The Novel Study By MLRS And SRμCT Methods For Rabbit Bone-Tendon Junction Structure And Assessing The Effect Of LIPUS On The Healing By MLRS

OBJECTIVE(1) A new method was developed to evaluate the healing process in the model of rabbit patellar-patellar tendon (PPT) junction injury using micro Laser Raman Spectrometer (MLRS), and proceeded to be observed the therapeutic effects of low intensity pulsed ultrasound (LIPUS) application on the rabbit injured PPT model.(2) High-resolution Synchrotron Radiation micro Computed Tomography (SRμCT) technique was firstly used to examine the3D morphological characteristics of the rabbit patellar-patellar tendon junction.METHODS(1) MLRS detection:A total of44healthy bone matured male New Zealand rabbits were employed in this experiment.40animals were subjected to partially patellectomy as the PPT injury model, followed by partitions into LIPUS treatment group (Gl) and Contral group (G2), and the other4animals as sham group (G3) without operation. G1was under the LIPUS therapy for20minutes a day postinjury, and the G2was operated similarly except for the treatment simulation without LIPUS. At the time of1/2/4/8/16weeks postinjury, all the PPT samples were harvested.The specimen was sectioned crossing the median sagittal plane. The MLRS was used to scan the PPT sections along the X-axis orientation which was set as the longest line of the new bone. Several parameters including the intensity of calcium, collagen peak, the calcified matrix ratio, the length of new bone, calcified fibrocartilage and uncalcified fibrocartilage were calculated for the comparatively analysis of the organic matrix content and the mineralization degree.(2) SRμCT imaging:The normal rabbit PPT samples were subjected to the formalin fixation and alcohol dehydration, followed by the SRμCT scanning in the SSRF. An image post-processing procedure of tomo projecting, slice reconstruction and3D image rendering were sequentially performed to obtain the best3D digital images, and to characterize the morphology of the3D PPT structure in rabbit.RESULTS(1)MLRS results:(1.1) The normal rabbit PPT results:The rabbit PPT along the X-axis orientation was sectioned into four typical areas including the subchondral bone (SCB) zone, calcified fibrocartilage (CFC) zone, uncalcified fibrocartilage (UCFC) zone and the tendon (TF) zone. The intensities of the calcium peak characterized as960Acm-1in these four zones were respectively1280cnt,480cnt,0cnt and0cnt, and the intensities of the collagen peak (2940△cm-1) were1300cnt,1100cnt,1700cnt and900cnt in sequence. The ratio of calcification/collagen matrix intensity was respectively0.985,0.44,0and0. The mineralization contents were homogeneously distributed in the SCB area. The mineralization substances gradually disappeared from the CFC toward to the UCFC zone. And that, there was no mineralization distribution in the TF zone. Similarly, the collagen matrix was homogeneously distributed in the TF zone. However, the matrix contents were significantly lower than that in UCFC zone. The length of the CFC,UCFC and the total fibrocartilage in normal rabbit PPT was1006.25±112.50μm,1037.50±192.03μm and2043.75±272.62μm.(1.2) the results of the rabbit PPT in healing process:At the time of1week postinjury, new calcification layer and new UCFC appeared between the residual patella and patellar tendon; At the time of2weeks, new calcification layer could be divided into NB and CFC;The length of NB, CFC and UCFC gradually increased with the healing time progress, in which the CFC restored approximately62%close to the normal sample, and35%in the UCFC at the time of16weeks postinjury.(1.3) The therapeutic effects of LIPUS on the healing process of the injured rabbit PPTCompared with the control group, the length of CFC, UCFC, CFC+UCFC, NB+CFC and NB+CFC+UCFC were significantly increased in the LIPUS treatment group at the time of8and16weeks postmjury.(2) SRμCT resultsThe3D image of the rabbit PPT was successfully rendered by SRuCT, The best imaging parameters were first established, such as spatial resolution (0.74μm), imaging distance (2.5cm), in which the four-layer structures including the SCB, CFC, UCFC and TF were clearly differentiated. Many cartilage cells with7-10μm diameter were linearly bedded in the fibrocartilage zone.CONCLUSIONS(1) MLRS was a useful method for the detection of the mineralization degree and organic matrix content in the healing process of the rabbit PPT injury.(2) LIPUS therapy significantly promoted the healing progress in the rabbit PPT injury model.(3) SRμCT allowed the high-resolution imaging of the rabbit PPT microscopic3D structure, and proceeded to evaluate the3D morphological characteristics in the PPT healing interface.