VEGF and bFGF expression and histological characteristics of the bone-tendon junction during acute injury healing.

Author:Wang, Lin
Position::Research article - Report
 
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Introduction

Chronic bone-tendon junction (BTJ) injury is common in athletes and some professional workers (Torkki et al., 2002). The major causes of chronic BTJ injury include repeated micro-trauma during exercise and delayed healing of an acute injury (Qin et al., 2006; Wang et al., 2008). The injury may affecte an athlete's routine training or result in an employee's work loss (Ergen, 2004; Fordham et al., 2004). Many researchers have established BTJ injury animal models for studying tendinosis or tendinopathy based on cyclical loading (Nakama et al., 2005; Wang et al., 2012), tendon enthesis section (Lu et al., 2006; Soeki et al., 2000; Sonnabend et al., 2010), acute exercise (Solem et al., 2011), and injection of chemical agents (Hsu et al., 2004; Vinores et al., 2001). Although these models have provided a great deal of information regarding BTJ injury healing and treatment, a reproducible model that can be easily established is still needed for the study of BTJ injury. In cases of BTJ injury caused by acute injury, such as a micro-tear or a laceration of the BTJ, the overuse or delayed healing of the injury results in minor acute injuries accumulating (Riley, 2008). The poor treatment outcome may result from the poor regeneration capacity of the BTJ (Hamilton and Purdam, 2004; Wang et al., 2005) and the lack of understanding of the acute BTJ injury healing characteristics.

During the BTJ injury healing, the basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were two important growth factors that affected injury healing (Lu et al., 2008; Thomopoulos et al., 2005; Wurgler-Hauri et al., 2007). The bFGF was found to significantly increase the cell density, collagen diameter, stiffness and loading capacity in the early stage of tenotomy, but no significant improvement in the biomechanical properties was reported (Chan et al., 2000). The increased VEGF expression that is associated with the acute injury caused inflammation, tissue metabolism promotion and hypoxia (Wang et al., 2007). It contributed to the healing of acute BTJ injury, but high VEGF expression levels were also found in injuries from BTJ overuse (Pufe and Petersen, 2005). Therefore, the high expression levels of bFGF and VEGF had different roles in the different stages of injury healing. In this study, a simple and repeatable BTJ injury model was established by using 7 plum-blossom needles to vertically puncture the patella-patellar tendon junction. Post injury time points of 1, 2, 4, and 8 weeks were observed to histologically and immunohistologically investigate the natural healing process. The aim of this study was to reveal the BTJ acute injury healing characteristics and provide theory for the post injury training management.

Methods

Thirty-three female rabbits (aged 18 weeks, 2.8 [+ or -] 0.24 kg) were divided into post-injury groups at 1 (ND1W, n = 6), 2 (ND2W, n = 6), 4 (ND4W, n = 7), and 8 weeks (ND8W, n = 7) and a normal control group (CON, n = 7). This experiment only used the right hindlimbs of the rabbits; the left limbs were used in another study (Wang et al., 2012). Under general anesthesia with 0.25% pentobarbital sodium (2 ml/kg, intraperitoneal injection) (Sigma Chemicals Co., St Louis, MO, USA), the patellar tendon enthesis (TE) area was damaged using 7 plum-blossom needles (0.1 mm diameter) via vertical puncture (Figure 1). The CON group had no injury. The animals were free in their cages and were provided with standard rabbit chow and water ad libitum. Animal research ethics approval was obtained from the China Agricultural University (ref: CUA4345/03M). The animals were euthanized with a 25% sodium pentobarbital overdose at weeks 1, 2, 4, and 8 post-injury. The patella-patellar tendon (PPT) complex of the knee was then harvested for the histological and immunohistological evaluations.

[FIGURE 1 OMITTED]

Evaluation

Histological evaluation

The harvested PPT complexes were decalcified and embedded in paraffin. Subsequently, 5-[micro]m-thick sections from the mid-sagittal plane of the PPT complex were stained with Safranin O to examine the BTJ proteoglycan profile and with hematoxylin & eosin (H&E) to examine the general morphology; the latter analysis included an assessment of the tendon collagen fibers under a polarized microscope (Nikon Eclipse 50i, Nikon Inc., Japan).

Quantitative evaluation of the tendon cell density and the thickness of the fibrocartilage zone

The tendon cell density and fibrocartilage zone thickness (FZT) are measured using our established protocols (Fu et al., 2008; Wang et al., 2008). Briefly, the cell density was calculated by counting the number of cells in 5 random standardized rectangular fields (100 x 100 [micro]m) along the junction of the calcified and uncalcified fibrocartilage within the H&E sections. The FZT of the sagittal sections was calculated by dividing the sectional area of the fibrocartilage zone by the corresponding length. All of the quantitative evaluations were performed at a magnification of 100 using an image analysis system (Metemorph 7.1).

Immunohistochemistry

For the immunostaining of the VEGF and bFGF protein, mouse anti-VEGF (Beijing ZhongYuan Ltd. (Abcam agency), Beijing, China) and anti-bFGF (Beijing biosynthesis biotechnology CO., LTD, Beijing, China) were used. Immunohistochemistry was performed according to the manufacturers' instructions. Briefly, 5 [micro]m sections were mounted on slides coated with a poly-L-lysine solution (Beijing Zhong Shan-Golden Bridge Biological Technology CO., LTD, Beijing, China). These sections were dewaxed and rehydrated before immersion in 3% hydrogen peroxide (to block the endogenous peroxidases) followed by rinsing in phosphate buffered saline (PBS). The sections were then digested with 0.1% trypsin for 30 min at 37[degrees]C and washed in PBS. Non-specific sites were saturated with normal goat serum (Beijing Zhong Shan Golden Bridge Biological Technology CO., LTD, Beijing, China) for 10 min at room temperature. The sections were incubated with the specific antisera 1 (VEGF: rat, anti-rabbit 1:50; Abcam, Cambridge, UK; bFGF: rabbit, anti- rabbit, 1:300; Biosynthesis biotechnology CO., LTD,...

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