Effects of Focal Cerebellar Injury on Fracture Healing and Oxidative Stress in Rat Model: An Experimental Animal Study.

AIM: To examine the effect of cerebellar damage on the process of fracture healing. MATERIAL AND METHODS: A total of forty-two male rats were selected at random and subsequently allocated into three distinct groups. The experimentals were divided into two subgroups within each group, with the intention of sacrificing them during the third and sixth weeks. Group 1 had isolated femoral fracture, Group 2 had femoral fracture after craniotomy, and Group 3 had femoral fracture accompanying cerebellar injury after craniotomy. Left femoral fractures in rats in all groups were treated using an intramedullary Kirschner wire. Radiological, histological, and biochemical evaluations were conducted at 3 and 6 weeks to assess the processes of fracture healing. To determine the effects of fracture healing and cerebellar injury on oxidant-antioxidant systems, catalase (CAT), malondialdehyde, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were measured. RESULTS: Between the time frame of 3 to 6 weeks, Group 3 had higher radiography scores, alkaline phosphatase levels, callus/ diaphyse ratio, callus improvement, and bone mineral density in comparison to the other groups. The activity of SOD was found to be statistically negligible in all groups, suggesting that SOD does not have a substantial impact on fracture healing in cerebellar injury. However, notable increases in the activity of GPx and CAT enzymes were observed, showing their considerable involvement in the process of fracture healing. CONCLUSION: Cerebellar injury reduces the oxidative stress in the fracture area and contributes positively to fracture healing by means of radiologically, biochemically and histopathologically.

MiR-22-3p facilitates bone marrow mesenchymal stem cell osteogenesis and fracture healing through the SOSTDC1-PI3K/AKT pathway.

Bone fractures are the most common form of musculoskeletal trauma worldwide. Numerous microRNAs (miRNAs) have been suggested to be participants in regulating bone-related diseases. Recent studies revealed the regulatory role of miR-22-3p in osteogenic differentiation, but its role in fracture healing has not been investigated previously. Here, a rat femoral fracture model was established, Bone marrow mesenchymal stem cells (BMSCs) were isolated to detect the specific function and underlying mechanisms of miR-22-3p. MiR-22-3p and sclerostin domain-containing 1 (SOSTDC1) expression was determined by RT-qPCR and immunohistochemistry staining. The levels of proteins associated with osteogenic differentiation were assessed by western blotting. Flow cytometry was conducted to identify the isolated rat BMSCs. Alizarin red staining, alkaline phosphatase staining and Oil Red O staining were used to evaluate the osteogenic and adipogenic differentiation of rat BMSCs. The interaction between miR-22-3p and SOSTDC1 was verified using a luciferase reporter assay. Haematoxylin and Eosin (H&E) staining of the bone tissues was performed to analyse the effect of miR-22-3p on histopathological changes in vivo. MiR-22-3p was downregulated in the callus tissues of rat femoral fracture, while the expression of SOSTDC1 was upregulated. The isolated rat BMSCs had the capacity for both osteogenic and adipogenic differentiation. The differentiation capacity of BMSCs into osteoblasts was increased by miR-22-3p overexpression. MiR-22-3p activated the PI3K/AKT pathway by targeting SOSTDC1. SOSTDC1 overexpression and PI3K/AKT signalling inhibitor LY294002 abolished the enhancing effect of miR-22-3p overexpression on the osteogenesis of BMSCs. Thus MiR-22-3p facilitated the femoral fracture healing in rats. MiR-22-3p overexpression promoted fracture healing via the activation of PI3K/AKT pathway by targeting SOSTDC1.

Myosin heavy chain 2 (MYH2) expression in hypertrophic chondrocytes of soft callus provokes endochondral bone formation in fracture.

AIMS: Muscle-bone interactions during fracture healing are rarely known. Here we investigated the presence and significance of myosin heavy chain 2 (MYH2), a component of myosin derived from muscles, in fracture healing. MAIN METHODS: We collected five hematoma and seven soft callus tissues from patients with distal radius fractures patients, randomly selected three of them, and performed a liquid chromatography-mass spectrometry (LC-MS) proteomics analysis. Proteomic results were validated by histological observation, immunohistochemistry, and immunofluorescence for MYH2 expression. These findings were further confirmed in a murine femoral fracture model in vivo and investigated using various methods in vitro. KEY FINDINGS: The LC-MS proteomics analysis showed that MYH proteins were enriched in human soft calluses compared to hematoma. Notably, MYH2 protein is upregulated as high rank in each soft callus. The histological examination showed that MYH2 expression was elevated in hypertrophic chondrocytes within the human soft callus. Consistent with human data, Myh2 were significantly co-localized with Sox9 in hypertrophic chondrocytes of murine femoral fracture, in comparison to pre-hypertrophic and proliferating chondrocytes. Soluble MYH2 protein treatment increased MMP13 and RUNX2 expression in chondrocytes. In soluble MYH2 treatment, proliferation of chondrocytes was not altered, but the osteogenic and chondrogenic features of chondrocytes increased and decreased during differentiation, respectively. SIGNIFICANCE: These findings indicate the potential of soluble MYH2 protein as a promising therapeutic strategy for promoting endochondral bone formation in chondrocytes following fracture.

Persicae Semen Promotes Bone Union in Rat Fractures by Stimulating Osteoblastogenesis through BMP-2 and Wnt Signaling.

Fractures cause extreme pain to patients and impair movement, thereby significantly reducing their quality of life. However, in fracture patients, movement of the fracture site is restricted through application of a cast, and they are reliant on conservative treatment through calcium intake. Persicae semen (PS) is the dried mature seeds of Prunus persica (L.) Batsch, and in this study the effects of PS on osteoblast differentiation and bone union promotion were investigated. The osteoblast-differentiation-promoting effect of PS was investigated through alizarin red S and Von Kossa staining, and the regulatory role of PS on BMP-2 (Bmp2) and Wnt (Wnt10b) signaling, representing a key mechanism, was demonstrated at the protein and mRNA levels. In addition, the bone-union-promoting effect of PS was investigated in rats with fractured femurs. The results of the cell experiments showed that PS promotes mineralization and upregulates RUNX2 through BMP-2 and Wnt signaling. PS induced the expression of various osteoblast genes, including Alpl, Bglap, and Ibsp. The results of animal experiments show that the PS group had improved bone union and upregulated expression of osteogenic genes. Overall, the results of this study suggest that PS can promote fracture recovery by upregulating osteoblast differentiation and bone formation, and thus can be considered a new therapeutic alternative for fracture patients.

Bone marrow-derived dedifferentiated fat cells exhibit similar phenotype as bone marrow mesenchymal stem cells with high osteogenic differentiation and bone regeneration ability.

BACKGROUND: Mesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model. METHODS: BM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice. RESULTS: BM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model. CONCLUSIONS: We showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

Landscape of Well-Coordinated Fracture Healing in a Mouse Model Using Molecular and Cellular Analysis.

The success of fracture healing relies on overlapping but coordinated cellular and molecular events. Characterizing an outline of differential gene regulation throughout successful healing is essential for identifying crucial phase-specific markers and may serve as the basis for engineering these in challenging healing situations. This study analyzed the healing progression of a standard closed femoral fracture model in C57BL/6N (age = 8 weeks) wild-type male mice. The fracture callus was assessed across various days post fracture (D = days 0, 3, 7, 10, 14, 21, and 28) by microarray, with D0 serving as a control. Histological analyses were carried out on samples from D7 until D28 to support the molecular findings. Microarray analysis revealed a differential regulation of immune response, angiogenesis, ossification, extracellular matrix regulation, mitochondrial and ribosomal genes during healing. In-depth analysis showed differential regulation of mitochondrial and ribosomal genes during the initial phase of healing. Furthermore, the differential gene expression showed an essential role of Serpin Family F Member 1 over the well-known Vascular Endothelial Growth Factor in angiogenesis, especially during the inflammatory phase. The significant upregulation of matrix metalloproteinase 13 and bone sialoprotein from D3 until D21 asserts their importance in bone mineralization. The study also shows type I collagen around osteocytes located in the ossified region at the periosteal surface during the first week of healing. Histological analysis of matrix extracellular phosphoglycoprotein and extracellular signal-regulated kinase stressed their roles in bone homeostasis and the physiological bone-healing process. This study reveals previously unknown and novel candidates, that could serve as a target for specific time points in healing and to remedy cases of impaired healing.

Unveiling the Time Course Mechanism of Bone Fracture Healing by Transcriptional Profiles.

BACKGROUND: Bone fracture healing is a time-consuming and high-priority orthopedic problem worldwide. OBJECTIVE: Discovering the potential mechanism of bone healing at a time course and transcriptional level may better help manage bone fracture. METHODS: In this study, we analyze a time-course bone fracture healing transcriptional dataset in a rat model (GSE592, GSE594, and GSE1371) of Gene Expression Omnibus (GEO). RNA was obtained from female Sprague-Dawley rats with a femoral fracture at the initial time (day 3) as well as early (week 1), middle (week 2), and late (week 4) time periods, with nonfracture rats used as control. Gene Ontology (GO) functional analysis and pathway examinations were performed for further measurements of GSEA and hub genes. RESULTS: Results indicated that the four stages of bone fracture healing at the initial, early, middle, and late time periods represent the phases of hematoma formation, callus formation, callus molding, and mature lamellar bone formation, respectively. Extracellular organization was positively employed throughout the four stages. At the hematoma formation phase, the muscle contraction process was downregulated. Antibacterial peptide pathway was downregulated at all phases. The upregulation of Fn1 (initial, early, middle, and late time periods), Col3a1 (initial, early, and middle time periods), Col11a1 (initial and early time periods), Mmp9 (middle and late time periods), Mmp13 (early, middle, and late time periods) and the downregulation of RatNP-3b (initial, early, middle, and late time periods) were possible symbols for bone fracture healing and may be used as therapeutic targets. CONCLUSION: These findings suggest some new potential pathways and genes in the process of bone fracture healing and further provide insights that can be used in targeted molecular therapy for bone fracture healing.

Wnt10b-overexpressing umbilical cord mesenchymal stem cells promote fracture healing via accelerated cartilage callus to bone remodeling.

The aim of this study was to investigate whether HUCMSCsWnt10b could promote long bone fracture healing. Commercially-available HUCMSCsEmp (human umbilical cord mesenchymal stem cells transfected with empty vector) in hydrogel, HUCMSCsWnt10b in hydrogel and HUCMSCsWnt10b with the Wnt signaling pathway inhibitor IWR-1 were transplanted into the fracture site in a rat model of femoral fracture. We found that transplantation of HUCMSCsWnt10b significantly accelerated bone healing in a rat model of femoral fracture. Meanwhile, three-point bending test proved that the mechanical properties of the bone at the fracture site in the HUCMSCWnt10b treatment group were significantly better than those of the other treatment groups. To understand the cellular mechanism, we explored the viability of periosteal stem cells (PSCs), as they contribute the greatest number of osteoblast lineage cells to the callus. In line with in vivo data, we found that conditioned medium from HUCMSCsWnt10b enhanced the migration and osteogenic differentiation of PSCs. Furthermore, conditioned medium from HUCMSCsWnt10b also induced endothelial cells to form capillary-like structures in a tube formation assay, which was blocked by SU5416, an angiogenesis inhibitor, suggesting that enhanced vessel formation and growth also contribute to accelerated hard callus formation. In summary, our study demonstrates that HUCMSCsWnt10b promote fracture healing via accelerated hard callus formation, possibly due to enhanced osteogenic differentiation of PSCs and vessel growth. Therefore, HUCMSCsWnt10b may be a promising treatment for long bone fractures.

Expression of nerve growth factor in the callus during fracture healing in a fracture model in aged mice.

BACKGROUND: Impaired fracture healing results in extensive and prolonged disability and long-term pain. Previous studies reported that nerve growth factor (NGF) was expressed during fracture healing and that anti-NGF antibody improves physical activity associate with facture pain. However, NGF expression levels in delayed or non-union are not fully understood. OBJECTIVE: We compared chronological changes in NGF in the callus of young mice after femur fracture with those in aged mice after femur fracture as a model of bone fracture in the elderly. METHODS: We used young (age 8 weeks) and aged (age 10 months) male C57BL/6J mice. A fracture was generated in the femur. At 5, 7, 10, 14, 17, and 21 days after creation of a fracture, mRNA expression levels of Col2a1, Col10a1, NGF were evaluated using quantitative PCR. We examined NGF protein expression levels and localization in the callus at day 14 using ELISA and immunohistochemistry, respectively. RESULTS: Expression of NGF in the callus after femur fracture in aged mice was significantly greater than that in young mice at days 5, 7, 10, 17, and 21 days. NGF protein levels in the callus of aged mice were also significantly higher than that in young mice. Immunohistochemical staining showed that NGF was heavily expressed in hypertrophic chondrocytes in the callus in aged mice. CONCLUSIONS: It is suggested that delayed Col2a1 and Col10a1 expression reflects delayed chondrocyte formation and delayed chondrocyte maturation in aged mice and that higher NGF expression in aged mice at day 14 may be associated with the presence of remaining hypertrophic chondrocytes in callus with delaying endochondral ossification.

Altered canalicular remodeling associated with femur fracture in mice.

We previously showed that femur fracture in mice caused a reduction in bone volume at distant skeletal sites within 2 weeks post-fracture. Osteocytes also have the ability to remodel their surrounding bone matrix through perilacunar/canalicular remodeling (PLR). If PLR is altered systemically following fracture, this could affect bone mechanical properties and increase fracture risk at all skeletal sites. In this study, we investigated whether lacunar-canalicular microstructure and the rate of PLR are altered in the contralateral limb following femoral fracture in mice. We hypothesized that femoral fracture would accelerate PLR by 2 weeks postfracture, followed by partial recovery by 4 weeks. We used histological evaluation and high-resolution microcomputed tomography to quantify the morphology of the lacunar-canalicular network at the contralateral tibia, and we used quantitative real-time polymerase chain reaction (RT-PCR) and RNA-seq to measure the expression of PLR-associated genes in the contralateral femur. We found that at both 2 and 4 weeks postfracture, canalicular width was significantly increased by 18.6% and 16.6%, respectively, in fractured mice relative to unfractured controls. At 3 days and 4 weeks post-fracture, we observed downregulation of PLR-associated genes; RNA-seq analysis at 3 days post-fracture showed a deceleration of bone formation and mineralization in the contralateral limb. These data demonstrate notable canalicular changes following fracture that could affect bone mechanical properties. These findings expand our understanding of systemic effects of fracture and how biological and structural changes at distant skeletal sites may contribute to increased fracture risk following an acute injury.

Differences in femur geometry and bone markers in atypical femur fractures and the general population.

This study aimed to identify differences in femur geometry between patients with subtrochanteric/shaft atypical femur fractures (AFFs) and the general population, and to evaluate the biomechanical factors related to femoral bowing in AFFs. We retrospectively reviewed 46 patients. Data on age, and history and duration of bisphosphonate use were evaluated. Femur computed tomography images were reconstructed into a 3D model, which was analyzed with a geometry analysis program to obtain the femur length, femur width and length, and femoral bowing. Patients were divided into two groups according to fracture location: the subtrochanteric and shaft AFF groups. We compared all parameters between groups, and also between each group and a general population of 300 women ≥ 60 years. Thirty-five patients had a history of bisphosphonate use (average duration, 6.1 years; range, 0.8-20 years). There was no statistical difference in bone turnover markers between the two groups. The shaft AFF group had a lower radius of curvature (ROC) (P = 0.001), lower bone mineral density (BMD, T score) (P = 0.020), and lower calcium (P = 0.016). However, other parameters and rate of bisphosphonate use were not significantly different. There were no significant differences in the parameters of the subtrochanter AFF group and the general population, but the shaft AFF group demonstrated a wider femur width (P < 0.001), longer anteroposterior length (P = 0.001), and lower ROC (P < 0.001) than the general population. Femoral bowing and width increased in shaft AFFs, but similar to subtrochanter AFFs compared to the general population. Our results highlight the biomechanical factors of femur geometry in AFFs.

A Population of M2 Macrophages Associated With Bone Formation.

We previously identified transient brown adipocyte-like cells associated with heterotopic ossification (HO). These ancillary cells support new vessel synthesis essential to bone formation. Recent studies have shown that the M2 macrophage contributes to tissue regeneration in a similar way. To further define the phenotype of these brown adipocyte-like cells they were isolated and characterized by single-cell RNAseq (scRNAseq). Analysis of the transcriptome and the presence of surface markers specific for macrophages suggest that these cells are M2 macrophages. To validate these findings, clodronate liposomes were delivered to the tissues during HO, and the results showed both a significant reduction in these macrophages as well as bone formation. These cells were isolated and shown in culture to polarize towards either M1 or M2 similar to other macrophages. To confirm that these are M2 macrophages, mice received lipopolysacheride (LPS), which induces proinflammation and M1 macrophages. The results showed a significant decrease in this specific population and bone formation, suggesting an essential role for M2 macrophages in the production of bone. To determine if these macrophages are specific to HO, we isolated these cells using fluorescence-activated cell sorting (FACS) from a bone defect model and subjected them to scRNAseq. Surprisingly, the macrophage populations overlapped between the two groups (HO-derived versus callus) suggesting that they may be essential ancillary cells for bone formation in general and not selective to HO. Of further note, their unique metabolism and lipogenic properties suggest the potential for unique cross talk between these cells and the newly forming bone.

Functional Analyses of Four CYP1A1 Missense Mutations Present in Patients with Atypical Femoral Fractures.

Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.

Fibrous Demineralized Bone Matrix (DBM) Improves Bone Marrow Mononuclear Cell (BMC)-Supported Bone Healing in Large Femoral Bone Defects in Rats.

Regeneration of large bone defects is a major objective in trauma surgery. Bone marrow mononuclear cell (BMC)-supported bone healing was shown to be efficient after immobilization on a scaffold. We hypothesized that fibrous demineralized bone matrix (DBM) in various forms with BMCs is superior to granular DBM. A total of 65 male SD rats were assigned to five treatment groups: syngenic cancellous bone (SCB), fibrous demineralized bone matrix (f-DBM), fibrous demineralized bone matrix densely packed (f-DBM 120%), DBM granules (GDBM) and DBM granules 5% calcium phosphate (GDBM5%Ca2+). BMCs from donor rats were combined with different scaffolds and placed into 5 mm femoral bone defects. After 8 weeks, bone mineral density (BMD), biomechanical stability and histology were assessed. Similar biomechanical properties of f-DBM and SCB defects were observed. Similar bone and cartilage formation was found in all groups, but a significantly bigger residual defect size was found in GDBM. High bone healing scores were found in f-DBM (25) and SCB (25). The application of DBM in fiber form combined with the application of BMCs shows promising results comparable to the gold standard, syngenic cancellous bone. Denser packing of fibers or higher amount of calcium phosphate has no positive effect.

Amlodipine accelerates bone healing in a stable closed femoral fracture model in mice.

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.

Hydrogen sulfide inhibits calcium and phosphorus loss after fracture by negatively regulating glucocorticoid/glucocorticoid receptor α.

AIMS: Post-fracture calcium and phosphorus excretion is greater than influx, which might be caused by stress. Glucocorticoid is known to enhance calcium and phosphorous excretion, and hydrogen sulfide (H2S) has been shown to exert inhibitory effects on glucocorticoid. Therefore, this study explored whether H2S could inhibit calcium and phosphorus loss after fracture by regulating glucocorticoid and/or its receptor. MAIN METHODS: The following properties were analyzed in rats with femur fractures: serum and urinary calcium and phosphorus (by colorimetry); bone turnover markers alkaline phosphatase, serum type 1 collagen amino terminal peptide, type 1 procollagen carboxy terminal peptide, and anti-tartaric acid phosphatase (by ELISA); factors related to calcium-phosphorus metabolism including glucocorticoid, parathyroid hormone, calcitonin, fibroblast growth factor 23, and 1,25(OH)2D3 (by ELISA); and sulfhydration of glucocorticoid receptor α in the kidney (by immunoprecipitation linked biotin-switch assay), after supplementing with mifepristone, the H2S donor GYY4137 or H2S generating enzyme inhibitors aminooxyacetic acid and propargylglycine. KEY FINDINGS: Serum H2S decreased and glucocorticoid secretion increased in rats post-fracture. The glucocorticoid receptor inhibitor mifepristone partly blunted calcium and phosphorus loss. Furthermore, supplementation with GYY4137 reduced glucocorticoid secretion; inhibited glucocorticoid receptor α activity by sulfhydration; downregulated vitamin D 1α-hydroxylase expression; and upregulated 24-hydroxylase, calbindin-D28k, and sodium phosphate cotransporter 2a expression in the kidney; thereby inhibiting calcium and phosphorus loss induced by fracture. Moreover, inhibiting endogenous H2S generation showed opposite effects. SIGNIFICANCE: Our findings suggest that H2S antagonized calcium and phosphorus loss after fracture by reducing glucocorticoid secretion and inhibiting glucocorticoid receptor α activity by sulfhydration.

Role of microRNA-335 carried by bone marrow mesenchymal stem cells-derived extracellular vesicles in bone fracture recovery.

Mesenchymal stem cells (MSCs) have the potential to reduce healing time and treat nonunion in fracture patients. In this study, bone marrow MSCs-derived extracellular vesicles (B-EVs) were firstly extracted and identified. CD9-/- and normal mice were enrolled for the establishment of fracture models and then injected with B-EVs. Osteoblast differentiation and fracture recovery were estimated. The levels of osteoblast-related genes were detected, and differentially expressed microRNAs (miRs) in B-EVs-treated normal fracture mice were screened and verified. The downstream mechanisms of miR were predicted and assessed. The loss-of functions of miR-335 in B-EV and gain-of-functions of VapB were performed in animal and cell experiments to evaluate their roles in bone fracture. Collectively, B-EVs promoted bone fracture recovery and osteoblast differentiation by releasing miR-335. miR-335 downregulation in B-EVs impaired B-EV functions in fracture recovery and osteoblast differentiation. miR-335 could target VapB, and VapB overexpression reversed the effects of B-EVs on osteoblast differentiation. B-EV treatment activated the Wnt/ß-catenin pathway in fracture mice and osteoblasts-like cells. Taken together, the study suggested that B-EVs carry miR-335 to promote bone fracture recovery via VapB and the Wnt/ß-catenin pathway. This study may offer insights into bone fracture treatment.

Probiotic Lactobacillus rhamnosus GG (LGG) restores intestinal dysbacteriosis to alleviate upregulated inflammatory cytokines triggered by femoral diaphyseal fracture in adolescent rodent model.

OBJECTIVE: The aim of the study was to examine the influence of femoral shaft fracture on systemic inflammation and gut microbiome in adolescent rats and evaluate the anti-inflammatory effect of Lactobacillus rhamnosus GG (LGG) and its regulation of intestinal flora, as well as illustrate the mechanism by which LGG ameliorates the inflammatory response and restores intestinal dysbacteriosis. MATERIALS AND METHODS: Twenty-four male Sprague Dawley rats of 5 to 6 weeks of age were subjected to a standard femoral shaft fracture and internally fixed with LGG supplementation in advance or on the same day of injury or with saline solution for 1 week. The levels of TNF-α, IL-6, IL-10, and CRP were assessed using standard protocols. Furthermore, gut microbiota composition was analyzed in the fecal samples using 16S rDNA gene sequencing, and the relationship between gut microbiota variation and inflammatory response was tested. RESULTS: The serum indices of the above-mentioned inflammatory cytokines were significantly increased, and the gut microbial balance was significantly disturbed in adolescent rats by diaphyseal fractures of the femur and surgery. Moreover, L. rhamnosus strains manipulated the gut microbiota by decreasing the relative abundance of Proteobacteria and increasing that of Firmicutes, Actinobacteria and Bacteroidetes, which in turn increased the levels of IL-10 and alleviated the levels of IL-6, CRP, and TNF-α. CONCLUSIONS: LGG exhibited anti-inflammatory effects by alleviating the inflammatory response and regulating the gut microbiota in adolescent rats who underwent skeletal fracture and surgery. Our results suggested that the L. rhamnosus strains could be considered as an alternative dietary supplement for the prevention or treatment of skeletal injury and its associated complications.

The effect of different irrigation solutions on fracture healing in a rat femur fracture model.

OBJECTIVES: This study aims to evaluate and compare radiological, biomechanical, histopathological, histomorphometric and immunohistochemical effects of povidone iodine (PVP-I), hydrogen peroxide (HPO) and chlorhexidine gluconate (CHG) on fracture healing in their minimum cytotoxic and most efficient concentrations. MATERIALS AND METHODS: This experimental animal study, conducted between April 2018 and January 2019, included 48 male Sprague Dawley® rats (aging 9 weeks; weighing 356 g) which were randomly divided into four groups: control (saline), HPO, PVP-I and CHG. Rat model of femoral fracture was established and intramedullary fixation was applied. Solutions were applied to fracture region in determined concentration and time, and all subjects were sacrificed on Day 28. Extracted femurs were investigated radiologically by micro-computed tomography. Then, all groups were divided into two random groups to be evaluated biomechanically, histopathologically, histomorphometrically and immunohistochemically. RESULTS: In histopathological evaluation, inflammation score of CHG group was significantly lower than other groups, and inflammation score of PVP-I group was significantly lower than control and HPO groups (p<0.05). Biomechanically, flexural strength (σbend) (megapascal) values of CHG and control groups showed similar results, but there was no significant difference between all groups (p>0.05). In immunohistochemical localization of bone morphogenic protein (BMP)-4, osteoblast and chondroblast histoscores (H-scores) of HPO group were significantly lower than other groups, and chondroblast H-score in CHG group was lower than control and PVP-I groups (p<0.05). In immunohistochemical localization of BMP-7, osteoblast H-score was significantly higher in CHG group than other groups (p<0.05). CONCLUSION: We determined that CHG 0.05% solution had no negative effect on the fourth week of fracture healing histopathologically, immunohistochemically and biomechanically, and is an alternative irrigative to normal saline.