3.1 Ethical compliance
In total, forty-two healthy New Zealand rabbits of 2 months of age are provided by the animal center of Shanxi medical university. All the treatment of animals is conducted in laboratory animal institute, under the supervision and assessment by the Laboratory Animal Ethics Committee of The second hospital of Shanxi medical university.
3.2 Preparation of seed cells
Six rabbits are randomly divided into two groups, of with each group contained 3 rabbits. USPIO (20mg/kg) is given to the experiment group via auricular vein injection, while none is given for the control group. 1.5T MRI scan is performed at 0h, 12h, 24h, and 48h after injection to detect the signal of bone marrow in the long bone of the hind limb. Anesthetizing by injecting simazine hydrochloride 30 min before MRI, the rabbits are fixed and scanned by a sagittal T2 weighted FSE sequence in the coil of the knee in the prone position and the feet in an advanced position at the parameters are shown in Table 1.
Table 1
MRI scan parameters of the long bone in rabbit
Scan parameters | FSE T2WI sequences |
The repeat time | 2610 |
The echo time | 120 |
Thick(mm) | 2.0 |
Layer(mm) | 0.2 |
Matrix | 448×384 |
Field of vision | 18×18 |
Incentive frequency | 8 |
3.3 BMSCs isolation, culture, and proliferation in rabbits with USPIO markers
The rabbits are executed at 48hrs after USPIO injection, and both lower limbs are collected [14, 15, 16]. Both ends of the selected bones are cut to expose the bone marrow, the primary bone marrow cells are collected by soaking using DMEM/F12 with 10% fetal bovine serum (FBS) with 10ml syringe repeatedly, and they are seeded. BMSCs are obtained with several days of culture at 37℃ 5%CO2. When the bottles are covered with rabbit BMSCs, we poured out old culture and washed the bottle 2–3 times using a moderate amount of PBS buffer, added 0.5-1 ml 0.25% trypsin solution. Let it stand in the oven for about 1 min and observed most of the cells in the microscope. Make it become suspended, then we added 2 times the amount of nutrient solution termination of trypsin digestion. We blew the pipe and blend cell suspension and the number of partial shipments in two culture bottles. When the suspension cells are adhered to the wall and covered with the bottom of the culture bottle, the first generative cells (P1) are obtained. The second generative cells (P2) and the third generative cells (P3) can be obtained according to the same passage method, and the morphological changes and growth of the cells are recorded, as well as the images are collected, and the third generative cells (P3) is taken as the follow-up research object.
3.4 Identification of the effectiveness and safety of USPIO labeled BMSCs in rabbits
USPIO is an iron agent. After reaction with Prussian blue staining, potassium ferrocyanide can bind to ferric iron in cells or interstitium to form ferrocyanide which is an insoluble blue compound. Iron particles are stained blue and nuclei are stained red with other tissues.
All groups of P3 BMSCs are cultured with 6-well plates. When the cells are uniformly overgrown, they are fixed by 4% paraformaldehyde. The tap water is rinsed twice, 2min each. The distilled water is rinsed twice, 2min each. The sections are stained with Perl’s stain. After the distilled water is fully rinsed, the nucleus is put into the solid red dye solution and the lightly stained nucleus. The tap water is rinsed, the routine dehydration is transparent, and the neutral gum is sealed. Under the microscope, 100 cells are observed in a randomly selected field, and the labeled rate of USPIO cells = the number of labeled cells/(labeled cells + unlabeled cells) x 100%.
P3 BMSCs are taken and the cells of each group are inoculated on the 96-well plate according to the standard of 200ul culture medium and 2x103 in each well. The 10ul enhanced cck-8 solution is added to each hole and incubated in the culture box for 1h. The absorbance value OD450 is measured at the wavelength of 450nm with the enzyme marker. The 10-ul enhanced cck-8 solution is added at the same time point for 7 consecutive days, and the absorbance value is determined by the same method mentioned above. The cell proliferation curve of each group is plotted with days (d) as the abscissa and OD value as the ordinate (the number of cells is positively correlated with OD value).
When P3 BMSCs cell alignment of 80 ~ 90%, we digest it using pancreatic enzyme and make the density become 2 x 104 / cm2. Then vaccinate it in the prior six orifice plate with gelatin package. When the degree of cell fusion reached 60–70%, the cell culture medium in the hole is carefully absorbed, and 2ml of rabbit bone marrow mesenchymal stem cells are added to each hole to complete the osteoblastic differentiation culture medium. Induced by 2 ~ 4 weeks later, we observe the cell growth by alizarin red staining. The detail operations are as follows: we suck the osteogenetic differentiation medium completely in the six orifices and add 1 x PBS fluid to flush with 1–2 times. We add 2 ml 4% neutral formalin to fix 30 min in each hole. suck neutral formaldehyde solution and add 1 x PBS fluid to flush with 2 times. Then we add alizarin red dyeing 1 ml (3 ~ 5 min) in each hole and suck alizarin red after dyeing, add 1 x PBS fluid to flush with 2–3 times. We observe the osteogenesis dyeing effect by microscope.
When P3 BMSCs cell alignment of 80 ~ 90%, we digest it using pancreatic enzyme and make the density become 2 x 104 / cm2. Then vaccinate it in the prior six orifice plate with gelatin package. Put into the culture box and change the liquid every 3 days until the cell fusion degree reaches 100% or overfusion.2ml of rabbit bone marrow mesenchymal stem cells are added to each hole to form lipid-induced differentiation culture medium A, which is removed after 3 days. 2ml of rabbit bone marrow mesenchymal stem cells are added to each hole to form lipid-induced differentiation culture medium B. Liquid A and liquid B are used alternately for 3 to 5 times. After observing the lipid drops are large enough and round under the microscope, we continue to use liquid B for 4 to 7 days for culture, during which fresh liquid B is replaced every 2 to 3 days. After the completion of the induction, we stain cells by oil red O, suck the fat differentiation medium completely in the six orifices and add 1 x PBS fluid to flush with 1–2 times. Then we add 2 ml 4% neutral formalin to fix 30 min in each hole. suck neutral formaldehyde solution and add 1 x PBS fluid to flush with 2 times. Then we add oil red O dyeing 1 ml (3 ~ 5 min) in each hole and suck it, add 1 x PBS fluid to flush with 2–3 times. We observe the fat dyeing effect by microscope.
Take P3 BMSCs with pancreatic enzyme digestion, add cell cultures of termination the digestion. Then we centrifugal it 5 min under 1000 r/min speed and add 1 x PBS liquid to wash, add cell culture to blend the cells and count cells to make the cell concentration 1 x 106 /ml. Moving 1 ml cell suspension liquid into 15 ml centrifugal tube with a gun, we centrifugal it 5 min under 1000 r/min speed, and the cluster of white cell mass can be seen at the bottom of the centrifuge tube. After incubating 24 hours, the rabbit bone marrow mesenchymal stem cell chondrogenic differentiation is completely cultured, and the new induction solution is replaced twice a week. After the inducting 4 weeks, the induction solution in the centrifuge tube is poured, and then 1ml of the polyformaldehyde solution containing the sodium dime thriller phosphate is added to fix for 1h. It is found that the cell microcluster is floating on the liquid surface, followed by dehydration: 75% ethanol, 60min-95% ethanol, 60min-anhydrous ethanol, 30min/ time-xylene for 15 minutes. After dehydration, the paraffin is dipped in wax for 30min and embedded in paraffin. Paraffin sections are made according to the thickness of 3 mm, and Safranin 'o staining is conducted.
3.5 Preparation of cell-rich alginate gel spheres
A proper amount of sodium alginate solid powder is weighed and prepared into a 1.5ml sodium alginate gel liquid with a concentration of 1.2%, which is divided into three 50ml centrifugal tubes on average and each tube is filled with 1.5ml for reserve. After the USPIO-labeled P3 BMSCs and unlabeled P3 BMSCs obtained from the separation and culture above, they are counted with a counting plate [17]. Two centrifuge tubes containing sodium alginate gel are added to each tube to make the final density of cells in each tube 5.8x106 /ml, while the other tube of sodium alginate gel did not receive any treatment. We add sodium alginate gel 25uL into the appliance containing a 1.13% CaCl2 solution and let it stand for 10 min. Then we use the curved tweezers to set for skin frozen samples of sodium alginate gel ball slowly and move into a container containing 0.9% NaCl solution, let it stand for 2 min. Then slowly move to contain DMEM liquid container and let it stand for 10s. Eventually, spherical USPIO labeling BMSCs-sodium alginate gel compound, not mark BMSCs-compound sodium alginate gel and pure sodium alginate gel ball are obtained.
3.6 Establishment of the rabbit model of knee cartilage defect
Thirty-six rabbits are divided into four groups, each consisting of nine. Anesthesia by intravenous injection of 10% chloral hydrate L/kg (1.0 m) intramuscular sierra qin hydrochloride injection (0.1 mL/kg), rabbits are fixed by supine position. After disinfection, we do the double knee medial longitudinal incision about 2–3 cm long, cut each layer, mobile patellar lateral dislocation, fully expose the block surface of the femur with 4 mm diameter bit, drill depth of 3 mm and form a full-thickness defect of articular cartilage [18]. Then we flush the articular cavity by saline: The BMSCs-alginate gel complex labeled with USPIO is implanted in group A, unlabeled BMSCs-alginate gel complex is implanted in group B, pure alginate gel ball is implanted in group C, and joint defects in group D are not treated. Figure 1 shows the distribution in vivo after USPIO injection. It can be seen from Fig. 1 that all layers of tissue are sutured with surgical suture, the joint cavity is closed, and iodine fu is disinfected. Intramuscular injection of penicillin (100,000 U/ only, Bid) is performed for 3 consecutive days after the operation to prevent wound infection. After 3 days, the defect is observed by double knee MRI.
3.7 Observation on the repair effect of cartilage defect in rabbit knee
We detected the four groups of rabbits at 4, 8, and 12 weeks after transplantation by MRI. The diameter and depth of cartilage defect areas in each group at different periods are measured by AW4. 4 work station and the signal intensity of the defect areas at different periods in group A (USPIO labeled cell-alginate complex transplantation group) and group B (unlabeled cell-alginate complex transplantation group) is measured, and the data are collected for statistical analysis.
At 4 weeks, 8 weeks, and 12 weeks after transplantation, the rabbits in each group are sacrificed and the specimens of femoral pulley are extracted. The cartilage repair in each group is generally observed at different stages, including repair degree, correlation with surrounding normal tissues, and surface smoothness.
Fixation, decalcification, and dehydration are performed as usual. Paraffin embedding and slicing are completed as follows: the specimens are embedded with paraffin embedding machine for 1h, and the specimens are embedded with paraffin embedding technology using the embedding box. According to the thickness of 5um, the slicer is used for 10 consecutive slices, followed by another 10 slices with an interval of 100 m, and the sections of each group are numbered for HE staining and Safranino-solid-green staining. Then we complete the staining by HE and Safranin 'o - solid green staining to observe the morphology under a microscope.
3.8 Statistical analysis
All experimental data are treated with SPSS17.0 software, and the results are expressed by mean ± standard deviation (in line with normal distribution). Univariate analysis of variance is used for multi-group comparison, and a t-test is used for comparison between two samples. P < 0.05 is considered statistically significant.