direct-mouse-traumaburn-model-of-heterotopic-ossification

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

University of Michigan Medical School

Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, occurs in over 60% of major burn injuries and blast traumas. A significant need exists to improve the current diagnostic modalities for HO which are inadequate to diagnose and intervene on HO at early time-points. Raman spectroscopy has been used in previous studies to report on changes in bone composition during bone development but has not yet been applied to burn induced HO. In this study, we validate transcutaneous, in-vivo Raman spectroscopy as a methodology for early diagnosis of HO in mice following a burn injury.

Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy.

To demonstrate the pro-osteogenic effect of burn injury on heterotopic bone formation using a novel burn ossicle in vivo model.

Burn injury enhances bone formation in heterotopic ossification model.

Extracellular adenosine triphosphate (ATP), present in thermally injured tissue, modulates the inflammatory response and causes significant tissue damage. The authors hypothesize that neutrophil infiltration and ensuing tissue necrosis would be mitigated by removing ATP-dependent signaling at the burn site. Mice were subjected to 30% TBSA partial-thickness scald burn by dorsal skin immersion in a water bath at 60 or 20°C (nonburn controls). In the treatment arm, an ATP hydrolyzing enzyme, apyrase, was applied directly to the site immediately after injury. Skin was harvested after 24 hours and 5 days for hematoxylin and eosin stain, elastase, and Ki-67 staining. Tumor necrosis factor (TNF)-α and interferon (IFN)-β expression were measured through quantitative real-time polymerase chain reaction. At 24 hours, the amount of neutrophil infiltration was different between the burn and burn + apyrase groups (P < .001). Necrosis was less extensive in the apyrase group when compared with the burn group at 24 hours and 5 days. TNF-α and IFN-β expression at 24 hours in the apyrase group was lower than in the burn group (P < .05). However, Ki-67 signaling was not significantly different among the groups. The results of this study support the role of extracellular ATP in neutrophil activity. The authors demonstrate that ATP hydrolysis at the burn site allays the neutrophil response to thermal injury and reduces tissue necrosis. This decrease in inflammation and tissue necrosis is at least partially because of TNF-α and IFN-β signaling. Apyrase could be used as topical inflammatory regulators to quell the injury caused by inflammation.

Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice.

Heterotopic ossification (HO) is the abnormal formation of bone in soft tissue. Current diagnostics have low sensitivity or specificity to incremental progression of mineralization, especially at early time points. Without accurate and reliable early diagnosis and intervention, HO progression often results in incapacitating conditions of limited range of motion, nerve entrapment, and pain. We hypothesized that non-invasive near-infrared (NIR) optical imaging can detect HO at early time points and monitor heterotopic bone turnover longitudinally. C57BL6 mice received an Achilles tenotomy on their left hind limb in combination with a dorsal burn or sham procedure. A calcium-chelating tetracycline derivative (IRDye 680RD BoneTag) was injected bi-weekly and imaged via NIR to measure accumulative fluorescence for 11 wk and compared to in vivo microCT images. Percent retention of fluorescence was calculated longitudinally to assess temporal bone resorption. NIR detected HO as early as five days and revealed a temporal response in HO formation and turnover. MicroCT could not detect HO until 5 wk. Confocal microscopy confirmed fluorophore localization to areas of HO. These findings demonstrate the ability of a near-infrared optical imaging strategy to accurately and reliably detect and monitor HO in a murine model.

Early detection of heterotopic ossification using near-infrared optical imaging reveals dynamic turnover and progression of mineralization following Achilles tenotomy and burn injury.

Heterotopic ossification (HO) is a common and debilitating complication of burns, traumatic brain injuries, and musculoskeletal trauma and surgery. Although the exact mechanism of ectopic bone formation is unknown, mesenchymal stem cells (MSCs) capable of osteogenic differentiation are known to play an essential role. Interestingly, the prevalence of HO in the elderly population is low despite the high overall occurrence of musculoskeletal injury and orthopedic procedures. We hypothesized that a lower osteogenicity of MSCs would be associated with blunted HO formation in old compared with young mice. In vitro osteogenic differentiation of adipose-derived MSCs from old (18-20 months) and young (6-8 weeks) C57/BL6 mice was assessed, with or without preceding burn injury. In vivo studies were then performed using an Achilles tenotomy with concurrent burn injury HO model. HO formation was quantified using μCT scans, Raman spectroscopy, and histology. MSCs from young mice had more in vitro bone formation, upregulation of bone formation pathways, and higher activation of Smad and nuclear factor kappa B (NF-κB) signaling following burn injury. This effect was absent or blunted in cells from old mice. In young mice, burn injury significantly increased HO formation, NF-κB activation, and osteoclast activity at the tenotomy site. This blunted, reactive osteogenic response in old mice follows trends seen clinically and may be related to differences in the ability to mount acute inflammatory responses. This unique characterization of HO and MSC osteogenic differentiation following inflammatory insult establishes differences between age populations and suggests potential pathways that could be targeted in the future with therapeutics.

Effects of aging on osteogenic response and heterotopic ossification following burn injury in mice.

Patients with severe burns suffer functional, structural, and esthetic complications. It is important to explore reconstructive options given that no ideal treatment exists. Transfer of adipose and adipose-derived stem cells (ASCs) has been shown to improve healing in various models. The authors hypothesize that use of fat isografts and/or ASCs will improve healing in a mouse model of burn injury. Twenty 6 to 8 week old C57BL/6 male mice received a 30% surface area partial-thickness scald burn. Adipose tissue and ASCs from inguinal fat pads were harvested from a second group of C57BL/6 mice. Burned mice received 500 μl subcutaneous injection at burn site of 1) processed adipose, 2) ASCs, 3) mixed adipose (adipose and ASCs), or 4) sham (saline) injection (n = 5/group) on the first day postinjury. Mice were followed by serial photography until being killed at days 5 and 14. Wounds were assessed for burn depth and healing by hematoxylin and eosin (H&E) and immunohistochemistry. All treated groups showed improved healing over controls defined by decreased wound depth, area, and apoptotic activity. After 5 days, mice receiving ASCs or mixed adipose displayed a non-significant improvement in vascularization. No significant changes in proliferation were noted at 5 days. Adipose isografts improve some early markers of healing postburn injury. The authors demonstrate that addition of these grafts improves specific structural markers of healing. This improvement may be because of an increase in early wound vascularity postgraft. Further studies are needed to optimize use of fat or ASC grafts in acute and reconstructive surgery.

Wound healing after thermal injury is improved by fat and adipose-derived stem cell isografts.

Access to readily available autogenous tissue that regenerates bone would greatly improve clinical care. We believe the osteogenic phenotype caused by mutations in ALK2 can be harnessed in adipose-derived stem cells (ASCs) to improve bone tissue engineering. We set out to demonstrate that ALK2 may serve as a novel target to (a) improve in vitro ASC osteogenic differentiation and (b) enhance in vivo bone regeneration and calvarial healing. Transgenic mice were designed using the Cre/lox system to express constitutively active ALK2 (caALK2) with ubiquitously inducible Cre expression after tamoxifen exposure. ASCs from caALK2+/- and caALK2-/- (control) mice were exposed to tamoxifen and assessed for pro-osteogenic gene expression, bone morphogenetic protein (BMP) signaling, and osteogenic differentiation. Next, ASCs collected from these transgenic mice were analyzed in vivo using a calvarial defect model and analyzed by micro-computed tomography (micro-CT) and histology. ASCs from caALK2+/- mice had increased BMP signaling as demonstrated by upregulation of pSmad 1/5. ASCs from caALK2+/- mice had enhanced bone signaling and osteogenic differentiation compared with caALK2-/- mice (n = 4, p < .05). Transcription of pro-osteogenic genes at day 7 was significantly higher in ASCs from caALK2-overexpressing mice (Alp, Runx2, Ocn, Opn) (n = 4, p < .05). Using micro-CT and histomorphometry, we found that bone formation was significantly higher in mice treated with caALK2-expressing ASCs in vivo. Using a novel transgenic mouse model, we show that expression of constitutively active ALK2 receptor results in significantly increased ASC osteogenic differentiation. Furthermore, we demonstrate that this increased ASC differentiation can be harnessed to improve calvarial healing.

Targeting of ALK2, a Receptor for Bone Morphogenetic Proteins, Using the Cre/lox System to Enhance Osseous Regeneration by Adipose-Derived Stem Cells.

Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation.

Treatment of heterotopic ossification through remote ATP hydrolysis.

BMP signaling mediated by ACVR1 plays a critical role for development of multiple structures including the cardiovascular and skeletal systems. While deficient ACVR1 signaling impairs normal embryonic development, hyperactive ACVR1 function (R206H in humans and Q207D mutation in mice, ca-ACVR1) results in formation of heterotopic ossification (HO). We developed a mouse line, which conditionally expresses ca-ACVR1 with Nfatc1-Cre(+) transgene. Mutant mice developed ectopic cartilage and bone at the distal joints of the extremities including the interphalangeal joints and hind limb ankles as early as P4 in the absence of trauma or exogenous bone morphogenetic protein (BMP) administration. Micro-CT showed that even at later time points (up to P40), cartilage and bone development persisted at the affected joints most prominently in the ankle. Interestingly, this phenotype was not present in areas of bone outside of the joints - tibia are normal in mutants and littermate controls away from the ankle. These findings demonstrate that this model may allow for further studies of heterotopic ossification, which does not require the use of stem cells, direct trauma or activation with exogenous Cre gene administration.

BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints.

Bony non-unions arising in the aftermath of collateral radiation injury are commonly managed with vascularized free tissue transfers. Unfortunately, these procedures are invasive and fraught with attendant morbidities. This study investigated a novel, alternative treatment paradigm utilizing adipose-derived stem cells (ASCs) combined with angiogenic deferoxamine (DFO) in the rat mandible.

Article	Year
Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy. Bone\| PubMed ID: 23314070	2013
Burn injury enhances bone formation in heterotopic ossification model. Annals of surgery\| PubMed ID: 23673767	2014
Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice. Journal of burn care & research : official publication of the American Burn Association\| PubMed ID: 23877144	2014
Early detection of heterotopic ossification using near-infrared optical imaging reveals dynamic turnover and progression of mineralization following Achilles tenotomy and burn injury. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 25087685	2014
Effects of aging on osteogenic response and heterotopic ossification following burn injury in mice. Stem cells and development\| PubMed ID: 25122460	2015
Wound healing after thermal injury is improved by fat and adipose-derived stem cell isografts. Journal of burn care & research : official publication of the American Burn Association\| PubMed ID: 25185931	2015
Targeting of ALK2, a Receptor for Bone Morphogenetic Proteins, Using the Cre/lox System to Enhance Osseous Regeneration by Adipose-Derived Stem Cells. Stem cells translational medicine\| PubMed ID: 25232183	2014
Treatment of heterotopic ossification through remote ATP hydrolysis. Science translational medicine\| PubMed ID: 25253675	2014
BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints. Developmental biology\| PubMed ID: 25722188	2015
Translational treatment paradigm for managing non-unions secondary to radiation injury utilizing adipose derived stem cells and angiogenic therapy. Head & neck\| PubMed ID: 25917284	2015

Jonathan R. Peterson

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