1 Experimental animals and materials
Wistar rats of 6–8 weeks old were purchased from Changsha Tianqin Biotechnology Co. Ltd (License No.: SCXK(Xiang)2019-0013). The materials and instruments used in this study include normal medical tape; 1×PBS; 3% H2O2; Modified Safranine O-Fast Green FCF Cartilage Stain Kit (G1371, Solarbio); neutral resin (CW0136, CWBIO); VEGF (19003-1-AP, Proteintech); collagen X (DF13214, Affinity); horseradish enzyme-labeled goat anti-rabbit IgG (H + L) (ZB-2301, ZSGB-BIO); DAB chromogenic kit (CW0125, CWBIO); hematoxylin staining (AR1180-1, Boster Bio); drug refrigerator (SF-477S, Orbotech); microscope (CX41, OLYMPUS); tissue slicer (BQ-318D, Bona); electrothermal blast drying oven (DHG-9070A); drug refrigerator (BYC-310, Shandong Boke Bio); electrothermal constant temperature blast drying oven (HGZF-101-1, Shanghai Yuejin Medical Equipment Co. Ltd); thermostatic incubator (DHP-9054, Shandong Boke Bio); microscope (BX43, OLYMPUS); Pipette gun (Research plus 0.5–10 µL, eppendorf); Pipette gun (Research plus 20–200 µL, eppendorf); Pipette gun (Research plus 100–1000 µL, eppendorf); Skyscan1276 X-Ray Microtomograph (Micro CT) (Skyscan1276, Bruker, Belgium).
2 Experimental Methods
2.1 Grouping and modeling of experimental animals
Male rats and female rats were combined with a ratio of Male:female = 2:1, and after 22 days gestation, 40 newborn rats were selected and randomly divided into control and experiment groups. In the experimental group, both lower limbs of newborn rats (6–8 hours after birth) were wrapped and fixed with 0.5 cm wide medical tape for 10 days (Fig. 1). The lower limbs in the control group were not treated. The rats of the experimental group were gently immobilized to avoid violent dislocation, and the tape was unfastened once a day for 1–3 minutes and then was fixed again. The above treatment, on the one hand, simulated the position of infants swaddled with both lower limbs straight after birth, and on the other hand, it allowed the hip and knee joints to slight extension and flexion, which was close to the natural state of humans swaddled with straight legs after birth.
On the 10th day, 10 young rats (half male and half female) were randomly selected from each of the experimental group and control group. The young rats were executed by over anesthesia, and the femoral trochlear specimens were taken. Bone tissue was conducted with HE staining, saffron O-solid green staining and immunohistochemistry. The remaining experimental rats were unswaddled and placed in a clean cabinet of a sterile laminar-flow room, together with the remaining control rats for free movement and feeding. At the end of 8 weeks, the rats were executed by over anesthesia. Then, HE staining of bone tissues was performed, and the patellofemoral joints were scanned by micro-CT (with the knees flexed at 30°). In addition, the angle, width, and depth of the trochlear grooves were measured. The rats were dissected, and knee joint specimens were taken. The bilateral knee joint capsules were opened, and the development of the femoral trochlear and cartilage changes were visually observed and measured.
2.2 Saffron O-solid green staining
The tissues were rinsed under running water for several hours, and they were dehydrated by 70%, 80%, and 90% ethanol solution, pure alcohol, xylene equal mixture for 15min, xylene Ⅰ for 15min, xylene Ⅱ for 15min (until transparent). Then the tissues were put into a mixture of half of xylene and paraffin for 15min, and were put into paraffin Ⅰ, paraffin Ⅱ permeable wax for 50-60min each. The tissues were embedded by paraffin and were sectioned. The paraffin sections were baked, dewaxed, and hydrated. The sections were washed by PBS 3 times for 5 min/time, immersed by solid green staining solution for 5 min, washed with distilled water for 1 min, immersed with Safranin O stain for 10 min, washed with distilled water for 1 min, washed with acetic acid solution for 1 min in order to remove the residual solid green, and washed with distilled water for 1 min. Subsequently, the sections were clamped out and dehydrated with 95% ethanol, anhydrous ethanol rapidly, treated with xylene to transparency, and finally were sealed with optical resin.
2.3 Immunohistochemistry
Immunohistochemistry was used to detect the expression of vascular endothelial growth factor (VEGF) and Collagen X. The paraffin sections were prepared for baking: tissue sections were baked in an oven at 65℃ for 2 h; dewaxing: the sections were placed in xylene for 10 min, replaced with xylene for another 10 min; hydration: the sections were placed in 100% ethanol, 100% ethanol, 95% ethanol, 80% ethanol, and purified water in turn for 5 min each. Then the sections were repaired by placing them in a wet box, adding pepsin, and incubating for 40 min at 37℃, and then rinsed with PBS. Elimination of endogenous peroxidase: To eliminate endogenous peroxidase, the sections were transferred into a wet box and added with freshly prepared 3% hydrogen for 10 min incubation at room temperature, and then washed with PBS. Non-specific block: The sections were washed 3 times with PBS for 5 min each time; blotting paper was used to absorb the PBS around the tissues; 5% BSA was added dropwise to the slides for 30 min at 37℃. Immunoreaction of primary antibody: the blocking solution around the tissues was absorbed with blotting paper without washing, and a sufficient amount of diluted primary antibodies (VEGF (1:200) collagen X (1:100)) were added dropwise to each slide, and the slides were incubated in a wet box overnight at 4℃. Apply secondary antibody: the wet box incubated at 4℃ was taken out at room temperature for 45 min, and the slides were washed with PBS for 3 times with each time for 5 min. Horseradish enzyme-labeled goat anti-rabbit IgG (H + L) (1:100) was dropped onto the slides and after 30 min incubation at 37℃, the slides were rinsed with PBS. Color development and redyeing: DAB was used for color development for 5–10 min and the degree of staining was managed under the microscope. The slides were rinsed by PBS or tap water for 1 min, re-stained with hematoxylin for 3 min, treated with hydrochloric acid alcohol for differentiation and return to the blue; Finally, the slides were rinsed by tap water for 1 min, and treated by dehydration, transparency, sealing, and examined by microscopy.
2.4 HE staining
Paraffin section: the fixed tissue samples were dehydrated by gradient of ethanol solution, put into xylene for transparency, embedded by paraffin, and sectioned. Dewaxing paraffin sections to water: the sections were baked and put into xylene, anhydrous ethanol, alcohol and pure water sequentially. Hematoxylin staining: the sections were soaked by hematoxylin staining solution for 3-5min, washed with running water, soaked with 1% hydrochloric acid alcohol for differentiation, washed with running water, soaked with returned blue solution, and rinsed with running water. Eosin staining: the sections were stained with Eosin staining solution for 3-5min, rinsed with running water. Dewatering and sealing: the sections were put into 80% alcohol, 95% alcohol, anhydrous ethanol, anhydrous ethanol II in order of rapid dehydration, and sealed with ultra-clean high-level sealing gel. Microscopic examination: image acquisition.
2.5 Micro-CT
The Scaner software of Micro CT (version: Skyscan 1276) was used to scan the knee joint of each rat under conditions of voltage 70 kV, current 200 µA, with 10.2 µm scanning resolution and 4032×2688 field of view size (Fig. 2). The width, depth, and angle of the femoral talocalcaneal groove were measured.
2.6 Statistical analysis
The statistical analysis for all data was conducted by SPSS 19.0. The significant differences were tested by t-test, with P < 0.05 as significant difference indicated by *.