Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging

AbstractFracture healing is regulated by mechanical loading. Understanding the underlying mechanisms during the different healing phases is required for targeted mechanical intervention therapies. Here, the influence of individualized cyclic mechanical loading on the remodelling phase of fracture healing was assessed in a non-critical-sized mouse femur defect model. After bridging of the defect, a loading group (n = 10) received individualized cyclic mechanical loading (8–16 N, 10 Hz, 5 min, 3 × /week) based on computed strain distribution in the mineralized callus using animal-specific real-time micro-finite element analysis with 2D/3D visualizations and strain histograms. Controls (n = 10) received 0 N treatment at the same post-operative time-points. By registration of consecutive scans, structural and dynamic callus morphometric parameters were followed in three callus sub-volumes and the adjacent cortex showing that the remodelling phase of fracture healing is highly responsive to cyclic mechanical loading with changes in dynamic parameters leading to significantly larger formation of mineralized callus and higher degree of mineralization. Loading-mediated maintenance of callus remodelling was associated with distinct effects on Wnt-signalling-associated molecular targets Sclerostin and RANKL in callus sub-regions and the adjacent cortex (n = 1/group). Given these distinct local protein expression patterns induced by cyclic mechanical loading during callus remodelling, the femur defect loading model with individualized load application seems suitable to further understand the local spatio-temporal mechano-molecular regulation of the different fracture healing phases.

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Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging

10.1101/2020.09.15.297861 ◽

2020 ◽

Cited By ~ 1

Author(s):

Esther Wehrle ◽

Graeme R Paul ◽

Duncan C Tourolle né Betts ◽

Gisela A Kuhn ◽

Ralph Müller

Keyword(s):

Fracture Healing ◽

Mechanical Loading ◽

Callus Formation ◽

Expression Patterns ◽

Element Analysis ◽

Spatio Temporal ◽

Underlying Mechanisms ◽

Mechanical Intervention ◽

Local Protein

AbstractFracture healing is regulated by mechanical loading. Understanding the underlying mechanisms during the different healing phases is required for targeted mechanical intervention therapies. Here, the influence of individualized cyclic mechanical loading on the remodelling phase of fracture healing was assessed in a mouse femur defect model. After bridging of the defect, a loading group (n=10) received individualized cyclic mechanical loading (8-16 N, 10 Hz, 5 min, 3x/week) based on computed strain distribution in the callus using animal-specific real-time micro-finite element analysis. Controls (n=10) received 0 N treatment at the same post-operative time-points. By registration of consecutive scans, structural and dynamic callus morphometric parameters were followed in three callus sub-volumes and the adjacent cortex showing that the remodelling phase of fracture healing is highly responsive to cyclic mechanical loading with changes in dynamic parameters leading to significantly larger callus formation and mineralization. Loading-mediated maintenance of callus remodelling was associated with distinct effects on Wnt-signalling-associated molecular targets Sclerostin and Rankl in callus sub-regions and the adjacent cortex. Given these distinct local protein expression patterns induced by cyclic mechanical loading, the femur defect loading model with individualized load application seems suitable to understand the local spatio-temporal mechano-molecular regulation of the different fracture healing phases.

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A piezoelectric bone fixation plate for in vivo application and monitoring of mechanical loading during fracture healing

Measurement Science and Technology ◽

10.1088/1361-6501/ab8c6c ◽

2020 ◽

Vol 31 (9) ◽

pp. 095703

Author(s):

Bradley D Nelson ◽

Salil Sidharthan Karipott ◽

Robert E Guldberg ◽

Keat Ghee Ong

Keyword(s):

Fracture Healing ◽

Mechanical Loading ◽

Bone Fixation ◽

Fixation Plate

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In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

eLife ◽

10.7554/elife.46181 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Li He ◽

Richard Binari ◽

Jiuhong Huang ◽

Julia Falo-Sanjuan ◽

Norbert Perrimon

Keyword(s):

Fluorescent Protein ◽

Fluorescent Proteins ◽

Expression Patterns ◽

Cell Type ◽

Dynamic Information ◽

New Genes ◽

Dual Color ◽

Dynamic Expression ◽

Spatio Temporal

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

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RPK1 and BAK1 sequentially form complexes with OST1 to regulate ABA-induced stomatal closure

Journal of Experimental Botany ◽

10.1093/jxb/erz489 ◽

2019 ◽

Author(s):

Yun Shang ◽

Dami Yang ◽

Yunmi Ha ◽

Hyun-young Shin ◽

Kyoung Hee Nam

Keyword(s):

Water Status ◽

Stomatal Closure ◽

Expression Patterns ◽

Cell Movement ◽

Guard Cells ◽

Aba Signaling ◽

Signaling Systems ◽

Underlying Mechanisms

Abstract Regulation of plant water status occurs via abscisic acid (ABA)-induced stomatal closure. Open Stomata 1 (OST1) is a critical ABA signaling component regulating this process in guard cells. We previously reported that BRI1-associated receptor kinase 1 (BAK1) positively regulates ABA-induced stomatal closure by interacting with, and phosphorylating OST1. Here, we show that the Receptor-like Protein Kinase 1 (RPK1), previously known to be induced by ABA, is a positive ABA-signaling component in guard cell movement, and interacts with OST1. ABA-inducible expression patterns were observed in RPK1 and OST1, but not in BAK1. We investigated the underlying mechanisms by which the RPK1/OST1 and BAK1/OST1 complexes interact in stomatal guard cells by monitoring the complex formation continuously using FRET analyses. We found that the BAK1/OST1 complex was formed earlier than the RPK1/OST1 complex in response to ABA. In vitro and semi-in vivo kinase assays revealed that a trans-phosphorylation event occurred in the RPK1/OST1 complex, which differs from that in the BAK1/OST1 complex, wherein only OST1 phosphorylation occurred via BAK1. ABA Insensitive 1 (ABI1) only dephosphorylated OST1 in the BAK1/OST1 complex, but dephosphorylated both RPK1 and OST1 proteins in the RPK1/OST1 complex. Our results suggest that there are multiple coordinated ABA signaling systems to regulate stomatal movement.

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Fracture Healing Research—Shift towards In Vitro Modeling?

Biomedicines ◽

10.3390/biomedicines9070748 ◽

2021 ◽

Vol 9 (7) ◽

pp. 748

Author(s):

Moritz Pfeiffenberger ◽

Alexandra Damerau ◽

Annemarie Lang ◽

Frank Buttgereit ◽

Paula Hoff ◽

...

Keyword(s):

Fracture Healing ◽

Ex Vivo ◽

3D Models ◽

In Vitro Models ◽

Human Patient ◽

In Vivo Models ◽

Underlying Mechanisms ◽

Biological Situation

Fractures are one of the most frequently occurring traumatic events worldwide. Approximately 10% of fractures lead to bone healing disorders, resulting in strain for affected patients and enormous costs for society. In order to shed light into underlying mechanisms of bone regeneration (habitual or disturbed), and to develop new therapeutic strategies, various in vivo, ex vivo and in vitro models can be applied. Undeniably, in vivo models include the systemic and biological situation. However, transferability towards the human patient along with ethical concerns regarding in vivo models have to be considered. Fostered by enormous technical improvements, such as bioreactors, on-a-chip-technologies and bone tissue engineering, sophisticated in vitro models are of rising interest. These models offer the possibility to use human cells from individual donors, complex cell systems and 3D models, therefore bridging the transferability gap, providing a platform for the introduction of personalized precision medicine and finally sparing animals. Facing diverse processes during fracture healing and thus various scientific opportunities, the reliability of results oftentimes depends on the choice of an appropriate model. Hence, we here focus on categorizing available models with respect to the requirements of the scientific approach.

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Cancellous bone adaptation to tibial compression is not sex dependent in growing mice

Journal of Applied Physiology ◽

10.1152/japplphysiol.00210.2010 ◽

2010 ◽

Vol 109 (3) ◽

pp. 685-691 ◽

Cited By ~ 66

Author(s):

Maureen E. Lynch ◽

Russell P. Main ◽

Qian Xu ◽

Daniel J. Walsh ◽

Mitchell B. Schaffler ◽

...

Keyword(s):

Bone Mass ◽

Mechanical Loading ◽

Cancellous Bone ◽

Adaptive Response ◽

Bone Adaptation ◽

Micro Computed Tomography ◽

Element Analysis ◽

Male And Female ◽

And Control

Mechanical loading can be used to increase bone mass and thus attenuate pathological bone loss. Because the skeleton's adaptive response to loading is most robust before adulthood, elucidating sex-specific responses during growth may help maximize peak bone mass. This study investigated the effect of sex on the response to controlled, in vivo mechanical loading in growing mice. Ten-week-old male and female C57Bl/6 mice underwent noninvasive compression of the left tibia. Peak loads of −11.5 N were applied, corresponding to +1,200 με at the tibial midshaft in both sexes. Cancellous bone mass, architecture, and dynamic formation in the proximal metaphysis were compared between loaded and control limbs via micro-computed tomography and histomorphometry. The strain environment of the proximal metaphysis during loading was characterized using finite element analysis. Both sexes responded to tibial compression through increased bone mass and altered architecture. Cancellous bone mass and tissue density were enhanced in loaded limbs relative to control limbs in both sexes through trabecular thickening and reduced separation. Changes in mass were due to increased cellular activity in loaded limbs compared with control limbs. Adaptation to loading increased the proportion of load transferred by the cancellous bone in the proximal metaphysis. For all cancellous measures, the response to tibial compression did not differ between male and female mice. When similar strains are engendered in males and females, the adaptive response in cancellous bone to mechanical loading does not depend on sex.

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In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

10.1101/564690 ◽

2019 ◽

Author(s):

Li He ◽

Richard Binari ◽

Jiuhong Huang ◽

Julia Falo-Sanjuan ◽

Norbert Perrimon

Keyword(s):

Fluorescent Protein ◽

Fluorescent Proteins ◽

Expression Patterns ◽

Cell Type ◽

Dynamic Information ◽

New Genes ◽

Dual Color ◽

Dynamic Expression ◽

Spatio Temporal

AbstractFluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

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