Intermittently Administered Parathyroid Hormone [1–34] Promotes Tendon-Bone Healing in a Rat Model

The objective of this study was to investigate whether intermittent administration of parathyroid hormone [1–34] (PTH[1–34]) promotes tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in vivo. A rat model of ACL reconstruction with autograft was established at the left hind leg. Every day, injections of 60 μg PTH[1–34]/kg subcutaneously were given to the PTH group rats (n = 10) for four weeks, and the controls (n = 10) received saline. The tendon-bone healing process was evaluated by micro-CT, biomechanical test, histological and immunohistochemical analyses. The effects of PTH[1–34] on serum chemistry, bone microarchitecture and expression of the PTH receptor (PTH1R) and osteocalcin were determined. Administration of PTH[1–34] significantly increased serum levels of calcium, alkaline phosphatase (AP), osteocalcin and tartrate-resistant acid phosphatase (TRAP). The expression of PTH1R on both osteocytes and chondrocyte-like cells at the tendon-bone interface was increased in the PTH group. PTH[1–34] also enhanced the thickness and microarchitecture of trabecular bone according to the micro-CT analysis. The results imply that systematically intermittent administration of PTH[1–34] promotes tendon-bone healing at an early stage via up-regulated PTH1R. This method may enable a new strategy for the promotion of tendon-bone healing after ACL reconstruction.


Introduction
The anterior cruciate ligament (ACL) is an important intra-articular ligament in maintaining the stability and normal motion of the knee joint. ACL injury is a common knee-related injury in the orthopedic surgery department, which influences sport activities, especially in young athletes. It was estimated that over 100,000 ACL reconstructions were performed in the United States each year [1]. ACL reconstruction is the golden standard for the treatment of ACL injury, because of its positive results. Due to reduced donor-site complications compared to bone-patellar tendon-bone graft, the hamstring tendon grafts have been widely used in ACL reconstructions [2][3][4], which are inserted into bone tunnels in the tibia and femur and are anchored at each end by a variety of methods. However, the healing process after ACL reconstruction with hamstring tendon graft, which takes nearly 9 to 12 months, is significantly slower than bone-patellar tendon healing [5][6][7].
ACL reconstruction creates a new tendon-bone interface along the bone tunnel. A successful surgery largely depends on the biological healing at the interface between the tendon graft and the bone tunnel. The fixation site of the tendon within the bone tunnels is the weakest point in the reconstructed knee [8]. Therefore, how to improve tendon-bone healing for functional exercises, daily activities, even sports as early as possible has already become the focus of the field of study. The tendon-bone junction heals slowly due to the bone loss at the site of injury and the avascularity of the fibrocartilage zone [9]. The native direct tendon-bone interface with four typical tissue zones is not reformed during healing, but rather a structurally and mechanically inferior interface [10]. This deficit of microarchitecture in the insertion site raises the subsequent risk of failure after reconstruction surgery [11]. During the past several years, strategies to accelerate and promote tendon-bone healing have been studied in the field of orthopedic basic science.
Parathyroid hormone (PTH) is an anabolic endocrine regulator of calcium and phosphorus homeostasis, and its actions on osteocytes and chondrocytes are mediated by the PTH receptor (PTH1R) [12][13][14]. PTH can activate bone lining cells, prevent osteoblast apoptosis and enhance bone resorption in vivo [15]. Recombinant human PTH , or teriparatide, was approved by the FDA in 2002 for the therapy of osteoporosis in men with high risk of fracture and postmenopausal women [16]. Intermittent PTH  injections greatly stimulate bone formation and resorption simultaneously, with a greater effect on bone formation than resorption, leading to a net bone gain [17]. PTH  demonstrates its effects on bone formation by increasing bone volume, connectivity and microarchitecture [18,19]. Evidence from animal model studies has shown that factors that positively impact bone formation and fracture healing, such as osteoinductive growth factors (bone morphogenetic protein (BMP) [20], transforming growth factor (TGF) [21], granulocyte colony-stimulating factor (G-CSF) [22]), osteoconductive materials (injectable calcium phosphate [23], magnesium-based bone adhesives [11]) and low-intensity pulsed ultrasound [24], also positively affect tendon-bone healing [25]. Several studies suggested that intermittently administered PTH  can promote the remolding and rebuilding process of fracture healing [26,27]. However, its effect on the tendon-bone healing process after ACL reconstruction has not been evaluated.
In this study, we attempted to determine whether PTH  could be used to improve tendon-bone healing after ACL reconstruction. Firstly, an ACL reconstruction animal model with autograft was established. PTH  was intermittently administered after surgery for four weeks. We found that daily injections of PTH  could improve tendon-bone healing, which was proven by micro-CT, biomechanical test and histological assessment.

Serum Chemistry
After four weeks of PTH  administration, serum levels for calcium, alkaline phosphatase (AP), osteocalcin and tartrate-resistant acid phosphatase (TRAP) were significantly increased in the PTH group compared with the controls (Table 1). The averages of the bone tunnel area in the PTH group were significantly smaller than those in the control group (PTH, 2.35 ± 0.38 mm 2 vs. control, 2.88 ± 0.32 mm 2 ; p = 0.043, n = 5). Compared to controls, PTH  application significantly increased the bone mineral density (BMD), bone volume fraction (BV/TV), bone surface/volume ratio (BS/BV) and trabecular thickness (Tb.Th) of the area we selected. A significant decrease of trabecular separation (Tb.Sp) in the PTH group was observed. No differences in bone surface density (BS/TV) and trabecular number (Tb.N) were detected between the two groups ( Table 2).

Biomechanical Test
As the evaluation of failure load, all grafts in both groups failed by total pullout from the bone tunnels. The average of failure load in the PTH group was significantly greater than that in the control group at four weeks after operation (PTH, 16.49 ± 4.90 N vs. control, 8.17 ± 0.59 N; p = 0.019, n = 5). There was no significant difference in stiffness between the two groups (PTH, 4.32 ± 1.95 N/mm vs. control, 2.48 ± 0.47 N/mm; p = 0.102, n = 5; Table 3). In the two groups, the fibrous connective tissue was aligned, and the tendon-bone interface was covered by the woven bone. In the PTH group, there were more aligned chondrocyte-like cells than in the control group. At the interface of the control group, chondrocyte-like cells were barely observed ( Figure 1). Staining with Safranin O showed larger collection of cartilage cells and more abundant proteoglycan production at the tendon-bone interface in the PTH group than in the control group. However, these cartilage cells were immature, and the typical four histological transitions were not observed ( Figure 2).

Immunohistochemistry Staining
After administration of PTH  for four weeks, a consecutive up-regulation of PTH1R expression on both osteocytes in the trabecular bone and chondrocyte-like cells at the tendon-bone interface was observed. Immunoreactivity to osteocalcin of the PTH group was significantly higher than that of the control group ( Figure 3).

Discussion
In the present study, we elucidated the effectiveness of PTH , which was intermittently administered after operation for early tendon-bone healing in ACL reconstruction using a rat model. Radiological, biomechanical and histological assessment at the site of the tendon-bone interface demonstrated the promoted healing in the PTH group. We noted that application of PTH  directly affected the cells mediating tendon-bone healing, as PTH1R expression was significantly up-regulated at the tendon-bone interface. The study demonstrated a therapeutic strategy using PTH  for tendon-bone healing after ACL reconstruction. The dosages of PTH  are various among different experimental investigations [28]. In our study, 60 μg PTH[1-34]/kg/day was chosen, because it was the median among several investigations in which bone formation was improved in a rat model [29,30]. Intermittent and short-term administration of PTH  could favor bone formation in animals [31] and human [32], and high levels of dosage induced a significant increase in serum calcium and AP concentration [33]. PTH not only strongly stimulates bone formation during the bone remodeling process, but also promotes bone resorption [34]. In our present study, a significant increase in the serum concentration of osteocalcin and TRAP was observed.
PTH  increases BMD, BV/TV and connectivity, enhancing bone strength and reducing the fracture risk in the animal models and human patients [28,32,35]. Bellido et al. [36] found that PTH  increased the volume and thickness of cortical and trabecular bone through evaluation of bone microarchitecture by micro-CT. These results fit in with the present study in which PTH  exerted its effects on BMD, BV/TV, BS/TV and Tb.Sp at the tendon-bone interface evaluated by micro-CT. Remarkably, the impact on the bone microarchitecture was accompanied by the increase in serum levels of calcium, AP, osteocalcin and TRAP. Accordingly, much more chondrocyte-like cells were observed at the tendon-bone interface in the PTH group than in the control group under microscopy. Solid tendon-bone healing is a prerequisite for successful ACL reconstruction [37]. In our present study, the failure load in the PTH group was larger than that in the control group, but there was no significant difference in stiffness between the two groups. This was because the remolding and rebuilding of the tendon-bone interface was just at an early phase when failures were generally via graft pullout [38]. The chondral tendon-bone healing was observed more in the PTH group, but incompletely matured chondral healing could not strengthen the biomechanical properties effectively [39].
The cell types that initiate and regulate the tendon-bone healing process have not been concretely identified. It has been reported that in the first two weeks following ACL reconstruction, graft cells do not survive and the graft undergoes necrosis [40]. It seems that the host cells from the surrounding bone marrow, which contains bone mesenchymal stem cells (BMSCs) rather than graft cells, contribute to the repair of the tendon-bone interface [40,41]. Several studies suggested that MSCs could promote tendon-bone healing after ACL reconstruction. Lim et al. [42] used hamstring tendon autograft coated with MSCs to reconstruct ACL in adult rabbits and found that MSC-enhanced grafts had significantly higher failure loads and stiffness at eight weeks after surgery. Soon et al. [43] found that the application of MSCs at the allograft tendon-bone interface during ACL reconstruction could develop an intervening zone of fibrocartilage. PTH1R is the only receptor for PTH in bone tissue and is expressed in osteoblast lineage cells, including BMSCs [44], osteoblasts [45] and osteocytes [46]. During the tendon-bone healing process, PTH could bind to PTH1R in MSCs and osteoblasts and accelerate the tendon-bone healing. Chen et al. [47] demonstrated that PTH could increase the fluorescence intensity of calcium ions and the proliferation of MSC in vitro. Alternatively, PTH could affect the tendon-bone healing process through regulating the secretion of certain factors from MSCs, osteocytes or osteoblasts. The significant up-regulation of PTH1R on osteocytes and chondrocytes at the tendon-bone interface indicated that systemic application of PTH  was capable of directly stimulating these cells. Chondrocytes and osteocytes express PTH1R [12,14], and binding of PTH  to the receptor may enhance the expression and production of growth factors [48,49]. In our present study, the coexistent increase in BMD, BV/TV and the improvement of tendon-bone healing most likely is ascribed to the up-regulated PTH1R expression on osteocytes and chondrocyte-like cells. Osteocalcin is a marker of terminal osteoblast differentiation and influences bone mineralization. In the PTH group, higher osteocalcin density indicated the positive effectiveness of PTH on osteogenesis. However, more studies are needed to make the underlying mechanisms clear.
There are some limitations in our study. Our specimens were harvested at only one time point (four weeks) postoperatively. The long-term effect of PTH on tendon-bone healing could not be observed. In a future study, the long-term effect of the administration of PTH will be investigated.

Study Design
Twenty male Sprague Dawley rats (12 weeks of age) were used in this study. Animals were allocated to 2 equal numbered groups randomly (control group and PTH group), and surgery was performed. Animals of the control group and PTH group received vehicle and recombinant PTH  (60 μg/kg/day, Bachem, Bubendorf, Switzerland), respectively, daily from Day 1 to 4 weeks postoperatively. At 4 weeks postoperatively, all animals were euthanized, and the blood and tibia-tendon-femur complex of the left hind leg were harvested for analyses. The study protocol was approved by the Zhejiang University Institutional Animal Care and Use Committee (Register ID No.: ZJU2014032006; Date: 6 May 2014).

Surgical Procedure
A unilateral rat ACL reconstruction model was adopted as described previously [41] and slightly modified [50]. The surgical procedure was performed under strictly aseptic conditions. After general anesthesia with an intraperitoneal injection of pentobarbital sodium solution (Kyoritsu-seiyaku, 50 mg/kg body weight), a 2-cm skin incision was made just posterior to the ankle and ending at the medial and palmar side of the first metatarsal at the right hind leg. The flexor digitorum longus tendon was identified and cut transversely at the midmetatarsal level. The full length of the tendon was about 4 cm after being stripped from the posterior tibia, and then, both ends were sutured using 4-0 Ethibond (Ethicon, Somerville, NJ, USA). A 2-cm longitudinal skin incision was used to expose the left knee joint. The patella was dislocated medially, and then, the native ACL was excised. The tibial and femoral bone tunnels were created with a 1.2-mm diameter Kirschner wire (K-wire; Zimmer, Warswa, IN, USA). The full length of the bone tunnel from anteromedial tibia, across the joint, to the anterolateral femur was about 20 mm. The flexor digitorum longus tendon was passed through the tunnel, and both ends were sutured to the periosteum and surrounding soft tissue using the 4-0 Ethibond suture (Figure 4). The animals were then returned to their cages and allowed to move freely.

Serum Chemistry
Blood was collected from aorta abdominalis at the time of euthanization. Serum was separated with centrifugation at 3000 r/min for 10 min. Serum levels of calcium and alkaline phosphatase (AP) were analyzed by the automatic biochemical analyzer (Beckman Coulter Au5400, Beckman Coulter Inc., Brea, CA, USA). Serum osteocalcin and TRAP level were analyzed by a Rat Osteocalcin ELISA Kit (Rapidbio Inc., Los Angeles, CA, USA) and a Rat TRAP ELISA Kit (Rapidbio).

Micro-CT
The left knee joints (n = 5 for each group) were collected at 4 weeks postoperatively and kept at −80 °C immediately. Before testing, the specimens were thawed overnight at 4 °C. Gross observation and radiographs were used to determine the mineralized tissue formation in bone tunnels and the areas of the vertical plane of the axis of the bone tunnel at 3-mm inferior to the tibial joint surface (micro-CT, 36-μm thickness; Skyscan1176, BRUKER, Antwerp, Belgium). Each area was measured an average of three times with Image-Pro Plus 6.0 software (

Biomechanical Test
After micro-CT assessment, the biomechanical test was performed (n = 5 for each group). All soft tissue, except for the ACL graft, was dissected to create a femur-graft-tibia complex. The tibia and femur were embedded in 15 mL centrifugal tubes with dental cement. Additionally, the two ends of the complex were fixed in the tailor-made cylinder with screws. The cylinders were clamped in an Instron 553A material testing system (Instron, Boston, MA, USA) at 45° flexion. The biomechanical test was performed by increasing the tensile load with a crosshead speed of 5 mm/min. The failure load (N) and elongation were recorded, and the stiffness (N/mm) was calculated from the slop of the load-deformation curve. The specimens were kept always moist with normal saline (NS).

Histological and Immunohistochemical Analyses
The specimens (n = 5 for each group) were harvested and fixed in 4% paraformaldehyde for 48 h and then proceeded to decalcification in 10% ethylenediaminetetraacetic acid (EDTA) with 0.1 M phosphate-buffered saline (PBS) for 3 weeks. After the dehydration with a graded alcohol solution and being embedded in paraffin wax, the specimens were sectioned parallel to the longitudinal axis of the bone tunnel. Sections were cut at 5 μm and stained with hematoxylin and eosin (H&E staining) for traditional light microscopy. Safranin O staining was also performed to observe the fibrocartilage formation in the tendon-bone interface with light microscopy.
Prior to application of primary antibodies, the Ultra V Block (Lab Vision Corporation, Fremont, CA, USA) was used to incubate in sections to reduce the non-specific background staining. For immunohistochemistry staining, monoclonal antibodies for PTH1R (Abcam, Cambridge, UK) and osteocalcin (Abcam) were applied for 12 h at 4 °C. The incubation with biotinylated goat anti-rabbit antibody (Lab Vision Corporation) for 1 h at room temperature was followed. Streptavidin peroxidase was applied for 45 min, and 3,3'-diaminobenzidine was added as a chromogenic agent. Counterstaining was done with hematoxylin, and the negative control was set by PBS instead of the primary antibodies.
For the evaluation of PTH1R immunostaining, histological section images taken at 200-fold were digitalized, and five standardized regions of interest (ROIs) were defined at the trabecular bone and tendon-bone interface. PTH1R expression was determined as the percentage of positive stained cells in the standardized ROIs. Immunoreactivity to osteocalcin in the tendon-bone interface of PTH group was compared with that of the control group. The ROI was defined as the area of the whole image selected. The integrated option density (IOD) and the density mean (IOD/ROI) were analyzed by the Image-Pro Plus 6.0 software (IPP 6.0)

Statistical Analysis
The data were expressed as the mean ± standard deviation (SD). All data were analyzed using SPSS 16.0 software (SPSS, Chicago, IL, USA), and p < 0.05 was considered statistically significant. An independent t-test or nonparametric Wilcoxon-Mann-Whiney test was used to detect the difference between the two groups.

Conclusions
In conclusion, the systematically intermittent administration of PTH  promotes tendon-bone healing at an early stage. This method may enable a new strategy for the promotion of tendon-bone healing after ACL reconstruction.

Author Contributions
Conceived and designed the experiment: Shigui Yan; Performed the experiments: Fanggang Bi, Zhongli Shi, Shuai Jiang and Peng Guo; Analyzed the data: Fanggang Bi; Contributed reagents/materials/analysis tools: Fanggang Bi and Zhongli Shi; Wrote the manuscript: Fanggang Bi.

Conflicts of Interest
The authors declare no conflict of interest.