CN103638558B - In vitro construction method for bionic ligament-bone tissue engineering connector - Google Patents

Abstract

The invention belongs to the technical field of interface tissue engineering, and relates to an in vitro construction method of a bionic ligament-bone connector. The technical problem to be solved by the present invention is to provide a new option for reconstructing the continuous ligament-bone interface between the ligament-bone interface and the heterogeneous transitional structure after ACL injury. The technical solution of the present invention is an in vitro construction method of a bionic ligament-bone connector, comprising the following steps: a, preparation of decellularized Achilles tendon; b, preparation of seed cells: including preparation of fibroblasts, chondrocytes, and osteoblasts ; c, planting. The invention provides a new strategy for tissue engineering regeneration of anterior cruciate ligament injury.

Description

In Vitro Construction Method of Biomimetic Ligament-Bone Tissue Engineering Connector

technical field

The invention belongs to the technical field of interface tissue engineering, and relates to an in vitro construction method of a bionic ligament-bone connector.

Background technique

The anterior cruciate ligament (ACL) is an important structure to maintain the stability and function of the knee joint. However, the anatomical characteristics of cell distribution and poor blood supply determine that the cruciate ligament is difficult to heal itself after injury, leading to joint instability, cartilage damage, osteoarthritis and other complications, which seriously affect the function of the knee joint. Simon M et al. conducted an epidemiological study on knee ligament injuries in their country from January 2000 to June 2005. The results showed that 80% of knee ligament surgeries were related to ACL injuries. The mechanism of ACL injuries can be roughly divided into two types: Both indirect injuries caused by valgus stress and non-contact injuries caused by, for example, bone injuries.

The commonly used mainstream treatment strategy for ACL injury is autologous bone-patellar tendon-bone and hamstring tendon transplantation. However, the original bone-patellar tendon-bone and hamstring tendon are damaged (or sacrificed) due to autologous tissue transplantation. In recent years, allografts have been used to treat ACL injuries, but their sources are limited and expensive, and the long-term efficacy is not satisfactory, with a failure rate of 10%-25%. In the revision of failed cases, it was found that the reconstruction structure of the hamstring tendon was biased towards fibrous features, while the reconstruction structure of the patellar tendon was biased towards cartilaginous features. The reasons for the failure are mostly attributed to the poor healing caused by the lack of good blood supply between the normal ACL-bone fibrocartilage in the reconstructed structure; or the "wiper effect" that enlarges the bone tunnel and causes the graft to wear and loosen or even break. Another study observed the histological changes of the tendon-bone tunnel interface after reconstruction: scar tissue filled the gap at 2 weeks, evolved into dense connective tissue after 1 month, and distributed a large amount of type I collagen after 6 weeks. Significant new bone and cartilage formation was found.

The study found that there is a complex, continuous distribution of transition zone (continuous gradients layers), or transition complex (heterogeneous transition complex): from superficial to deep structure and its corresponding The composition is as follows: fibrous layer (fibroblasts and type I collagen), non-calcified fibrocartilage (oval chondrocytes and type II collagen), calcified fibrocartilage (hypertrophic chondrocytes and type X collagen), and subchondral bone. That is, the gradient distribution of heterogeneous cells, extracellular matrix, and mineralization degree. This buffer structure can redistribute complex loads and strains between different types of tissues, disperse shear, avoid stress concentration, and ensure ligament-cartilage Anatomically complete and mechanically stable inferior bony connections. Studies by Kurosaka M et al. have shown that the mechanical stability of ACL grafts will be greatly affected if there is no structurally gradual fibrocartilage transition zone formation and proper tissue integration between the surgical reconstruction graft and the bone tunnel.

According to the above discussion, the biological goal of ACL injury reconstruction should be to reconstruct the continuous ligament-bone interface with heterogeneous transitional structure of the ligament-bone connector.

Contents of the invention

The technical problem to be solved by the present invention is to provide a new option for reconstructing the continuous ligament-bone interface between the ligament-bone interface and the heterogeneous transitional structure after ACL injury.

The technical solution of the present invention is a method for constructing a bionic ligament-bone connector in vitro, comprising the following steps:

a. Preparation of decellularized Achilles tendon;

b. Preparation of seed cells: including preparation and gene transfection of fibroblasts, Sox-9 gene-enhanced chondrocytes, and Runx-2 gene-enhanced osteoblasts;

c. Implantation: The decellularized Achilles tendon is divided into three structural and functional areas according to the longitudinal and transverse axes of the fibroblast FB-chondrogenic CH-osteogenetic OS, and the fibroblasts and Runx-2 genes are enhanced Osteoblasts and Sox-9 gene-enhanced chondrocytes were planted in layers and divided into fibroblasts, chondrocytes, and osteoblasts, cultured for 5 to 7 days after histological verification, and then cultured for 1 week for use; The layered planting described above refers to the transplantation from the surface of the Achilles tendon to the center of the cross-section, so that the fibroblasts, the osteoblasts enhanced by the Runx-2 gene, and the chondrocytes enhanced by the Sox-9 gene are located in the inner, middle, and outer parts of the cross-section respectively. 1/3 area.

Wherein, the decellularized Achilles tendon scaffold in step a is derived from the Achilles tendon tissue of rabbits, dogs, and cattle.

Wherein, the preparation of osteoblasts in step b includes the following steps: constructing an adenovirus vector expressing early osteogenic marker RUNX-2, and transfecting osteoblasts.

Wherein, the preparation of chondrocytes in step b includes the following steps: constructing an adenovirus vector expressing early cartilage marker SOX-9, transfecting chondrocytes, and then dissolving in collagen hydrogel.

Wherein, the preparation of the fibroblasts in step b includes the following steps: dissolving the fibroblasts in the collagen hydrogel.

Wherein, before planting in step c, the osteogenic segment of the decellularized Achilles tendon is treated with recombinant fibronectin/cadherin rFN/CDH.

The present invention also provides the tissue engineering cell-tendon complex prepared by the above method.

Beneficial effects of the present invention:

The present invention adopts a decellularization method based on tissues and organs and a co-cultivation strategy of cell scaffolds. The decellularization method not only effectively removes the cells in the Achilles tendon but also retains the general shape of the scaffold. Moreover, the microstructure of the obtained scaffold is obvious, and the pore diameter and porosity are obviously increased, which is beneficial to the proliferation and endotropic growth of cells. The present invention has high potential and application prospect in inducing tissue engineered tendon to differentiate in the direction of fiber, cartilage and bone tissue in a targeted manner, and then forming a "transition structure".

Description of drawings

Figure 1 shows the preparation of decellularized rabbit Achilles tendon scaffold

(a,b) The general morphology of Achilles tendon before and after decellularization;

(c, e, d, f) H&E staining (hematoxylin-eosin staining) of Achilles tendon before and after decellularization (×100);

(g, i, h, j) SEM (scanning electron microscope) scanning electron microscope results of transverse and longitudinal sections of Achilles tendon before and after decellularization (×1000 times).

Figure 2 shows the schematic diagram of gene-enhanced adenovirus vector construction and transfection efficiency

(a) Schematic diagram of the construction of sox-9 and runx-2 high-expression adenoviral vectors;

(b) DNA electrophoresis showed that the pAdtrack-CMV-GFP-sox-9/RunX-2 shuttle plasmid was successfully constructed;

(c) DNA electrophoresis showed that the pAdEasy-1-sox-9/RunX-2 plasmid was successfully constructed;

(d, f) Efficient transfection of pAdEasy-1-sox-9 plasmid to chondrocytes;

(e,g) Efficient transfection of osteoblasts with pAdEasy-1-RunX-2.

Figure 3 shows the principle of in vitro construction of "migration structured" engineered tendon, detection of cytocompatibility of decellularized scaffold, preparation of collagen hydrogel and rFN/CDH

(a) Schematic diagram of the in vitro construction of "migrating structured" engineered tendon;

(,b) Functional division of "transition structured" engineered tendon: FB is the fibroblast area, CH is the chondrogenic area, and OS is the osteogenic area;

(c) Appearance of the collagen hydrogel-cell mixture before and after gel curing;

(d) Cytocompatibility detection of decellularized scaffolds by MTT method, the abscissa represents the culture time (days), and the ordinate represents the optical density (OD) value (control and scaffold represent the tendon tissue before and after decellularization, respectively.)

(e) XPS (X photoelectron spectroscopy analysis) confirmed that rFN/CDH was successfully cross-linked with the surface of the decellularized tendon scaffold;

(f) SEM confirmed the successful adhesion of cells on the surface of scaffolds mediated by rFN/CDH.

Figure 4 shows the statistical chart of the gene protein expression of the corresponding markers in tissue engineered tendon in each group at different periods (*P<0.05, ﹟P<0.01)

(a) mRNA expression of RunX2 at day 5 and osteogenic markers (OCN, OPN and BSP) at day 10 in each experimental group;

(b) The mRNA expression of sox-9 and cartilage markers (COL2A1, COMP and Aggrecan) in each experimental group on day 5 and day 10;

(c) Protein expression of osteogenic markers (OCN, OPN and BSP) and cartilage markers (COL2A1, COMP and Aggrecan) in each experimental group at 2 weeks.

Group Ⅰ, pure decellularized scaffold group; group Ⅱ, decellularized scaffold+fibroblast+chondrocyte+osteoblast group; group Ⅲ, decellularized scaffold+fibroblast+SOX-9 gene enhanced chondrocyte+osteoblast Group; group Ⅳ, acellular scaffold + fibroblasts + chondrocytes + RUNX-2 gene enhanced osteoblast group; group Ⅴ, acellular scaffold + fibroblasts + SOX-9 gene enhanced chondrocytes + RUNX-2 gene enhanced Osteoblast group.

Figure 5 shows specific staining of bone/cartilage/fibrous tissue (transverse section, 200 times): (a-e) Alizarin red staining analysis of OS segment of tissue engineered tendon: representing groups Ⅰ-Ⅴ (specifically the same as in Figure 4c); (f-j) tissue Alician blue staining of CH segment of engineered tendon: representing groups Ⅰ-Ⅴ (see Figure 4c for details); (k-o) H&E staining analysis of FB segment of tissue engineered tendon, representing groups Ⅰ-Ⅴ (see Figure 4c for details).

Figure 6 shows the immunofluorescence analysis of bone/cartilage markers (transverse section, 400 times): (a-e) Osteocalcin immunofluorescence in the OS segment, representing groups Ⅰ-Ⅴ (specifically the same as Figure 4c)); type II collagen in the CH segment Immunofluorescence, representing groups Ⅰ-Ⅴ (specifically the same as in Figure 4c)).

Figure 7 shows the results of osteogenesis and mineralization of the OS segment of the tissue engineered tendon: (a-e) The distribution of mineralized nodules in the OS segment, positive findings were found only in groups Ⅱ, Ⅳ, and Ⅴ. The arrows indicate the spherical distribution of calcium and phosphorus on the surface of the OS segment Tissue-engineered tendons; (f-j) relative content percentages of C, N, O, Ca, and P: respectively represent groups Ⅰ-Ⅴ (see Figure 4c for details). The abscissa in the figure represents the electron volts of the elements, and the ordinate represents the relative content.

Detailed ways

The tissue-engineered cell-tendon tissue-engineered tendon of the present invention uses decellularized Achilles tendon as a scaffold, simulates the fiber-cartilage-bone physiological structure of the anterior cruciate ligament-bone junction, and forms fibroblasts, osteoblasts and chondrocytes.

The construction process of the present invention is described in detail below in conjunction with embodiment:

Example 1 Preparation of Decellularized Rabbit Achilles Tendon Scaffold

(1) Steps: Aseptically cut out 5-6 cm of the Achilles tendon of rabbit limbs, mechanically remove the attached tissue, and wash repeatedly with sterile PBS. Freeze in a deep low-temperature refrigerator for 10 minutes (-70°C) and then place it in a constant temperature water bath for 10 minutes (37°C). Repeat this for 5 times. After fully rinsing with PBS, perform the following treatments in turn at 37°C: shake with 0.5% trypsin for 2 hours, Fully rinse with PBS 3 times (30min/time) to remove residual trypsin, shake with 10% Triton X-100 for 12h, fully rinse with PBS 3 times (30min/time) to remove residual Triton X-100, 5% Triton X-100 Sodium dodecyl sulfonate was shaken for 2 hours, rinsed with PBS for 3 times (30 min/time) to remove residual sodium dodecyl sulfonate, and finally treated with 1× phosphate buffered saline, which was the desorption solution required for this experiment. Cell rabbit Achilles tendon scaffold. The appearance, hardness and elasticity of fresh Achilles tendon and decellularized Achilles tendon were observed respectively, and specimens were collected for histological and electron microscopic examination.

(2) Results: As shown in Figure 1, the Achilles tendon was hard before decellularization (a); after decellularization, the Achilles aponeurosis was complete, soft and tough (b); H&E staining: Achilles tendon before decellularization The inner cell nuclei were deeply stained blue and clearly visible (c, e, ×100 times); after decellularization, no cellular components were found in the Achilles tendon, and the collagen fibers were loosely arranged, and there were many pores (d, f, ×100 times) ;SEM: Electron microscope scanning results of the transverse and longitudinal sections of Achilles tendon before decellularization, it can be seen that the collagen fibers are densely arranged, with fewer pores (g,i,×1000 times); the results of electron microscope scanning of the transverse and longitudinal sections after decellularization, collagen fibers The arrangement is loose, and the three-dimensional network structure between the tendon bundles is obvious (h, j, ×1000 times).

Example 2 Detection of Cytocompatibility of Decellularized Scaffold

The cell proliferation rate and viability of fibroblasts on the surface of decellularized Achilles tendon were evaluated by CCK-8 method.

(1) Steps: First, soak the decellularized rabbit Achilles tendon scaffold in 70% ethanol for one day, then rinse with sterile PBS overnight. Then the scaffold was cut into several small pieces along the cross-section, and spread in a 96-well plate, and then the human-derived fibroblast cell line (3T3, Shanghai Bogu) was transferred to the surface of the scaffold in the 96-well plate. Culture at 37°C, 5% CO ₂ . On days 0, 1, 2, 3, 5, 7, and 9, 10% CCK-8 was added to 96 wells and incubated for 2 hours, and the color (yellow) of the liquid was observed and detected by Beckman DU-70 at a wavelength of 450nm. The OD value (n=6) was detected under the spectrophotometer, and the cell proliferation curve was drawn. The results are shown in Figure 3. On days 0, 1, 2, 3, 4, 5, 7, and 9, cell expansion and viability analysis showed that the color (yellow) and OD value of the liquid gradually increased after adding CCK-8, indicating that the decellularized scaffold is suitable for Proliferation and inward growth of heterogeneous cells.

(2) Statistical analysis: n=6 samples in each group in the experiment, and all the data are represented by mean ± standard deviation. The difference between the data of each group was analyzed by t distribution test with SPSS10.0 software. It is statistically significant when P<0.05.

(3) Results: The results of cell proliferation and viability analysis showed that the color (yellow) and optical density OD value of the liquid increased gradually when detected by CCK-8 at 0, 1, 2, 3, 5, 7, and 9 days. After 7 to 9 days, it tended to be stable (Figure 4), indicating that the acellular Achilles tendon scaffold has good biocompatibility with cells, and is suitable for the inward growth and expansion of cells in the acellular Achilles tendon scaffold.

Example 3 Construction of Adenoviral Vector and Transfection of Seed Cells

(1) Steps: Take chondrocytes (RAT-CELL-0096, primary primary from Wuhan) and osteoblasts (RAT-CELL-0055, primary primary from Wuhan) that have been stably cultured for 4 to 5 passages and have a fusion rate of 80%. Total RNA was extracted by phenol-chloroform method (TaKaRa total RNA extraction kit, Dalian), and the upstream and downstream primers listed in Table 1 were used to clone the full-length cds of Sox-9 (cartilage regulation gene) and RunX-2 (osteogenesis regulation gene). Select the PI-SceI and I-CeuI restriction sites, and insert the cDNA fragments of Sox-9 and RunX-2 target genes into the multiple cloning site of the shuttle plasmid pshuttle-CMV plasmid (see Table 2 for the gene sequence). The formation of recombinant plasmids is identified by enzyme digestion or sequencing. Amplify the recombinant plasmid, purify and prepare enough shuttle plasmid pshuttle-CMV containing the target gene. The linearized recombinant shuttle plasmid was digested with PmeI, and the plasmid was completely cut as identified by electrophoresis. Gel recovered linearized plasmids for co-transformation. The schematic diagram and detection map of the constructed vector are shown in Fig. 2a, 2b and 2c. Add the above-mentioned linearized shuttle plasmid (about 1 μg) and virus backbone plasmid (pAdEasy-1, about 100ng, Stratagene, USA) to the electroporation-competent EP tube containing E. 1500V/mm, 5ms). Add SOC or LB culture medium to recover. Take an appropriate volume of cell fluid transformed by electroshock and apply it to several kanamycin-resistant plates for culture. Pick the colony (the smallest colony) grown on the plate the next day and inoculate LB medium (kanamycin resistance) to expand the culture. After the plasmid was extracted by alkaline lysis of bacteria, positive clones were screened by gel electrophoresis. Take the positive plasmid and transform it into DH5α Escherichia coli cells to amplify the bacteria and purify the plasmid. Recombined viral plasmids were digested with PacI, ethanol precipitated after complete linearization, and dissolved in ddH ₂ O. Encapsulate the plasmid with liposomes (approximately 20 μL Lipofectamine per 4 μg PacI). Add the Lipofectamine-DNA mixture to 2× ¹⁰⁶ human kidney epithelial cells transfected with adenovirus E1A gene (293T cells, self-storage in the laboratory) with a growth density of about 50-70% in ^{a 25cm} square flask. After transduction Add DMEM complete medium (Hyclone, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA) for routine culture, and observe the cell growth during the process. Cytopathic changes (CPE) can be seen after about 2 weeks, and can be observed Green fluorescence (pAdTrack-CMV plasmid containing GFP reporter gene, Stratagene, USA). After 10-14 days of transduction, collect the cell pellet, suspend in PBS, freeze and thaw the cells repeatedly, collect the supernatant after centrifugation, and infect 293T cells with a confluence of 50-70% (self-storage in the laboratory). After 2 to 3 days, obvious cytopathic changes appeared. 3-5 days after infection, the virus was collected when 1/3-1/2 of the cells were floating. Cells were harvested and viral supernatants were prepared. Recombinant adenovirus production was identified by Western-blot. An appropriate amount of virus supernatant was added to 293T cells with an infection growth density of 90%. After 3 to 4 days, all cells were collected, resuspended in PBS, and repeatedly frozen and thawed 4 times. Three consecutive dialysis (10mM Tris, pH8.0+2mM MgC+5% sucrose) can basically remove CsCl, and the virus is stored at -80°C. Prepare 293T cells in a 96-well plate (10 ⁴ cells/well), dilute the virus solution to 8 groups of higher concentrations (10 ^-3 ～10 ^-10 ) with 2% DMEM medium, and add 100 μL of diluted virus liquid. In addition, two rows of wells were left without virus dilution as negative controls. Under the condition of 37°C, culture in an incubator for 10 days, observe the morphological changes of the cells, count the number of wells with CPE on the 96-well plate, and calculate the lesion rate of the cells. (If all the cells in each well are pathological under a certain concentration, the ratio is recorded as 1, and if there is no cell pathology, the ratio is recorded as 0). The calculation formula is as follows: T=101×10+(S-0.5)d/mL, (d=Log10 dilution, S=sum of cytopathic ratio of each concentration). Transfection of chondrocytes and osteoblasts with 3-5 generations of subculture and stable morphology was achieved by the same method as above

(2) Results: The recombinant shuttle plasmid pAdtrack-CMV-GFP-sox-9/RunX-2 was digested by KpnIII, HindIII and pAdEasy-1-sox-9/RunX-2 by PacI and then analyzed by SDS-PAGE. Products consistent with the expected target were shown, all of which were 30.0 kbp in size, proving that the recombination was successful (Fig. 2(ac)). The virus titer was determined to be 1×10 ⁹ PFU/mL. The pAdEasy-1-sox-9 and pAdEasy-1-RunX-2 plasmids were digested with PacI and then transfected into chondrocytes and osteoblasts respectively. are about 83% and 87% (Fig. 2(dg)).

Table 1 is used for the primer design of Sox-9 and RunX-2 gene cloning

Table 2 The cDNA gene sequence used for Sox-9 and RunX-2 clones in this project

The preparation of embodiment 4 collagen hydrogel

First, dissolve 100 mg of acid-soluble bovine skin type I collagen (Sigma, USA) in 40 mL of 0.1% sterile acetic acid solution (prepared with sterile double distilled water, sterilized by 220 μm bacterial filter) to prepare for storage solution (solution A), followed by preparing 0.34M NaOH and 10×DMEM (Hyclone, USA) solutions respectively, and after sterilizing with a 220 μm bacterial filter, mix 0.34M NaOH and 10×DMEM at a volume ratio of 1:2 Mix DMEM solution (solution B), and finally mix solution A and solution B at a volume ratio of 4:1 (solution C), filter and sterilize with a 220 μm bacterial filter, add 1 % penicillin/streptomycin is the collagen hydrogel solution required for this experiment. The hydrogel was applied to shape the seed cells in the scaffold to avoid mutual influence caused by cell migration, and the preparation effect is shown in Figure 3d.

Preparation of osteoblast/chondrocyte collagen hydrogel suspension:

(1) Take osteoblasts (6×10 ⁶ cells) with a cell fusion degree of about 90%, and observe under an inverted microscope: the field of vision is clear and there is no pollution;

(2) Discard the medium in the bottle, add PBS, wash the cells gently, and discard the PBS;

(3) Add 1 mL of 0.25% trypsin to digest for about 1 min;

(4) Add 2 mL of 10% FBS DMEM/F12 high-glucose complete medium to stop digestion, and blow down the cells with a pipette, then transfer to a centrifuge tube, centrifuge at 500 rpm for 5 min;

(5) After centrifugation, the white cell precipitate at the bottom of the tube can be seen, and 1 mL of collagen hydrogel is added to resuspend the cells to obtain a single-cell collagen hydrogel suspension;

(6) The cell count is 1×10 ⁶ cells/mL.

Example 5 Construction of "Migration Structured" Engineering Tendon in Vitro

(1) Steps: Soak the decellularized rabbit Achilles tendon scaffold (length 40 mm) in 70% ethanol for 48 hours, then rinse with sterile PBS overnight. One end (about 10 mm in length) of each group (n=4) of the scaffold was placed in DEME/F12 medium (Hyclone, USA) containing rFN/CDH 50 μg/mL and incubated at room temperature for 4 h to allow rFN/CDH to fully adsorb on the surface of the scaffold (For the preparation method of rFN/CDH, please refer to the National Invention Patent, "Fusion Protein of Fibronectin and Cadherin-11, Preparation Method and Application", Patent No.: ZL200910103100.6).

For the FB+sox-9-CC+RunX-2-OB experimental group: first place the end of the scaffold soaked in rFN/CDH in RunX-2-OB, 10% FBS, DMEM with a density of 1×10 ⁶ /mL /F12 high glucose complete medium suspension, static culture overnight. The next day, take 1 mL of 2×10 ⁶ cells/mL of fibroblasts or 1×10 ⁶ cells/mL of sox-9-CC or 1×10 ⁶ cells/mL of RunX-2-OB collagen hydrogel to suspend Using a 1mL syringe, inject into the FB (fibroblast) segment, CH (chondrogenic) segment and OS (osteogenic) segment of the stent along the longitudinal axis of the stent. After the hydrogel is completely solidified, place the tendon scaffold in a solution containing 10% fetal bovine serum, 1.5mg/mL β-glycerol phosphate, 100u/mL penicillin, 100g/mL streptomycin, TGF-P10ng/mL, ascorbic acid 50μg/mL The DMEM/F12 high-glucose culture medium, static culture for 2 weeks, is the ligament substitute constructed in this experiment.

Each step of the simple scaffold group was treated with the same amount of 10% FBS DMEM/F12 high-glucose complete medium or collagen hydrogel as the experimental group; each step of the group II was the same as the experimental group; the group III only used the same amount of 10 Except for %FBSDMEM/F12 high-glucose complete medium or collagen hydrogel treatment, the rest of the steps were the same as the experimental group; in group IV, except that the chondrocytes were only treated with the same amount of collagen hydrogel, the rest of the steps were the same as the experimental group.

(2) Results: The engineered tendon scaffold was successfully constructed by standard decellularization technology, and the expression of chondrogenic and osteogenic genes in seed cells was successfully enhanced by genetic engineering, and collagen hydrogel and rFN/CDH recombinant protein were used to promote The seed cells were planted in layers and three-dimensionally cultured in the tissue-engineered tendon by sticky means, successfully simulating the transitional tissue structure of the normal ligament-bone junction.

Example 6 Gene Expression of Cartilage/Osteogenic Markers in Tissue Engineered Tendon

Sox-9 was selected as an early cartilage marker, RunX-2 was an early osteogenic marker; COL2A1 (collagen2A1, COL2A1), COMP (Cartilage oligomeric interstitial protein, cartilage oligomeric interstitial protein) and Aggrecan (cartilage proteoglycan) were selected as Late cartilage markers; OCN (osteocalcin, osteocalcin), OPN (osteopontin, osteopontin), BSP (Bone Sialoprotein, bone sialoprotein) are late osteogenesis markers.

(1) Total RNA was extracted as follows: Grind the tissue with liquid nitrogen at one week and two weeks respectively, transfer it into a 1.5mLEP tube, add 1mL TRNzol, lyse on ice for 20min, add 1/5 volume of chloroform (0.2mL) , use the vortex condition to shake and mix it, place it at 4°C, and then centrifuge at 15,000 rpm/5 minutes; then transfer the upper aqueous phase (about 400 μL) to another 1.5mL EP tube; add the same volume of After isopropanol (about 400 μL), shake and mix well; put it in a centrifuge at 4 °C at 15,000 rpm/20 minutes; suck off the supernatant with a straw, and add 1 mL of 75% ethanol cooled with ice cubes in advance; Centrifuge in a centrifuge at 15,000 rpm for 5 minutes; suck off the supernatant with a straw, add 1 mL of 100% alcohol; put it in a centrifuge at 4°C for 15,000 rpm for 5 minutes; suck off the supernatant with a straw , placed in the air hole for 5-10 minutes to dry; then put in 40 μL of 1‰ DEPC water, and stored at -70°C for later use. Take 2 μL of RNA and dilute it 50 times, measure the ratio of OD value at 260nm/280nm by UV spectrophotometer, and multiply the result by 50 to get the actual concentration of RNA. A part of the extracted RNA was randomly selected for gel electrophoresis, and the gel was prepared for electrophoresis. Add 1g of agarose to 100mL of TAE buffer, heat it in a microwave oven to dissolve it, then cool it down to 60°C, add 5μL of EB dye, and finally pour it into the gel tank with the comb inserted , and wait for it to cool and solidify before use. Take 1 μg of the extracted RNA and electrophoresis at 5V/1cm. According to the primers designed in Table 2, the "relative quantification" method (Relative Quantitation) of the ABI7500 system was used to detect the gene expression of cartilage, bone and other early and late markers in real time. Confirm the amplification and melting curve after the reaction, and use the 2-ΔΔCt analysis method for relative quantitative analysis: ΔCt=target gene Ct value-GAPDH Ct value; -ΔΔCt=normal control group ΔCt average value-each sample ΔCt, 2-ΔΔCt reflects The relative expression level of the target gene in each sample relative to the normal control group.

(2) Statistical analysis: n=4 samples in each group in the experiment, and all data are represented by mean ± standard deviation. The difference between the data of each group was analyzed by one-way analysis of variance with SPSS10.0 software. It is statistically significant when P<0.05.

(3) Result analysis:

The relative quantitative real-time fluorescent RT-PCR method was used to analyze the mRNA expression of cartilage/osteogenesis regulation genes sox-9 and RunX-2 genes and their downstream marker molecules in tissue-engineered tendon-cell tissue-engineered tendon. The results are shown in Figure 4(a). When the expression level of group I (stent-only group) was calibrated to 0, the RunX-2 mRNA expression of group V was 2.91 times that of group II (P<0.05) and 6.77 times that of group IV. times (P<0.01), with significant statistical difference. The results are shown in Figure 4(b), the increase of sox-9 mRNA expression in group V was 195% and 743% of group II and group III respectively (P<0.05), which was statistically significant (P<0.05) .

Correspondingly, the present invention detected the classic downstream regulatory molecules of sox-9 (COL2A1, COMP and Aggrecan) and the classic downstream regulatory molecules of RunX-2 (OCN, OPN and BSP) at 10 days of culture, and the results are shown in Figure 4 (a-b) The results showed that the expression of cartilage marker COL2A1 in group V was 2.55 times that of group II and 9.44 times that of group IV, both of which were statistically significant (P<0.05); it was 1.15 times that of group III, without statistical significance (P =0.13); the mRNA expression patterns of COMP and Aggrecan were similar to those of COL2A1.

The evaluation results of osteogenic gene expression showed that when compared with group b and group c, the increase of OCN mRNA expression in group V was 139% and 409% of group II and group III (P<0.05); The mRNA expression of OCN was not statistically significant between group Ⅴ and group Ⅳ; the mRNA expression of OPN and BSP was similar to that of OCN.

The above data show that the mRNA expression of sox-9 and RunX-2 and its classic downstream regulatory molecules were significantly increased in the scaffold tissue engineered tendon cultured for two weeks, thus confirming that the osteogenic-chondrogenic ability of tissue engineered tendon has been significantly improved .

Table 2 is used for the primer design of gene expression of cartilage and bone marker molecules

Example 7 Protein Expression of Cartilage/Osteogenic Markers in Tissue Engineered Tendon

1. Using the method in Example 7, the composite tissue ligament cultured for two weeks was shredded, thoroughly homogenized, and filtered.

2. Extract the total protein according to the following method:

Add 3μL aprotinin to 1mL lysis solution, 10μL 0.1M PMSF (phenylmethylsulfonyl fluoride) and 5μL 0.1M Na ₃ VO ₄ (sodium orthovanadate) for later use (shake the PMSF until there is no crystallization before adding the lysis solution ). Add the prepared liquid into the bottle at a ratio of 100-200 μL lysate/mL, and place it on ice to lyse for 30 minutes. The bottle should be blown or shaken frequently to fully lyse the cells. After lysing, use a dropper to gently wash and pipette the liquid, and then use a dropper to transfer the lysate to a 10mL centrifuge tube (the entire operation should be performed on ice as much as possible). Centrifuge at 12,000 rpm for 15 minutes at 4°C (turn on the centrifuge to pre-cool). After centrifugation, take the supernatant and put it into an EP tube, take a part to measure the concentration, store it at -80°C, and distribute it appropriately. Do not freeze and thaw it repeatedly.

3. Determination of protein content:

(1) The making of standard curve;

(2) Take out BSA (1mg/mL) from -20°C, melt at room temperature, and set aside;

(3) Dilute BSA with deionized water according to Table 3 to the following concentration (mg/mL):

Table 3 BSA dilution ratio

BSA(mL) 0 1 2 4 6 8 10 Deionized water (mL) 10 9 8 6 4 2 0

(4) Add 100 μL protein concentration determination working solution (micro-BCA, Pierce, USA) to each tube

(5) Place in a 37°C water bath for 30 minutes.

(6) Measure the absorbance with an ultraviolet spectrophotometer at a wavelength of 562nm

(7) The computer automatically generates a standard curve, save the standard curve and exit.

4. Detection of the protein content of the sample

(1) Dilute the sample according to a certain ratio;

(2) BCA reagent B: A=1:50;

(3) The ratio of diluted sample to BCA working solution is 1:10;

(4) Place in a water bath at 37°C for 30 minutes;

(5) Measure the absorbance with an ultraviolet spectrophotometer at a wavelength of 562nm;

(6) Save the experimental data.

5. SDS-PAGE electrophoresis

Make the separating gel:

(1) The molecular weight of the target protein is: COMP (105KD), and 8% separating gel is used. For BSP (35KD), use 12% separating gel. For OPN (55KD), use 10% separating gel. Aggrecan (200KD), select 6% separating gel for use. For COL2A1 (190KD), use 6% separating gel. For OCN (35KD), use 12% separating gel. GAPDH (37KD), choose 12% separating gel;

(2) Make the concentration of stacking gel as 5%;

(3) Clean the glass plate, align the glass plate and put it into the splint to clamp it tightly. In order to prepare for glue filling, clamp it vertically on the shelf;

(4) After fully mixing according to the above ratio, pour the components of the separating gel into the gap between the glass plates;

(5) Seal the gap with n-butanol and wait for 20 to 30 minutes. Pour off n-butanol and rinse it twice with deionized water;

(6) Make concentrated gel according to the ratio of 5%, add it after mixing and insert it into the comb immediately;

(7) Wait for 30 minutes to 1 hour to take out the comb and assemble the electrophoresis device. Add the electrophoresis liquid to the electrophoresis device, first add half of it and tilt to remove the bottom air bubbles;

(8) Sample treatment: Take an appropriate amount of protein sample (according to the protein content of the test sample), add an appropriate amount of 5×SDSLoading Buffer, and cook in boiling water for 3-5 minutes;

(9) Load the sample after cooling to room temperature. Generally, the first hole is reserved as a maker, and an appropriate amount of 1×SDSLoading Buffer pressure tape is added to the remaining holes;

(10) Electrophoresis: ①, 30V for five minutes, ②, 80V stacking gel, ③, 120V separating gel (can be changed to 120V after the marker appears);

(11) When running to about 0.5cm from the edge, turn off the power;

(12) Membrane transfer: first pour the electrotransfer fluid into a 2.5L flat-bottomed tray, and soak the sponge pad and filter paper in the electrotransfer fluid. The membrane used in our laboratory is PVDF (polyvinylidene fluoride) membrane. After taking it out according to the habit, cut off a corner of the membrane and mark it, and then soak it in anhydrous methanol for 5-10s. Then take it out and soak it in the electrotransfer solution for 5 minutes, and cut the strip according to the size of the target band. Lay it flat on the PVDF membrane, and put the filter paper on it after aligning. Then clamp the clips, pay attention to the correct order of assembly (positive and negative). 100v, 1.5~2h, you can add an ice box when transferring the membrane, and use a magnetic stirrer to stir to ensure that the membrane system is at a lower temperature and prevent the transfer effect from being affected.

6. Western blot

(1) Western-blot semi-quantitative analysis of COL2A1, COMP, Aggrecan, OCN, BSP, OPN protein expression (n=3). Use goat anti-COMP, Aggrecan (1:1000dilution, Sigma) antibody; rabbit anti-BSP, OCN (1:1000dilution, Sigma) antibody; and GAPDH, OPN, COL2A1 (1:1000dilution, Sigma) antibody as the primary antibody, and the secondary antibody Rabbit anti-goat antibody (1:1000dilution, Sigma); goat anti-rabbit antibody (1:1000dilution, Sigma); and goat anti-mouse antibody (1:1000dilution, Sigma) were secondary antibodies.

(2) Use image analysis software to measure the gray ratio of the bands, and perform statistical analysis in parallel. In the experiment, n=4 samples in each group, and all data are represented by mean ± standard deviation. The difference between the data of each group was analyzed by one-way analysis of variance with SPSS10.0 software. It is statistically significant when P<0.05.

(3) Result analysis:

The protein translation levels of COL2A1, COMP, Aggrecan, OCN, OPN and BSP in tissue engineered tendon protein extracts were detected by Western blotting. The results are shown in Figure 4(c), the protein expression of the cartilage marker COL2A1 in group V was 1.87 times that of group II and 2.8 times that of group IV, both of which were statistically significant (P<0.05); There was no statistical significance between group III (1.12 times, P>0.05), and the expression levels of COMP and Aggrecan were similar to those of COL2A1.

The protein expression of osteogenic marker OCN in group Ⅴ increased by 230% and 407% respectively compared with group Ⅱ and group Ⅲ, which was statistically significant (P<0.05); there was no statistical significance between group Ⅴ and group Ⅳ ( P>0.05), the protein expression of OPN and BSP was similar to that of OCN.

Example 8 Histological identification of cartilage/osteogenesis/fibroblast in tissue engineered tendon

1. Special histological staining

(1) The composite tissue engineered ligaments cultured for 2 weeks were rinsed with PBS, fixed with 10% paraformaldehyde, embedded in paraffin, serially sectioned with a thickness of 5um (transverse section), and then immunofluorescent and histologically stained (n=4). According to the instructions of the reagents, the continuous distribution of fibroblasts and fibrous tissues, GAGs and calcium nodules in the composite tissue engineered ligaments were observed with hematoxylin-eosin, alicin blue and alizarin red staining, respectively.

(2) Image acquisition: The prepared slices were observed under an ordinary light microscope and the original images were obtained.

(3) Result analysis:

Alizarin red staining results showed that the osteogenic segments of group V and groups II and IV were all stained orange (Fig. 5(a-e)), however, no obvious orange was visible in the CH and FB areas and other groups ( Results not provided), the generation of calcium nodules was mainly confined to the OS region, presumably because expanded osteoblasts accelerated the secretion of a representative extracellular matrix. However, compared with group II, groups V and IV had more mineral tissue-engineered tendon production, a result that showed that RunX-2 gene transfection accelerated the osteogenic mineralization of corresponding osteoblasts.

As a result of Alician blue staining, it was found that the positive staining (blue) was mainly concentrated in the CH region of group V, group II and group III (Fig. 5(f-j)), and the staining of group V and group IV was obviously stronger than that of group II. The results indicated that sox-9 promoted the production of GAGs in chondrocytes.

The results of H&E staining showed the distribution of fibroblasts and collagen fibers in the FB segment (Figure 5(k-o)). Compared with group Ⅰ, the expanded cells and newly generated extracellular matrix covered the surface of the interconnected pores, group Ⅰ There was no obvious morphological difference between Ⅱ-Ⅴ.

2. Immunofluorescence analysis

(1) The composite tissue engineered ligaments cultured for 2 weeks were rinsed with PBS, fixed with 10% paraformaldehyde, embedded in paraffin, serially sectioned with a thickness of 5um (transverse section), and then immunofluorescent and histologically stained (n=4). According to the instructions of the reagents, the expression and distribution of osteocalcin and COL2A1 in the tissue engineered tendon were detected, and mouse anti-human osteocalcin (osteocalcin, OCN, diluted 1:100, abcam) and rabbit anti-human COL2A1 (collagen2A1, COL2A1, The dilution concentration is 1:50, abcam) was used as the primary antibody to label the tissue section, and the donkey anti-mouse (the dilution concentration was 1:50, abcam) labeled with Cy3 and the donkey anti-rabbit labeled with FITC (fluorescein isothiocyanate) ( The dilution concentration was 1:100, Abcam) fluorescent antibody was used as the secondary antibody, and the nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) (blue).

(2) Image acquisition: The prepared slices were observed under a fluorescence microscope and the original images were obtained.

(3) Analysis of the results: Figure 6 (a-e) The red-colored area of Liang is the OCN fluorescent labeling area, and the blue is the nucleus. The semi-quantitative results indicate that the expression level of OCN in group Ⅴ is significantly higher than that of the other groups, indicating that the tissue engineered tendon is more effective in each group. The bone formation ability is the strongest. Figure 6 (f-j) The bright green colored area is the OCN fluorescent labeling area, and the blue is the cell nucleus. The semi-quantitative results indicate that the expression level of COL2A1 in group V is significantly higher than that in the other groups, indicating that the tissue-engineered tendon has the strongest chondrogenic ability in each group .

3. SEM and EDX analysis of the surface of tissue engineered tendon OS segment

(1) Rinse the composite tissue engineered ligament co-cultured for 2 weeks with PBS three times, fix with 2.5% glutaraldehyde for 24-48 hours, gradient ethanol: dehydrate in 30% ethanol for 10 minutes→dehydrate in 50% ethanol for 10 minutes→ Dehydration in 70% ethanol for 10 minutes → dehydration in 90% ethanol for 10 minutes → dehydration in absolute ethanol for 10 minutes → dehydration in absolute ethanol for 10 minutes and tert-propanol: dehydration in 30% tert-butanol for 10 minutes → 50% tert-butyl alcohol Dehydration in alcohol for 10 minutes → dehydration in 70% tert-butanol for 10 minutes → dehydration in 90% tert-butanol for 10 minutes → dehydration in anhydrous tert-butanol for 10 minutes → dehydration in anhydrous tert-butanol for 10 minutes After dehydration (note this During each liquid change process, it is not advisable to stay in the air for too long), fix the specimen on the electron microscope holder, and use energy dispersive X-ray energy spectrometer (EDX, Horiba EX-220) to semi-quantify calcium, phosphorus and other minerals on the surface of tissue engineering tendon After the distribution of chemical elements, the sample holder was taken out and the surface of the scaffold was plated with gold (Hitachi E-1030ion sputterer), and then placed under a scanning electron microscope (S-3400N) to observe the appearance of the tissue-engineered tendon.

(2) Statistical analysis: n=4 samples in each group in the experiment, and all data are represented by mean ± standard deviation. The difference between the data of each group was analyzed by t distribution test with SPSS10.0 software. It is statistically significant when P<0.05.

(3) Result analysis:

After 2 weeks of tissue-engineered tendon culture in vitro, the surface elements were detected, and the results are shown in Figure 7: under SEM, except for Group I and Group III (Figure 7(a-e)), the surface elements of tissue-engineered tendons in other groups were widely distributed. The nodular or patchy distribution of mineralized substances, especially in group V, was the highest, and these substances were confined to the surface of the OB segment where osteoblasts adhered via rFN/CDH.

In addition, the elemental composition of the surface of the samples in each group was analyzed by EDX, and the precise distribution of calcium and phosphorus was detected on the surface of the OS segment (Fig. 7(f-j)). Group Ⅱ and group Ⅳ without RunX-2 gene transfer also proved that high-efficiency RunX-2 transfection in tissue-engineered tendon accelerated the osteogenic differentiation effect in the corresponding area. The relative weight percentages of calcium and phosphorus in each group are marked in the figure.

Claims (13)

1. the in vitro construction method of bionics ligament-bone connector, is characterized in that: comprise the steps: a. Preparation of decellularized Achilles tendon; b. Preparation of seed cells: including preparation and gene transfection of fibroblasts, Sox-9 gene-enhanced chondrocytes, and Runx-2 gene-enhanced osteoblasts; c. Implantation: The decellularized Achilles tendon is divided into three structural and functional areas according to the longitudinal and transverse axes of the fibroblast FB-chondrogenic CH-osteogenetic OS, and the fibroblasts and Runx-2 genes are enhanced Osteoblasts and Sox-9 gene-enhanced chondrocytes were planted in layers and divided into fibroblasts, chondrocytes, and osteoblasts, cultured for 5 to 7 days after histological verification, and then cultured for 1 week for use; The layered planting described above refers to the transplantation from the surface of the Achilles tendon to the center of the cross-section, so that the fibroblasts, the osteoblasts enhanced by the Runx-2 gene, and the chondrocytes enhanced by the Sox-9 gene are located in the inner, middle, and outer parts of the cross-section respectively. 1/3 area. 2. The method according to claim 1, characterized in that: the decellularized Achilles tendon scaffold in step a is derived from the Achilles tendon tissue of rabbits, dogs, and cattle. 3. The method according to claim 1 or 2, wherein the preparation of osteoblasts in step b comprises the following steps: constructing an adenoviral vector expressing the early osteogenic marker RUNX-2, and transfecting osteoblasts. 4. The method according to claim 1 or 2, characterized in that: the preparation of chondrocytes in step b comprises the following steps: constructing an adenoviral vector expressing early cartilage marker SOX-9, transfecting chondrocytes, and then dissolving in collagen hydrogel. 5. The method according to claim 3, characterized in that: the preparation of chondrocytes in step b comprises the steps of: constructing an adenoviral vector expressing early cartilage marker SOX-9, transfecting chondrocytes, and then dissolving in collagen water in the gel. 6. The method according to claim 1 or 2, characterized in that the preparation of the fibroblasts in step b comprises the following steps: dissolving the fibrous cells in the collagen hydrogel. 7. The method according to claim 3, characterized in that: the preparation of the fibroblasts in step b comprises the following steps: dissolving the fibrous cells in the collagen hydrogel. 8. The method according to claim 4, characterized in that: the preparation of the fibroblasts in step b comprises the following steps: dissolving the fibrous cells in the collagen hydrogel. 9. The method according to claim 1 or 2, characterized in that: before planting in step c, the osteogenic segment of the decellularized Achilles tendon is treated with recombinant fibronectin/cadherin rFN/CDH. 10. The method according to claim 3, characterized in that: before planting in step c, the osteogenic segment of the decellularized Achilles tendon is treated with recombinant fibronectin/cadherin rFN/CDH. 11. The method according to claim 4, characterized in that: before planting in step c, the osteogenic segment of the decellularized Achilles tendon is treated with recombinant fibronectin/cadherin rFN/CDH. 12. The method according to claim 6, characterized in that: before planting in step c, the osteogenic segment of the decellularized Achilles tendon is treated with recombinant fibronectin/cadherin rFN/CDH. 13. The tissue engineering cell-tendon complex prepared by the method according to any one of claims 1-12.