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Annals of Biomedical Engineering Apr 2021Bone lengthening and bone transport are regeneration processes that commonly rely on distraction osteogenesis, a widely accepted surgical procedure to deal with numerous...
Bone lengthening and bone transport are regeneration processes that commonly rely on distraction osteogenesis, a widely accepted surgical procedure to deal with numerous bony pathologies. Despite the extensive study in the literature of the influence of biomechanical factors, a lack of knowledge about their mechanobiological differences prevents a clinical particularization. Bone lengthening treatments were performed on sheep metatarsus by reproducing the surgical and biomechanical protocol of previous bone transport experiments. Several in vivo monitoring techniques were employed to build an exhaustive comparison: gait analysis, radiographic and CT assessment, force measures through the fixation, or mechanical characterization of the new tissue. A significant initial loss of the bearing capacity, quantified by the ground reaction forces and the limb contact time with the ground, is suffered by the bone lengthening specimens. The potential effects of this anomaly on the musculoskeletal force distribution and the evolution of the bone callus elastic modulus over time are also analyzed. Imaging techniques also seem to reveal lower bone volume in the bone lengthening callus than in the bone transport one, but an equivalent mineralization rate. The simultaneous quantification of biological and mechanical parameters provides valuable information for the daily clinical routine and numerical tools development.
Topics: Animals; Biomechanical Phenomena; Bone Regeneration; Bony Callus; Elastic Modulus; Female; Gait; Metatarsal Bones; Osteogenesis, Distraction; Sheep; Tomography, X-Ray Computed
PubMed: 33111968
DOI: 10.1007/s10439-020-02665-z -
Frontiers in Endocrinology 2022Catecholamine signaling is known to influence bone tissue as reuptake of norepinephrine released from sympathetic nerves into bone cells declines with age leading to...
Catecholamine signaling is known to influence bone tissue as reuptake of norepinephrine released from sympathetic nerves into bone cells declines with age leading to osteoporosis. Further, β-adrenoceptor-blockers like propranolol provoke osteoprotective effects in osteoporotic patients. However, besides systemic adrenal and sympathetic catecholamine production, it is also known that myeloid cells can synthesize catecholamines, especially under inflammatory conditions. To investigate the effects of catecholamines produced by CD11b myeloid cells on bone turnover and regeneration, a mouse line with specific knockout of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis, in CD11b myeloid cells (TH/CD11b-Cre, referred to as TH) was generated. For bone phenotyping, male mice were sacrificed at eight and twelve weeks of age and harvested bones were subjected to bone length measurement, micro-computed tomography, fluorescence-activated cell sorting of the bone marrow, gene expression analysis, histology and immunohistochemistry. Support for an age-dependent influence of myeloid cell-derived catecholamines on bone homeostasis is provided by the fact that twelve-week-old, but not eight-week-old TH mice, developed an osteopenic phenotype and showed increased numbers of neutrophils and T lymphocytes in the bone marrow, while CCL2, IL-6, IL-4 and IL-10 mRNA expression was reduced in sorted myeloid bone marrow cells. To investigate the influence of myeloid cell-derived catecholamines on fracture healing, mice received a diaphyseal femur osteotomy. Three days post-fracture, immunohistochemistry revealed an increased number of macrophages, neutrophils and cytotoxic T lymphocytes in the fracture hematoma of TH mice. Micro-computed tomography on day 21 showed a decreased tissue mineral density, a reduced bone volume and less trabeculae in the fracture callus indicating delayed fracture healing, probably due to the increased presence of inflammatory cells in TH mice. This indicates a crucial role of myeloid cell-derived catecholamines in immune cell-bone cell crosstalk and during fracture healing.
Topics: Animals; Bone Remodeling; Bony Callus; Catecholamines; Fractures, Bone; Interleukin-10; Interleukin-4; Interleukin-6; Macrophages; Male; Mice; Norepinephrine; Propranolol; RNA, Messenger; Receptors, Adrenergic; Tyrosine 3-Monooxygenase; X-Ray Microtomography
PubMed: 36187089
DOI: 10.3389/fendo.2022.997745 -
Journal of Visualized Experiments : JoVE Dec 2022Micro-computed tomography (µCT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture...
Micro-computed tomography (µCT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.
Topics: Fracture Healing; X-Ray Microtomography; Bony Callus; Osteogenesis
PubMed: 36571411
DOI: 10.3791/64262 -
Journal of Orthopaedic Research :... Jan 2021Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC)....
Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC). Identification of new markers for periosteal MSCs is essential for the development of fracture therapeutics. Expression of the matricellular protein thrombospondin-2 (TSP2) increases during early fracture healing; however, it is currently unknown what cell population expresses TSP2. Using a TSP2 GFP reporter mouse and a stabilized murine fracture model, we characterized the expression of TSP2 during the inflammatory, soft callus formation, and hard callus formation phases of fracture healing. In addition, using TSP2 GFP positive cells harvested from reporter mouse cells, we characterized the cell population using flow cytometry and colony formation assays. In uninjured diaphyseal bone, we observed TSP2 expression in the cells located along the inner periosteum. We also observed a population of TSP2 expressing cells in undifferentiated regions of early fracture callus and along the periphery of the callus. Later in callus development, TSP2 cells were broadly distributed in the undifferentiated callus, but GFP was not expressed by chondrocytes. Flow cytometry confirmed that the majority of TSP2 expressing cells were positive for traditional murine MSC markers. Our in vitro assays further supported these findings by demonstrating all adherent and colony-forming cells expressed TSP2. Taken together, our results suggest that TSP2 is expressed by undifferentiated MSCs, but downregulated in chondrocytes. Clinical significance: expression of the matricellular protein TSP2 is a promising new marker to identify MSCs in early fracture healing.
Topics: Animals; Bony Callus; Cell Proliferation; Chondrocytes; Chondrogenesis; Fractures, Bone; Genes, Reporter; Mice; Thrombospondins
PubMed: 32437051
DOI: 10.1002/jor.24749 -
Development (Cambridge, England) Mar 2022Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous...
Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.
Topics: Animals; Bone Regeneration; Bony Callus; Fracture Healing; Mammals; Mandible; Zebrafish
PubMed: 35178545
DOI: 10.1242/dev.199995 -
Injury Jun 2021The process of bone healing largely recapitulates bone development in the embryo and ideally achieves complete restoration of bone shape and structure. However, because... (Review)
Review
The process of bone healing largely recapitulates bone development in the embryo and ideally achieves complete restoration of bone shape and structure. However, because successful fracture healing requires tight interactions of numerous cell types and signaling molecules, any disruption of this highly coordinated processes can result in delayed healing or even non-union formation. The rate of fracture healing complications in orthopedic patients is reported to be 5-20%. Therefore, there is a need for new therapeutic strategies to improve fracture healing in patients with healing complications. One treatment strategy would include the easy and safe application of a pharmacological agent inducing osteoanabolic effects during fracture healing. One potential promising molecular target is the osteoanabolic WNT signaling pathway. This pathway plays an important role during embryonic bone development, homeostasis, mechanotransduction, development of osteoporosis and bone regeneration. This review focuses on preclinical studies targeting WNT signaling molecules to accelerate fracture healing. The three main investigated antagonists of the WNT signaling pathway, which can be blocked experimentally by antibodies, are Sclerostin, Dickkopf-1 and Midkine. Treating animals with antibodies against these proteins enhanced bone formation in the fracture callus. This indicates a therapeutic potential for these antibodies to accelerate fracture healing in patients with orthopedic complications.
Topics: Animals; Bony Callus; Fracture Healing; Fractures, Bone; Humans; Mechanotransduction, Cellular; Wnt Signaling Pathway
PubMed: 33234263
DOI: 10.1016/j.injury.2020.11.051 -
Calcified Tissue International Feb 2022Objective and accurate assessment of bone union after a fracture, arthrodesis, or osteotomy is relevant for scientific and clinical purposes. Bone union is most... (Review)
Review
Objective and accurate assessment of bone union after a fracture, arthrodesis, or osteotomy is relevant for scientific and clinical purposes. Bone union is most accurately imaged with computed tomography (CT), but no consensus exists about objective assessment of bone union from CT images. It is unclear which CT-generated parameters are most suitable for bone union assessment. The aim of this review of animal studies is to find which CT-generated parameters are associated most strongly with actual bone union. Scientific databases were systematically searched. Eligible studies were studies that (1) were animal studies, (2) created a fracture, (3) assessed bone union with CT, (4) performed mechanical or histological testing as measure of actual bone union, and (5) associated CT-generated outcomes to mechanical or histological testing results. Two authors selected eligible studies and performed risk of bias assessment with QUADAS-2 tool. From 2567 studies that were screened, thirteen studies were included. Most common CT parameters that were investigated were bone mineral density, bone volume, and total callus volume. Studies showed conflicting results concerning the associations of these parameters with actual bone union. CT-assessed torsional rigidity (assessed by three studies) and callus density (assessed by two studies) showed best results. The studies investigating these two parameters reported moderate to strong associations with actual bone union. CT-assessed torsional rigidity and callus density seem the most promising parameters to represent actual bone union after a fracture, arthrodesis, or osteotomy.Prospero trial registration number: CRD42020164733.
Topics: Animals; Bone Density; Bony Callus; Fracture Healing; Fractures, Bone; Osteotomy; Tomography, X-Ray Computed
PubMed: 34417861
DOI: 10.1007/s00223-021-00904-6 -
Bone Feb 2021Diabetics are at increased risk for fracture, and experience severely impaired skeletal healing characterized by delayed union or nonunion of the bone. The periosteum...
Diabetics are at increased risk for fracture, and experience severely impaired skeletal healing characterized by delayed union or nonunion of the bone. The periosteum harbors osteochondral progenitors that can differentiate into chondrocytes and osteoblasts, and this connective tissue layer is required for efficient fracture healing. While bone marrow-derived stromal cells have been studied extensively in the context of diabetic skeletal repair and osteogenesis, the effect of diabetes on the periosteum and its ability to contribute to bone regeneration has not yet been explicitly evaluated. Within this study, we utilized an established murine model of type I diabetes to evaluate periosteal cell differentiation capacity, proliferation, and availability under the effect of a diabetic environment. Periosteal cells from diabetic mice were deficient in osteogenic differentiation ability in vitro, and diabetic mice had reduced periosteal populations of mesenchymal progenitors with a corresponding reduction in proliferation capacity following injury. Additionally, fracture callus mineralization and mature osteoblast activity during periosteum-mediated healing was impaired in diabetic mice compared to controls. We propose that the effect of diabetes on periosteal progenitors and their ability to aid in skeletal repair directly impairs fracture healing.
Topics: Animals; Bony Callus; Cell Differentiation; Diabetes Mellitus, Experimental; Fracture Healing; Mice; Osteogenesis; Periosteum
PubMed: 33221502
DOI: 10.1016/j.bone.2020.115764 -
Journal of Bone and Mineral Research :... May 2023Despite considerable improvement in fracture care, 5%-10% of all fractures still heal poorly or result in nonunion formation. Therefore, there is an urgent need to...
Despite considerable improvement in fracture care, 5%-10% of all fractures still heal poorly or result in nonunion formation. Therefore, there is an urgent need to identify new molecules that can be used to improve bone fracture healing. One activator of the Wnt-signaling cascade, Wnt1, has recently gained attention for its intense osteoanabolic effect on the intact skeleton. The aim of the present study was to investigate whether Wnt1 might be a promising molecule to accelerate fracture healing both in skeletally healthy and osteoporotic mice that display a diminished healing capacity. Transgenic mice for a temporary induction of Wnt1 specifically in osteoblasts (Wnt1-tg) were subjected to femur osteotomy. Non-ovariectomized and ovariectomized Wnt1-tg mice displayed significantly accelerated fracture healing based on a strong increase in bone formation in the fracture callus. Transcriptome profiling revealed that Hippo/yes1-associated transcriptional regulator (YAP)-signaling and bone morphogenetic protein (BMP) signaling pathways were highly enriched in the fracture callus of Wnt1-tg animals. Immunohistochemical staining confirmed increased activation of YAP1 and expression of BMP2 in osteoblasts in the fracture callus. Therefore, our data indicate that Wnt1 boosts bone formation during fracture healing via YAP/BMP signaling both under healthy and osteoporotic conditions. To further test a potential translational application of Wnt1, we applied recombinant Wnt1 embedded into a collagen gel during critical-size bone-defect repair. Mice treated with Wnt1 displayed increased bone regeneration compared to control mice accompanied by increased YAP1/BMP2 expression in the defect area. These findings are of high clinical relevance because they indicate that Wnt1 could be used as a new therapeutic agent to treat orthopedic complications in the clinic. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Topics: Mice; Animals; Fracture Healing; Osteogenesis; Fractures, Bone; Bony Callus; Mice, Transgenic; Wnt Signaling Pathway
PubMed: 36891752
DOI: 10.1002/jbmr.4797 -
Journal of Orthopaedic Research :... Aug 2023Fracture burden has created a need to better understand bone repair processes under different pathophysiological states. Evaluation of structural and material properties...
Fracture burden has created a need to better understand bone repair processes under different pathophysiological states. Evaluation of structural and material properties of the mineralized callus, which is integral to restoring biomechanical stability is, therefore, vital. Microcomputed tomography (micro-CT) can facilitate noninvasive imaging of fracture repair, however, current methods for callus segmentation are only semiautomated, restricted to defined regions, time/labor intensive, and prone to user variation. Herein, we share a new automatic method for segmenting callus in micro-CT tomograms that will allow for objective, quantitative analysis of the bone fracture microarchitecture. Fractured and nonfractured mouse femurs were scanned and processed by both manual and automated segmentation of fracture callus from cortical bone after which microarchitectural parameters were analyzed. All segmentation and analysis steps were performed using CTAn (Bruker) with automatic segmentation performed using the software's image-processing plugins. Results showed automatic segmentation reliably and consistently segmented callus from cortical bone, demonstrating good agreement with manual methods with low bias: tissue volume (TV): -0.320 mm , bone volume (BV): 0.0358 mm , and bone volume/tissue volume (BV/TV): -3.52%, and was faster and eliminated user-bias and variation. Method scalability and translatability across rodent models were verified in scans of fractured rat femora showing good agreement with manual methods with low bias: TV: -3.654 mm , BV: 0.830 mm , and BV/TV: 7.81%. Together, these data validate a new automated method for segmentation of callus and cortical bone in micro-CT tomograms that we share as a fast, reliable, and less user-dependent tool for application to study bone callus in fracture, and potentially elsewhere.
Topics: Rats; Mice; Animals; X-Ray Microtomography; Rodentia; Bony Callus; Femur; Femoral Fractures
PubMed: 36582023
DOI: 10.1002/jor.25507