Abstract
Background:
Rotator cuff tears are a common source of pain and disability, and poor healing after repair leads to high retear rates. Bone loss in the humeral head before and after repair has been associated with poor healing. The purpose of the current study was to mitigate bone loss near the repaired cuff and improve healing outcomes.
Methods:
Sclerostin antibody (Scl-Ab) treatment, previously shown to increase bone formation and strength in the setting of osteoporosis, was used in the current study to address bone loss and enhance rotator cuff healing in an animal model. Scl-Ab was administered subcutaneously at the time of rotator cuff repair and every 2 weeks until the animals were sacrificed. The effect of Scl-Ab treatment was evaluated after 2, 4, and 8 weeks of healing, using bone morphometric analysis, biomechanical evaluation, histological analysis, and gene expression outcomes.
Results:
Injury and repair led to a reduction in bone mineral density after 2 and 4 weeks of healing in the control and Scl-Ab treatment groups. After 8 weeks of healing, animals receiving Scl-Ab treatment had 30% greater bone mineral density than the controls. A decrease in biomechanical properties was observed in both groups after 4 weeks of healing compared with healthy tendon-to-bone attachments. After 8 weeks of healing, Scl-Ab-treated animals had improved strength (38%) and stiffness (43%) compared with control animals. Histological assessment showed that Scl-Ab promoted better integration of tendon and bone by 8 weeks of healing. Scl-Ab had significant effects on gene expression in bone, indicative of enhanced bone formation, and no effect on the expression of genes in tendon.
Conclusions:
This study provides evidence that Scl-Ab treatment improves tendon-to-bone healing at the rotator cuff by increasing attachment-site bone mineral density, leading to improved biomechanical properties.
Clinical Relevance:
Scl-Ab treatment may improve outcomes after rotator cuff repair.
Rotator cuff tears do not heal spontaneously, can progress in size over time, and motivate >250,000 surgical repairs in the United States annually1. Poor tendon-to-bone healing after repair results in an alarmingly high rate of retears at the site of attachment, ranging from 20% in young healthy patients with small tears to 94% in older patients with massive tears2,3. Rotator cuff tears are associated with loss of bone at the healing interface and a lack of regeneration of the functionally graded, mineralized fibrocartilage found in the healthy attachment4. Bone loss has been observed at healing tendon-to-bone interfaces at multiple anatomic sites5-11. The loss of mineralized tissue is likely caused by mechanical unloading during the period from tearing through surgical repair and by high osteoclast activity during the healing period after repair10,12. Chronic rotator cuff tears lead to unloading-induced osseous changes to the humeral head13. Chronic degeneration of the muscle before repair is associated with greater bone loss in the humeral head and leads to low cellular remodeling and poor extracellular matrix formation14. However, Ditsios et al. showed that mechanical unloading is not the only factor accountable for the reduction in bone mineral density (BMD) at the healing tendon-to-bone attachment15. Injury to the flexor digitorum profundus tendon in an animal model without any alteration of limb loading resulted in a sevenfold increase in osteoclast surface after 7 days, leading to a 7% decrease in BMD after 21 days.
To address the bone loss that occurs during tendon-to-bone healing, investigators in a previous study suppressed osteoclast activity using bisphosphonate treatment8,9,16. Treatment led to improved mechanical properties in the treatment group compared with the control group. However, therapy with bisphosphonates is not ideal, especially in the younger population, because of its association with reduced bone turnover and increased risk for bone fracture17-19. Another approach to increase bone mass at the site of healing is to administer a bone-anabolic agent to stimulate new bone formation. Sclerostin antibodies that block sclerostin, a negative regulator of bone formation produced largely by osteocytes, systemically increase bone formation and bone mass in animal models and osteoporotic patients20-22. In the current study, a novel application of sclerostin antibody (Scl-Ab) treatment was tested for enhancing tendon-to-bone healing. Using a well-established animal model of the rotator cuff, we tested the hypothesis that Scl-Ab treatment would prevent bone loss during tendon-to-bone healing, leading to improved outcomes.
Materials and Methods
Animal Model and Study Design
Eighty-seven adult male Sprague-Dawley rats (approximately 4 months old and weighing approximately 350 g) were used in this study, as approved by the Institutional Animal Care and Use Committee. Fifty-three rats received surgical injury and repair, and 34 rats were used as uninjured controls (the normal group). Of the 53 injured-and-repaired rats, 10 (5 that received Scl-Ab [the Scl-Ab group] and 5 that had no treatment [the control (CTL) group]) were used to study 2 weeks of healing, 20 (10 in the Scl-Ab treatment group and 10 in the CTL group) were used to study 4 weeks of healing, and 23 (12 in the Scl-Ab treatment group and 11 in the CTL group) were used to study 8 weeks of healing. Of the 34 uninjured control animals, 17 were used to study the effect of Scl-Ab treatment on the healthy rotator cuff and 17 were used as healthy untreated controls. For all animals, 1 shoulder was used for bone morphometry and/or biomechanical evaluation and the contralateral shoulder was used for histological analysis or gene expression.
For rats in the injury-and-repair groups, the supraspinatus tendon was sharply severed from the humeral head and was repaired bilaterally in each animal, as previously described23. Operatively and nonoperatively treated animals either were left untreated (the CTL group) or were administered Scl-Ab (Scl-Ab VI; Amgen). The Scl-Ab was delivered via subcutaneous injections (25 mg/kg) at the onset of the study (i.e., at the time of injury and repair for the operatively treated animals and at an equivalent age for the nonoperatively treated animals) and every 2 weeks until they were killed (the Scl-Ab group). Animals in the operatively treated CTL and Scl-Ab groups were allowed cage activity and were sacrificed after 2, 4, or 8 weeks. Animals in the nonoperatively treated CTL and Scl-Ab groups were allowed cage activity and were sacrificed after 4 weeks. Postmortem supraspinatus muscle-tendon-bone samples were dissected and assessed using bone morphometry, biomechanical evaluation, histological analysis, and gene expression. As surgery was performed bilaterally and the treatment was systemic, each animal had 2 samples available for analysis. Samples were allocated to either bone morphometry followed by biomechanical evaluation or bone morphometry followed by either histological analysis or gene expression.
At the time that the animals were killed, body weight was not significantly different between treatment groups (mean [and standard deviation] across time points, 520 ± 54 g for Scl-Ab group and 510 ± 55 g for the CTL group). No repair site failure or gap was observed in the Scl-Ab and CTL groups at the time of dissection.
Bone Morphometric Analysis
After the animals were sacrificed, the humerus with the supraspinatus tendon and muscle attached was dissected for bone morphometric analysis (17 per group of nonoperatively treated rats, 5 per group of operatively treated rats after 2 weeks of healing, and 20 to 23 per group of operatively treated rats after 4 or 8 weeks of healing). The humeral head and tendon enthesis region (approximately 5 mm) were scanned using micro-computed tomography (microCT) at a resolution of 20 μm, 45 kVp, and 177 μA (μCT 40; SCANCO Medical)13,24. The region of interest included trabecular bone within the humeral head near the tendon attachment (within approximately 1 mm) and proximal to the growth plate and was determined by evaluators blinded to the treatment groups. The amount of bone in the region of interest was calculated to determine bone volume fraction (BV/TV; bone volume to total volume). BMD, trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp) were also determined.
Biomechanical Evaluation
After killing, dissection, and microCT scanning of the animals, the supraspinatus tendon-to-bone attachment was mechanically tested in uniaxial tension (17 per group for nonoperatively treated animals and 10 to 12 per group for operatively treated animals)13. The repair-site suture was released to remove its mechanical contribution, the humeri were potted in polymethylmethacrylate, and specimens were tested in a 0.9% saline solution water bath at 37°C (Instron 5866). Samples were preconditioned for 5 cycles (5% strain, 0.2%/s), allowed 300 seconds for recovery, and pulled to failure at 0.2%/s. Strain was determined from grip-to-grip displacement. Cross-sectional area near the attachment site was measured from microCT scans by evaluators blinded to treatment group. Load-deformation curves were used to determine maximum load and stiffness. Stress-strain curves were used to determine strength (maximum stress), modulus, and resilience (area under the curve from 0% to yield strain). The mechanism of failure was determined visually. Specimens were excluded from the data analysis if the tendon slipped out of the grip or failure occurred at the growth plate of the humeral head.
Histological Analysis
Histological analysis was performed for the CTL and Scl-Ab-treated groups following 2, 4, and 8 weeks of healing (3 animals per group). Humerus-supraspinatus constructs were fixed in 4% paraformaldehyde for 24 hours. Samples were decalcified in 14% EDTA (ethylenediaminetetraacetic acid), dehydrated in graded ethanol, and embedded in paraffin. Five-micrometer coronal sections were stained with hematoxylin and eosin, toluidine blue, or Goldner trichrome. Additional samples were fixed for 24 hours in 4% paraformaldehyde and embedded in plastic, and 5μm-thick coronal sections were stained with von Kossa stain. The sections were semiquantitatively analyzed by 1 blinded observer (N.H.) using a tendon-to-bone maturity score adapted from Ide et al.24,25. Insertion continuity, bone resorption, matrix quality, cell and fiber alignment, and cellularity were part of 9 factors evaluated on a scale from 1 to 4 (Table I). A lower score is indicative of improved tendon-to-bone healing, with a score of 9 equivalent to a healthy attachment24,25.
TABLE I.
The Modified Tendon-to-Bone Maturity Score with Evaluation of 9 Individual Outcomes on a Scale of 1 to 4*
| Score |
||||
| Individual Outcomes | 1 | 2 | 3 | 4 |
| Cellularity (inflammation) | Minimal | Mild | Moderate | Marked |
| Presence of fibrocytes (%) | >75 | 51-75 | 26-50 | ≤25 |
| Proportion of cells oriented parallel (%) | >75 | 51-75 | 26-50 | ≤25 |
| Proportion of fibers oriented parallel (%) | >75 | 51-75 | 26-50 | ≤25 |
| Presence of matrix | Marked | Moderate | Mild | Minimal |
| Insertion integrity | C(+), R(+), F(+), tidemark(+) | C(+), R(+), F(+), tidemark(−) | C(+), R(+), F(−) | C(+), R(−) |
| Insertion continuity (%) | >75 | 51-75 | 26-50 | <25 |
| Bone resorption at enthesis (%) | ≤25 | 26-50 | 51-75 | >75 |
| Epiphyseal bone modeling (%) | ≤25 | 26-50 | 51-75 | >75 |
Modification of the system of Ide et al.25. A healthy (uninjured) enthesis has a combined score of 9. C = continuity, R = regularity, and F = fibrocartilage.
Gene Expression
For gene expression studies (4 per group), humerus-supraspinatus samples were frozen in liquid nitrogen. RNA was isolated separately from the supraspinatus tendon and the portion of the humeral head proximal to the growth plate near the tendon attachment (RNeasy Kit; Qiagen). RNA was quantified using a NanoDrop 2000 (ThermoFisher Scientific). Quantitative real-time polymerase chain reaction (PCR) was performed using a Biomark HD System (Fluidigm) for tendon and bone RNA on a panel of 25 genes related to bone, tendon, and fibrocartilage (Table II). TaqMan gene expression assays (ThermoFisher Scientific) were used for the analysis. Rpl13a was used as a housekeeping gene, as expression of Rpl13a did not vary more than ±0.5 Ct (cycle threshold value) among groups. Results are presented as relative expression compared with Rpl13a expression (2-ΔCt).
TABLE II.
Names of Genes, Associated Categories, and TaqMan Assay Identification Numbers*
| Gene Name | Category | TaqMan Assay ID |
| Tnfrsf11b (tumor necrosis factor receptor superfamily, member 11b), OPG (osteoprotegerin) | Bone, inhibits osteoclast activity | Rn00563499_m1 |
| Sost (sclerostin) | Bone, osteoclast activity | Rn00577971_m1 |
| Dmp1 (dentin matrix acidic phosphoprotein) | Bone, osteoclast activity | Rn01450122_m1 |
| RankL (receptor activator of nuclear factor kappa-B ligand), Tnfsf11 (tumor necrosis factor [ligand] superfamily, member 11) | Bone, osteoblast activity | Rn00589289_m1 |
| Bglap (bone gamma-carboxyglutamate protein), OCN (osteocalcin) | Bone, osteoblast activity | Rn00566386_g1 |
| Pth1r (parathyroid hormone 1 receptor) | Bone, osteoblast activity | Rn00571596_m1 |
| Ctsk (cathepsin K) | Bone, bone resorption | Rn00580723_m1 |
| Dkk1 (dickkopf WNT signaling pathway inhibitor 1) | Bone, Wnt signaling antagonist | Rn01501537_m1 |
| Lrp5 (low density lipoprotein receptor-related protein 5) | Bone, Wnt signaling | Rn01451428_m1 |
| Runx2 (runt-related transcription factor 2) | Bone, osteoprogenitor | Rn01512298_m1 |
| Sp7 (Osterix) | Bone, osteoprogenitor | Rn02769744_s1 |
| Scx (scleraxis) | Tendon | Rn01504576_m1 |
| Tnmd (tenomodulin) | Tendon | Rn00574164_m1 |
| Col1a1 (collagen, type I, alpha 1) | Tendon | Rn01463848_m1 |
| Col1a2 (collagen, type I, alpha 2) | Tendon | Rn01526721_m1 |
| Col3a1 (collagen, type III, alpha 1) | Tendon | Rn01437681_m1 |
| Col2a1 (collagen, type II, alpha 1) | Fibrocartilage | Rn01637087_m1 |
| Acan (aggrecan) | Fibrocartilage | Rn00573424_m1 |
| Tgfb1 (transforming growth factor, beta 1) | Fibrocartilage | Rn00572010_m1 |
| Tgfb3 (transforming growth factor, beta 3) | Fibrocartilage | Rn00565937_m1 |
| Mmp2 (matrix metalloproteinase 2) | Fibrocartilage | Rn01538170_m1 |
| Sox9 (SRY [sex determining region Y]-box 9) | Fibrocartilage | Rn01751069_mH |
| Smo (smoothened, frizzled class receptor) | Development | Rn00563043_m1 |
| Notch1 | Development | Rn01758633_m1 |
| Rpl13a (ribosomal protein L13A) | Housekeeping | Rn00821946_g1 |
TaqMan Gene Expression Assays are manufactured by ThermoFisher Scientific.
Statistical Analysis
The primary outcome tested in this study was the effect of Scl-Ab treatment. To incorporate the effect of healing duration, a 2-factor analysis of variance (ANOVA), in which the factors analyzed were Scl-Ab treatment and healing time, was performed. When the ANOVA was significant, Tukey post hoc tests were performed to determine specific effects of treatment (CTL or Scl-Ab) and duration of healing (2, 4, or 8 weeks). A secondary outcome tested in this study was the effect of injury and repair. Comparisons between the uninjured control group (the normal group) and the injury-and-repair groups (CTL or Scl-Ab group) were performed using 2-tailed Student t tests. A p value of <0.05 was considered significant for all comparisons. SYSTAT 13 software (Systat Software) was used for all statistical analyses. Error bars in the plotted data represent 1 standard deviation from the mean. Due to the semiquantitative nature of the histological analysis and the low sample size in each group, statistical analysis was not performed on the histological results.
Results
Bone Morphometry
There was significant bone loss in the CTL and Scl-Ab groups by 4 weeks of healing, with recovery in the Scl-Ab group by 8 weeks of healing (Fig. 1). Specifically, BV/TV, BMD, and Tb.N in the CTL group and BMD in the Scl-Ab group were significantly decreased compared with the uninjured group at 4 weeks. However, after 8 weeks of healing, in the Scl-Ab treatment group compared with the CTL group, the BV/TV was increased by 34%, BMD was increased by 30%, Tb.N was increased by 17%, Tb.Th was increased by 24%, and TbSp was decreased by 21% (Fig. 1; see Appendix), reaching levels comparable with those of the uninjured control group. Treatment with Scl-Ab also led to increased BV/TV, BMD, and Tb.Th in the uninjured groups as well. When evaluating the overall effect of Scl-Ab treatment using an ANOVA, Scl-Ab-treated animals had significantly higher BV/TV, BMD, and Tb.Th compared with CTL animals, by 19%, 18%, and 20%, respectively.
Fig. 1.
Treatment with Scl-Ab increased bone volume fraction (BV/TV) (Fig. 1-A), bone mineral density (BMD) (Fig. 1-B), trabecular number (Tb.N) (Fig. 1-C), and/or trabecular thickness (TbTh) (Fig. 1-D) in the normal (uninjured) and 8-week healing groups. A significant effect of Scl-Ab is indicated by a line over bars (p < 0.05; ANOVA followed by Tukey post hoc test compared with CTL within group). A significant difference compared with normal is indicated by an “a” within a bar (p < 0.05; ANOVA followed by the Tukey post hoc test compared with normal within a particular treatment group). CTL = control.
Biomechanical Evaluation
Eleven of 77 samples were excluded from analysis because they did not fail at the tendon attachment; the excluded samples included 4 operatively treated shoulders from the Scl-Ab group and 4 from the CTL group and 2 nonoperatively treated shoulders from the Scl-Ab group and 1 from the CTL group. Injury caused a significant increase in cross-sectional area (mean and standard deviation, 15.9 ± 3.9 mm2 compared with 6.6 ± 1.9 mm2). There was a significant decrease in mechanical properties in the CTL and Scl-Ab groups by 4 weeks of healing, with improved mechanical properties in the Scl-Ab group by 8 weeks of healing (Fig. 2; see Appendix). After 8 weeks of healing, comparison of the Scl-Ab treatment and CTL groups demonstrated that failure load was increased by 48%, strength was increased by 38%, and stiffness was increased by 43%. Treatment with Scl-Ab also led to significant decreases in stiffness and modulus in the uninjured groups.
Fig. 2.
Treatment with Scl-Ab led to increased attachment-site failure load (Fig. 2-A), strength (Fig. 2-B), and stiffness (Fig. 2-C) after 8 weeks of healing, and stiffness and modulus (Fig. 2-D) were decreased in normal (uninjured) attachments. A significant effect of Scl-Ab is indicated by a line over bars (p < 0.05; ANOVA followed by Tukey post hoc test compared with CTL within group). A significant difference compared with normal is indicated by an “a” within a bar (p < 0.05; ANOVA followed by the Tukey post hoc test compared with normal within a particular treatment group). CTL = control.
Histological Analysis
Supraspinatus tendon healing to humeral head bone occurred via a fibrovascular scar, with bone loss evident in the humeral heads of the CTL group (Fig. 3). Based on a blinded analysis of 9 features of the healing tendon-to-bone attachment, the CTL and Scl-Ab healing attachments appeared similar at 2 and 4 weeks of healing (Table III). However, after 8 weeks of healing, the Scl-Ab treatment led to improved insertion continuity, integrity, and fiber alignment compared with the CTL group. The analysis demonstrated a more mature tendon-to-bone attachment and new bone formation in the Scl-Ab group compared with CTL group at 8 weeks (Table III).
Fig. 3.
Photomicrographs made after 8 weeks of healing show that Scl-Ab treatment (Figs. 3-B and 3-D) improved insertion continuity, integrity, and fiber alignment compared with CTL (Figs. 3-A and 3-C). The enthesis area is outlined with a white dashed box. Scale bars = 1 mm for Figs. 3-A and 3-B and 250 μm for Figs. 3-C and 3-D. Slides were stained with Masson trichrome.
TABLE III.
The Modified Tendon-to-Bone Maturity Scores*
| 2 Weeks |
4 Weeks |
8 Weeks |
||||
| CTL | Scl-Ab | CTL | Scl-Ab | CTL | Scl-Ab | |
| Cellularity | 2 (1, 3) | 3 (2, 3) | 1 (1, 1) | 1 (1, 2) | 1 (1, 1) | 1 (1, 1) |
| Fibroblasts | 2 (1, 2) | 2 (2, 2) | 1 (1, 1) | 1.5 (1.5, 2) | 1 (1, 2) | 1 (1, 1) |
| Matrix | 4 (1, 4) | 2 (2, 3) | 3 (2.5, 3) | 2.5 (1, 3) | 2 (2, 3) | 1.5 (1, 2.5) |
| Cell orientation | 3 (1, 3) | 2 (2, 3) | 2 (2, 3) | 1.5 (1, 3) | 2 (1, 2) | 1 (1, 1) |
| Collagen orientation | 3 (1, 3) | 2 (2, 3) | 2 (2, 3) | 1.5 (1, 3) | 2 (1, 2) | 1 (1, 1) |
| Insertion integrity | 3 (1, 4) | 1.5 (1.5, 2) | 2.5 (2.5, 3) | 2.5 (1, 2.5) | 2.5 (1.5, 3) | 1.5 (1, 2.5) |
| Insertion continuity | 3 (1, 4) | 2 (2, 3) | 3 (3, 3) | 3 (1, 3) | 2 (1, 3) | 1 (1, 2) |
| Bone resorption | 3 (2, 4) | 4 (3, 4) | 3 (2, 4) | 3 (3, 4) | 3 (3, 4) | 4 (4, 4) |
| Epiphyseal bone modeling | 3 (1, 4) | 4 (3, 4) | 4 (3, 4) | 4 (3, 4) | 4 (4, 4) | 4 (4, 4) |
| Overall maturity | 24 (14, 29) | 22.5 (22, 23.5) | 21 (19.5, 25) | 20.5 (15.5, 24.5) | 21.5 (16.5, 22) | 16 (15, 19) |
The results are shown as the median (minimum, maximum). The modified tendon-to-bone maturity scores improved over time for all groups. Scl-Ab treatment resulted in a more mature attachment at 8 weeks of healing compared with CTL.
Gene Expression
Mineralized Tissues (Bone and Fibrocartilage)
The Scl-Ab treatment had a significant effect on the expression of a number of genes in mineralized tissue near the tendon enthesis, including Sclerostin, Dkk1 (dickkoph-related protein 1), RankL (receptor activator of nuclear factor kappa-B ligand), Dmp1 (dentin matrix acidic phosphoprotein 1), and Runx2 (runt-related transcription factor-2) (Fig. 4). After 8 weeks of healing, expression of Sclerostin and Dkk1 was 3.3 times and 2.5 times greater in the Scl-Ab group compared with the CTL group, respectively. Expression of Lrp5 (low-density lipoprotein receptor-related protein 5) was not affected by treatment or healing time. Osteocalcin (Ocn), a marker of osteoblast activity, was significantly increased following injury, while osteoprotegerin (Opg), a marker of osteoclast inhibition, was significantly decreased after injury in all groups. Expression of RankL and Dmp1 was increased with Scl-Ab treatment (Fig. 4; see Appendix). There was no consistent effect of Scl-Ab treatment on the fibrocartilage-related genes Tgf (transforming growth factor)-β1, Tgf-β3, Mmp (matrix metalloproteinase 2), Col2a1 (collagen type II alpha 1), and Sox9 (Fig. 4; see Appendix). Expression of Smo and Notch1, members of the hedgehog signaling pathway, was not affected by treatment.
Fig. 4.
Gene expression in mineralized tissue adjacent to the tendon enthesis relative to the housekeeping gene Rpl13a. A significant effect of Scl-Ab is indicated by a line over bars (p < 0.05; ANOVA followed by Tukey post hoc test compared with CTL within group). A significant difference compared with normal is indicated by an “a” within a bar (p < 0.05; ANOVA followed by Tukey post hoc test compared with normal within a particular treatment group). A significant effect of Scl-Ab compared with normal in the CTL group is indicated by a “b” within a bar (p < 0.05; ANOVA followed by the Tukey post hoc test). CTL = control, Pth1r = parathyroid hormone 1 receptor, and Ctsk = cathepsin K.
Tendon Gene Expression
Scl-Ab treatment had no significant effect on tendon gene expression (Fig. 5; see Appendix). Healing time, however, significantly affected all tendon-related genes: Scleraxis, Tenomodulin, Col1a1 (collagen type I alpha 1), Col1a2 (collagen type I alpha 2), and Col3a1 (collagen type III alpha 1). Changes were most apparent at the 2-week healing time point and trended toward normal by the 8-week healing time point. Additionally, expression of aggrecan was 20 times greater in the CTL group and 17 times greater with Scl-Ab treatment after 2 weeks of healing. Similarly, expression of Mmp2 was 4.1 times greater in both the CTL and Scl-Ab treatment groups after 2 weeks of healing.
Fig. 5.
Gene expression in the tendon relative to the housekeeping gene Rpl13a. A significant difference compared with normal is indicated by an “a” within a bar (p < 0.05; ANOVA followed by the Tukey post hoc test compared with normal within a particular treatment group).
Discussion
Rotator cuff injury and repair led to bone loss at the tendon-to-bone interface. A decrease in bone quantity and quality at the healing interface contributes to the high rates of retear following surgical repair3. Scl-Ab treatment increased bone volume fraction and BMD of the trabecular bone in the humeral head nearest to the healing tendon attachment. Although injury-associated bone loss remained in the treatment group after 8 weeks of healing, rapid recovery toward normal bone was seen by 8 weeks of healing in the treated animals. The improvement in bone morphology at the healing interface had functional consequences, as demonstrated by improved attachment strength. Qualitative histological assessment further confirmed the benefit of Scl-Ab treatment, with a more mature tendon-to-bone interface after 8 weeks of healing in the treated animals compared with control animals.
Delivery of osteoinductive agents such as bone morphogenetic protein-2 to repaired tendon-to-bone attachments has been ineffective in improving healing24,26. However, bisphosphonates have previously shown success in improving tendon-to-bone healing by reducing bone resorption16,27. In a canine model of flexor tendon-to-bone healing, tendon injury caused BMD near the tendon-to-bone interface to decrease by 29% compared with normal after 21 days16. An oral dose of alendronate was effective in preventing bone resorption, leading to only a 6% decrease in BMD compared with normal. The prevention of bone loss resulted in a significant improvement in the failure load of the repair after 21 days of healing. In a separate study, subcutaneous injections of zoledronic acid to ovariectomized rats resulted in a 23% increase in BMD of the humeral head near the supraspinatus tendon insertion compared with the control27. The increased BMD was associated with a 24% increase in failure load at the interface following treatment. In the current study, after 8 weeks of healing, Scl-Ab treatment caused a 30% increase in BMD and a 48% increase in failure load compared with the control, consistent with the prior bisphosphonate treatment approaches.
The Scl-Ab used in the current study has been previously shown to neutralize sclerostin by preventing sclerostin binding to the Lrp5 receptor28-30. To determine whether improvements in bone morphology during tendon-to-bone healing were achieved through this mechanism, we measured expression of Wnt signaling-related genes in the mineralized tissues at the healing attachment. Scl-Ab treatment increased expression of the Wnt-target genes sclerostin and Dkk1 relative to control during tendon-to-bone-healing, in contrast to decreases observed in controls relative to normal, uninjured attachments. The changes observed in the Scl-Ab group indicate a compensatory cellular response to chronic use of Scl-Ab treatment, as described by Taylor et al.31. Further analysis of bone-related gene expression showed a reduction of osteoclast inhibition (as demonstrated by an increase in Opg relative to RankL) and an increase in osteoblast activity (as demonstrated by increased osteocalcin) with Scl-Ab, consistent with the observed improvements in bone morphology. Genes related to osteoprogenitors (Osterix and Runx2) were significantly increased with injury compared with normal, uninjured groups. These genes, however, were not affected by treatment in healing bone, although Runx2 was increased by Scl-Ab in normal, uninjured bone. Increased expression of these factors, which are also associated with differentiation of mesenchymal cells into osteoblasts32,33, suggests the possible induction of bone formation via progenitors as well as mature osteoblasts.
The strength of the tendon attachment is in large part dictated by the quality of the mineralized tissue at the interface34,35. The healthy tendon-to-bone attachment has a gradient of mineral content across the fibrocartilaginous insertion and into the trabecular bone36. The increase in mechanical strength at the attachment because of Scl-Ab treatment is likely the result of improved mineralization in not only the trabecular bone compartment (as measured by microCT) but also the fibrocartilage at the healing interface. Expression of aggrecan, an extracellular matrix marker of cartilage and fibrocartilage, was significantly higher with Scl-Ab treatment after 8 weeks of healing. Furthermore, blinded evaluation of histological sections showed improvements in tendon-to-bone attachment maturity, including insertion integrity, after 4 and 8 weeks of healing with Scl-Ab treatment compared with controls.
Scl-Ab was administered by subcutaneous injection; therefore, all tissues were exposed to the antibody, including the tendon adjacent to the healing interface. To evaluate possible effects of Scl-Ab on nonmineralized tissues, gene expression was examined in the supraspinatus tendon adjacent to the healing interface. Scl-Ab treatment did not have a significant effect on expression of genes in tendon tissue, alleviating the concern of possible effects of treatment on off-target tissue. However, Scl-Ab treatment did lead to a decrease in modulus and stiffness in healthy tendon-to-bone attachments. This result is consistent with a previous finding that bisphosphonate treatment during tendon-to-bone healing can cause a decrease in stiffness16. Due to the high mechanical safety factor of tendons and ligaments for typical physiologic activities37, the relatively small decreases in stiffness and modulus may not predispose healthy tendons to injury. However, possible consequences of the reduced stiffness and modulus at uninjured attachments should be further considered.
There were several limitations to the current study. The animal model consisted of an acute injury and repair. In contrast, most rotator cuff tears in the clinical population occur after chronic tendon degeneration. Bone loss in these cases may therefore be more severe than in the current animal study. It is unclear whether Scl-Ab treatment would be effective in the chronic injury scenario, although studies have shown efficacy of the treatment approach in the context of osteoporosis20-22. Additional animal and clinical studies are necessary to test this premise. A second limitation of this study was the use of subcutaneous (i.e., systemic) high-dose injections of Scl-Ab. This dosing regimen (25 mg/kg every 2 weeks) has been reported in proof-of-concept studies across various animal models38-40, although future studies would be required to determine the dose-response to Scl-Ab for tendon-to-bone healing and potential application of Scl-Ab via local delivery. Finally, an additional control group consisting of vehicle injection would have strengthened the study design. In the current study, treatment was compared with the equivalent of “standard of care,” i.e., repair only without additional biologic treatment.
In conclusion, bone loss was observed at the healing tendon-to-bone interface, contributing to poor outcomes. Treatment with Scl-Ab improved healing by increasing attachment-site BMD, leading to improved biomechanical properties. Considering recent evidence that Scl-Ab (i.e., romosozumab, for human use) is safe and effective for treating osteoporosis in humans20,41, the results from the current study support the use of the treatment in combination with repair of acute rotator cuff tears. Treatment is expected to enhance tendon-to-bone repair, by improving bone morphology and tendon insertion maturity, leading to improved tendon attachment strength.
Appendix
Figures demonstrating the effect of treatment with Scl-Ab and 8 weeks of healing on trabecular spacing and resilience, gene expression in mineralized tissue adjacent to the tendon enthesis, and gene expression in the tendon are available with the online version of this article as a data supplement at jbjs.org (http://links.lww.com/JBJS/D273).
Footnotes
Investigation performed at Washington University in St. Louis, St. Louis, Missouri
A commentary by Brian C. Werner, MD, is linked to the online version of this article at jbjs.org.
Disclosure: The study was supported by National Institutes of Health (NIH) grants F31-AR066452 and NIH R01-AR057836. Sclerostin antibody was provided by Amgen, Inc. One of the authors was an employee of Amgen, Inc. at the time of the study. On the Disclosure of Potential Conflicts of Interest forms, which are provided with the online version of the article, one or more of the authors checked “yes” to indicate that the author had a relevant financial relationship in the biomedical arena outside the submitted work and “yes” to indicate that the author had a patent planned for the treatment that is the subject of this article (http://links.lww.com/JBJS/D272).
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