The effects of bone morphogenetic protein 2 and thrombopoietin treatment on angiogenic properties of endothelial cells derived from the lung and bone marrow of young and aged, male and female mice.

With an aging world population, there is an increased risk of fracture and impaired healing. One contributing factor may be aging-associated decreases in vascular function; thus, enhancing angiogenesis could improve fracture healing. Both bone morphogenetic protein 2 (BMP-2) and thrombopoietin (TPO) have pro-angiogenic effects. The aim of this study was to investigate the effects of treatment with BMP-2 or TPO on the in vitro angiogenic and proliferative potential of endothelial cells (ECs) isolated from lungs (LECs) or bone marrow (BMECs) of young (3-4 months) and old (22-24 months), male and female, C57BL/6J mice. Cell proliferation, vessel-like structure formation, migration, and gene expression were used to evaluate angiogenic properties. In vitro characterization of ECs generally showed impaired vessel-like structure formation and proliferation in old ECs compared to young ECs, but improved migration characteristics in old BMECs. Differential sex-based angiogenic responses were observed, especially with respect to drug treatments and gene expression. Importantly, these studies suggest that NTN1, ROBO2, and SLIT3, along with angiogenic markers (CD31, FLT-1, ANGPT1, and ANGP2) differentially regulate EC proliferation and functional outcomes based on treatment, sex, and age. Furthermore, treatment of old ECs with TPO typically improved vessel-like structure parameters, but impaired migration. Thus, TPO may serve as an alternative treatment to BMP-2 for fracture healing in aging owing to improved angiogenesis and fracture healing, and the lack of side effects associated with BMP-2.

The Effects of SRT1720 Treatment on Endothelial Cells Derived from the Lung and Bone Marrow of Young and Aged, Male and Female Mice.

Angiogenesis is critical for successful fracture healing. Age-related alterations in endothelial cells (ECs) may cause impaired bone healing. Therefore, examining therapeutic treatments to improve angiogenesis in aging may enhance bone healing. Sirtuin 1 (SIRT1) is highly expressed in ECs and its activation is known to counteract aging. Here, we examined the effects of SRT1720 treatment (SIRT1 activator) on the growth and function of bone marrow and lung ECs (BMECs and LECs, respectively), derived from young (3-4 month) and old (20-24 month) mice. While aging did not alter EC proliferation, treatment with SRT1720 significantly increased proliferation of all LECs. However, SRT1720 only increased proliferation of old female BMECs. Vessel-like tube assays showed similar vessel-like structures between young and old LECs and BMECs from both male and female mice. SRT1720 significantly improved vessel-like structures in all LECs. No age, sex, or treatment differences were found in migration related parameters of LECs. In males, old BMECs had greater migration rates than young BMECs, whereas in females, old BMECs had lower migration rates than young BMECs. Collectively, our data suggest that treatment with SRT1720 appears to enhance the angiogenic potential of LECs irrespective of age or sex. However, its role in BMECs is sex- and age-dependent.

Review of Secondary Causes of Osteoporotic Fractures Due to Diabetes and Spinal Cord Injury.

PURPOSE OF REVIEW: The aim of this review is to gain a better understanding of osteoporotic fractures and the different mechanisms that are driven in the scenarios of bone disuse due to spinal cord injury and osteometabolic disorders due to diabetes. RECENT FINDINGS: Despite major advances in understanding the pathogenesis, prevention, and treatment of osteoporosis, the high incidence of impaired fracture healing remains an important complication of bone loss, leading to marked impairment of the health of an individual and economic burden to the medical system. This review underlines several pathways leading to bone loss and increased risk for fractures. Specifically, we addressed the different mechanisms leading to bone loss after a spinal cord injury and diabetes. Finally, it also encompasses the changes responsible for impaired bone repair in these scenarios, which may be of great interest for future studies on therapeutic approaches to treat osteoporosis and osteoporotic fractures.

Minced muscle autografting improves bone healing but not muscle function in a porcine composite injury model.

Composite tissue injuries (CTIs) in extremities include segmental bone defects (SBDs) and volumetric muscle loss. The objective of this study was to determine if skeletal muscle autografting with minced muscle grafts (MMGs) could improve healing in an SBD and improve muscle function in a porcine CTI model that includes an SBD and adjacent volumetric muscle loss injury. Adult Yucatan Minipigs were stratified into three groups including specimens with an isolated SBD, an SBD with volumetric muscle loss (CTI), and an SBD with volumetric muscle loss treated with MMG (CTI + MMG). Bone healing was quantified with serial x-rays and postmortem computed tomography scanning. Muscle function was quantified with a custom in vivo force transducer. Muscle tissue content was determined by biochemical analyses and histology. Anterior cortex-modified Radiographic Union Score for Tibia fractures (mRUSTs) decreased from 2.7 to 1.9 (p = 0.003) in CTI versus SBD animals. MMG improved anterior mRUST scores to 2.5 in CTI + MMG specimens (p = 0.030 compared to CTI specimens) and overall mRUST scores increased from 9.4 in CTI specimens to 11.1 in CTI + MMG specimens (p = 0.049). Residual strength deficits at euthanasia were 42% in SBD (p < 0.001), 44% in CTI (p < 0.001), and 48% in CTI + MMG (p < 0.001) compared to preoperative values. There were no differences in strength deficits between the three groups. Biochemical and histologic analyses demonstrated scattered differences between the three groups compared to contralateral muscle. MMG improved bone healing. However, the primary cause of muscle dysfunction and biochemical changes was the presence of an SBD. Clinical significance: Early mitigation of SBDs may be necessary to prevent muscle damage and weakness in patients sustaining composite extremity trauma.

Effects of diet, BMP-2 treatment, and femoral skeletal injury on endothelial cells derived from the ipsilateral and contralateral limbs.

Type 2 diabetes (T2D) results in physiological and structural changes in bone, contributing to poor fracture healing. T2D compromises microvascular performance, which can negatively impact bone regeneration as angiogenesis is required for new bone formation. We examined the effects of bone morphogenetic protein-2 (BMP-2) administered locally at the time of femoral segmental bone defect (SBD) surgery, and its angiogenic impacts on endothelial cells (ECs) isolated from the ipsilateral or contralateral tibia in T2D mice. Male C57BL/6 mice were fed either a low-fat diet (LFD) or high-fat diet (HFD) starting at 8 weeks. After 12 weeks, the T2D phenotype in HFD mice was confirmed via glucose and insulin tolerance testing and echoMRI, and all mice underwent SBD surgery. Mice were treated with BMP-2 (5 µg) or saline at the time of surgery. Three weeks postsurgery, bone marrow ECs were isolated from ipsilateral and contralateral tibias, and proliferation, angiogenic potential, and gene expression of the cells was analyzed. BMP-2 treatment increased EC proliferation by two fold compared with saline in LFD contralateral tibia ECs, but no changes were seen in surgical tibia EC proliferation. BMP-2 treatment enhanced vessel-like structure formation in HFD mice whereas, the opposite was observed in LFD mice. Still, in BMP-2 treated LFD mice, ipsilateral tibia ECs increased expression of CD31, FLT-1, ANGPT1, and ANGPT2. These data suggest that the modulating effects of T2D and BMP-2 on the microenvironment of bone marrow ECs may differentially influence angiogenic properties at the fractured limb versus the contralateral limb.

Validation of the modified radiographic union score for tibia fractures (mRUST) in murine femoral fractures.

Bony union is a primary predictor of outcome after surgical fixation of long bone fractures. Murine models offer many advantages in assessing bony healing due to their low costs and small size. However, current fracture recovery investigations in mice frequently rely on animal sacrifice and costly analyses. The modified Radiographic Union Score for Tibia fractures (mRUST) scoring system is a validated metric for evaluating bony healing in humans utilizing plain radiographs, which are relatively inexpensive and do not require animal sacrifice. However, its use has not been well established in murine models. The aim of this study was to characterize the longitudinal course of mRUST and compare mRUST to other conventional murine fracture analyses. 158 mice underwent surgically created midshaft femur fractures. Mice were evaluated after fracture creation and at 7, 10, 14, 17, 21, 24, 28, 35, and 42 days post-injury. mRUST scoring of plain radiographs was performed by three orthopaedic surgeons in a randomized, blinded fashion. Interrater correlations were calculated. Micro-computed tomography (µCT) was analyzed for tissue mineral density (TMD), total callus volume (TV), bone volume (BV), trabecular thickness, trabecular number, and trabecular separation. Histomorphometry measures of total callus area, cartilage area, fibrous tissue area, and bone area were performed in a blinded fashion. Ultimate torque, stiffness, toughness, and twist to failure were calculated from torque-twist curves. A sigmoidal log-logistic curve fit was generated for mRUST scores over time which shows mRUST scores of 4 to 6 at 7 days post-injury that improve to plateaus of 14 to 16 by 24 days post-injury. mRUST interrater correlations at each timepoint ranged from 0.51 to 0.86, indicating substantial agreement. mRUST scores correlated well with biomechanical, histomorphometry, and µCT parameters, such as ultimate torque (r=0.46, p<0.0001), manual stiffness (r=0.51, p<0.0001), bone percentage based on histomorphometry (r=0.86, p<0.0001), cartilage percentage (r=-0.87, p<0.0001), tissue mineral density (r=0.83, p<0.0001), BV/TV based on µCT (r=0.65, p<0.0001), and trabecular thickness (r=0.78, p<0.0001), among others. These data demonstrate that mRUST is reliable, trends temporally, and correlates to standard measures of murine fracture healing. Compared to other measures, mRUST is more cost-effective and non-terminal. The mRUST log-logistic curve could be used to characterize differences in fracture healing trajectory between experimental groups, enabling high-throughput analysis.

The effects of high fat diet, bone healing, and BMP-2 treatment on endothelial cell growth and function.

Angiogenesis is a vital process during the regeneration of bone tissue. The aim of this study was to investigate angiogenesis at the fracture site as well as at distal locations from obesity-induced type 2 diabetic mice that were treated with bone morphogenetic protein-2 (BMP-2, local administration at the time of surgery) to heal a femoral critical sized defect (CSD) or saline as a control. Mice were fed a high fat diet (HFD) to induce a type 2 diabetic-like phenotype while low fat diet (LFD) animals served as controls. Endothelial cells (ECs) were isolated from the lungs (LECs) and bone marrow (BMECs) 3 weeks post-surgery, and the fractured femurs were also examined. Our studies demonstrate that local administration of BMP-2 at the fracture site in a CSD model results in complete bone healing within 3 weeks for all HFD mice and 66.7% of LFD mice, whereas those treated with saline remain unhealed. At the fracture site, vessel parameters and adipocyte numbers were significantly increased in BMP-2 treated femurs, irrespective of diet. At distal sites, LEC and BMEC proliferation was not altered by diet or BMP-2 treatment. HFD increased the tube formation ability of both LECs and BMECs. Interestingly, BMP-2 treatment at the time of surgery reduced tube formation in LECs and humeri BMECs. However, migration of BMECs from HFD mice treated with BMP-2 was increased compared to BMECs from HFD mice treated with saline. BMP-2 treatment significantly increased the expression of CD31, FLT-1, and ANGPT2 in LECs and BMECs in LFD mice, but reduced the expression of these same genes in HFD mice. To date, this is the first study that depicts the systemic influence of fracture surgery and local BMP-2 treatment on the proliferation and angiogenic potential of ECs derived from the bone marrow and lungs.

The effects of ethyl-2-cyanoacrylate and butyl-2-cyanoacrylate in the process of bone healing in rats. A controlled experimental study.

OBJECTIVE: Synthetic adhesives are used by various medical specialties, especially in surgery; however, studies reporting their use in orthopedic practice are scarce. The aim of this study was to compare the results in using ethyl-2-cyanoacrylate or butyl-2-cyanoacrylate in the treatment of fractures in rats. METHODS: This was an experimental prospective controlled study in 90 rats, with humerus, femur, and tibia fractures, treated with ethyl-2-cyanoacrylate (SB group; n = 45) or butyl-2-cyanoacrylate (HA group; n = 45). Biomechanical and histomorphometric analyses were performed at three different moments (60, 120, and 180 days); besides a clinical study performed weekly by measurement of the animals body mass. RESULTS: No differences were observed regarding body mass (p = 0.07). In both groups, there were no significant differences regarding maximum load (p = 0.6), yield point strength (p = 0.6), and stiffness coefficient (p = 0.4) of the femurs. The same was observed in tibias for maximum load (p = 0.4), yield point strength (p = 0.7), and stiffness coefficient (p = 0.6). The humerus from both groups had similar bone callus area (p = 0.66). In both groups, there were no statistical differences related to inflammatory cells (p = 0.4), osteoblasts (p = 0.2), and osteoclasts (p = 0.2). CONCLUSION: Ethyl-2-cyanoacrylate was more effective than butyl-2-cyanoacrylate in the treatment of fractures in rats.

OBJETIVO: Os adesivos sintéticos são usados em várias especialidades médicas cirúrgicas, contudo, os estudos que relatam seu uso na prática ortopédica são escassos. O objetivo deste trabalho foi comparar os resultados do uso do etil-2-cianoacrilato e do butil-2-cianoacrilato no tratamento de fraturas em ratos. MÉTODOS: Foi realizado um estudo experimental, prospectivo e controlado em 90 ratos com fraturas de úmero, fêmur e tíbia, tratados com etil-2-cianoacrilato (grupo SB; n = 45) ou butil-2-cianoacrilato (grupo HA; n = 45). Foram realizadas análises biomecânicas e histomorfométricas em três momentos (60, 120 e 180 dias), além do estudo clínico pela aferição semanal da massa corporal dos animais. RESULTADOS: Não foram observadas diferenças relacionadas à massa corporal dos animais (p = 0,07). Os fêmures de ambos os grupos não apresentaram diferença com relação à carga máxima (p = 0,6), limite de elasticidade (p = 0,6) e coeficiente de rigidez (p = 0,4). Analisando-se as tíbias, o mesmo foi observado com relação à carga máxima (p = 0,4), ao limite de elasticidade (p = 0,7) e ao coeficiente de rigidez (p = 0,6). Os úmeros de ambos os grupos apresentaram a mesma área de calo ósseo formado (p = 0,66). Em ambos os grupos, não houve diferença estatística relacionada ao número de osteoblastos (p = 0,2), osteoclastos (p = 0,2) e células inflamatórias (p = 0,4). CONCLUSÃO: O etil-2-cianoacrilato foi mais eficaz do que o butil-2-cianoacrilato no tratamento de fraturas em ratos.

The effects of ethyl-2-cyanoacrylate and butyl-2-cyanoacrylate in the process of bone healing in rats. A controlled experimental study / Efeitos do etil-2-cianoacrilato e do butil-2-cianoacrilato no processo de consolidação óssea em ratos. Estudo experimental controlado

ABSTRACT Objective: Synthetic adhesives are used by various medical specialties, especially in surgery; however, studies reporting their use in orthopedic practice are scarce. The aim of this study was to compare the results in using ethyl-2-cyanoacrylate or butyl-2-cyanoacrylate in the treatment of fractures in rats. Methods: This was an experimental prospective controlled study in 90 rats, with humerus, femur, and tibia fractures, treated with ethyl-2-cyanoacrylate (SB group; n = 45) or butyl-2-cyanoacrylate (HA group; n = 45). Biomechanical and histomorphometric analyses were performed at three different moments (60, 120, and 180 days); besides a clinical study performed weekly by measurement of the animals body mass. Results: No differences were observed regarding body mass (p = 0.07). In both groups, there were no significant differences regarding maximum load (p = 0.6), yield point strength (p = 0.6), and stiffness coefficient (p = 0.4) of the femurs. The same was observed in tibias for maximum load (p = 0.4), yield point strength (p = 0.7), and stiffness coefficient (p = 0.6). The humerus from both groups had similar bone callus area (p = 0.66). In both groups, there were no statistical differences related to inflammatory cells (p = 0.4), osteoblasts (p = 0.2), and osteoclasts (p = 0.2). Conclusion: Ethyl-2-cyanoacrylate was more effective than butyl-2-cyanoacrylate in the treatment of fractures in rats.

RESUMO Objetivo: Os adesivos sintéticos são usados em várias especialidades médicas cirúrgicas, contudo, os estudos que relatam seu uso na prática ortopédica são escassos. O objetivo deste trabalho foi comparar os resultados do uso do etil-2-cianoacrilato e do butil-2-cianoacrilato no tratamento de fraturas em ratos. Métodos: Foi realizado um estudo experimental, prospectivo e controlado em 90 ratos com fraturas de úmero, fêmur e tíbia, tratados com etil-2-cianoacrilato (grupo SB; n = 45) ou butil-2-cianoacrilato (grupo HA; n = 45). Foram realizadas análises biomecânicas e histomorfométricas em três momentos (60, 120 e 180 dias), além do estudo clínico pela aferição semanal da massa corporal dos animais. Resultados: Não foram observadas diferenças relacionadas à massa corporal dos animais (p = 0,07). Os fêmures de ambos os grupos não apresentaram diferença com relação à carga máxima (p = 0,6), limite de elasticidade (p = 0,6) e coeficiente de rigidez (p = 0,4). Analisando-se as tíbias, o mesmo foi observado com relação à carga máxima (p = 0,4), ao limite de elasticidade (p = 0,7) e ao coeficiente de rigidez (p = 0,6). Os úmeros de ambos os grupos apresentaram a mesma área de calo ósseo formado (p = 0,66). Em ambos os grupos, não houve diferença estatística relacionada ao número de osteoblastos (p = 0,2), osteoclastos (p = 0,2) e células inflamatórias (p = 0,4). Conclusão: O etil-2-cianoacrilato foi mais eficaz do que o butil-2-cianoacrilato no tratamento de fraturas em ratos.