Abstract
Purpose:
Systemic and intrinsic mechanisms in bone and soft tissues help promote biomineralization to the skeleton, while preventing it in soft tissues. However, severe injury can disrupt this homeostatic biomineralization tropism, leading to adverse patient outcomes due to a paradoxical decrease of biomineralization in bone and increased biomineralization in soft tissues. There remains a need for therapeutics that restore the natural tropism of biomineralization in severely injured patients. Bisphosphonates can elicit potent effects on biomineralization, though with variable impact on musculoskeletal repair. Thus, a critical clinical question remains as to the optimal time to initiate bisphosphonate therapy in patients following a polytrauma, in which bone and muscle are injured in combination with a severe injury, such as a burn.
Methods:
To test the hypothesis that the dichotomous effects of bisphosphonates are dependent upon the time of administration relative to the ongoing biomineralization in reparative bone and soft tissues, this study utilized murine models of isolated injury or polytrauma with a severe injury, in conjunction with sensitive, longitudinal measure of musculoskeletal repair.
Results:
This study demonstrated that if administered at the time of injury, bisphosphonates prevented severe injury-induced bone loss and soft tissue calcification, but did not interfere with bone repair or remodeling. However, if administered between 7–21 days post-injury, bisphosphonates temporally and spatially localized to sites of active biomineralization, leading to impaired fracture callus remodeling and permanence of soft tissue calcification.
Conclusion:
There is a specific pharmacologic window following polytrauma that bisphosphonates can prevent the consequences of dysregulated biomineralization, yet not impair musculoskeletal regeneration.
Keywords: Severe Injury, Polytrauma, Fracture, Bisphosphonate, Skeletal Muscle Calcification, Osteoporosis
Mini Abstract:
Following severe injury, biomineralization is disrupted and limited therapeutic options exist to correct these pathologic changes. This study utilized a clinically relevant murine model of polytrauma including a severe injury with concomitant musculoskeletal injuries to identify when bisphosphonate administration can prevent the paradoxical decrease of biomineralization in bone and increased biomineralization in soft tissues, yet not interfere with musculoskeletal repair.
INTRODUCTION:
In an ‘isolated’ musculoskeletal injury, such as a bone fracture with injury to the adjacent muscle in a single extremity, numerous systemic and intrinsic mechanisms exist that promote biomineralization in the repairing bone yet prevent it from occurring in repairing muscle. Contrarily, these mechanisms are often dysregulated in the setting of a polytrauma that includes an injury to bone and muscle in an extremity, in combination with an anatomically remote severe injury. In this context, a severe injury is defined as one that provokes a robust systemic acute phase response, leading to global, pathologic changes in distant organs and tissues. In polytraumas that include a severe injury, such as burn, blast, head, or a spinal cord injury, this severe acute phase response can markedly dysregulate biomineralization tropism leading to the paradoxical decrease of biomineralization in bone(1–7), with increased biomineralization in soft tissues(8–11). Together, these pathologic events result in adverse outcomes including bone loss (osteopenia and osteoporosis), impaired fracture healing, and soft tissue calcification (dystrophic calcification potentially leading to heterotopic ossification). To overcome these pathologic changes and improve patient outcomes following polytrauma, there is a need for therapeutics that restore the natural tropism of biomineralization to the bone and away from soft tissues.
Bisphosphonates are a class of pharmacologics that elicit potent effects on biomineralization. Bisphosphonates are primarily administered to treat osteoporosis or osteopenia, given their high affinity for hydroxyapatite (the main analog of bone mineral) and capacity to inhibit osteoclast function; thereby supporting increased bone formation and reduced risk of fragility fractures (12–15). However, given bisphosphonates anti-catabolic properties, prior studies have likewise demonstrated that bisphosphonates administration in the context of a bone injury can uncouple the bone remodeling unit, leading to a delay in fracture union, callus maturation, and remodeling (Online Resource Table 1), with variable clinical relevance. Furthermore, like their parent molecule pyrophosphate, bisphosphonates also possess anti-mineralization properties leading them to be examined clinically as a means to inhibit soft tissue mineralization following severe injury, though with varying success (Online Resources Table 2). Yet, given their high affinity for hydroxyapatite, bisphosphonates have been shown to bind and stabilize calcification, influencing its permanence in soft tissues(13). Considering the above, along with the fact that bisphosphonates can have a remarkably long half-life in mineralized tissues (up to ~10 years), differing recommendations have been put forward regarding the use of bisphosphonates following musculoskeletal injury. This study aims to answer the critical clinical question as to the appropriate time to initiate bisphosphonate therapy in patients following a polytrauma that includes a severe injury and necessitates both bone and soft tissue repair(16–19).
The overarching hypothesis is that there exists a pharmacologic window in which bisphosphonate administration will prevent the undue consequences of severe injury-induced dysregulation of biomineralization, yet not interfere with bone and muscle regeneration. First, utilizing an isolated musculoskeletal injury model in animals genetically prone to the formation of soft tissue calcification(20–22), we tested the hypothesis that the dichotomous effects of bisphosphonates on fracture repair and muscle calcification are dependent upon the time of administration relative to the stage of bone or muscle regeneration. Through these experiments, we identified a temporal window in which bisphosphonate administration did and did not interfere with either fracture repair or regression of soft tissue calcification. Guided by these results, we next examined bisphosphonate administration in a polytrauma model, in which a severe injury full-thickness dorsal burn injury is conducted in conjunction with an anatomically remote femur fracture and skeletal muscle injury (i.e. isolated injury model)(7). This model effectively phenocopied a polytraumatic injury, given that the severe burn provoked a systemic response, sufficient to dysregulate biomineralization in distant sites(7). Here, administration of bisphosphonates to the polytrauma model demonstrated that, if administered at the time of injury, bisphosphonates can prevent the undue consequences of severe injury-induced dysregulation of biomineralization, including severe injury-induced osteoporosis and muscle calcification. However, if administered at 7 days post-injury (DPI), while still mildly effective at reducing severe injury-induced osteoporosis, bisphosphonates did not prevent muscle calcification, but rather lead to its permanence within damaged tissues through 42DPI. Together, these results demonstrate that there is a specific pharmacologic window following a polytrauma that bisphosphonates can prevent the undue consequences of severe injury induced dysregulated biomineralization, yet not impair musculoskeletal regeneration.
METHODS:
Isolated Musculoskeletal Injury Model- Combined Murine Fracture and Cardiotoxin Skeletal Muscle Injury.
Male 6-week-old BALB/cJ mice were purchased directly from The Jackson Laboratory for use in the isolated injury model to help screen the pharmacologic window for bisphosphonate administration. BALB/cJ mice were selected for these experiments given their previously described genetic predisposition to soft tissue calcification following injury (Online Resource Fig1)(20–22). At 8 weeks of age, mice received a pre-injury injection of 0.3-mg/mL buprenorphine (0.5-mg/kg). 30-min prior to the procedure, mice were anesthetized with Isoflurane, the fur overlying the medial thigh was removed, and the skin was prepped with betadine prior to making a 10 mm long incision overlying the midshaft of the femur. Once exposed, a cardiotoxin-induced skeletal muscle injury was induced in the quadriceps by injecting 40 μL of 10 μM cardiotoxin(10, 23). Following cardiotoxin injury, a transverse femur fracture was induced and fixed with a 23-gauge needle(24, 25). The incision was then closed with 4-0 nylon sutures and mice were provided analgesia every 12 hours for 3DPI.
Polytraumatic injury model- Combined Murine Burn, Fracture, and Cardiotoxin Skeletal Muscle Injury.
A murine model of polytrauma that includes injury to bone and muscle in the extremity in combination with a severe injury (dorsal burn), was employed. Male 6-week old C57BL/6J mice were purchased directly from The Jackson Laboratory for use in the polytrauma model at 8 weeks of age. C57BL/6J mice were utilized for these experiments given that they have been previously shown to not possess a genetic predisposition to soft tissue calcification following focal muscle injury, allowing us to examine the effects of the polytrauma on instigating soft tissue calcification (10). Following subcutaneous injection of buprenorphine (0.5-mg/kg) 30-min prior to the procedure, mice were anesthetized with isoflurane and the dorsal hair was removed. Following hair removal, mice first underwent cardiotoxin injury of the quadricep followed by a femur fracture, as described above. Finally, mice receive a full-thickness cutaneous burn covering approximately 30% of the total body surface area, as previously described(7). Immediately following the burn, the mouse was dried and injected with 2 ml of intraperitoneal fluid resuscitation with lactated Ringer’s solution. Mice were provided analgesia every 8 hours for 3DPI.
Therapeutic Administration:
In the isolated injury model, zoledronate, a nitrogen-containing bisphosphonate, was administered subcutaneously at 200 μg/kg of body weight. Zoledronate was administered in three unique treatment strategies in the isolated injury model to assess the optimal therapeutic window: 1) Administration of zoledronate 7 days before the injury and at the time of injury (pre-dosing), 2) Administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI (continual dosing), 3) or Single-dose administration of zoledronate at, 7, 14, 21, 28, or 35 DPI (single dosing). Based on results from the isolated injury model, single-dose zoledronate (200 μg/kg) was administered at the time of injury or at 7 DPI in the polytrauma model. For all treatment cohorts, sterile saline was administered at equal volume/weight as a vehicle control. An intravenous dose of ~100 μg/kg zoledronate has been reported to be an equivalent dose of 5mg administered every 21–28 days in human. Given that zoledronate is administered subcutaneously as part of this study, the concentrations selected following drug uptake represent a clinically-relevant dose, aligning with prior small animal studies and drug metabolism/pharmacokinetic data available for Zoledronate (26, 27).
Radiographic Analysis:
Digital radiographic imaging was utilized longitudinally to assess both fracture healing and skeletal muscle calcification, as previously described(10, 24).
Modified Soft Tissue Calcification Scoring System Scale:
The soft tissue calcification scoring system (STiCSS) is an ordinal scoring system previously developed to longitudinally quantify skeletal muscle calcification from digital radiographic images(28). This ordinal scoring system, which was first designed for use in the gastrocnemius and soleus muscles, was modified for use in the quadriceps (Online Resource Fig2). Briefly, a STiCSS Score of 4 represents that greater than 75% of the quadriceps area is mineralized, a score of 3 represents that 50–74% of the quadriceps area is mineralized, a score of 2 represents that 25–49% of the quadriceps area is mineralized, a score of 1 represents that less than 25% of the quadriceps area is mineralized, while a score of 0 represents that no mineralization is detected in the quadriceps.
X-Ray Micro-Computed Tomography (μCT):
Following sacrifice, μCT imaging was performed to visualize skeletal muscle calcification, bone healing at the fracture site, and bone quality within the uninjured contralateral femur for assessment of severe injury-induced osteoporosis. Images were acquired at 55 kVp, 145 μA, 200 ms integration, 1000 projections per full rotation, with a 20 μm isotropic voxel size (μCT40, Scanco Medical AG). Three-dimensional reconstruction of skeletal muscle calcification was segmented from soft tissue using a Gaussian noise filter with a variance of 0.2 and support of 1 and a threshold of 220 per mille (or 450.7 mgHA/cm3)(23, 24, 29).
Finite element analysis (FEA):
micro-FEA was utilized to predict the stability of the fracture callus in silico. Briefly, the segmented μCT images were converted to finite element models (direct bone voxel-to-hexahedral element conversion) for an elastic FEA solver using built-in code (FE-software v1.13) from Scanco Medical AG. After ensuring that each fracture callus was aligned in the z-direction (i.e. along the anatomical long axis), the displacements of the nodes located at the distal cross-section (z=zmin) were fixed in all directions. At the proximal cross-section (z=zmax), nodal forces were applied which resulted in a unit torque at an orientation that rotated the bone around the central axis, simulating a torsion test. Isotropic material properties (Young’s modulus = 10 GPa & Poisson’s ratio = 0.3) were assigned to each element. The predicted failure moment was the resultant moment at the distal end when 2% of the total volume exceeded a von Mises equivalent strain of 0.7 (Online Resource Fig3). Torsional rigidity was the unit torque divided by the corresponding displacement at the proximal end.
Histological analysis:
Following sacrifice at the designated time point, samples were fixed with 10% neutral buffered formalin, decalcified with 0.1M EDTA, and processed for paraffin sectioning and Hematoxylin and Eosin (H&E) staining as previously described(23).
Fluorescent Imaging of BP Localization:
To assess bisphosphonate localization, OsteoSense800 (NEV11105, PerkinElmer), an NIR-labeled nitrogen-containing bisphosphonate, was used. 24 hours before imaging, mice were injected intraperitoneally with 2 nmols of OsteoSense800 in a total volume of 100 μL. A Pearl small animal imaging system and image studio software (LI-COR Biotechnology) were utilized to measure fluorescence at an excitation wavelength of 780nm and an emission wavelength of 805nm following sacrifice.
Statistical analysis and Data Handling:
Differences in modified STiCSS scores among the groups over DPI were statistically evaluated using a repeated measure two-way ANOVA with a Sidak’s or Tukey multiple comparison test were appropriate. Functional testing 42 days and assessment of osteoporosis following injury were evaluated by the non-parametric Kruskal-Wallis Test with a Dunn’s test for multiple comparisons against a control group. P values reported are adjusted for multiplicity. For all analysis, alpha=0.05. All statistical analysis was performed in GraphPad V9. Calculation for sample size was based upon previously published investigations (10, 28), with all groups exceeding the minimum of 3 animals per group. Animals possessed individual numeric identifier, allowing for blinded during the data analysis phase. No animals were excluded during this study.
RESULTS:
Effect of Bisphosphonate Dosing on Fracture Repair and Skeletal Muscle Calcification.
Utilizing an isolated musculoskeletal injury model in animals genetically prone to the formation of soft tissue calcification (Online Resource Fig1)(20–22), we examined two independent dosing strategies of the bisphosphate, zoledronate: 1) pre-dosing beginning 7 days prior to injury and continuing until the time of injury (n=5) or 2) continual dosing beginning 7DPI through 42DPI (n=5). An equivalent volume of saline was administered as a vehicle control (n=16). Longitudinal analysis by radiographic imaging was utilized to assess both muscle calcification within the quadriceps and fracture healing of the femur (Fig1a). A standard longitudinal course of fracture healing and regression of muscle calcification was observed in control (PBS) treated animals. Importantly, this temporal course of healing was the same as individual injury events (fracture alone or muscle injury alone), as previously reported(23, 25). When comparing control mice to those receiving zoledronate in a pre-dosing strategy, fracture healing was comparable, measured by fracture union, fracture callus formation, and callus remodeling across 42DPI (Fig1a). While muscle calcification within the injured quadricep was not prevented in this genetically-prone model with a pre-dosing of zoledronate, calcification did regress from damaged muscle over 42DPI in both control and zoledronate pre-dosing animals (Fig1a&b).
Figure 1: Continuous Zoledronate Dosing in an Isolated Injury Model Delays Fracture Repair and Regression of Soft Tissue Calcification.
A) Representative longitudinal xray of mice administered saline (n=16), zoledronate pre-dosed (n=5), or zoledronate continuous dosing (n=5). Yellow outline; soft tissue calcification within the quadriceps. Black arrow; delay in fracture remodeling. White arrow; delay in regression of soft tissue calcification in mice administered zoledronate beginning at 7 DPI through 42 DPI. B) Quantification of skeletal muscle calcification longitudinally. Points represent median and interquartile range. Given the ordinal nature of the data, repeated measures two-way ANOVA with a Sidak’s or Tukey multiple comparison test were used to assess changes in muscle calcification (STiCSS) Score. ns, p>0.05 *, p≤0.05; **, p≤0.01; ***, p≤0.001. * Indicates comparison between control and BP Pre-dosing. # Indicates comparison between control and zoledronate continual dosing. C) 3D μCT reconstruction of fractured femur and adjacent soft tissue calcification of the quadriceps. White arrows indicate marked residual soft tissue calcification and a delay in fracture callus remodeling at 42 DPI. BP: bisphosphonate. Pre-dosing: administration of zoledronate 7 days before injury and at the time of injury (pre-dosing). Continual dosing: administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI.
Alternatively, when comparing control mice to those who received zoledronate in a continual dosing strategy beginning at 7DPI, a significant change in fracture healing was observed at 42DPI, characterized by reduced hard callus remodeling. Furthermore, we likewise observed marked permanence of muscle calcification within damaged tissues by 42DPI, as measured by radiographic analysis, X-ray micro-computed tomographic imaging (μCT)(Fig1), and histologic sections (Online Resource Fig4).
Effect of Bisphosphonate Dosing on Fracture Callus Size and Predicted Strength.
Given the impairment of fracture callus remodeling observed, the structural and biomechanical properties of the fracture callus between cohorts were assessed. Through evaluation with μCT at 42DPI, fracture callus size trended towards being larger in mice treated continuously with zoledronate compared to saline controls (Online Resource Table3 & Online Resource Fig5), corresponding with a significant increase in polar moment of inertia (p=0.005)(Fig2a). Micro-finite element analysis (μFEA) demonstrated that in addition to being larger, calluses from mice treated with zoledronate trended towards having greater estimated failure torque, with continual zoledronate treatment through 42DPI reaching statistical significance compared to control-treated animals (P=0.027)(Fig2b&c). Together, these results demonstrate that continuous zoledronate treatment beginning at 7DPI leads to a larger fracture callus and greater resistance to torsion at 42DPI.
Figure 2: Structural and Biomechanical Properties of Healing Fractures Following Continuous Zoledronate Dosing.
At 42 DPI, fractured femurs were isolated from mice and assessed for fracture callus size (Supplemental Table 3) and A) polar moment of inertia (pMOI) to assess the torsional resistance of the fracture callus. B) μFEA for the scanned fractured femurs estimated the failure torque of the callus assuming homogeneous tissue modulus of 10 GPa, a failure strain of 0.7%, and a failure volume of 2%. 3D renderings of tissue strain. Scale bar represents 1mm. C) Following normalization to total callus area, estimated failure torque (Nmm) was calculated. Box plot represents median and 25 and 75 percentiles. Whiskers represent data range. ns, p>0.05 *, p≤0.05; **, p≤0.01; ***, p≤0.001.Non-parametric Kruskal-Wallis test with a Dunn’s test for multiple comparisons was utilized to assess changes in structural or biomechanical properties relative to a control group. BP: bisphosphonate. Pre-dosing: administration of zoledronate 7 days before injury and at the time of injury (pre-dosing). Continual dosing: administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI.
Narrowing the Pharmacologic Window- Impact of Single Dose Zoledronate Administration in an Isolated Musculoskeletal Injury Model.
To examine the effects on fracture healing and soft tissue calcification regression through a narrower therapeutic window (one-week intervals), zoledronate was administered to the isolated injury model as a single dose at 7, 14, 21, 28, or 35DPI (N= 5, 5, 4, 5, 4 respectively) (Fig3 and Online Resource Table 4). Single-dose of zoledronate at 7DPI markedly impaired fracture callus remodeling and resulted in permanence of soft tissue calcification to a comparable degree seen with continuous zoledronate dosing previously. When zoledronate was administered at 14 or 21DPI, while limited permanence of soft tissue calcification was noted (Online Resource Table 4), impairment of fracture callus remodeling was observed (Fig3c and Online Resource Table 3). Finally, when administered at 28 or 35DPI, there was no significant impairment in fracture callus remodeling or regression of soft tissue calcification compared to vehicle control (Figure 3 and Online Resource Table 4). Taken together, these data suggest that starting zoledronate administration at the time of injury (Fig1) or after 28DPI does not impact fracture callus remodeling or lead to permanence of soft tissue calcification, while administration between these timepoint will impact one or both of these biomineralization processes.
Figure 3: Single Dose Bisphosphonate Administration Following Isolated Musculoskeletal Injury.
A) Representative longitudinal xray of mice administered a single dose of zoledronate at 7, 14, 21, 28, or 35DPI (N= 5, 5, 4, 5, 4 respectively). Yellow outline; soft tissue calcification within the quadriceps. Black arrow; delay in fracture remodeling. White arrows; delays in regression of soft tissue calcification. B) Quantification skeletal muscle calcification longitudinally. Points represent median and interquartile range. Supplemental Tables 4 contains statistical analysis between groups at each time point. At 42DPI, fractured femurs from mice administered a single dose of zoledronate at 21DPI were analyzed by μCT and FEA. In addition to being significantly larger (Supplemental Table 3), C) polar moment of inertia (pMOI) and FEA assessment of callus strength were assessed. Results are compared to mice receiving continual zoledronate dosing beginning at 7DPI (Figure 2). Box plot represents median and 25 and 75 percentiles. Whiskers represent data range. ns, p>0.05 *, p≤0.05; **, p≤0.01; ***, p≤0.001. Non-parametric Mann-Whitney Test was utilized to assess changes in structural or biomechanical between groups. BP: bisphosphonate. Continual dosing: administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI.
Temporal-Spatial Localization of Bisphosphonate Following Isolated Injury.
Given the above results of single dose bisphosphonate effects on fracture healing and permanence of soft tissue calcification, we likewise assessed the temporal-spatial localization of bisphosphonates, relative to the biomineral processes occurring during musculoskeletal healing in our isolated injury model. Using a fluorescently labeled bisphosphonate, marked localization was observed at the physis in non-injured limbs aligning with high bone turnover in this area. When administered immediately following injury, little to no localization of the bisphosphonate was observed in either the soft tissue or fracture site; yet, when administered at 7DPI, marked fluorescent signal was observed at both the fracture site and within the mineralized soft tissue (Fig4, white arrow- fracture; yellow arrow- soft tissue calcification). Analysis at 14 and 21DPI demonstrated marked bisphosphonate localization to the fracture callus, but reduced localization to the mineralized skeletal muscle aligning with the ongoing regression (Fig4). Finally, analysis at 28, 35, or 42DPI demonstrated limited localization of the bisphosphonate to either the fracture callus or the remaining skeletal muscle calcification. At no point was labeled bisphosphonate observed within non-calcified skeletal muscle. Together, these findings directly correlated with the observed effects on callus remodeling and permanence of soft tissue calcification (Fig3).
Figure 4: Temporal Assessment of Bisphosphonate Localization Following Isolated Musculoskeletal Injury.
To assess the temporal-spatial tropism of bisphosphonates following injury, a fluorescently labeled BP (OsteoSense800, pamidronate) was administered at distinct DPI. White arrows; focal localization of bisphosphonate to skeletal muscle calcification (7DPI) and/or fracture site (7, 14, 21DPI). Yellow outline; skeletal muscle calcification.
Examining Bisphosphonates Pharmacologic Window in a Murine Model of Polytrauma.
Guided by the results from bisphosphonate utilization in the isolated injury model genetically predisposed to the formation of soft tissue calcification, we next assessed bisphosphonate administration in a clinically relevant, polytraumatic injury model(7). Aligning with prior results, when administered at 7DPI (N=5), a single dose of zoledronate treatment led to permanence of soft tissue calcification within the injured quadriceps. Alternatively, when zoledronate was administered as a single dose at the time of injury (N=5), a significant reduction in the amount of soft tissue calcification formed by 7DPI was observed, with no changes in regression over 28DPI (Fig5a&b). In this polytraumatic injury model, reduced fracture callus formation was observed in both the control (N=4) and zoledronate treatment groups. As such, no impairment in callus remodeling was detected when zoledronate was administered at the time of injury or 7DPI, yet fracture union was observed radiographically in all samples (Fig5a).
Figure 5: Examining Bisphosphonates Pharmacologic Window in a Murine Model of Polytraumatic Injury.
A) Representative longitudinal xray of mice undergoing polytrauma (burn+ fracture(fx)+muscle injury) followed by a single dose of zoledronate at the time of injury (N=5) or 7DPI (N=5), compared to animals administered saline (N=4). Yellow outline; soft tissue calcification within the quadriceps. White arrows; delays in regression of soft tissue calcification. B) Quantification skeletal muscle calcification. Point represents median and interquartile range. At 28DPI, the bone quality of the distal metaphysis of non-injured contralateral limbs was evaluated by μCT. * indicates comparison between control and BP at 0DPI. # Indicates comparison between control and bisphosphonate at 7DPI. C) 2D-cross section of the distal metaphysis of the non-injured, contralateral femur. Yellow arrows; reduced trabecular bone. D) bone volume/ tissue volume (BV/TV), E) trabecular space, F) trabecular number, and G) trabecular thickness were evaluated. No injury controls- N=10, burn injury alone- N=10, polytrauma with PBS- N=4, polytrauma with single dose of zoledronate at time of injury (0DPI)- N=5, polytrauma with single dose of zoledronate at 7DPI- N=5. Box plot represents median and 25 and 75 percentiles. Whiskers represent data range. ns, p>0.05 *, p≤0.05; **, p≤0.01; ***, p≤0.001. BP: bisphosphonate.
In addition to the above measures, the use of the murine polytraumatic injury model allowed for the assessment of zoledronate’s effects on preventing severe injury-induced osteoporosis. Aligning with prior reports(7), this murine polytraumatic injury model develops significant severe injury-induced osteoporosis by 28DPI, indicated by a significant loss in metaphyseal bone volume/tissue volume, reduced trabecular thickness and trabecular number, and increase in trabecular space (Fig5c–g). When administered as a single dose at the time of injury, zoledronate treatment significantly improved bone parameters in the contralateral, non-fractured, limb to levels comparable to that of non-injured animals at 28DPI (Fig5d–h). When administered as a single dose at 7DPI, zoledronate treatment likewise significantly improved bone parameters in the contralateral limb by 28DPI, however, given that significant bone loss does occur within the first 7DPI(7), the delay zoledronate treatment resulted in reduce bone parameters compared to mice receiving zoledronate at the time of injury.
DISCUSSION:
This study utilized clinically relevant murine models of musculoskeletal injury to identify the pharmacologic window in which bisphosphonate administration can broadly impact biomineralization occurring as part of fracture repair, soft tissue calcification, and severe injury-induced osteoporosis. These experiments demonstrated that, depending on the time of administration, bisphosphonates can be administered following a polytrauma that includes a severe injury to prevent the undue consequences of severe injury-induced dysregulation of biomineralization yet not interfere with musculoskeletal repair. Prior clinical reports have suggested that the anatomic distribution of bisphosphonates may rapidly change during fracture repair, altering the systemic anti-catabolic properties of the medication and their effect on bone quality(16). Aligning with this clinical intuition, this study found that a pharmacologic window exists in which nitrogen-containing bisphosphonate, such as zoledronate, bind to sites of injury (e.g. fracture callus or soft tissue calcification) and can alter the ongoing tissue regeneration. Taken together, these results illustrate that the potential dichotomous effects of bisphosphonates on musculoskeletal healing are dependent upon the biomineral processes occurring during the time of administration.
Numerous preclinical and clinical studies have examined the effects of bisphosphonate administration on fracture repair (Online Resource Table 1). Given that the type of bisphosphonate, dosage, the timing of administration relative to injury, and animal model or patient populations examined are markedly varied between studies, the relative effect of bisphosphonate on fracture repair are not unanimous. When considering fracture union, the majority of preclinical and clinical studies report that fracture union is not impaired following bisphosphonate administration, which was likewise observed in this study in both the isolated and polytraumatic injury model(30). Aligning with the majority of prior studies, larger callus size and impaired callus remodeling were observed when zoledronate was administered during active bone formation(31), as either a single or continuous dose in the isolated injury model. Prior studies suggest that a larger callus following bisphosphonate administration is beneficial to the strength of the fracture site(32). In this study, the use of μFEA corroborates the notion that zoledronate administration increased callus strength by increasing callus size. It remains however an open question as to whether the zoledronate-related impairment of callus remodeling eventually causes the repaired femur to have less fracture resistance. Further considerations of the biomechanical properties such as fatigue resistance and the clinical impact associated with impaired callus remodeling are still warranted.
Guided by the results from the isolated injury model, this study examined the effect of single-dose zoledronate at the time of injury or 7DPI in a polytraumatic injury model. Unlike the isolated injury model, in this model, no changes in fracture callus remodeling were observed when zoledronate was administered at 7DPI. This was due in part to the marked reduction in total callus size observed across all animals in the polytrauma model. This variance in total callus size may be due in part to reduce movement of these mice following severe injury, thereby decreasing strain across the fracture site, as well as the systemic impact of a severe injury on tissue healing. Future studies discerning this biology are warranted, yet importantly no changes in fracture union were observed radiographically, indicating that the change in callus size may not have a marked impact on bone healing overall in this model.
This study found that administering zoledronate at the time of injury effectively reduced the amount of muscle calcification formed by 7DPI following polytraumatic injury. However, when administered prior to injury in a model genetically predisposed to soft tissue calcification(20–22), zoledronate had no protective effect. This variance in outcome suggests that the molecular underpinning of genetically-associated soft tissue calcification (i.e. fibrodysplasia ossificans progressive (FOP), pseudoxanthoma elasticum (PXE), etc.) may differ from that of severe-injury induces soft tissue calcification, and therefore will necessitate different treatments and pharmacologic strategies to treat clinically. Aligning with this variability, clinical reports examining bisphosphonates for the prevention of soft tissue calcification have reported dichotomous outcomes (Online Resource Table 2). Specifically, while reports suggest that bisphosphonate administration may be advantageous for preventing severe injury-induced soft tissue calcification and heterotopic ossification, other clinical reports conclude no improved effects or even negative consequences associated with bisphosphonate administration leading to an increased risk of soft tissue calcification(33, 34). Considering the above results, these dichotomous outcomes can be due to variable underlying molecular mechanisms leading to soft tissue calcification, variance in patient populations (children vs adults), the diverse structure-activity relationships of the bisphosphonates examined, as well as variable administration strategies that alter the dose, frequency, and timing of the medication.
Beyond prevention of soft tissue calcification, the permanence of calcification within damaged skeletal muscle can markedly impair tissue healing(23). As such, as part of this study, we likewise examined the effect of zoledronate on soft tissue calcification regression. In the isolated injury model, we observed a temporal association between bisphosphonate localizing to regions of skeletal muscle calcification at 7DPI and the permanence of soft tissue calcification by 42DPI. This result may be in part due to bisphosphonates capacity to stabilize and prevent the dissolution of mineral once is it already formed(13, 35, 36). Furthermore, phagocytic cells, such as macrophages, are critical for clearing calcification from damaged skeletal muscle(23, 37); thus, the observed permanence of soft tissue calcification may be a result of BPs capacity to inhibit phagocytic cell function, aligning with their anti-osteoclastic properties observed in bone(38). While less defined clinically, the permanence of soft tissue calcification can have negative consequences on tissue function and repair as persistent calcific nodules can promote tissue inflammation and are sufficient to support the development of heterotopic ossification(23). Further studies assessing the biological effects of the permanence of soft tissue calcification within damaged skeletal muscle are warranted.
Complementing our investigation of bone and soft tissue repair in an isolated injury model, the use of a polytrauma model likewise allowed us to assess the efficacity of bisphosphonates for preventing severe injury-induced osteoporosis(7). Prior clinical studies have demonstrated that administration of pamidronate within 10 days of injury was effective at limiting bone turnover, thereby preserving bone mineral content in children following severe burn injury(6, 39, 40). Results from this study align with these prior clinical findings but demonstrate that when zoledronate was administered at 7DPI, it can likewise impact the ongoing regression of soft tissue calcification. Thus, given the temporal overlap between the development of severe injury-induced osteoporosis, fracture repair, and soft tissue calcification, physicians must consider the impact of a therapeutic like bisphosphonates relative to multiple ongoing biological processes. As such, future studies assessing the temporal effects of bisphosphonates on other musculoskeletal tissues injured, such as nerves, skin, tendons, blood vessels, and ligaments, in polytraumas associated with a severe injury are warranted to construct a broader pharmacologic profile to help inform clinical utilization.
Limitations of this study include the fact that only one bisphosphonate, zoledronate, was examined. Bisphosphonates are a variable class of molecules with a high affinity for bone. Nitrogen-containing bisphosphonate (i.e. zoledronate, pamidronate, alendronate, etc.) are most commonly used in clinical practice given their potent anti-resorptive properties, guiding selection for this study. Furthermore, OsteoSense800 utilized to examine bisphosphonate localization at different points following injury, while not zoledronate, is likewise produced from a nitrogen-containing bisphosphonate, pamidronate. An additional limitation of this study is that all experiments were conducted in young, male, animals. Given the variance in musculoskeletal repair and incidence of soft tissue calcification between pediatric and adult populations, additional studies in aged animals are warranted.
Prior chemical modifications made to the bisphosphonate structure have been in an attempt to improve their anti-resorptive properties, allowing for greater drug efficacy for treating osteoporosis at lower doses. However, as a result of greater efficacy, the anti-mineralization capacity of many nitrogen-containing bisphosphonates is reduced compared to that of non-nitrogen bisphosphonates, given the lower required dosage(41). This variability in anti-mineralization capacity between bisphosphonates may be in part responsible for the limited efficacy at preventing soft tissue calcification in a genetically predisposed model, as well as the variable results from prior clinical studies (Online Resource Table 2). Further investigations of alternative bisphosphonate and variable dosing are warranted for the treatment of genetically-predisposed or severe injury-induced soft tissue calcification.
Importantly, following tissue injury the acute phase response in activated proportional to the severity of the injury in order to resolve bleeding, risk of infection and tissue hypoxia(42). A severe injury can provoke a systemic response that imposes global, pathologic changes on organ systems and tissue repair(42–44). When a severe injury, such as a burn, occurs in association with injury to musculoskeletal tissue, such as a femur fracture or muscle injury, the severe acute phase response can pathologically affect the healing of the musculoskeletal tissue. While controlled in this pre-clinical model, variation in the type and extent of polytrauma experienced by patients may influence the survival and reparative phases of the acute phase response, therefore altering the treatment window in which bisphosphonates administration is optimal for musculoskeletal repair. To help guide future bisphosphonate treatment relative to the on the ongoing biological processes of repair, identification of circulating markers that correlate with temporal points in healing, along with imaging, may prove useful.
In conclusion, this study demonstrated that early bisphosphonate administration at the time of a polytrauma that includes a severe injury was effective at limiting bone loss from the skeleton, helped to reduce soft tissue calcification, but did not notably interfere with fracture healing or the permanence of soft tissue calcification. Together, these findings support future clinical trials aimed at administering bisphosphonate to help prevent the undue consequence of severe injury-induced dysregulated biomineralization, but not sacrifice musculoskeletal regeneration.
Supplementary Material
Supplemental Figure 1: BALB/cJ mice Develop Soft Tissue Calcification Following Injury. Aligning with the previously reported genetic predisposition for soft tissue calcification (20–22), when skeletal muscle is injured, BALB/cJ mice develop marked dystrophic calcification that progressivly regresses over 28DPI. A) Logitudinal xray and end-point μCT analysis (Scale bar: 1mm) allow for the sensitive detection of calcification within skeletal muscle. Yellow outlines; soft tissue calcification within the injured lower limb. Comparatively, wild type C57BL/6J mice that possess no known genetic prediposition to soft tissue calcification do not develop detectable soft tissue calcification following muscle injury alone (7, 10). B) Soft tissue calcification detected in BALB/cJ mice by xray can be quantified via the soft tissue calcification scoring system (STiCSS)(28). Different colored lines denote individual, 6-week-old animals (N=5). C) Example histologic images (20x magnification) at 7 and 28 DPI illustratating marked soft tissue calcification in areas of damaged skeletal muscle in BALB/cJ mice that regress over 28 days. Hematoxylin and eosin (H&E) staining is utilized to denote skeletal muscle morphology. Von kossa staining denotes calcium deposits (black). Scale bar denotes 100 microns.
Supplemental Figure 2: Modified Soft Tissue Calcification Scoring System for use in Quadriceps. Images represent an ordinal scale from 0–4, with “4” representing robust calcification of greater or equal to 75% of the visible quadriceps area becoming mineralized, “3” representing 50–74% calcification of the visible quadriceps, “2” represent 25–49% calcification of the visible quadriceps, “1” representing less than 25% calcification of the quadriceps, and 0 representing no visible calcification within the quadriceps.
Supplemental Figure 3: μCT-FEA of Fracture Callus on femur mid-shaft. Boundary conditions prescribed a unit torque around the vertical axis. Displacement of nodes at the distal face (z=zmin) were fixed in all directions. At the proximal face (z=zmax), force vectors in the transverse plane (i.e. x- and y-directions) were distributed to impart a unit torque that twists the bone about center.
Supplemental Figure 4: Histological analysis of injured quadriceps at 42 DPI. Marked nodules of dystrophic calcification are still observed at 42 DPI within the damaged skeletal muscle of mice administered zoledronate beginning 7 DPI and continuing weekly through 42 DPI. N=3 animals assessed per treatment cohort. Pre-dosing: administration of zoledronate 7 days before injury and at the time of injury (pre-dosing). Continual dosing: administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI.
Supplemental Figure 5: Two Dimensional Images of Fracture Femur at 42 DPI. Axial, coronal and sagittal Images obtained following μCT imaging of two-dimensional planes of section. White arrows indicate the detection of calcified skeletal muscle, as previously visualized in 3D reconstructions (Figure 3C); yellow arrows indicate enlarged fracture callus as quantified in Figure 4A&B. Images are representative of the cohort. Scale bare = 1.0mm. BP: bisphosphonate. Pre-dosing: administration of zoledronate 7 days before injury and at the time of injury (pre-dosing). Continual dosing: administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI.
ACKNOWLEDGMENTS:
The authors would like to thank the members of the Schoenecker lab, in particular Mr. Zachary Backstrom, Mr. J. Court Reese, Dr. Joey Barnett, Dr. Rivka Ihejirika, Dr. Alex Hysong, and Dr. Deke Blum, for their experimental assistance, and help in critically reviewing this manuscript. We would also like to acknowledge our family, friends, and academic colleagues for supporting this work. Finally, we would like to acknowledge the Vanderbilt Small Animal-Imaging Core and the Vanderbilt animal care staff for supplying and maintaining the imaging equipment and our animal facility, respectively.
Funding:
Funding for this work was provided by the National Institutes of Health ([1R01GM126062-01A1, NIGMS, JGS], [T32GM007628, NIGMS, SNML], [T32AR059039, NIAMS, BGHY], [1F31HL149340, NHLBI, BGHY]), the Vanderbilt University Medical Center Department of Orthopaedics and Rehabilitation (JGS), the Jeffrey W. Mast Chair in Orthopaedics Trauma and Hip Surgery (JGS), the Department of Veterans Affairs (JSN, BX005062), and the Caitlin Lovejoy Fund (JGS). Use of the Translational Pathology Shared Resource was supported by NCI/NIH Cancer Center Support Grant (2P30 CA068485-14) and the Vanderbilt Mouse Metabolic Phenotyping Center Grant (5U24DK059637-13). μCT imaging and analysis were supported in part by the Center for Small Animal Imaging at the Vanderbilt University Institute of Imaging Sciences (S10RR027631) from the NIH. Grant 1S10OD021804-01A1 supported the Replacement and Upgrade of an Optical Imaging System for Small Animals, housed in the Vanderbilt Center for Small Animal Imaging, and used in this proposal. Funding sources for this project had no involvement in study design, collection, and analysis of data, writing of the report, or decision in submitting this article for publication.
Conflict of interest:
JGS receives research funding and research support from IONIS Pharmaceuticals, PXE International, OrthoPediatrics, the United States Department of Defense, and the National Institutes of health. SNML receives research funding unrelated to this study from the American Society of Bone and Mineral Research (ASBMR). SLP is a member of the U.S. Air Force. The views expressed in this article are those of the author and do not reflect the official policy or position of the United States Air Force, Department of Defense, or the U.S. Government. All other authors have declared that no conflict of interest exists.
Footnotes
Ethics Approval: Two murine models were examined as part of this study. All animal procedures were approved by the Vanderbilt Institutional Animal Care and Use Committee (M1600231 and M1600225). No human studies were conducted as part of this study.
Availability of Data and Material:
All pertinent data can be found within the following manuscript. Raw data will be provided by the senior author upon request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental Figure 1: BALB/cJ mice Develop Soft Tissue Calcification Following Injury. Aligning with the previously reported genetic predisposition for soft tissue calcification (20–22), when skeletal muscle is injured, BALB/cJ mice develop marked dystrophic calcification that progressivly regresses over 28DPI. A) Logitudinal xray and end-point μCT analysis (Scale bar: 1mm) allow for the sensitive detection of calcification within skeletal muscle. Yellow outlines; soft tissue calcification within the injured lower limb. Comparatively, wild type C57BL/6J mice that possess no known genetic prediposition to soft tissue calcification do not develop detectable soft tissue calcification following muscle injury alone (7, 10). B) Soft tissue calcification detected in BALB/cJ mice by xray can be quantified via the soft tissue calcification scoring system (STiCSS)(28). Different colored lines denote individual, 6-week-old animals (N=5). C) Example histologic images (20x magnification) at 7 and 28 DPI illustratating marked soft tissue calcification in areas of damaged skeletal muscle in BALB/cJ mice that regress over 28 days. Hematoxylin and eosin (H&E) staining is utilized to denote skeletal muscle morphology. Von kossa staining denotes calcium deposits (black). Scale bar denotes 100 microns.
Supplemental Figure 2: Modified Soft Tissue Calcification Scoring System for use in Quadriceps. Images represent an ordinal scale from 0–4, with “4” representing robust calcification of greater or equal to 75% of the visible quadriceps area becoming mineralized, “3” representing 50–74% calcification of the visible quadriceps, “2” represent 25–49% calcification of the visible quadriceps, “1” representing less than 25% calcification of the quadriceps, and 0 representing no visible calcification within the quadriceps.
Supplemental Figure 3: μCT-FEA of Fracture Callus on femur mid-shaft. Boundary conditions prescribed a unit torque around the vertical axis. Displacement of nodes at the distal face (z=zmin) were fixed in all directions. At the proximal face (z=zmax), force vectors in the transverse plane (i.e. x- and y-directions) were distributed to impart a unit torque that twists the bone about center.
Supplemental Figure 4: Histological analysis of injured quadriceps at 42 DPI. Marked nodules of dystrophic calcification are still observed at 42 DPI within the damaged skeletal muscle of mice administered zoledronate beginning 7 DPI and continuing weekly through 42 DPI. N=3 animals assessed per treatment cohort. Pre-dosing: administration of zoledronate 7 days before injury and at the time of injury (pre-dosing). Continual dosing: administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI.
Supplemental Figure 5: Two Dimensional Images of Fracture Femur at 42 DPI. Axial, coronal and sagittal Images obtained following μCT imaging of two-dimensional planes of section. White arrows indicate the detection of calcified skeletal muscle, as previously visualized in 3D reconstructions (Figure 3C); yellow arrows indicate enlarged fracture callus as quantified in Figure 4A&B. Images are representative of the cohort. Scale bare = 1.0mm. BP: bisphosphonate. Pre-dosing: administration of zoledronate 7 days before injury and at the time of injury (pre-dosing). Continual dosing: administration of zoledronate weekly beginning 7 DPI and continuing through 42 DPI.
Data Availability Statement
All pertinent data can be found within the following manuscript. Raw data will be provided by the senior author upon request.