Diminished Chondrogenesis and Enhanced Osteoclastogenesis in Leptin-Deficient Diabetic Mice (ob/ob) Impair Pathologic, Trauma-Induced Heterotopic Ossification

Diabetic trauma patients exhibit delayed postsurgical wound, bony healing, and dysregulated bone development. However, the impact of diabetes on the pathologic development of ectopic bone or heterotopic ossification (HO) following trauma is unknown. In this study, we use leptin-deficient mice as a model for type 2 diabetes to understand how post-traumatic HO development may be affected by this disease process. Male leptin-deficient (ob/ob) or wild-type (C57BL/6 background) mice aged 6?8 weeks underwent 30% total body surface area burn injury with left hind limb Achilles tenotomy. Micro-CT (?CT) imaging showed significantly lower HO volumes in diabetic mice compared with wild-type controls (0.70 vs. 7.02?mm3, P?Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140207/1/scd.2015.0135.pd

Scleraxis‐Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136325/1/stem2515_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136325/2/stem2515.pd

Coordinating Tissue Regeneration Through Transforming Growth Factorâ β Activated Kinase 1 Inactivation and Reactivation

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factorâ β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment (â drug onâ ), the impact of drug withdrawal (â drug offâ ) implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dualâ inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment (â drug onâ ) and subsequent withdrawal (â drug offâ ) through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the â drug onâ (Creâ mediated inactivation) and â drug offâ (Flpâ mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766â 778Manipulating transforming growth factor βâ activated kinase 1 for cell and scaffold free tissue regeneration using a dualâ inducible Combinational Sequential Inversion Engineering mouse model.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149573/1/stem2991_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149573/2/stem2991.pd

Effects of Aging on Osteogenic Response and Heterotopic Ossification Following Burn Injury in Mice

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 ?CT 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-?B) 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-?B 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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140205/1/scd.2014.0291.pd

Characterization of Heterotopic Ossification Using Radiographic Imaging: Evidence for a Paradigm Shift.

Heterotopic ossification (HO) is the growth of extra-skeletal bone which occurs following trauma, burns, and in patients with genetic bone morphogenetic protein (BMP) receptor mutations. The clinical and laboratory evaluation of HO is dependent on radiographic imaging to identify and characterize these lesions. Here we show that despite its inadequacies, plain film radiography and single modality microCT continue to serve as a primary method of HO imaging in nearly 30% of published in vivo literature. Furthermore, we demonstrate that detailed microCT analysis is superior to plain film and single modality microCT radiography specifically in the evaluation of HO formed through three representative models due to its ability to 1) define structural relationships between growing extra-skeletal bone and normal, anatomic bone, 2) provide accurate quantification and growth rate based on volume of the space-occupying lesion, thereby facilitating assessments of therapeutic intervention, 3) identify HO at earlier times allowing for evaluation of early intervention, and 4) characterization of metrics of bone physiology including porosity, tissue mineral density, and cortical and trabecular volume. Examination of our trauma model using microCT demonstrated two separate areas of HO based on anatomic location and relationship with surrounding, normal bone structures. Additionally, microCT allows HO growth rate to be evaluated to characterize HO progression. Taken together, these data demonstrate the need for a paradigm shift in the evaluation of HO towards microCT as a standard tool for imaging

Effects of Aging on Osteogenic Response and Heterotopic Ossification Following Burn Injury in Mice

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 ?CT 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-?B) 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-?B 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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140205/1/scd.2014.0291.pd

MicroCT can evaluate HO metrics in addition to volume.

<p><i>(</i>A) Average tissue mineral density (TMD) of tibia, soft tissue HO and bone-associated HO evaluated at 5 and 9 weeks post-op in burn/tenotomy model. (B) Representative cross sectional images of soft tissue HO (<i>top two rows</i>) and bone-associated HO (<i>bottom row</i>) at 5 and 9 weeks in burn/tenotomy model. (C) Tibial bone, soft tissue HO, and bone-associated HO in burn/tenotomy model evaluated for average porosity using 1250 HU threshold. (D) Burn/tenotomy HO shell and marrow space average volumes isolated with manual, trabecular contours (<i>white</i>) or by automated 800 HU threshold (<i>blue</i>). (E) Average TMD of soft tissue and bone-associated HO in burn/tenotomy mice that were: untreated, celecoxib-injected, or apyrase-injected. (F) Representative cross sectional images of soft tissue HO (<i>top two rows</i>) and bone-associated HO (<i>bottom row</i>) of untreated and treated burn/tenotomy groups at 9 weeks after injury. (G) Average porosity of soft tissue (<i>left</i>) and calcaneal (<i>right</i>) HO in burn/tenotomy untreated and treated groups.</p

MicroCT allows clear delineation of HO from normal anatomic bone.

<p><b>(</b>A) Plain film radiograph of 9-week burn/tenotomy of mouse hindlimb to demonstrate obscuring effect by normal anatomic bone. (B) High resolution microCT image of burn/tenotomy mouse hindlimb 9 weeks post-op (<i>red arrow</i>: axial plane of cross section; <i>red outline</i>: <i>bone-associated HO growth at calcaneus</i>). (C) Plain film radiograph of day 22 Ad.cre/cardiotoxin-induced mouse hindlimb to demonstrate obscuring effect by normal anatomic bone (D) High resolution microCT image of day 22 Ad.cre/cardiotoxin-induced hindlimb with representative serial cross sections (<i>orange outline</i>: Ad.cre/cardiotoxin-induced HO). (E) Histologic cross sections of uninjured, contralateral mouse hindlimb stained with aniline blue with comparable microCT cross sections depicting normal tibia, talus, and calcaneal bones. (F) Experimental burn/tenotomy hindlimb histologic cross sections stained with aniline blue with comparable microCT cross sections depicting normal tibia, talus, and calcaneal bones with HO sites. (G) MicroCT evaluation of HO with blanking technique demonstrated to remove normal bone (<i>orange dotted circles</i>) from cross-sections of burn/tenotomy model. H) MicroCT evaluation of HO with blanking technique demonstrated to remove normal bone (<i>orange dotted circles</i>) from cross-sections of Ad.cre/cardiotoxin-induced model.</p

MicroCT allows for superior volume and growth quantification of HO.

<p>(A) Representative 3D reconstructions of normal, anatomic bone, bone-associated HO <i>(red outline</i>), and soft tissue HO (<i>yellow outline</i>) of burn/tenotomy model at 5 and 9 weeks; HO visual rendered at three density-dependent thresholds. (B) Total and density-dependent volumes of HO in burn/tenotomy model at 9 weeks after injury. (C) HO volume following trauma in two separate anatomic sites (bone-associated or soft tissue) at separate time points. (D) Mean daily growth rate of HO in burn/tenotomy model based on longitudinal, total volume quantification, (E) Representative 3D reconstructions (800 HU) of normal, anatomic bone and genetic HO (<i>green outline</i>) at four timepoints. (F) Density-dependent volumes of day 40 genetic HO model. (G) Genetic HO 800HU volume at separate timepoints. (H) Mean daily growth rate of HO in genetic model based on longitudinal, 800HU volume quantification. (In graphs: * = p<0.05; # = p<0.07).</p