Despite generally favorable results with mini-open and arthroscopic rotator cuff repairs, both have relatively high rates of failure to heal (13–93%) when evaluated using MRI and ultrasound.1, 2, 3 A recent study found that only 69% of repairs in the mini-open rotator cuff repair group and 53% of repairs in the arthroscopic group remained intact. Shoulders with intact rotator cuff repairs had greater strength of forward elevation and external rotation.2
Successful tendon repair depends on secure tendon-to-bone healing. The native insertion site is a highly specialized transition zone that functions in the transmission of mechanical load from soft tissue to bone. The normal insertion site consists of four distinct regions: tendon, unmineralized fibrocartilage, mineralized fibrocartilage, and bone. The structure and composition of the native insertion site is not reformed following rotator cuff tendon-to-bone repair.
Recombinant human parathyroid hormone (rhPTH) has been shown in multiple studies to accelerate bone healing in both osteoporotic and non-osteoporotic animal and human studies.4, 5, 6 In addition to being osteogenic, recent studies have also shown that rhPTH is chondrogenic by increasing chondrocyte recruitment and rate of differentiation.7, 8, 9, 10 For example, rhPTH increases cartilaginous growth in the avian sternum, and increases hyaline cartilage regeneration in animal models.11, 12 Other studies have shown an increase in chondrogenesis relative to osteogenesis.7, 13
As a result of rhPTH being both osteogenic and chondrogenic, it has the potential to positively affect both aspects of healing at the tendon-to-bone interface. We hypothesized that rhPTH would improve tendon-to-bone healing in a rat rotator cuff tendon repair model.
After obtaining approval from the Institutional Animal Care and Use Committee, 114 male Sprague Dawley rats were obtained with a mean preoperative weight of 282.6 g. Each animal underwent detachment and immediate repair of the right supraspinatus tendon using bone tunnel suture fixation. Fifty seven rats underwent repair alone and 57 rats received daily subcutaneous injections of 10 µg/kg of Forteo (rhPTH, Eli Lilly, Indianapolis, IN) beginning on the date of surgery. Post-operatively the rats were housed in pairs and allowed ad lib activity. Four rats in each group were sacrificed at 3, 7, 14, 28, and 56 days for histologic analysis. Twelve rats in each group were sacrificed at 14 and 28 days for biomechanical testing, three of which also underwent micro CT analysis. In the 56-day group, 13 rats underwent biomechanical testing.
The rats were anesthetized with 100 mg/ml of ketamine and 20 mg/ml of xylazine administered intraperitoneally into the right lower abdominal quadrant. In seven rats, anesthesia was supplemented by administration of 2% isoflourane via nose cone. A deltoid splitting incision was made and the acromioclavicular joint divided to visualize the rotator cuff. The supraspinatus tendon was identified, and a curved clamp passed underneath. The tendon was then sharply dissected off the greater tuberosity. The tuberosity was debrided of all soft tissue, and a 3-0 Ethibond suture (Ethicon, Piscataway, NJ) was passed through the supraspinatus tendon in Mason–Allen fashion. Bone tunnels were drilled using a 21-gauge needle into the greater tuberosity. The suture ends were passed through the bone tunnels and tied over the humeral cortex, repairing the supraspinatus to its native footprint. The deltoid split was re-approximated with 3-0 Ethibond (Ethicon, Piscataway, NJ), and the skin was closed with 3-0 Vicryl (Ethicon, Piscataway, NJ) subcutaneous suture. Cyanoacrylate skin adhesive (Nexaband, Abbott Laboratories, North Chicago, IL) was applied to further protect the wound. Buprenorphine 0.05 mg/kg was administered subcutaneously for post-operative pain control.
The rats were assigned to either the rhPTH or control group pre-operatively (57 animals per group). The rhPTH dose (10 mcg/kg) was chosen based on previous studies14 as being the lowest effective dose, similar to the dose taken by humans. The animals in the experimental group were given daily subcutaneous injections beginning on the day of surgery.
Each specimen was examined visually to assess for tendon detachment and any gross differences between groups.
The specimens allocated for histological analysis were fixed in 10% neutral buffered formalin for 48 h. After fixation, tissues were decalcified in Immunocal (Decal Chemical Corporation, Tallman, NY) and washed in a phosphate buffered solution. The tissues were then dehydrated and embedded in paraffin. Five-micrometer thick sections were cut in the coronal plane. The specimens were evaluated in a blinded fashion by three investigators under polarized and non-polarized light microscopy.
Immunohistochemistry was used to identify type-I collagen expressing cells and blood vessels within the tendon–bone interface and the midsubstance of the supraspinatus tendon. Hydrated serial sections were treated with 3% H2O2 to quench endogenous peroxidase activity. Non-specific antibody binding was blocked with serum-free protein block (Dako, Carpinteria, CA). Primary antibodies against rat type-I procollagen were applied to serial sections overnight at room temperature. Primary antibodies against rat Factor VIII were applied for 1 h at room temperature. Bound antibodies were visualized by employing a goat avidin–biotin peroxidase system with 3,3′-diaminobenzidine (DAB; Dako, Carpinteria, CA) as a chromagen.
The area of fibrocartilage tissue at the tendon insertion site was analyzed using safranin O-stained sections at 4× magnification. Safranin O stains sulfated proteoglycans which are found in the fibrocartilage transition zone between tendon and bone. The area of fibrocartilage was carefully outlined and measured using Image J software (NIH, Bethesda, MD). The measured areas were averaged between the four samples for treated animals and controls at each time point.
Picrosirius red staining was used for semi-quantitative analysis of collagen content as described previously.15, 16, 17 To evaluate the organization of collagenous tissue at the healing tendon bone interface, sections were stained with picrosirius red and illuminated with monochromatic polarized light. Measurements were obtained by rotating the polarization plane until maximum brightness was obtained to control for variations in specimen orientation on the slide. To facilitate comparisons between groups, all tissues were embedded and cut in exactly the same orientation, and sections were cut to a uniform thickness. The images were obtained with a Nikon Eclipse E800 light microscope (Melville, NY) which was interfaced to a CCD video camera mounted on an eyepiece tube. For all specimens, the images were taken under identical conditions of illumination during a single sitting. The images then underwent 8-bit digitization using Image J software (NIH, Bethesda, MD) with a resolution of 640 × 480 pixels, yielding an image in which non-collagenous tissue was dark and collagen was depicted by gray scale from 1 to 255. Ten rectangular areas (50 × 50 µm) were measured and the measurement of gray scale was performed using the Image J software. The values from the 10 fields examined were then averaged to obtain a value of average ‘brightness’ per specimen, with increased brightness interpreted as more organized collagen.
Type-I procollagen expression was quantified in the healing tendon. Images centered over the area of insertion were taken at 100× magnification and imported into Photoshop (Photoshop CS2 2005, Adobe Systems Inc., San Jose, CA). A standard transparent grid of seven (1000 µm2) squares were layered onto the digital image and oriented over the area of insertion (Fig. 1). Cells staining positively for the expression of procollagen that were located within the squares or crossed by their border were counted. Cells were counted in a blinded manner, by three of the authors independently, and the numbers averaged.
Tartrate-resistant acid phosphatase (TRAP) expression at the tendon–bone interface was measured to evaluate the number of osteoclasts at the area of insertion and within the subchondral bone. Digital images were obtained at 100× magnification centered at the supraspinatus tendon insertion. The number of TRAP positive cells was determined within each 100× microscope field. Cells were counted in a blinded manner, by three of the authors independently, and the numbers averaged.
Factor VIII staining was used to evaluate angiogenesis within the supraspinatus tendon and tendon–bone interface. Microscope field images at 40× magnification were centered over the area of insertion and over the midsubstance of the supraspinatus tendon. The vessels within these areas were counted and the number of vessels per mm2 was calculated. Vessels were counted in a blinded manner, by three of the authors independently, and the numbers averaged.
Each rat shoulder was kept in a −80°C freezer until biomechanical testing was performed. The specimen was thawed at room temperature and the humerus with attached supraspinatus was carefully dissected from surrounding tissues. The supraspinatus muscle was then bluntly removed from the supraspinatus tendon. The cross sectional area of the supraspinatus tendon was measured using a digital micrometer in the midsubstance of the tendon. The specimen was placed into a Materials Testing System (Eden Prairie, MN) with a 45 N load cell which was aligned to allow uni-axial tensile testing. The end of the tendon was secured in a screw grip using sandpaper and ethyl cyanoacrylate (Krazy Glue, Elmer's Products, Inc, Columbus, OH). The humerus was then placed in a custom-designed vice grip that prevented fracture through the humeral physis. The specimen was preloaded to 0.10 N and then loaded to failure at a rate of 14 µm/s, corresponding to approximately 0.4% strain. The load-to-failure data were recorded and stiffness calculated from the linear portion of the load–deformation curves using Sigma Plot 8.0 (SPSS Inc, Chicago, IL). The site of graft failure (pull-out from bone versus mid-substance rupture) was recorded.
Bone density and new bone formation at the tendon insertion site on the greater tuberosity was assessed with micro-computed tomography (micro-CT) (eXplore Locus SP, GE Healthcare, London, Ontario, Canada). Micro-CT was done immediately after sacrifice, with the specimens in formalin for fixation. Each sample was placed in the holder surrounded by formalin and scanned at 80 V and 80 mA. The scans included a phantom containing air, saline, and a bone reference material for calibration of Hounsfield units to tissue mineral density. The images were thresholded to distinguish bone voxels using a global threshold for each specimen. After thresholding, the total bone mineral content, bone volume fraction (BV/TV), and mineral distribution was calculated for a volume of interest (VOI) at the greater tuberosity. The BV is the total number of thresholded bone voxels within the total volume (TV) of the VOI.
Independent sample Student's t-test was used to compare load-to-failure, tendon cross sectional area, and the quantitative histology. Statistical analyses were performed using SPSS 16.0 (Chicago, IL), with consultation of a statistician.
There were no gross failures in either group.
At 14 days, the controls had a significantly higher load to failure and stiffness than the rhPTH specimens (p < 0.05). At 28 days, there was no significant difference between groups. At 56 days, there was no significant difference in the load to failure, but the rhPTH group had significantly higher stiffness (p < 0.01). Data can be seen in Figure 2. In all groups the failure site was at the tendon–bone interface, except for the 8-week rhPTH group where 11/13 failed at the tendon midsubstance.
MicroCT analysis showed that the rhPTH group had significantly greater total mineral content at all time points (p < 0.05), as well as significantly higher BV at 2 and 4 weeks. At 14 days the rhPTH group had significantly higher total mineral density and BV. Data can be seen in Table 1.
Table 1. MicroCT Data for rhPTH and Control Groups
At 3 days post-operatively there was significantly more fibrocartilage in the rhPTH group (p < 0.01). There was no significant difference in the collagen fiber organization, procollagen/osteoblasts, osteoclasts, or Factor 8 expression between groups at 3 days.
At 7 days there was significantly more fibrocartilage (p < 0.05) and procollagen/osteoblasts (p < 0.02) in the rhPTH group. There was no significant difference in collagen fiber organization, osteoclasts, or Factor 8 expression between groups.
At 14 days there was significantly more fibrocartilage (p < 0.05), and significantly more vessels per mm2 in the tendons of the rhPTH group (p < 0.01). The control specimens had significantly more osteoclasts (p < 0.05). There were no significant differences in collagen fiber organization or in procollagen/osteoblast staining between groups.
At 28 days there was significantly more fibrocartilage (p < 0.05) and procollagen/osteoblast staining (p < 0.05) in the rhPTH group. There were significantly more vessels per mm2 in the tendons of the rhPTH group (p < 0.02), but there was no difference in vessel density at the insertion site. The rhPTH specimens also had significantly better collagen fiber organization (p < 0.05). There was no difference in TRAP staining between the groups.
At 56 days there was significantly more fibrocartilage (p < 0.01) and vessels per mm2 in the insertion of the rhPTH group (p < 0.05). There was also better collagen fiber organization in the rhPTH group (p < 0.02). There were no differences in procollagen/osteoblasts, osteoclasts, or vessel density in the tendon between groups.
Graphs depicting the fibrocartilage, osteoblasts, vessel density, and collagen fiber organization can be seen in Figures 3–6.
PTH has a well-established positive effect on bone and cartilage formation. Based on this, we hypothesized that PTH could improve healing between tendon and bone. We used a well-characterized rat model of rotator cuff tendon repair. Although treatment with rhPTH resulted in an increase in bone and mineralized fibrocartilage formation in this model, consistent with prior studies5, 7, 18 this did not translate into improved biomechanical properties. In fact, the load to failure was lower in the rhPTH-treated animals at 2 weeks.
There are several possible reasons that may explain why the improved tissue formation at the healing tendon–bone interface did not translate into improvements in biomechanical properties. Simple formation of both bone and fibrocartilage will not strengthen the attachment if the newly formed tissue is not well integrated with the tendon. The newly formed tissue needs to be integrated at the microstructural level, with re-establishment of collage fiber continuity, which was not evaluated here. Other ultrastructural parameters such as collagen cross-linking will also affect biomechanical properties.
An alteration in the balance of osteoclastic resorption versus osteoblastic bone formation may have an adverse effect on tissue material properties. The rhPTH group had significantly more osteoblasts and procollagen formation at 7 and 28 days, suggesting a possible imbalance between bone resorption and formation. Although rhPTH resulted in more new bone formation, this newly formed bone needs to undergo remodeling in order to gain strength. Exposure to appropriate mechanical loading signals is likely required to direct optimal remodeling of this newly formed tissue. Longer time points may demonstrate a positive effect of rhPTH via improved bone maturation and remodeling over time.
Excessive angiogenesis may also have an adverse effect on material properties. The significantly increased angiogenesis within the tendon at 14 days coincided with the decreased load to failure, which had resolved by 56 days. Although vascularity is clearly important for the initial phases of healing, diminished vascularity typically occurs during the remodeling phase of connective tissue healing. Again, longer time points may demonstrate a beneficial effect of rhPTH by allowing further regression of the initial excessive vascularity.
Further studies with longer observation times are clearly required to better understand the biology of healing and the potential for anabolic agents such as PTH to improve healing. For example, better collagen fiber organization at later time points and increased stiffness in the tendon at 8 weeks could indicate improved healing in the rhPTH group. The failure of the specimens at 8 weeks was in the tendon midsubstance in 11/13 specimens, indicating that the insertion site is likely stronger, but failure occurred at the tendon first. This could imply that rhPTH actually weakened the tendon, which could occur with excessive vascularity. Other studies have shown continued improvement in biomechanical properties for up to 16 weeks,19 however, our longest time point was 8 weeks.
This study has the limitations of any animal study, with uncertain correlation between healing in humans and small rodent models. We have only studied healing out to 8 weeks. Importantly, we only studied one dosing regimen, and further studies are needed to see if alternative doses and timing of administration would have a different effect. For example, rhPTH may work differently if given in a delayed fashion, after resolution of the initial post-operative inflammation.
In conclusion, rhPTH has a positive effect on tissue formation at the healing tendon–bone interface. However, the positive structural changes do not translate into improved biomechanical function. These results show that simple formation of more bone and fibrocartilage at the healing tendon–bone interface is not sufficient, and suggest that further integration of the newly formed tissue at the microstructural level is necessary to improve biomechanics. Further studies are necessary to determine if parathyroid hormone can positively affect tendon-to-bone healing.
This study was funded internally by the Hospital for Special Surgery. Dr. Lane is on the speaker's bureau for Eli Lilly. There are no other conflicts of interest.