Site Specific Effects of Zoledronic Acid during Tibial and Mandibular Fracture Repair

Numerous factors can affect skeletal regeneration, including the extent of bone injury, mechanical loading, inflammation and exogenous molecules. Bisphosphonates are anticatabolic agents that have been widely used to treat a variety of metabolic bone diseases. Zoledronate (ZA), a nitrogen-containing bisphosphonate (N-BP), is the most potent bisphosphonate among the clinically approved bisphosphonates. Cases of bisphosphonate-induced osteonecrosis of the jaw have been reported in patients receiving long term N-BP treatment. Yet, osteonecrosis does not occur in long bones. The aim of this study was to compare the effects of zoledronate on long bone and cranial bone regeneration using a previously established model of non-stabilized tibial fractures and a new model of mandibular fracture repair. Contrary to tibial fractures, which heal mainly through endochondral ossification, mandibular fractures healed via endochondral and intramembranous ossification with a lesser degree of endochondral ossification compared to tibial fractures. In the tibia, ZA reduced callus and cartilage formation during the early stages of repair. In parallel, we found a delay in cartilage hypertrophy and a decrease in angiogenesis during the soft callus phase of repair. During later stages of repair, ZA delayed callus, cartilage and bone remodeling. In the mandible, ZA delayed callus, cartilage and bone remodeling in correlation with a decrease in osteoclast number during the soft and hard callus phases of repair. These results reveal a more profound impact of ZA on cartilage and bone remodeling in the mandible compared to the tibia. This may predispose mandible bone to adverse effects of ZA in disease conditions. These results also imply that therapeutic effects of ZA may need to be optimized using time and dose-specific treatments in cranial versus long bones

Age-related changes to macrophages are detrimental to fracture healing in mice.

The elderly population suffers from higher rates of complications during fracture healing that result in increased morbidity and mortality. Inflammatory dysregulation is associated with increased age and is a contributing factor to the myriad of age-related diseases. Therefore, we investigated age-related changes to an important cellular regulator of inflammation, the macrophage, and the impact on fracture healing outcomes. We demonstrated that old mice (24 months) have delayed fracture healing with significantly less bone and more cartilage compared to young mice (3 months). The quantity of infiltrating macrophages into the fracture callus was similar in old and young mice. However, RNA-seq analysis demonstrated distinct differences in the transcriptomes of macrophages derived from the fracture callus of old and young mice, with an up-regulation of M1/pro-inflammatory genes in macrophages from old mice as well as dysregulation of other immune-related genes. Preventing infiltration of the fracture site by macrophages in old mice improved healing outcomes, with significantly more bone in the calluses of treated mice compared to age-matched controls. After preventing infiltration by macrophages, the macrophages remaining within the fracture callus were collected and examined via RNA-seq analysis, and their transcriptome resembled macrophages from young calluses. Taken together, infiltrating macrophages from old mice demonstrate detrimental age-related changes, and depleting infiltrating macrophages can improve fracture healing in old mice

Stimulating Fracture Healing in Ischemic Environments: Does Oxygen Direct Stem Cell Fate during Fracture Healing?

Bone fractures represent an enormous societal and economic burden as one of the most prevalent causes of disability worldwide. Each year, nearly 15 million people are affected by fractures in the United States alone. Data indicate that the blood supply is critical for fracture healing; as data indicate that concomitant bone and vascular injury are major risk factors for non-union. However, the various role(s) that the vasculature plays remains speculative. Fracture stabilization dictates stem cell fate choices during repair. In stabilized fractures stem cells differentiate directly into osteoblasts and heal the injury by intramembranous ossification. In contrast, in non-stable fractures stem cells differentiate into chondrocytes and the bone heals through endochondral ossification, where a cartilage template transforms into bone as the chondrocytes transform into osteoblasts. One suggested role of the vasculature has been to participate in the stem cell fate decisions due to delivery of oxygen. In stable fractures, the blood vessels are thought to remain intact and promote osteogenesis, while in non-stable fractures, continual disruption of the vasculature creates hypoxia that favors formation of cartilage, which is avascular. However, recent data suggests that non-stable fractures are more vascularized than stable fractures, that oxygen does not appear associated with differentiation of stem cells into chondrocytes and osteoblasts, that cartilage is not hypoxic, and that oxygen, not sustained hypoxia, is required for angiogenesis. These unexpected results, which contrast other published studies, are indicative of the need to better understand the complex, spatio-temporal regulation of vascularization and oxygenation in fracture healing. This work has also revealed that oxygen, along with the promotion of angiogenesis, may be novel adjuvants that can stimulate healing in select patient populations

Stimulating Fracture Healing in Ischemic Environments: Does Oxygen Direct Stem Cell Fate during Fracture Healing?

Bone fractures represent an enormous societal and economic burden as one of the most prevalent causes of disability worldwide. Each year, nearly 15 million people are affected by fractures in the United States alone. Data indicate that the blood supply is critical for fracture healing; as data indicate that concomitant bone and vascular injury are major risk factors for non-union. However, the various role(s) that the vasculature plays remains speculative. Fracture stabilization dictates stem cell fate choices during repair. In stabilized fractures stem cells differentiate directly into osteoblasts and heal the injury by intramembranous ossification. In contrast, in non-stable fractures stem cells differentiate into chondrocytes and the bone heals through endochondral ossification, where a cartilage template transforms into bone as the chondrocytes transform into osteoblasts. One suggested role of the vasculature has been to participate in the stem cell fate decisions due to delivery of oxygen. In stable fractures, the blood vessels are thought to remain intact and promote osteogenesis, while in non-stable fractures, continual disruption of the vasculature creates hypoxia that favors formation of cartilage, which is avascular. However, recent data suggests that non-stable fractures are more vascularized than stable fractures, that oxygen does not appear associated with differentiation of stem cells into chondrocytes and osteoblasts, that cartilage is not hypoxic, and that oxygen, not sustained hypoxia, is required for angiogenesis. These unexpected results, which contrast other published studies, are indicative of the need to better understand the complex, spatio-temporal regulation of vascularization and oxygenation in fracture healing. This work has also revealed that oxygen, along with the promotion of angiogenesis, may be novel adjuvants that can stimulate healing in select patient populations

Local injections of β-NGF accelerates endochondral fracture repair by promoting cartilage to bone conversion.

There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local β-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from β-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local β-NGF injections. Finally, we demonstrate functional improvements to bone healing following local β-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate β-NGF\u27s ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation

Altered early immune response after fracture and traumatic brain injury

IntroductionClinical and preclinical data suggest accelerated bone fracture healing in subjects with an additional traumatic brain injury (TBI). Mechanistically, altered metabolism and neuro-endocrine regulations have been shown to influence bone formation after combined fracture and TBI, thereby increasing the bone content in the fracture callus. However, the early inflammatory response towards fracture and TBI has not been investigated in detail so far. This is of great importance, since the early inflammatory phase of fracture healing is known to be essential for the initiation of downstream regenerative processes for adequate fracture repair.MethodsTherefore, we analyzed systemic and local inflammatory mediators and immune cells in mice which were exposed to fracture only or fracture + TBI 6h and 24h after injury.ResultsWe found a dysregulated systemic immune response and significantly fewer neutrophils and mast cells locally in the fracture hematoma. Further, local CXCL10 expression was significantly decreased in the animals with combined trauma, which correlated significantly with the reduced mast cell numbers.DiscussionSince mast cells and mast cell-derived CXCL10 have been shown to increase osteoclastogenesis, the reduced mast cell numbers might contribute to higher bone content in the fracture callus of fracture + TBI mice due to decreased callus remodeling

Delays in hospital admissions in patients with fractures across 18 low-income and middle-income countries (INORMUS): a prospective observational study

© 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license Background: The Lancet Commission on Global Surgery established the Three Delays framework, categorising delays in accessing timely surgical care into delays in seeking care (First Delay), reaching care (Second Delay), and receiving care (Third Delay). Globally, knowledge gaps regarding delays for fracture care, and the lack of large prospective studies informed the rationale for our international observational study. We investigated delays in hospital admission as a surrogate for accessing timely fracture care and explored factors associated with delayed hospital admission. Methods: In this prospective observational substudy of the ongoing International Orthopaedic Multicenter Study in Fracture Care (INORMUS), we enrolled patients with fracture across 49 hospitals in 18 low-income and middle-income countries, categorised into the regions of China, Africa, India, south and east Asia, and Latin America. Eligible patients were aged 18 years or older and had been admitted to a hospital within 3 months of sustaining an orthopaedic trauma. We collected demographic injury data and time to hospital admission. Our primary outcome was the number of patients with open and closed fractures who were delayed in their admission to a treating hospital. Delays for patients with open fractures were defined as being more than 2 h from the time of injury (in accordance with the Lancet Commission on Global Surgery) and for those with closed fractures as being a delay of more than 24 h. Secondary outcomes were reasons for delay for all patients with either open or closed fractures who were delayed for more than 24 h. We did logistic regression analyses to identify risk factors of delays of more than 2 h in patients with open fractures and delays of more than 24 h in patients with closed fractures. Logistic regressions were adjusted for region, age, employment, urban living, health insurance, interfacility referral, method of transportation, number of fractures, mechanism of injury, and fracture location. We further calculated adjusted relative risk (RR) from adjusted odds ratios, adjusted for the same variables. This study was registered with ClinicalTrials.gov, NCT02150980, and is ongoing. Findings: Between April 3, 2014, and May 10, 2019, we enrolled 31 255 patients with fractures, with a median age of 45 years (IQR 31–62), of whom 19 937 (63·8%) were men, and 14 524 (46·5%) had lower limb fractures, making them the most common fractures. Of 5256 patients with open fractures, 3778 (71·9%) were not admitted to hospital within 2 h. Of 25 999 patients with closed fractures, 7141 (27·5%) were delayed by more than 24 h. Of all regions, Latin America had the greatest proportions of patients with delays (173 [88·7%] of 195 patients with open fractures; 426 [44·7%] of 952 with closed fractures). Among patients delayed by more than 24 h, the most common reason for delays were interfacility referrals (3755 [47·7%] of 7875) and Third Delays (cumulatively interfacility referral and delay in emergency department: 3974 [50·5%]), while Second Delays (delays in reaching care) were the least common (423 [5·4%]). Compared with other methods of transportation (eg, walking, rickshaw), ambulances led to delay in transporting patients with open fractures to a treating hospital (adjusted RR 0·66, 99% CI 0·46–0·93). Compared with patients with closed lower limb fractures, patients with closed spine (adjusted RR 2·47, 99% CI 2·17–2·81) and pelvic (1·35, 1·10–1·66) fractures were most likely to have delays of more than 24 h before admission to hospital. Interpretation: In low-income and middle-income countries, timely hospital admission remains largely inaccessible, especially among patients with open fractures. Reducing hospital-based delays in receiving care, and, in particular, improving interfacility referral systems are the most substantial tools for reducing delays in admissions to hospital. Funding: National Health and Medical Research Council of Australia, Canadian Institutes of Health Research, McMaster Surgical Associates, and Hamilton Health Sciences

Role of Matrix Metalloproteinase 13 in Both Endochondral and Intramembranous Ossification during Skeletal Regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13−/− mice is intrinsic to cartilage and bone. Mmp13−/− mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts