-
Frontiers in Cellular and Infection... 2023Diabetes mellitus (DM) impairs fracture healing and is associated with susceptibility to infection, which further inhibits fracture healing. While intermittent...
INTRODUCTION
Diabetes mellitus (DM) impairs fracture healing and is associated with susceptibility to infection, which further inhibits fracture healing. While intermittent parathyroid hormone (1-34) (iPTH) effectively improves fracture healing, it is unknown whether infection-associated impaired fracture healing can be rescued with PTH (teriparatide).
METHODS
A chronic diet-induced type 2 diabetic mouse model was used to yield mice with decreased glucose tolerance and increased blood glucose levels compared to lean-fed controls. Methicillin-resistant (MRSA) was inoculated in a surgical tibia fracture model to simulate infected fracture, after which mice were treated with a combination of antibiotics and adjunctive teriparatide treatment. Fracture healing was assessed by Radiographic Union Scale in Tibial Fractures (RUST), micro-computed tomography (μCT), biomechanical testing, and histology.
RESULTS
RUST score was significantly poorer in diabetic mice compared to their lean nondiabetic counterparts. There were concomitant reductions in micro-computed tomography (μCT) parameters of callus architecture including bone volume/total volume, trabecular thickness, and total mineral density in type 2 diabetes mellitus (T2DM) mice. Biomechanicaltesting of fractured femora demonstrated diminished torsional rigidity, stiffness, and toughness to max torque. Adjuvant teriparatide treatment with systemic antibiotic therapy improved numerous parameters of bone microarchitecture bone volume, increased connectivity density, and increased trabecular number in both the lean and T2DM group. Despite the observation that poor fracture healing in T2DM mice was further impaired by MRSA infection, adjuvant iPTH treatment significantly improved fracture healing compared to antibiotic treatment alone in infected T2DM fractures.
DISCUSSION
Our results suggest that teriparatide may constitute a viable adjuvant therapeutic agent to improve bony union and bone microarchitecture to prevent the development of septic nonunion under diabetic conditions.
Topics: Mice; Animals; Fracture Healing; Methicillin-Resistant Staphylococcus aureus; Teriparatide; Diabetes Mellitus, Type 2; Diabetes Mellitus, Experimental; X-Ray Microtomography; Parathyroid Hormone
PubMed: 37829606
DOI: 10.3389/fcimb.2023.1230568 -
Experimental Gerontology Jul 2023Nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, belong to the most prescribed analgesic medication after traumatic injuries. However, there is...
Nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, belong to the most prescribed analgesic medication after traumatic injuries. However, there is accumulating evidence that NSAIDs impair fracture healing. Because bone regeneration in aged patients is subject to significant changes in cell differentiation and proliferation as well as a markedly altered pharmacological action of drugs, we herein analyzed the effects of diclofenac on bone healing in aged mice using a stable closed femoral facture model. Thirty-three mice (male n = 14, female n = 19) received a daily intraperitoneal injection of diclofenac (5 mg/kg body weight). Vehicle-treated mice (n = 29; male n = 13, female n = 16) served as controls. Fractured mice femora were analyzed by means of X-ray, biomechanics, micro computed tomography (μCT), histology and Western blotting. Biomechanical analyses revealed a significantly reduced bending stiffness in diclofenac-treated animals at 5 weeks after fracture when compared to vehicle-treated controls. Moreover, the callus tissue in diclofenac-treated aged animals exhibited a significantly reduced amount of bone tissue and higher amounts of fibrous tissue. Further histological analyses demonstrated less lamellar bone after diclofenac treatment, indicating a delay in callus remodeling. This was associated with a decreased number of osteoclasts and an increased expression of osteoprotegerin (OPG) during the early phase of fracture healing. These findings indicate that diclofenac delays fracture healing in aged mice by affecting osteogenic growth factor expression and bone formation as well as osteoclast activity and callus remodeling.
Topics: Mice; Male; Female; Animals; Diclofenac; Fracture Healing; Anti-Inflammatory Agents, Non-Steroidal; X-Ray Microtomography; Bony Callus; Femoral Fractures; Biomechanical Phenomena
PubMed: 37169100
DOI: 10.1016/j.exger.2023.112201 -
International Journal of Molecular... Mar 2021Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1...
Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1's role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration.
Topics: Animals; Bony Callus; Cell Adhesion Molecules; Chondrocytes; Core Binding Factor Alpha 1 Subunit; Fracture Healing; Male; Mesenchymal Stem Cells; Nerve Tissue Proteins; Osteoblasts; Osteocytes; Osteogenesis; RNA-Binding Proteins; Rats; Rats, Wistar
PubMed: 33810326
DOI: 10.3390/ijms22073395 -
Scientific Reports Oct 2016Bone callus, generated during fracture healing, is commonly discarded during surgical procedures. The aim of this study was to investigate the osteogenic potential of...
Bone callus, generated during fracture healing, is commonly discarded during surgical procedures. The aim of this study was to investigate the osteogenic potential of bone callus and its possible use as autograft material for patients needing bone grafts. Histology, immunohistochemistry, micro-computed tomography, and biomechanics were performed to examine osteogenic cells, osteoinductive factors, and the osteoconductive structure of bone callus. Alkaline phosphatase-positive osteoblasts, osteoinductive factors (including BMP2, FGF2, TGFB1, and IGF1), and a porous structure were found in bone callus. Early-stage callus (within 3 months after fracture) presented significantly improved osteogenic properties compared to medium- (3-9 months) and late-stage (longer than 9 months) callus. The results revealed that bone callus induced new bone formation in a nude mouse model. Early-stage callus showed better performance to medium- and late-stage callus in the induction of new bone formation at both 8 and 12 weeks. These findings indicated that bone callus, especially early-stage callus, possesses osteogenic potential and can potentially serve as an alternative source of material for bone grafts.
Topics: Adult; Alkaline Phosphatase; Animals; Bone Transplantation; Bony Callus; Female; Humans; Male; Mice; Mice, Nude; Middle Aged; Osteoblasts; Osteogenesis; X-Ray Microtomography
PubMed: 27796345
DOI: 10.1038/srep36330 -
Nature Communications May 2021Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we...
Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma.
Topics: Animals; Bone Regeneration; Bony Callus; Cell Differentiation; Cells, Cultured; Fracture Healing; Fractures, Bone; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Muscle, Skeletal; Osteogenesis; Mice
PubMed: 34001878
DOI: 10.1038/s41467-021-22842-5 -
Frontiers in Endocrinology 2021Wnt signaling plays a critical role in bone formation, homeostasis, and injury repair. Multiple cell types in bone have been proposed to produce the Wnts required for...
Wnt signaling plays a critical role in bone formation, homeostasis, and injury repair. Multiple cell types in bone have been proposed to produce the Wnts required for these processes. The specific role of Wnts produced from cells of hematopoietic origin has not been previously characterized. Here, we examined if hematopoietic Wnts play a role in physiological musculoskeletal development and in fracture healing. Wnt secretion from hematopoietic cells was blocked by genetic knockout of the essential Wnt modifying enzyme PORCN, achieved by crossing transgenic mice with mice. Knockout mice were compared with their wild-type littermates for musculoskeletal development including bone quantity and quality at maturation. Fracture healing including callus quality and quantity was assessed in a diaphyseal fracture model using quantitative micro computer-assisted tomographic scans, histological analysis, as well as biomechanical torsional and 4-point bending stress tests. The hematopoietic knockout mice had normal musculoskeletal development, with normal bone quantity and quality on micro-CT scans of the vertebrae. They also had normal gross skeletal dimensions and normal bone strength. Hematopoietic Wnt depletion in the healing fracture resulted in fewer osteoclasts in the fracture callus, with a resultant delay in callus remodeling. All calluses eventually progressed to full maturation. Hematopoietic Wnts, while not essential, modulate osteoclast numbers during fracture healing. These osteoclasts participate in callus maturation and remodeling. This demonstrates the importance of diverse Wnt sources in bone repair.
Topics: Acyltransferases; Animals; Biomechanical Phenomena; Bony Callus; Female; Fracture Healing; Male; Membrane Proteins; Mice; Mice, Knockout; Osteoclasts; Osteogenesis; Wnt Signaling Pathway
PubMed: 34108937
DOI: 10.3389/fendo.2021.667480 -
Advanced Science (Weinheim,... Jun 2023The formation of a calcified cartilaginous callus (CACC) is crucial during bone repair. CACC can stimulate the invasion of type H vessels into the callus to couple...
The formation of a calcified cartilaginous callus (CACC) is crucial during bone repair. CACC can stimulate the invasion of type H vessels into the callus to couple angiogenesis and osteogenesis, induce osteoclastogenesis to resorb the calcified matrix, and promote osteoclast secretion of factors to enhance osteogenesis, ultimately achieving the replacement of cartilage with bone. In this study, a porous polycaprolactone/hydroxyapatite-iminodiacetic acid-deferoxamine (PCL/HA-SF-DFO) 3D biomimetic CACC is developed using 3D printing. The porous structure can mimic the pores formed by the matrix metalloproteinase degradation of the cartilaginous matrix, HA-containing PCL can mimic the calcified cartilaginous matrix, and SF anchors DFO onto HA for the slow release of DFO. The in vitro results show that the scaffold significantly enhances angiogenesis, promotes osteoclastogenesis and resorption by osteoclasts, and enhances the osteogenic differentiation of bone marrow stromal stem cells by promoting collagen triple helix repeat-containing 1 expression by osteoclasts. The in vivo results show that the scaffold significantly promotes type H vessels formation and the expression of coupling factors to promote osteogenesis, ultimately enhancing the regeneration of large-segment bone defects in rats and preventing dislodging of the internal fixation screw. In conclusion, the scaffold inspired by biological bone repair processes effectively promotes bone regeneration.
Topics: Rats; Animals; Osteogenesis; Biomimetics; Bone and Bones; Cartilage; Chloride Channels
PubMed: 36999832
DOI: 10.1002/advs.202207089 -
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 -
Cell Reports Nov 2022Skeletal stem cells (SSCs) fuel adult bone with stemness resources to maintain homeostasis and support regeneration, which depends on the precise determination of the...
Skeletal stem cells (SSCs) fuel adult bone with stemness resources to maintain homeostasis and support regeneration, which depends on the precise determination of the osteogenic lineage commitment of SSCs. In this study, using Cre-loxP reporter lineage tracking, we identified and characterized a population of NFATc1 SSCs in bone regeneration. Pre-existing NFATc1 SSCs are involved in early bone callus formation. Subsequently, these NFATc1 SSCs produce osteolineage descendants in the subsequent stages of regeneration. The Ca-triggered transcriptional activity of NFATc1 constitutes the pre-imprinted memory of the trajectory to intrinsically orchestrate osteogenesis of SSCs. Inhibition of Ca/NFATc1 signaling in SSCs directly impairs osteogenesis and bone regeneration. In summary, our findings provide a mechanistic understanding of adult bone regeneration through the regulation of NFATc1 SSCs.
Topics: Humans; Adult; Bone Regeneration; Stem Cells; Osteogenesis; Bone and Bones; Transcription Factors; Cell Differentiation; NFATC Transcription Factors
PubMed: 36351390
DOI: 10.1016/j.celrep.2022.111599 -
Stem Cells (Dayton, Ohio) May 2023Regulator of G protein signaling 5 (RGS5) is a GTPase activator for heterotrimeric G-protein α-subunits, shown to be a marker of pericytes. Bone marrow stromal cell...
Regulator of G protein signaling 5 (RGS5) is a GTPase activator for heterotrimeric G-protein α-subunits, shown to be a marker of pericytes. Bone marrow stromal cell population (BMSCs) is heterogeneous. Populations of mesenchymal progenitors, cells supportive of hematopoiesis, and stromal cells regulating bone remodeling have been recently identified. Periosteal and bone marrow mesenchymal stem cells (MSCs) are participating in fracture healing, but it is difficult to distinguish the source of cells within the callus. Considering that perivascular cells exert osteoprogenitor potential, we generated an RGS5 transgenic mouse model (Rgs5-CreER) which when crossed with Ai9 reporter animals (Rgs5/Tomato), is suitable for lineage tracing during growth and post-injury. Flow cytometry analysis and histology confirmed the presence of Rgs5/Tomato+ cells within CD31+ endothelial, CD45+ hematopoietic, and CD31-CD45- mesenchymal/perivascular cells. A tamoxifen chase showed expansion of Rgs5/Tomato+ cells expressing osterix within the trabeculae positioned between mineralized matrix and vasculature. Long-term chase showed proportion of Rgs5/Tomato+ cells contributes to mature osteoblasts expressing osteocalcin. Following femoral fracture, Rgs5/Tomato+ cells are observed around newly formed bone within the BM cavity and expressed osterix and osteocalcin, while contribution within periosteum was low and limited to fibroblastic callus with very few positive chondrocytes. In addition, BM injury model confirmed that RGS5-Cre labels population of BMSCs expands during injury and participates in osteogenesis. Under homeostatic conditions, lineage-traced RGS5 cells within the trabecular area demonstrate osteoprogenitor capacity that in an injury model contributes to new bone formation primarily within the BM niche.
Topics: Mice; Animals; Osteocalcin; Bony Callus; Osteogenesis; Fracture Healing; Chondrocytes; Mice, Transgenic; Osteoblasts; RGS Proteins
PubMed: 36888549
DOI: 10.1093/stmcls/sxad020