Abstract
Summary Statement
We successfully delineated the 3D micro morphology of chondrocytes in patella-patellar tendon using IL-XPCT for the first time. Compared with conventional histology, IL-XPCT can not only provide a higher resolution imgaing but also keep the 3D integrity of the specimen.
Introduction
The morphology of the bone-tendon junction was complex and quite different from other organs, which result the injured bone-tendon junction repair process too slowly. To study the micro morphology of the bone-tendon junction in 3D may have a great significant value to revealing the repair mechanisms of this pathological process and accelerating injured bone-tendon junction repair. However, it was hindered by the convention methods such as histologic section. In our study, a novel imaging tool, synchrotron radiation based in-line x-ray phase contrast imaging (IL-XPCT) was used to research the 3D micro morphology of the bone-tendon junction.
Methods
1) Sample Preparation: 3 patella-patellar tendons was harvested from the knee joint of New Zealand adult rabbits and was immediately fixed, rinsed in water for 2 hours. Dehydration was done using a series of graded ethanol. The sample was cut out for the CCD pixel resolution in sagittal section. 2) Image Acquisition: The IL-XPCT was performed at the BL13W1 of the Shanghai Synchrotron Radiation Facility (SSRF) in China. The CCD pixel resolution was 0.74 μm. Image Acquisition include three steps, such as the the acquisition of tomo projections, CT slices and and 3D reconstruction of patella-patellar tendon on full scale by using VG Studio Max version 2.1. 3) Histological characterization observation: After scanning, the specimen was cut to histologic sectioning and used for morphology staining by safranin O staining and H&E staining. The histological morphology then compared with the IL-XPCT imaging dateset.
Results
(1) The tissue gradations of patella-patellar tendon are clearly detected by IL-XPCT. (2) The 3D reconstruction image of patella-patellar tendon sample were largely match with the histological morphology stained by safranin O and H&E in sagittal view. (3) After the image segmentation, the 3D micro morphology of the bone-tendon junction could be vividly visualised in multi-angles. Through manipulate threshold of the 3D image, we can successfully obtained the 3D morphology of the chondrocyte, and the smallest diameter is approximately 5μm.
Discussion & Conclusion
In the present study, we successfully delineated the 3D micro morphological features of chondrocytes in normal patella-patellar tendon using SR-based IL-XPCT for the first time. Compared with conventional histology, IL-XPCT can not only provide a higher resolution ratio without distortion but also keep the three-dimensional integrity of the specimen. Above all, IL-XPCT opens access to a new dimension in the morphological investigation of bone-tendon junction tissues, giving important complementary information to the conventional morphological analyses in view of the three-dimensional composition of bone-tendon junction tissues, On the other hand, it could be helpful to understanding the repair processes of bone-tendon junction injury and promoting the injured bone-tendon junction repair fast and high quality.
