaw defects
1. Collecting cartilage-like microtissue
The collection procedure was the same as that used for the collection of cartilage-like microtissue in step 5 of example 2.
2. Preparation of gel containing cartilage-like micro-tissue
A5 mg/ml type I collagen gel was prepared according to the manufacturer's instructions. 100ul of 5mg/ml type I mouse tail collagen (Solarbio Co.) was added to 6ul of 0.1M NaOH, mixed well, and 11.5ul of 10xPBS was added. After mixing well, the mixture was added to a cell pellet containing 2500 cartilage-like micro-tissues. Blowing, beating and mixing evenly, and placing on ice for later use.
3. Establishing jaw bone defect model transplanted cartilage-like micro-tissue
C57BL/6 mice were anesthetized intraperitoneally with 75mg/kg of suetamol and 10mg/kg of thiamethoxam hydrochloride. After the induction of anesthesia was successful, the skin was prepared on the face and neck of the mice. Shaving the face and neck surgical area, and sterilizing with iodine. The skin was incised along the inferior border of the mandible, the masseter was exposed, the masseter was suspended blunt, and the local masseter was lifted up along the lateral facet of the mandible using a periosteal elevator. A dental slow-speed hand machine is used for connecting a split drill with the diameter of 1.5mm to drill a hole 2mm above the depression of the front edge of the mandibular angle, and the drill is stopped after a falling feeling occurs, so that soft tissues on the inner side surface of the jaw bone are prevented from being damaged (A in fig. 6 and B in fig. 6).
And (3) removing broken bone fragments by using gauze, fully stopping bleeding, sucking 5ul of the collagen gel containing the micro-tissues on the ice in the step (2) by using a liquid transfer gun, and then injecting the collagen gel into the defect. Allowing the gel to stand at the defect part for 10min, and allowing the gel to gradually solidify due to the increase of temperature.
The peeled masseter muscle is reset, and the masseter muscle is suspended and sutured by absorbable suture, and the skin is sutured. The wound is cleaned.
2.5mg/kg of flunixin meglumine is given after the operation for 5 days of anti-inflammation, and 5mg/kg of ceftiofur sodium and 15mg/kg of enrofloxacin are combined for anti-infection for 7 days.
4. Detecting bone defect repair
Jaw bone tissue was dissected at 7d post-surgery, no significant bone healing was observed (a in fig. 7), decalcification, section embedding, and alcain Blue staining revealed extensive cartilage callus formation after transplantation of cartilage-like micro-tissue (C in fig. 7).
Jaw bone tissue was dissected at 14d after surgery and a large amount of bone tissue was observed by scanning micro CT (B in fig. 7). Sections were embedded after decalcification and bone tissue formation was observed by Masson Trichrome staining (D in FIG. 7).
Claims (6)
1. A preparation method of cartilage-like microtissue for rapidly repairing jaw bone defect is characterized in that: the method comprises the following steps:
s1: separating and culturing jaw periosteum cells by enzyme digestion;
s2: culturing periosteum cell micro-tissues in a micro-pore array scale, inducing chondrogenesis and identifying cell functions;
s3: transplantation of cartilage-like micro-tissue into a jaw defect initiates endochondral osteogenesis to repair the bone defect.
2. The method for preparing cartilage-like micro-tissue for rapid repair of a jaw defect according to claim 1, wherein:
the jaw periosteum cell separation and culture method uses an agarose micropore array culture plate to induce cartilage-like micro-tissue formation, a jaw cavity defect construction operation method and a preparation method of a transplant containing micro-tissue.
3. The method for preparing cartilage-like micro-tissue for rapidly repairing a jaw defect according to claim 1 or 2, wherein: in the step S1, the jaw bone periosteum cells are separated and cultured, and the digestion solution components, the digestion step and time and the primary cell inoculation density are combined.
4. The method for preparing cartilage-like micro-tissue for rapidly repairing a jaw defect according to claim 3, wherein: and (3) inducing the cartilage-like micro tissue by using the agarose micropore array culture plate in the step (S2), copying the culture plate with the micropore array by using agarose on the basis of the PDMS micro-column array template prepared by using a soft lithography method, and embedding the culture plate into a common cell culture pore plate for use.
5. The method for preparing cartilage-like micro-tissue for rapid repair of a jaw defect according to claim 1, 2 or 4, wherein: and in the step S3, the jaw bone hole-shaped defect is constructed by an operation access for stripping masseter muscle and a drilling operation method of connecting a slow-speed mobile phone with a split drill.
6. The method for preparing cartilage-like micro-tissue for rapidly repairing a jaw defect according to claim 5, wherein: the step S3 includes a microtissue graft, and is prepared by collecting cartilage-like microtissue by a blow-and-punch centrifugation method, a collagen gel coating method, and a fixation method of transplanting the microtissue graft to a defect site.
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