Stages Of Fracture Healing Research Articles

Single nuclei transcriptomics reveal the differentiation trajectories of periosteal skeletal/stem progenitor cells in bone regeneration.

Bone regeneration is mediated by skeletal stem/progenitor cells (SSPCs) that are mainly recruited from the periosteum after bone injury. The composition of the periosteum and the steps of SSPC activation and differentiation remain poorly understood. Here, we generated a single-nuclei atlas of the periosteum at steady-state and of the fracture site during early stages of bone repair ( https://fracture-repair-atlas.cells.ucsc.edu ). We identified periosteal SSPCs expressing stemness markers ( Pi16 and Ly6a /SCA1) and responding to fracture by adopting an injury-induced fibrogenic cell (IIFC) fate, prior to undergoing osteogenesis or chondrogenesis. We identified distinct gene cores associated with IIFCs and their engagement into osteogenesis and chondrogenesis involving Notch, Wnt and the circadian clock signaling respectively. Finally, we show that IIFCs are the main source of paracrine signals in the fracture environment, suggesting a crucial paracrine role of this transient IIFC population during fracture healing. Overall, our study provides a complete temporal topography of the early stages of fracture healing and the dynamic response of periosteal SSPCs to injury, redefining our knowledge of bone regeneration.

Gallein increases the fibroblast growth factor 2-elicited osteoprotegerin synthesis in osteoblasts

Gallein is known as an inhibitor of Gβγ subunits, but roles of gallein in bone metabolism have not been reported. Fibroblast growth factor 2 (FGF-2) increases angiogenesis and promotes bone regeneration during the early stages of fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in the suppression of bone resorption. Our previous report demonstrated that FGF-2 activates the phosphorylation of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (JNK), and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. Additionally, FGF-2-activated phosphorylation of p38 MAPK and JNK but not p44/p42 MAPK is positively involved in OPG synthesis in these cells. This work aimed to investigate the effects of gallein on the FGF-2-elicited OPG synthesis in osteoblast-like MC3T3-E1 cells and the mechanism. Our findings demonstrated that gallein significantly increased the FGF-2-elicited OPG synthesis in MC3T3-E1 cells. By contrast, fluorescein, gallein-like compound that does not bind Gβγ, did not affect the FGF-2-elicited OPG synthesis. Gallein significantly enhanced the FGF-2-induced OPG mRNA expression levels. Gallein did not affect the FGF-2-activated phosphorylation of p38 MAPK and p44/p42 MAPK, but significantly increased the FGF-2-activated phosphorylation of JNK, while fluorescein did not affect JNK phosphorylation. SP600125, a specific JNK inhibitor, strongly inhibited gallein-induced enhancement of FGF-2-induced OPG synthesis and mRNA expression levels. Our results indicated that gallein increases the FGF-2-induced OPG synthesis due to the JNK activation in the osteoblast.

Mass Spectrometry Reveals Molecular Effects of Citrulline Supplementation during Bone Fracture Healing in a Rat Model.

Bone fracture healing is a complex process in which specific molecular knowledge is still lacking. The citrulline-arginine-nitric oxide metabolism is one of the involved pathways, and its enrichment via citrulline supplementation can enhance fracture healing. This study investigated the molecular effects of citrulline supplementation during the different fracture healing phases in a rat model. Microcomputed tomography (μCT) was applied for the analysis of the fracture callus formation. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid-chromatography tandem mass spectrometry (LC-MS/MS) were used for lipid and protein analyses, respectively. μCT analysis showed no significant differences in the fracture callus volume and volume fraction between the citrulline supplementation and control group. The observed lipid profiles for the citrulline supplementation and control group were distinct for the different fracture healing stages. The main contributing lipid classes were phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs). The changing effect of citrulline supplementation throughout fracture healing was indicated by changes in the differentially expressed proteins between the groups. Pathway analysis showed an enhancement of fracture healing in the citrulline supplementation group in comparison to the control group via improved angiogenesis and earlier formation of the soft and hard callus. This study showed the molecular effects on lipids, proteins, and pathways associated with citrulline supplementation during bone fracture healing, even though no effect was visible with μCT.

Biomechanical Effects of Mechanical Stress on Cells Involved in Fracture Healing.

Fracture healing is a complex staged repair process in which the mechanical environment plays a key role. Bone tissue is very sensitive to mechanical stress stimuli, and the literature suggests that appropriate stress can promote fracture healing by altering cellular function. However, fracture healing is a coupled process involving multiple cell types that balance and limit each other to ensure proper fracture healing. The main cells that function during different stages of fracture healing are different, and the types and molecular mechanisms of stress required are also different. Most previous studies have used a single mechanical stimulus on individual mechanosensitive cells, and there is no relatively uniform standard for the size and frequency of the mechanical stress. Analyzing the mechanisms underlying the effects of mechanical stimulation on the metabolic regulation of signaling pathways in cells such as in bone marrow mesenchymal stem cells (BMSCs), osteoblasts, chondrocytes, and osteoclasts is currently a challenging research hotspot. Grasping how stress affects the function of different cells at the molecular biology level can contribute to the refined management of fracture healing. Therefore, in this review, we summarize the relevant literature and describe the effects of mechanical stress on cells associated with fracture healing, and their possible signaling pathways, for the treatment of fractures and the further development of regenerative medicine.

Temporal dynamics of immune-stromal cell interactions in fracture healing.

Bone fracture repair is a complex, multi-step process that involves communication between immune and stromal cells to coordinate the repair and regeneration of damaged tissue. In the US, 10% of all bone fractures do not heal properly without intervention, resulting in non-union. Complications from non-union fractures are physically and financially debilitating. We now appreciate the important role that immune cells play in tissue repair, and the necessity of the inflammatory response in initiating healing after skeletal trauma. The temporal dynamics of immune and stromal cell populations have been well characterized across the stages of fracture healing. Recent studies have begun to untangle the intricate mechanisms driving the immune response during normal or atypical, delayed healing. Various in vivo models of fracture healing, including genetic knockouts, as well as in vitro models of the fracture callus, have been implemented to enable experimental manipulation of the heterogeneous cellular environment. The goals of this review are to (1): summarize our current understanding of immune cell involvement in fracture healing (2); describe state-of-the art approaches to study inflammatory cells in fracture healing, including computational and in vitro models; and (3) identify gaps in our knowledge concerning immune-stromal crosstalk during bone healing.

Biomechanical optimization of the magnesium alloy bionic cannulated screw for stabilizing femoral neck fractures: a finite element analysis.

The magnesium alloy bionic cannulated screw (MABCS) was designed in a previous study promoting cortical-cancellous biphasic healing of femoral neck fractures. The main purpose was to analyze the bore diameters that satisfy the torsion standards and further analyze the optimal pore and implantation direction for stabilizing femoral neck fractures. The MABCS design with bionic holes with a screw diameter of less than 20% met the torsion standard for metal screws. The MABCS was utilized to repair the femoral neck fracture via Abaqus 6.14 software, which simulated the various stages of fracture healing to identify the optimal biomechanical environment for bionic hole size (5%, 10%, 15%, and 20%) and implantation direction (0°, 45°, 90°, and 135°). The stress distribution of the MABCS fracture fixation model is significantly improved with an implantation orientation of 90°. The MABCS with a bionic hole and a screw diameter of 10% provides optimal stress distribution compared with the bionic cannulated screw with diameters of 5%, 15%, and 20%. In addition, the cannulated screw fixation model with a 10% bionic hole size has optimal bone stress distribution and better internal fixation than the MABCS fixation models with 5%, 15%, and 20% screw diameters. In summary, the MABCS with 10% screw diameter bionic holes has favorable biomechanical characteristics for stabilizing femoral neck fractures. This study provides a biomechanical foundation for further optimization of the bionic cannulated screw.

A new method regulates bone fracture tissue exosome lncRNA-mRNA to promote mesenchymal stem cell proliferation and migration

Post-injury adaptation (PIA) is a simple and convenient method to promote bone healing, but its mechanism is unclear. This study was to discuss the role of fracture site tissue exosomes lncRNAs-mRNAs networks on PIA promoting bone mesenchymal stem cells (BMSCs) proliferation and migration. Firstly, the effects of PIA accelerating BMSCs proliferation and migration were confirmed by rat fracture model and bone fracture environment in vitro. Besides, the fracture site tissue exosomes were isolated and authenticated. Then the tissue exosomes were the key factor in PIA promoting BMSCs proliferation and migration authenticated by in vitro and in vivo experiments. The high throughput sequencing and RT-PCR were used to analyze the tissue exosomes lncRNAs-mRNAs networks. It was found that PIA treatment upregulated 118 lncRNAs, 295 mRNAs, and downregulated 111 lncRNAs, 2706 mRNAs in tissue exosomes. A total 12,211 genes were the target genes. Akt1, Actb and Uba52 were the hub mRNAs in tissue exosomes. In additions, tissue-derived exosomes of PIA treated rats upregulated 49 genes, 3 lncRNAs and downregulated 28 genes, 1 lncRNA in BMSCs. Kif11 was the hub gene. Overall, PIA promoted BMSCs proliferation and migration in the early stage of fracture healing, which was closely related to the fracture site tissue exosomes. Akt1, Actb and Uba52 were the hub mRNAs in the exosomes. Besides, Kif11 might be the key gene in BMSC regulated by tissue-derived exosomes of PIA treated rats.

Transient Exposure of Tissue-Engineered Cartilage Analogs to Synovial Fluid Hematoma After Ankle Fracture Promotes Chondrocyte Death and Alters Gene Expression Toward an Osteoarthritic Phenotype

Category: Basic Sciences/Biologics; Ankle Arthritis Introduction/Purpose: The first stage of fracture healing consists of hematoma formation with recruitment of proinflammatory cytokines and matrix metalloproteinases. Unfortunately, when there is an intra-articular fracture, these inflammatory mediators are not retained at the fracture site, but instead, bathe the healthy cartilage of the entire joint via the synovial fluid fracture hematoma (SFFH). These inflammatory cytokines and matrix metalloproteinases are known factors in the progression of osteoarthritis and rheumatoid arthritis. Despite the known inflammatory contents of the SFFH, little research has been done on the effects of the SFFH on healthy cartilage with regard to cell death and alteration in gene expression that could lead to post- traumatic osteoarthritis (PTOA). Methods: SFFH was collected from 12 patients with intraarticular ankle fracture at the time of surgery. Separately, human chondrocytes were three-dimensionally cultured to create scaffold -free cartilage tissue analogues (CTAs) to simulate articular cartilage. Experimental CTAs (n=12) were exposed to 100% SFFH for 3 days, washed, and transferred to complete media for 3 days. Control CTAs (n=12) were simultaneously cultured in complete medium. Subsequently, CTAs were harvested and underwent biochemical, histological, and gene expression analysis. Results: Exposure of CTAs to ankle SFFH 3 days significantly decreased chondrocyte viability by 34% (p = .027). Gene expression of both COL2A1 and SOX9 were significantly decreased after exposure to SFFH (p= .012 and p=.0013 respectively). COL1A1 expression trended upward in the SFFH exposure group while there was no difference in RUNX2 and MMP13 gene expression. Quantitative analysis of Picrosirius red staining demonstrated increased collagen I deposition with poor ultrastructural organization in SFFH exposed CTAs. Conclusion: Exposure of an organoid model of healthy cartilage tissue to SFFH after intraarticular ankle fracture resulted in decreased chondrocyte viability, decreased expression of genes regulating normal chondrocyte phenotype, and altered matrix ultrastructure indicating differentiation toward an osteoarthritis phenotype.

Key Trace Elements Zinc and Magnesium Levels Decline After Fracture in a Rat Model

Category: Basic Sciences/Biologics; Basic Sciences/Biologics Introduction/Purpose: Zinc and magnesium are crucial for bone health, and their deficiencies can lead to impaired bone metabolism and repair. Although enhancing their bioavailability has been linked to better fracture healing, their levels during healing have not been studied. We hypothesized that zinc and magnesium levels transiently decrease at the fracture site compared to intact bone. This study is the first to explore temporal trace element levels during the initial stages of fracture healing. The findings will reveal when each element changes its concentration at the fracture site, which is associated with specific functions necessary for appropriate healing. Understanding the connection between trace element concentration and repair processes could lead to more effective treatment or supplementation during fracture healing. Methods: Following induction of a femur fracture in male BB Wistar rats, injured and intact contralateral femurs were harvested at 1- (n=3), 3- (n=4), and 7-days (n=3) after fracture. Fracture segments 10mm in length were resected 5mm from the center of the callus in either direction. Contralateral non-fractured femur segments were resected from the same region within the femoral diaphysis. Samples were analyzed via inductively coupled mass spectrometry using an Agilent ICP-MS 7900 (Agilent Technologies, Santa Clara, CA). Data was converted from ppb to percent weight zinc and magnesium per weight of bone (w/w%). The difference between zinc and magnesium in contralateral intact bone vs. fractured bone was calculated and averaged (∆) for each group. The average % change were also determined. An independent sample two-tailed t-test was performed comparing average w/w% of zinc and magnesium between fracture and contralateral samples. A p-value of < 0.05 was considered significant. Results: After 1-day post-fracture, there was a -29.09 average % change in zinc levels (∆ 6.90x10^-03 ± 5.05x10^-03) (p = 0.033) and a -25.76 average % change in magnesium levels (∆ 1.21x10^-01 ± 7.36x10^-02 w/w%) (p = 0.241) between fractured and non- fractured femurs. After 3-days post-fracture, there was a -22.51 average % change in zinc levels (∆ 5.45x10^-03 ± 3.95x10^-03 w/w%) (p = 0.022) and a -33.07 average % change in magnesium levels (∆ 1.84x10^-01 ± 1.05x10^-01 w/w%) (p = 0.025). After 7- days post-fracture, there was a -9.40 average % change in zinc levels (∆ 1.90x10^-03 ± 3.98x10^-03) (p = 0.377) and a -17.88 average % change in magnesium levels (∆ 9.04x10^-02 ± 1.24x10^-01 w/w%) (p = 0.159) between fractured and non-fractured femurs. Conclusion: Previous research has established the significance of trace elements, namely zinc and magnesium, in the process of fracture healing. Zinc has been shown to improve fracture healing, while magnesium has been found to play a role regulating inflammation during early fracture healing. The findings of this study corroborate our hypothesis that levels of these elements transiently decline following fracture. By evaluating these changes in trace element levels, we can gain a better understanding of their function in the fracture healing process. These observations may offer insight into potential therapeutic approaches utilizing zinc and magnesium for fracture healing in the future.

Inflammatory Processes Affecting Bone Health and Repair.

The purpose of this article is to review the current understanding of inflammatory processes on bone, including direct impacts of inflammatory factors on bone cells, the effect of senescence on inflamed bone, and the critical role of inflammation in bone pain and healing. Advances in osteoimmunology have provided new perspectives on inflammatory bone loss in recent years. Characterization of so-called inflammatory osteoclasts has revealed insights into physiological and pathological bone loss. The identification of inflammation-associated senescent markers in bone cells indicates that therapies that reduce senescent cell burden may reverse bone loss caused by inflammatory processes. Finally, novel studies have refined the role of inflammation in bone healing, including cross talk between nerves and bone cells. Except for the initial stages of fracture healing, inflammation has predominately negative effects on bone and increases fracture risk. Eliminating senescent cells, priming the osteo-immune axis in bone cells, and alleviating pro-inflammatory cytokine burden may ameliorate the negative effects of inflammation on bone.

Herbal Extracts of Ginseng and Maqui Berry Show Only Minimal Effects on an In Vitro Model of Early Fracture Repair of Smokers.

Smoking is a major risk factor for delayed fracture healing, affecting several aspects of early fracture repair, including inflammation, osteogenesis, and angiogenesis. Panax ginseng (GE) and maqui berry extract (MBE) were shown in our previous studies to reduce smoke-induced cellular damage in late bone-healing in vitro models. We aimed here to analyze their effects on the early fracture repair of smokers in a 3D co-culture model of fracture hematomas and endothelial cells. Both extracts did not alter the cellular viability at concentrations of up to 100 µg/mL. In early fracture repair in vitro, they were unable to reduce smoking-induced inflammation and induce osteo- or chondrogenicity. Regarding angiogenesis, smoking-induced stress in HUVECs could not be counteracted by both extracts. Furthermore, smoking-impaired tube formation was not restored by GE but was harmed by MBE. However, GE promoted angiogenesis initiation under smoking conditions via the Angpt/Tie2 axis. To summarize, cigarette smoking strikingly affected early fracture healing processes in vitro, but herbal extracts at the applied doses had only a limited effect. Since both extracts were shown before to be very effective in later stages of fracture healing, our data suggest that their early use immediately after fracture does not appear to negatively impact later beneficial effects.

Exposure of Tissue-Engineered Cartilage Analogs to Synovial Fluid Hematoma After Ankle Fracture Is Associated With Chondrocyte Death and Altered Cartilage Maintenance Gene Expression.

The first stage of fracture healing consists of hematoma formation with recruitment of proinflammatory cytokines and matrix metalloproteinases. Unfortunately, when there is an intra-articular fracture, these inflammatory mediators are not retained at the fracture site, but instead, envelop the healthy cartilage of the entire joint via the synovial fluid fracture hematoma (SFFH). These inflammatory cytokines and matrix metalloproteinases are known factors in the progression of osteoarthritis and rheumatoid arthritis. Despite the known inflammatory contents of the SFFH, little research has been done on the effects of the SFFH on healthy cartilage with regard to cell death and alteration in gene expression that could lead to posttraumatic osteoarthritis (PTOA). SFFH was collected from 12 patients with intraarticular ankle fracture at the time of surgery. Separately, C20A4 immortalized human chondrocytes were 3-dimensionally cultured to create scaffold-free cartilage tissue analogs (CTAs) to simulate healthy cartilage. Experimental CTAs (n = 12) were exposed to 100% SFFH for 3 days, washed, and transferred to complete media for 3 days. Control CTAs (n = 12) were simultaneously cultured in complete medium without exposure to SFFH. Subsequently, CTAs were harvested and underwent biochemical, histological, and gene expression analysis. Exposure of CTAs to ankle SFFH for 3 days significantly decreased chondrocyte viability by 34% (P = .027). Gene expression of both COL2A1 and SOX9 were significantly decreased after exposure to SFFH (P = .012 and P = .0013 respectively), while there was no difference in COL1A1, RUNX2, and MMP13 gene expression. Quantitative analysis of Picrosirius red staining demonstrated increased collagen I deposition with poor ultrastructural organization in SFFH-exposed CTAs. Exposure of an organoid model of healthy cartilage tissue to SFFH after intraarticular ankle fracture resulted in decreased chondrocyte viability, decreased expression of genes regulating normal chondrocyte phenotype, and altered matrix ultrastructure indicating differentiation toward an osteoarthritis phenotype. The majority of ankle fracture open reduction and internal fixation does not occur immediately after fracture. In fact, typically these fractures are treated several days to weeks later in order to let the swelling subside. This means that the healthy innocent bystander cartilage not involved in the fracture is exposed to SFFH during this time. In this study, the SFFH caused decreased chondrocyte viability and specific altered gene expression that might have the potential to induce osteoarthritis. These data suggest that early intervention after intraarticular ankle fracture could possibly mitigate progression toward PTOA.

Engineering 3D-Printed Strontium-Titanium Scaffold-Integrated Highly Bioactive Serum Exosomes for Critical Bone Defects by Osteogenesis and Angiogenesis.

Currently, healing of large bone defects faces significant challenges such as a bulk of bone regeneration and revascularization on the bone defect region. Here, a "cell-free scaffold engineering" strategy that integrates strontium (Sr) and highly bioactive serum exosomes (sEXOs) inside a three-dimensional (3D)-printed titanium (Ti) scaffold (Sc) is first developed. The constructed SrTi Sc can serve as a sophisticated biomaterial platform for maintaining bone morphological characteristics of the radius during the period of critical bone defect (CBD) repair and further accelerating bone formation and fibroblastic suppression via the controlled release of Sr from the superficial layer of the scaffold. Moreover, compared with sEXO from healthy donors, the sEXO extracted from the serum of the femoral fracture rabbit model at the stage of fracture healing, named BF EXO, is robustly capable of facilitating osteogenesis and angiogenesis. In addition, the underlying therapeutic mechanism is elucidated, whereby altering miRNAs shuttled by BF EXO enables osteogenesis and angiogenesis. Further, the in vivo study revealed that the SrTi Sc + BF EXO composite dramatically accelerated bone repair via osteoconduction, osteoinduction, and revascularization in radial CBD of rabbits. This study broadens the source and biomedical potential of specifically functionalized exosomes and provides a comprehensive clinically feasible strategy for therapeutics on large bone defects.

Ultrasensitive capacitive sensing system for smart medical devices with ability to monitor fracture healing stages

Bone fractures are a global public health problem. A sustained increase in the number of incident cases has been observed in the last few decades, as well as the number of prevalent cases and the number of years lived with disability. Current monitoring techniques are based on imaging techniques, which are highly subjective, radioactive, expensive and unable to provide daily monitoring of fracture healing stages. The development of reliable, non-invasive and non-subjective technologies is mandatory to minimize non-union risks. Delayed healing and non-union conditions require timely medical intervention, such that preventive procedures and shortened treatment periods can be carried out. This work proposes the development of an ultrasensitive capacitive sensing system for smart implantable fixation implants with ability to effectively monitor the evolution of bone fractures. Both in vitro experimental tests and numerical simulations highlight that networks of co-surface capacitive systems are able: (i) to detect four different bone healing phases, capacitance decrease patterns occurring as the healing process progresses and (ii) to monitor the callus evolution in multiple target regions. These are very promising results that highlight the potential of capacitive technologies to minimize the individual and social burdens related to fracture management, mainly when delayed healing or non-union conditions occur.

The effects of mechanical instability on PDGF mediated inflammatory response at early stage of fracture healing under diabetic condition

Background and ObjectiveMechanical stability plays an important role in fracture healing process. Excessive interfragmentary movement will continuously damage the tissue and newly formed capillaries at the fracture site, which leads to overproduction of platelet-derived growth factor (PDGF) that attracts more macrophages into fracture callus, ultimately persistent and enhanced inflammatory response happens. For diabetic condition, the impact of mechanical instability of fracture site on inflammatory response could be further compliciated and the relevant research in this field is relatively limited. MethodsBuilding on previous experimental studies, this study presents a numerical model consisting of a system of reactive-transport equations representing the transport as well as interactions of different cells and cytokines within the fracture callus. The model is initially validated by available experimental data, and then implemented to investigate the role of mechanical stability of fracture site in inflammatory response during early stage of healing. It is assumed that there is an increased release of PDGF due to the rupture of blood vessels resulting from mechanical instability, which leads to increased production of inflammatory cytokines (i.e., TNF-α). The bone healing process under three different conditions were investigated, i.e., mechanically stable condition with normal inflammatory response (Control, Case 1), mechanically unstable condition with normal inflammatory response (Case 2) and mechanically unstable condition with diabetes (Case 3). ResultsMechanical instability can promote the macrophage infiltration and thus induce an enhanced and prolonged inflammatory response, which could impede the MSCs proliferation during the early fracture healing stage (e.g., compared with the control condition, the MSCs concentration in unstable fracture with normal inflammatory response can be reduced by 3.2% and 5.2% on day 2 and day 10 post-fracture, respectively). Under diabetic condition, the mechanical instability of fracture site could lead to a significant increase of TNF-α concentration in fracture callus (Case 3) in comparison to control (Case 1) (e.g., three-fold increase in TNF-α concentration compared to control). In addition, the results show that the mechanical instability affects the cell differentiation and proliferation in fracture callus in a spatially dependent manner, e.g., for diabetic fracture patients, the mechanical instability could potentially decrease the concentration of MSCs, osteoblasts and chondrocytes by around 39%, 30% and 29% in cortical callus, respectively, in comparison to control. ConclusionThe mechanical instability together with diabetic condition can significantly affect the natural resolution of inflammation during early stage of healing by turning acute inflammation into chronic inflammation which is characterized by a continuously upregulated TNF-α pathway.

A timeseries analysis of the fracture callus extracellular matrix proteome during bone fracture healing.

While most bones fully self-heal, certain diseases require bone allograft to assist with fracture healing. Bone allografts offer promise as treatments for such fractures due to their osteogenic properties. However, current bone allografts made of decellularized bone extracellular matrix (ECM) have high failure rates, and thus grafts which improve fracture healing outcomes are needed. Understanding specific changes to the ECM proteome during normal fracture healing would enable the identification of key proteins that could be used enhance osteogenicity of bone allograft. Here, we performed a timeseries analysis of the fracture callus in mice to investigate proteomic and mineralization changes to the ECM at key stages of fracture healing. We found that changes to the ECM proteome largely coincide with the distinct phases of fracture healing. Basement membrane proteins (AGRN, COL4, LAMA), cartilage proteins (COL2A1, ACAN), and collagen crosslinking enzymes (LOXL, PLOD, ITIH) were initially upregulated, followed by bone specific proteoglycans and collagens (IBSP, COL1A1). Various tissue proteases (MMP2, 9, 13, 14; CTSK, CTSG, ELANE) were expressed at different levels throughout fracture healing. These changes coordinated with mineralization of the fracture callus, which increased steeply during the initial stages of healing. Interestingly the later timepoint was characterized by a response to wound healing and high expression of clotting factors (F2, 7, 9, 10). We identified ELANE and ITIH2 as tissue remodeling enzymes having no prior known involvement with fracture healing. This data can be further mined to identify regenerative proteins for enhanced bone graft design.

Tetracalcium phosphate treatment on experimental fracture model in rats.

Objectives This study aims to investigate the efficacy of tetracalcium phosphate (TTCP) on fracture healing in rat femurs.Materials and methods Forty-two female Wistar Albino rats were randomized into two groups (Group 1 and Group 2, n=21 for each). The left femur of all animals was fractured by osteotomy after deep anesthesia with ketamine. Additional procedure was not applied to the rats in Group 1. Rats in Group 2, following osteotomy, were applied to the fracture line approximately 2 mL TTCP. The animals were sacrificed at Weeks 1, 2, and 3 after surgery (seven animals were sacrificed from each group each week) and the broken femurs were removed. The femurs were examined first radiographically and second histopathologically.Results Radiologically, callus maturity and bone union increased with time in both groups. However, no significant differences were found regarding callus maturity and bone union in weekly comparisons (anteroposterior plain: p=0.53, p=0.37, p=0.42, lateral plain: p=0.26, p=0.42, p=0.87). Histopathologically, the fractures healed normally as the weeks progressed in both groups. The histological scores of Group 2 were higher at Weeks 1, 2, and 3. In the evaluation, no significant difference was found between the groups in terms of histological scores except for the first week (p=0.024, p=104, p=462, respectively).Conclusion Although there was no statistically significant difference in the histological evaluation of both groups, except for the first week, the histological scores of Group 2, which underwent TTCP in all weeks, were higher. According to the results of this study, we believe that TTCP may be beneficial, particularly in the early stages of fracture healing.

The Effects of Mechanical Instability on PDGF Mediated Inflammatory Response at Early Stage of Fracture Healing Under Diabetic Condition

The Effects of Mechanical Instability on PDGF Mediated Inflammatory Response at Early Stage of Fracture Healing Under Diabetic Condition

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

Local delivery and controlled release of miR-34a loaded in hydroxyapatite/mesoporous organosilica nanoparticles composite-coated implant wire to accelerate bone fracture healing

Immediate mechanical stability is a prerequisite for fracture healing. In addition to bringing immediate mechanical stability in fracture site, implants with bioactive coating can release active substance to accelerate bone-fracture healing. However, limited drug-loading capacity of established coatings weakens their biological functions, which urges the engineering of more effective coating biomaterials for accelerating fracture healing. Herein, mesoporous organosilica nanoparticles (MONs), as miR-34a delivers, are loaded onto hydroxyapatite (HA)-coated Kirschner wire to engineer a HA/MONs@miR-34a composite coating. The composite coating can effectively deliver miR-34a into osteoclasts, generate gene dose-dependent inhibiting effect on differentiation and resorptive activity of osteoclasts by regulating multiple downstream gene expression at the early stage of fracture healing, which additionally exhibits decent bone regeneration potentials as evidenced in rat tibial fracture model. In particular, differentially expressed genes regulated by miR-34a are identified using RNA-seq followed by bioinformatics analysis. Functional enrichment analysis reveals that genes with altered expression mainly distribute in mainly distribute in DNA replication and cell cycle, which are associated with the development of osteoclasts. This work not only demonstrates the high clinical translation potential of HA/MONs@miR-34a to accelerate fracture healing, but also reveals the underlying molecular mechanism of regulating physiological functions of osteoclasts based on analysis of singlecell RNA sequencing.