HHS Public Access Author manuscript Author Manuscript
J Burn Care Res. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: J Burn Care Res. 2016 ; 37(6): 367–378. doi:10.1097/BCR.0000000000000389.
Burn Injury has Skeletal Site-Specific Effects on Bone Integrity and Markers of Bone Remodeling Matthew Hoscheit, MD, Loyola University Medical Center
Author Manuscript
Grant Conner, BS, Loyola University Medical Center James Roemer, BS, Saint Louis University Aleksandra Vuckovska, BS, Loyola University Medical Center Pegah Abbasnia, DVM/PhD, Loyola University Medical Center Paul Vana, MD, Loyola University Medical Center
Author Manuscript
Ravi Shankar, PhD, Loyola University Medical Center Richard Kennedy, PhD, and Loyola University Medical Center John Callaci, PhD Loyola University Medical Center
Abstract
Author Manuscript
Objective—To further understand the mechanisms of perturbations in bone remodeling following severe burn injury, the biomechanical properties, genetic expression, and serological markers were evaluated in rodents at six time intervals within six weeks following injury. Moreover, these effects were observed in rodent tibia and lumbar vertebrae to explore possible skeletal-site localization of this pathologic bone loss. Methods—Rodents underwent either thermal injury (100°C water, 30 seconds, 30% BSA) or sham burn. Bone mineral density was evaluated though peripheral quantitative computer tomography, while specialized apparatus measured the weight bearing capacity of tibia and lumbar vertebrae. Markers of bone resorption (RANK-ligand, osteocalcin) and bone formation
Address for reprints: Matthew Hoscheit, CC: Dr. John Callaci, 2160 South 1st Avenue, Department of Orthopedic Surgery: Burn and Shock Trauma Institute, Maywood, IL 60153. Conflicts of Interest: None to disclose
Hoscheit et al.
Page 2
Author Manuscript
(osteoprotegrin, procollagenase type 1 alpha 2) were measured at 7, 14, and 21 days following injury, while serum RANK-ligand levels were observed at these time intervals. Results—Rodent body mass, bone mineral density, and weight bearing capacity were negatively influenced both acutely and several weeks following burn injury. Moreover, a genetic expression profile favoring increased bone resorption and lower bone formation was demonstrated. Our serum analysis findings of significantly increased RANKL 1 and 2 weeks following injury support the increased expression of bone resorption markers. Furthermore, these effects occurred sooner and were more pronounced in the rodent lumbar vertebrae than tibia.
Author Manuscript
Conclusions—These results suggest that severe burn injury results in perturbations in bone remodeling secondary to increased bone resorption and diminished bone formation, impacting both bone mineral density and weight bearing capacity. Furthermore, these processes had a skeletal-site effect more pronounced in the lumbar vertebrae. With a better understanding of the mechanisms of burn-injury bone loss, targeted therapies can be implemented to improve long term clinical outcomes.
Introduction
Author Manuscript
Severe burn injury associated-hypermetabolism is characterized by acute hyperdynamic circulatory, physiologic, catabolic, and immune system responses.1 The first 24 hours following injury is marked by a rise in inflammatory cytokines, specifically IL-1 and IL-6, enhancing osteoblast production of the ligand of the receptor activator of nuclear transcription factor κB (RANKL), and stimulation of the differentiation of marrow stromal cells into osteoclasts.2-4 Pathological stimulation of osteoclastogenesis results in bone resorption, also seen in other diseases of bone such as osteoporosis and Paget’s disease.5-8 Endogenous glucocorticoid production rises 8-fold two weeks following severe burn injury, furthering RANKL production and subsequently augmenting bone resorption.9 Urinary cortisol levels remain elevated three months following injury, suggesting that these physiologic mechanisms persist beyond recovery from the initial insult.10 Alongside increased bone resorption, reduced biochemical markers of osteoblast formation and absence of osteoblasts on bone surface two weeks following injury suggest reduced bone formation.9 Dysregulation of calcium homeostasis within days of burn injury results in hypocalcemia, serving yet as another possible mechanism for the perturbations in bone remodeling.11 This evidence suggests that severe burn injury is followed by inflammatory, hypermetabolic, and mineral homeostatic dysregulation resulting in increased bone resorption, decreased bone formation, and ultimately pathologic bone loss.
Author Manuscript
Several lines of evidence suggest that this pathologic bone loss following severe burn injury has implications of acute and prolonged clinical outcomes in children and adults.12-14 Decreased bone mineral density (BMD) in children has been observed within fourteen days of injury, with effects persisting up to five years.12,14 Anatomical findings of reduced BMD in the lumbar spine, specifically L1-L4, forearm, and proximal femur following severe burn injury show localized areas of more significant bone remodeling.15-16 Mineral density loss can be observed as early as fourteen days following burn injury in rodents and ten days in sheep.14,17 Significant loss of trabecular bone volume has been observed in an 8% total body surface area (TBSA) injury, suggesting that even non-severe burn injury may lead to J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
Page 3
Author Manuscript
perturbations in bone metabolism.18 Histologic analysis of bone after thermal injury has demonstrated substantially reduced indices of cancellous bone formation, whereas those indices in cortical bone were essentially nonexistent.15 These changes in bone metabolism have been shown to result in decreased weight bearing ability in rodent femurs as soon as fourteen days following injury.14 These findings have dramatic clinical implications; severe thermal injuries may result in nearly doubled annual increased risk of fracture in young adults while also predisposing them to failure in achieving predicted stature.19 The loss of and failure to gain bone mass in children and adults may lead to life-long osteoporosis.20
Author Manuscript
Though biomechanical experiments, genetic expression analyses, and serological investigations have evaluated the outcomes of burn-induced bone remodeling, the exact mechanisms involved remain unclear. Moreover, prior research has only examined certain variables of burn-induced bone remodeling at singular time points following burn injury. A prospective study observing multiple factors at multiple time intervals following injury has not been performed. The goal of the current investigation was to use a previously validated rodent paradigm of burn-injury and hypermetabolism to observe the time course of biomechanical properties, gene expression, and serological marker changes related to bone remodeling through the first six weeks following severe burn injury.21 More specifically, tibia and lumbar vertebrae BMD, weight bearing ability, bone formation and resorptionrelated gene expression, and serological markers were evaluated at multiple time intervals within 6 weeks following burn injury. Our hypothesis was that a 30% TBSA burn injury is associated with a skeletal site-specific decrease in bone mineral density leading to deficits in the biomechanical properties of tibia and lumbar vertebrae. We further hypothesized that severe burn injury will affect the expression of biological markers of bone remodeling.
Author Manuscript
Methods Animal Procedures
Author Manuscript
This investigation received approval from the Loyola University Chicago Institutional Animal Care and Use Committee. Adolescent male Sprague-Dawley rats were obtained at 10 weeks of age (300-350g body weight; Harlan, Indianapolis, IN). Prior to treatment, animals were randomly assigned into control (n = 3), sham controls (n = 6), and burn injury (n = 6) groups, chosen based on a power analysis performed using preliminary data. Animals were pair housed and acclimated for 1 week with a 12 hour light/dark cycle and fed and given water ad libitum. Animals in the treatment group were anesthetized with isoflurane inhalation + ketamine i.p., shaved, given additional isoflurane, and placed into a template that exposes a dorsal body area corresponding to 30% TBSA. Full thickness burn was then induced by immersion of the dorsal skin into 100°C boiling water for 30 seconds. Sham animals were anesthetized, shaved, and their dorsal surface immersed in tepid water. Animals were then resuscitated with lactated Ringer’s solution i.p. as per the Parkland formula (4ml/kg/percent burn) during the first 24 hours after burn injury. Animals were euthanized at either 3, 7, 14, 21, 28, or 42 days following burn injury. The lumbar spine and tibias were subsequently harvested from each animal. Fibrous connective tissue and muscle were manually removed from each sample using sterile equipment.
J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
Page 4
BMD Measurements
Author Manuscript Author Manuscript
Bone samples were stored in formalin following extraction. Both cortical + subcortical and trabecular BMD of the lumbar vertebral bodies and tibias of each animal were determined by peripheral quantitative computer tomography (pQCT) using a Norland Stratec bone densitometer (Orthometrix, Inc. White Plains, NY). For vertebrae, each vertebral segment was positioned uniformly on a support so that the instrument-scanning plane was perpendicular to the longitudinal axis of the vertebral body. Scout views were initially obtained to determine the midpoint of each vertebral body. Midpoint analysis was performed to ensure that each sample was analyzed at the same point to minimize variation due to density differences based on sampling area. Three consecutive measurements were performed at a resolution of 70mm/voxel 1 mm apart from this point. Using a predetermined peel algorithm, the cancellous area of each vertebral segment was defined as 45% of the total bone cross-sectional area of each measured plane of the vertebral body, the remaining fraction defined as cortical bone. For the tibia, three measurements were performed, beginning 1 mm from the proximal endplate and proceeding distally. The three measurements obtained for both the tibia and vertebrae were then averaged separately and reported as the overall mineral density of each respective sample. The cancellous compartment was defined as 42% of the total tibial cross sectional area. The instrument was set to use the threshold contour mode (soft-tissue threshold set at 220mg/cm3). Scans were made at 50 kV and 0.3 mA. A solid phantom was used prior to CT image acquisition to ensure proper calibration. Biomechanical Structural Properties of Rodent Tibia and Lumbar Vertebrae
Author Manuscript
Compressive strength analyses were performed on the tibias as well as L3 and L4 vertebral bodies of each rat using a Biomaterials testing machine (Model 5544, Instron Crop., Cantom, MA). For each tibia, a custom 4-point compression sequence designed in-house was used to determine the load to failure. For each vertebrae, endplates were potted in bone cement using a previously described method that results in two parallel loading surfaces necessary to perform a uniform compression test on individual rodent vertebrae.22,23 The specimens were prepared so that the posterior elements of the vertebra did not contact the loading platforms. Compression testing was performed at a crosshead speed of 0.5 mm/min to eliminate any strain rate effects. A 100kg load-cell was used to monitor the compressive load and a precision sensor was used to measure the axial deformation of the specimen. This load-deformation data was analyzed to obtain the compressive strength of the vertebrae, defined as the maximum load sustained before material failure. Load to failure measurements for the L3 and L4 vertebrae of each animal were recorded, averaged, and reported as a single value.
Author Manuscript
RNA Extraction and Quality Control For evaluation of bone formation and resorption markers, tibia and lumbar vertebrae samples were extracted, snap frozen in liquid nitrogen, and stored at −80°C. Bone samples were prepared for RNA extraction by first adding 3mL Tri-Reagent Solution (Ambion) and then pulverizing each bone sample individually using a 6770 Freezer/Mill (SPEX SamplePrep, NJ). RNA extraction from the pulverized samples was performed using RiboPure RNA
J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
Page 5
Author Manuscript
Purification kit (Life Technologies). RNA isolation was performed according to the manufacturer’s instructions. Samples were then quantified via Nanodrop spectroscopy and visualized for purity and integrity using the Agilent 2100 Bioanalyzer prior to gene array analysis. Gene Expression Array Analysis
Author Manuscript
Markers for bone formation and resorption were evaluated through Quantitative Real-Time PCR (qRT-PCR). qRT-PCR was performed using the 7500 Fast Real-Time RT-PCR Gene Expression System (ABI). Each cDNA sample was amplified in duplicates using gene specific and control Taqman Gene Expression Assay primer/probe sets (Life Technologies) in 96 well plates. Primer/probe sets included RANK-ligand (RANKL), osteocalcin (OPG), procollagenase type 1 alpha 2 (Col1a2), and osteoprotegerin (Bglap) (Life Technologies). SDS software 1.4 using the 2−DDCT relative quantification method was used to analyze the data. Serum Biomarker Analysis Serial blood samples were collected from the tail vein in sham and burn-injured rodents at 7, 14, and 21 days following treatment to evaluate serum levels of bone-resorption biomarker RANKL. 1mL of blood was collected into serum tubes following a 6 hour fast. Samples were allowed to clot for 2 hours at room temperature before centrifuging for 20 minutes at 2000rpm and stored at -80°C. An enzyme-linked immunosorbant assay kit was used to quantify RANKL activity (Quantikine ELISA, Minneapolis, MN) and read in a microplate reader. All assays were carried out in duplicates, according to the manufacturer’s instructions.
Author Manuscript
Statistical Analysis All statistical analyses were carried out using the PRISM 6 software program (San Diego, CA). All data are reported as mean ± standard error (SE), calculated using one-way ANOVA analysis. Variations in animal weights are expressed as a percentage change in body weight from day 0. SDS software 1.4 using the 2−DDCT relative quantification method was used to analyze mRNA level expression in markers of bone formation and resorption. Bar graphs show semi-quantitative RANKL & OPG normalized such that gene expression in the tibia and lumbar vertebrae of sham animals has a value of one, while mRNA expression of the experimental group was calculated to demonstrate expression relative to control animals. Level of significance was < 0.05 and < 0.0001.
Author Manuscript
Results General Observations Animals were weighed at the start and end of their respective observation periods. All sham rodents were observed to gain weight during the study except during the first three days of the study, in which they lost an average of 6 grams. The initial weights of the two groups were similar prior to treatment [starting weights of 342 ± 3.8 g in sham group and 339 ± 2.9 g in experimental group (P = .49)]. The experimental animals lost weight at 3, 7, and 14 days following treatment, with significant weight loss at 7 and 14 days (P < 0.05). The J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
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Author Manuscript
largest amount of weight loss was noted 7 days following burn injury, during which time animals lost an average of 7.1% of their initial weight (initial weight of 334.67 ± 4.0 g and final weight of 310.83 ± 7.9 g 7 days following burn injury). Experimental animals lost weight until 14 days following injury, after which their weight increased throughout the remainder of the study. Experimental animals reached their approximate pretreatment weights by 28 days following injury, surpassing their pretreatment weight 42 days postinjury [initial weight of 318.0 ± 2.1 g and final weight of 338.8 ± 5.8 g 7 days following burn injury (P < .05)]. Sham animals weighed significantly more than experimental rodents at 7 (P < .05), 14, 21, 28, and 42 (P < .001) days following thermal injury (Figure 1). Effects of Burn Injury on Tibia and Lumbar Vertebrae BMD
Author Manuscript Author Manuscript
Trabecular and cortical + subcortical BMD were evaluated from rodent tibial and lumbar vertebrae at 3, 7, 14, 21, and 42 days post-burn injury (Figure 2). Lumbar vertebral BMD was also evaluated 28 days following injury. Tibia trabecular BMD was similar in both groups at 3 days post-injury (P = .23). Significantly lower trabecular bone mineral density in the experimental group was first noted 21 days post-injury and again at 42 days, which demonstrated a 26% and 42% decrease in BMD from sham animals, respectively (P < .05). Sham animals demonstrated greater tibial trabecular BMD than burn-injured rodents throughout the study (Figure 2a). Tibia cortical + subcortical BMD analysis demonstrated significantly decreased BMD in the experimental group 14 and 42 days after burn injury in comparison to sham animals (P < .05). pQCT measurement of trabecular and cortical + subcortical bone mineral density L3 and L4 vertebrae found a decreased BMD in the burn rodents throughout the course of the study. Trabecular BMD in the experimental group vertebrae was lower also compared to sham animals throughout the study, with significance found 7, 21, 28 and 42 days following burn injury (P < .05 at 7 and 21 days, P < .0001 at 21 and 42 days, Figure 2c). A trend towards decreased trabecular BMD was found in the experimental groups 3 and 14 days following burn injury [11.4% reduction in BMD for burn-injury vs. control animals at day 3 (P = 0.08) and 19.9% reduction in BMD at day 14 (P = .07)]. Cortical + subcortical bone mineral density differences in sham and burn animals demonstrated a similar trend to the trabecular BMD findings. A lower cortical + subcortical bone mineral density in the experimental group was found at each time point during the study, with a significant difference seen 3, 7, 14, 28, and 42 days following burn injury [(P
J Burn Care Res. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: J Burn Care Res. 2016 ; 37(6): 367–378. doi:10.1097/BCR.0000000000000389.
Burn Injury has Skeletal Site-Specific Effects on Bone Integrity and Markers of Bone Remodeling Matthew Hoscheit, MD, Loyola University Medical Center
Author Manuscript
Grant Conner, BS, Loyola University Medical Center James Roemer, BS, Saint Louis University Aleksandra Vuckovska, BS, Loyola University Medical Center Pegah Abbasnia, DVM/PhD, Loyola University Medical Center Paul Vana, MD, Loyola University Medical Center
Author Manuscript
Ravi Shankar, PhD, Loyola University Medical Center Richard Kennedy, PhD, and Loyola University Medical Center John Callaci, PhD Loyola University Medical Center
Abstract
Author Manuscript
Objective—To further understand the mechanisms of perturbations in bone remodeling following severe burn injury, the biomechanical properties, genetic expression, and serological markers were evaluated in rodents at six time intervals within six weeks following injury. Moreover, these effects were observed in rodent tibia and lumbar vertebrae to explore possible skeletal-site localization of this pathologic bone loss. Methods—Rodents underwent either thermal injury (100°C water, 30 seconds, 30% BSA) or sham burn. Bone mineral density was evaluated though peripheral quantitative computer tomography, while specialized apparatus measured the weight bearing capacity of tibia and lumbar vertebrae. Markers of bone resorption (RANK-ligand, osteocalcin) and bone formation
Address for reprints: Matthew Hoscheit, CC: Dr. John Callaci, 2160 South 1st Avenue, Department of Orthopedic Surgery: Burn and Shock Trauma Institute, Maywood, IL 60153. Conflicts of Interest: None to disclose
Hoscheit et al.
Page 2
Author Manuscript
(osteoprotegrin, procollagenase type 1 alpha 2) were measured at 7, 14, and 21 days following injury, while serum RANK-ligand levels were observed at these time intervals. Results—Rodent body mass, bone mineral density, and weight bearing capacity were negatively influenced both acutely and several weeks following burn injury. Moreover, a genetic expression profile favoring increased bone resorption and lower bone formation was demonstrated. Our serum analysis findings of significantly increased RANKL 1 and 2 weeks following injury support the increased expression of bone resorption markers. Furthermore, these effects occurred sooner and were more pronounced in the rodent lumbar vertebrae than tibia.
Author Manuscript
Conclusions—These results suggest that severe burn injury results in perturbations in bone remodeling secondary to increased bone resorption and diminished bone formation, impacting both bone mineral density and weight bearing capacity. Furthermore, these processes had a skeletal-site effect more pronounced in the lumbar vertebrae. With a better understanding of the mechanisms of burn-injury bone loss, targeted therapies can be implemented to improve long term clinical outcomes.
Introduction
Author Manuscript
Severe burn injury associated-hypermetabolism is characterized by acute hyperdynamic circulatory, physiologic, catabolic, and immune system responses.1 The first 24 hours following injury is marked by a rise in inflammatory cytokines, specifically IL-1 and IL-6, enhancing osteoblast production of the ligand of the receptor activator of nuclear transcription factor κB (RANKL), and stimulation of the differentiation of marrow stromal cells into osteoclasts.2-4 Pathological stimulation of osteoclastogenesis results in bone resorption, also seen in other diseases of bone such as osteoporosis and Paget’s disease.5-8 Endogenous glucocorticoid production rises 8-fold two weeks following severe burn injury, furthering RANKL production and subsequently augmenting bone resorption.9 Urinary cortisol levels remain elevated three months following injury, suggesting that these physiologic mechanisms persist beyond recovery from the initial insult.10 Alongside increased bone resorption, reduced biochemical markers of osteoblast formation and absence of osteoblasts on bone surface two weeks following injury suggest reduced bone formation.9 Dysregulation of calcium homeostasis within days of burn injury results in hypocalcemia, serving yet as another possible mechanism for the perturbations in bone remodeling.11 This evidence suggests that severe burn injury is followed by inflammatory, hypermetabolic, and mineral homeostatic dysregulation resulting in increased bone resorption, decreased bone formation, and ultimately pathologic bone loss.
Author Manuscript
Several lines of evidence suggest that this pathologic bone loss following severe burn injury has implications of acute and prolonged clinical outcomes in children and adults.12-14 Decreased bone mineral density (BMD) in children has been observed within fourteen days of injury, with effects persisting up to five years.12,14 Anatomical findings of reduced BMD in the lumbar spine, specifically L1-L4, forearm, and proximal femur following severe burn injury show localized areas of more significant bone remodeling.15-16 Mineral density loss can be observed as early as fourteen days following burn injury in rodents and ten days in sheep.14,17 Significant loss of trabecular bone volume has been observed in an 8% total body surface area (TBSA) injury, suggesting that even non-severe burn injury may lead to J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
Page 3
Author Manuscript
perturbations in bone metabolism.18 Histologic analysis of bone after thermal injury has demonstrated substantially reduced indices of cancellous bone formation, whereas those indices in cortical bone were essentially nonexistent.15 These changes in bone metabolism have been shown to result in decreased weight bearing ability in rodent femurs as soon as fourteen days following injury.14 These findings have dramatic clinical implications; severe thermal injuries may result in nearly doubled annual increased risk of fracture in young adults while also predisposing them to failure in achieving predicted stature.19 The loss of and failure to gain bone mass in children and adults may lead to life-long osteoporosis.20
Author Manuscript
Though biomechanical experiments, genetic expression analyses, and serological investigations have evaluated the outcomes of burn-induced bone remodeling, the exact mechanisms involved remain unclear. Moreover, prior research has only examined certain variables of burn-induced bone remodeling at singular time points following burn injury. A prospective study observing multiple factors at multiple time intervals following injury has not been performed. The goal of the current investigation was to use a previously validated rodent paradigm of burn-injury and hypermetabolism to observe the time course of biomechanical properties, gene expression, and serological marker changes related to bone remodeling through the first six weeks following severe burn injury.21 More specifically, tibia and lumbar vertebrae BMD, weight bearing ability, bone formation and resorptionrelated gene expression, and serological markers were evaluated at multiple time intervals within 6 weeks following burn injury. Our hypothesis was that a 30% TBSA burn injury is associated with a skeletal site-specific decrease in bone mineral density leading to deficits in the biomechanical properties of tibia and lumbar vertebrae. We further hypothesized that severe burn injury will affect the expression of biological markers of bone remodeling.
Author Manuscript
Methods Animal Procedures
Author Manuscript
This investigation received approval from the Loyola University Chicago Institutional Animal Care and Use Committee. Adolescent male Sprague-Dawley rats were obtained at 10 weeks of age (300-350g body weight; Harlan, Indianapolis, IN). Prior to treatment, animals were randomly assigned into control (n = 3), sham controls (n = 6), and burn injury (n = 6) groups, chosen based on a power analysis performed using preliminary data. Animals were pair housed and acclimated for 1 week with a 12 hour light/dark cycle and fed and given water ad libitum. Animals in the treatment group were anesthetized with isoflurane inhalation + ketamine i.p., shaved, given additional isoflurane, and placed into a template that exposes a dorsal body area corresponding to 30% TBSA. Full thickness burn was then induced by immersion of the dorsal skin into 100°C boiling water for 30 seconds. Sham animals were anesthetized, shaved, and their dorsal surface immersed in tepid water. Animals were then resuscitated with lactated Ringer’s solution i.p. as per the Parkland formula (4ml/kg/percent burn) during the first 24 hours after burn injury. Animals were euthanized at either 3, 7, 14, 21, 28, or 42 days following burn injury. The lumbar spine and tibias were subsequently harvested from each animal. Fibrous connective tissue and muscle were manually removed from each sample using sterile equipment.
J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
Page 4
BMD Measurements
Author Manuscript Author Manuscript
Bone samples were stored in formalin following extraction. Both cortical + subcortical and trabecular BMD of the lumbar vertebral bodies and tibias of each animal were determined by peripheral quantitative computer tomography (pQCT) using a Norland Stratec bone densitometer (Orthometrix, Inc. White Plains, NY). For vertebrae, each vertebral segment was positioned uniformly on a support so that the instrument-scanning plane was perpendicular to the longitudinal axis of the vertebral body. Scout views were initially obtained to determine the midpoint of each vertebral body. Midpoint analysis was performed to ensure that each sample was analyzed at the same point to minimize variation due to density differences based on sampling area. Three consecutive measurements were performed at a resolution of 70mm/voxel 1 mm apart from this point. Using a predetermined peel algorithm, the cancellous area of each vertebral segment was defined as 45% of the total bone cross-sectional area of each measured plane of the vertebral body, the remaining fraction defined as cortical bone. For the tibia, three measurements were performed, beginning 1 mm from the proximal endplate and proceeding distally. The three measurements obtained for both the tibia and vertebrae were then averaged separately and reported as the overall mineral density of each respective sample. The cancellous compartment was defined as 42% of the total tibial cross sectional area. The instrument was set to use the threshold contour mode (soft-tissue threshold set at 220mg/cm3). Scans were made at 50 kV and 0.3 mA. A solid phantom was used prior to CT image acquisition to ensure proper calibration. Biomechanical Structural Properties of Rodent Tibia and Lumbar Vertebrae
Author Manuscript
Compressive strength analyses were performed on the tibias as well as L3 and L4 vertebral bodies of each rat using a Biomaterials testing machine (Model 5544, Instron Crop., Cantom, MA). For each tibia, a custom 4-point compression sequence designed in-house was used to determine the load to failure. For each vertebrae, endplates were potted in bone cement using a previously described method that results in two parallel loading surfaces necessary to perform a uniform compression test on individual rodent vertebrae.22,23 The specimens were prepared so that the posterior elements of the vertebra did not contact the loading platforms. Compression testing was performed at a crosshead speed of 0.5 mm/min to eliminate any strain rate effects. A 100kg load-cell was used to monitor the compressive load and a precision sensor was used to measure the axial deformation of the specimen. This load-deformation data was analyzed to obtain the compressive strength of the vertebrae, defined as the maximum load sustained before material failure. Load to failure measurements for the L3 and L4 vertebrae of each animal were recorded, averaged, and reported as a single value.
Author Manuscript
RNA Extraction and Quality Control For evaluation of bone formation and resorption markers, tibia and lumbar vertebrae samples were extracted, snap frozen in liquid nitrogen, and stored at −80°C. Bone samples were prepared for RNA extraction by first adding 3mL Tri-Reagent Solution (Ambion) and then pulverizing each bone sample individually using a 6770 Freezer/Mill (SPEX SamplePrep, NJ). RNA extraction from the pulverized samples was performed using RiboPure RNA
J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
Page 5
Author Manuscript
Purification kit (Life Technologies). RNA isolation was performed according to the manufacturer’s instructions. Samples were then quantified via Nanodrop spectroscopy and visualized for purity and integrity using the Agilent 2100 Bioanalyzer prior to gene array analysis. Gene Expression Array Analysis
Author Manuscript
Markers for bone formation and resorption were evaluated through Quantitative Real-Time PCR (qRT-PCR). qRT-PCR was performed using the 7500 Fast Real-Time RT-PCR Gene Expression System (ABI). Each cDNA sample was amplified in duplicates using gene specific and control Taqman Gene Expression Assay primer/probe sets (Life Technologies) in 96 well plates. Primer/probe sets included RANK-ligand (RANKL), osteocalcin (OPG), procollagenase type 1 alpha 2 (Col1a2), and osteoprotegerin (Bglap) (Life Technologies). SDS software 1.4 using the 2−DDCT relative quantification method was used to analyze the data. Serum Biomarker Analysis Serial blood samples were collected from the tail vein in sham and burn-injured rodents at 7, 14, and 21 days following treatment to evaluate serum levels of bone-resorption biomarker RANKL. 1mL of blood was collected into serum tubes following a 6 hour fast. Samples were allowed to clot for 2 hours at room temperature before centrifuging for 20 minutes at 2000rpm and stored at -80°C. An enzyme-linked immunosorbant assay kit was used to quantify RANKL activity (Quantikine ELISA, Minneapolis, MN) and read in a microplate reader. All assays were carried out in duplicates, according to the manufacturer’s instructions.
Author Manuscript
Statistical Analysis All statistical analyses were carried out using the PRISM 6 software program (San Diego, CA). All data are reported as mean ± standard error (SE), calculated using one-way ANOVA analysis. Variations in animal weights are expressed as a percentage change in body weight from day 0. SDS software 1.4 using the 2−DDCT relative quantification method was used to analyze mRNA level expression in markers of bone formation and resorption. Bar graphs show semi-quantitative RANKL & OPG normalized such that gene expression in the tibia and lumbar vertebrae of sham animals has a value of one, while mRNA expression of the experimental group was calculated to demonstrate expression relative to control animals. Level of significance was < 0.05 and < 0.0001.
Author Manuscript
Results General Observations Animals were weighed at the start and end of their respective observation periods. All sham rodents were observed to gain weight during the study except during the first three days of the study, in which they lost an average of 6 grams. The initial weights of the two groups were similar prior to treatment [starting weights of 342 ± 3.8 g in sham group and 339 ± 2.9 g in experimental group (P = .49)]. The experimental animals lost weight at 3, 7, and 14 days following treatment, with significant weight loss at 7 and 14 days (P < 0.05). The J Burn Care Res. Author manuscript; available in PMC 2017 November 01.
Hoscheit et al.
Page 6
Author Manuscript
largest amount of weight loss was noted 7 days following burn injury, during which time animals lost an average of 7.1% of their initial weight (initial weight of 334.67 ± 4.0 g and final weight of 310.83 ± 7.9 g 7 days following burn injury). Experimental animals lost weight until 14 days following injury, after which their weight increased throughout the remainder of the study. Experimental animals reached their approximate pretreatment weights by 28 days following injury, surpassing their pretreatment weight 42 days postinjury [initial weight of 318.0 ± 2.1 g and final weight of 338.8 ± 5.8 g 7 days following burn injury (P < .05)]. Sham animals weighed significantly more than experimental rodents at 7 (P < .05), 14, 21, 28, and 42 (P < .001) days following thermal injury (Figure 1). Effects of Burn Injury on Tibia and Lumbar Vertebrae BMD
Author Manuscript Author Manuscript
Trabecular and cortical + subcortical BMD were evaluated from rodent tibial and lumbar vertebrae at 3, 7, 14, 21, and 42 days post-burn injury (Figure 2). Lumbar vertebral BMD was also evaluated 28 days following injury. Tibia trabecular BMD was similar in both groups at 3 days post-injury (P = .23). Significantly lower trabecular bone mineral density in the experimental group was first noted 21 days post-injury and again at 42 days, which demonstrated a 26% and 42% decrease in BMD from sham animals, respectively (P < .05). Sham animals demonstrated greater tibial trabecular BMD than burn-injured rodents throughout the study (Figure 2a). Tibia cortical + subcortical BMD analysis demonstrated significantly decreased BMD in the experimental group 14 and 42 days after burn injury in comparison to sham animals (P < .05). pQCT measurement of trabecular and cortical + subcortical bone mineral density L3 and L4 vertebrae found a decreased BMD in the burn rodents throughout the course of the study. Trabecular BMD in the experimental group vertebrae was lower also compared to sham animals throughout the study, with significance found 7, 21, 28 and 42 days following burn injury (P < .05 at 7 and 21 days, P < .0001 at 21 and 42 days, Figure 2c). A trend towards decreased trabecular BMD was found in the experimental groups 3 and 14 days following burn injury [11.4% reduction in BMD for burn-injury vs. control animals at day 3 (P = 0.08) and 19.9% reduction in BMD at day 14 (P = .07)]. Cortical + subcortical bone mineral density differences in sham and burn animals demonstrated a similar trend to the trabecular BMD findings. A lower cortical + subcortical bone mineral density in the experimental group was found at each time point during the study, with a significant difference seen 3, 7, 14, 28, and 42 days following burn injury [(P
