Bone Blood Vessels Research Articles

Aβ -induced excessive mitochondrial fission drives type H blood vessels injury to aggravate bone loss in APP/PS1 mice with Alzheimer's diseases.

Alzheimer's diseases (AD) patients suffer from more serious bone loss than cognitively normal subjects at the same age. Type H blood vessels were tightly associated with bone homeostasis. However, few studies have concentrated on bone vascular alteration and its role in AD-related bone loss. In this study, APP/PS1 mice (4- and 8-month-old) and age-matched wild-type mice were used to assess the bone vascular alteration and its role in AD-related bone loss. Transmission electron microscopy, immunofluorescence staining and iGPS 1.0 software database were utilized to investigate the molecular mechanism. Mitochondrial division inhibitor (Mdivi-1) and GSK-3β inhibitor (LiCl) were used to rescue type H blood vessels injury and verify the molecular mechanism. Our results revealed that APP/PS1 mice exhibited more serious bone blood vessels injury and bone loss during ageing. The bone blood vessel injury, especially in type H blood vessels, was accompanied by impaired vascularized osteogenesis in APP/PS1 mice. Further exploration indicated that beta-amyloid (Aβ) promoted the apoptosis of vascular endothelial cells (ECs) and resulted in type H blood vessels injury. Mechanistically, Aβ-induced excessive mitochondrial fission was found to be essential for the apoptosis of ECs. GSK-3β was identified as a key regulatory target of Aβ-induced excessive mitochondrial fission and bone loss. The findings delineated that Aβ-induced excessive mitochondrial fission drives type H blood vessels injury, leading to aggravate bone loss in APP/PS1 mice and GSK-3β inhibitor emerges as a potential therapeutic strategy.

Lymphatic vessels in bone support regeneration after injury

Blood and lymphatic vessels form a versatile transport network and provide inductive signals to regulate tissue-specific functions. Blood vessels in bone regulate osteogenesis and hematopoiesis, but current dogma suggests that bone lacks lymphatic vessels. Here, by combining high-resolution light-sheet imaging and cell-specific mouse genetics, we demonstrate presence of lymphatic vessels in mouse and human bones. We find that lymphatic vessels in bone expand during genotoxic stress. VEGF-C/VEGFR-3 signaling and genotoxic stress-induced IL6 drive lymphangiogenesis in bones. During lymphangiogenesis, secretion of CXCL12 from proliferating lymphatic endothelial cells is critical for hematopoietic and bone regeneration. Moreover, lymphangiocrine CXCL12 triggers expansion of mature Myh11+ CXCR4+ pericytes, which differentiate into bone cells and contribute to bone and hematopoietic regeneration. In aged animals, such expansion of lymphatic vessels and Myh11-positive cells in response to genotoxic stress is impaired. These data suggest lymphangiogenesis as a therapeutic avenue to stimulate hematopoietic and bone regeneration.

Type II collagen-positive progenitors are important stem cells in controlling skeletal development and vascular formation

Type II collagen-positive (Col2+) cells have been reported as skeletal stem cells (SSCs), but the contribution of Col2+ progenitors to skeletal development both prenatally and postnatally during aging remains unclear. To address this question, we generated new mouse models with ablation of Col2+ cells at either the embryonic or postnatal stages. The embryonic ablation of Col2+ progenitors resulted in the death of newborn mice due to a decrease in skeletal blood vessels, loss of all vertebral bones and absence of most other bones except part of the craniofacial bone, the clavicle bone and a small piece of the long bone and ribs, which suggested that intramembranous ossification is involved in long bone development but does not participate in spine development. The postnatal ablation of Col2+ cells resulted in mouse growth retardation and a collagenopathy phenotype. Lineage tracing experiments with embryonic or postnatal mice revealed that Col2+ progenitors occurred predominantly in the growth plate (GP) and articular cartilage, but a limited number of Col2+ cells were detected in the bone marrow. Moreover, the number and differentiation ability of Col2+ progenitors in the long bone and knee joints decreased with increasing age. The fate-mapping study further revealed Col2+ lineage cells contributed to, in addition to osteoblasts and chondrocytes, CD31+ blood vessels in both the calvarial bone and long bone. Specifically, almost all blood vessels in calvarial bone and 25.4% of blood vessels in long bone were Col2+ lineage cells. However, during fracture healing, 95.5% of CD31+ blood vessels in long bone were Col2+ lineage cells. In vitro studies further confirmed that Col2+ progenitors from calvarial bone and GP could form CD31+ vascular lumens. Thus, this study provides the first demonstration that intramembranous ossification is involved in long bone and rib development but not spine development. Col2+ progenitors contribute to CD31+ skeletal blood vessel formation, but the percentage differs between long bone and skull bone. The number and differentiation ability of Col2+ progenitors decreases with increasing age.

SIGNIFICANCE OF INCREASED D-DIMER VALUES IN FRESH FRACTURES IN ORTHOPEDICS AND CORRELATIONS WITH THROMBOEMBOLIC COMPLICATIONS

Background/aim. At the end of the process of separating clots composed of fibrin we have a D-dimer. This process involves thrombin, which is formed during the process where fibrinogen is converted into fibrin, a factor whose role is to bind the basic units of clots and plasmin, the final participant in the breakdown of fibrin. In medicine, we should not use value of D-dimers as the only parameter for thrombosis. The aims of are research is to investigate correlation between clinical signs of venous thrombosis and D-dimer values in fresh bone fractures and demonstrate, that there is momentous correlation between high values of D-dimers and clinical signs of venous thrombosis, which is often a contraindication for surgical treatment of fractures. Methods: D-dimer levels of 211 patients with fresh bone fractures. They are classified into groups based on D-dimer values as follows: <250 ng/ml, 250–1000 ng/ml, 1000-5000 ng/ml, 5000-10 000ng/ml. Results: D-dimer values are not statistically significant with clinical symptoms of venous thrombosis in recent fractures in orthopedics. Discussion: D-dimer is an indicator related to fibrin degradation that has been used in the past as a prevalence in patients with chances of venous thrombosis. In inflammatory processes, we also have increased values of this parameter, which suggests the existence of some inflammatory change in the body or infection. Оur research is among the first to compare D-dimer values in recent fractures in orthopedics and thromboembolic complications. In our research, we showed that there is no significant correlation between elevated D-dimer values in hospitalizations of fresh fractures with clinical signs and a diagnosis of vascular thrombosis. We showed that there is no significant correlation between elevated D-dimer values in hospitalizations of fresh fractures with clinical signs and a diagnosis of vascular thrombosis. With this study, we proved that elevated D dimer is a consequence of trauma and disruption of the continuity and integrity of bone blood vessels, both endosteal and periosteal, which results in thrombosis of small blood vessels. Conclusions: There is no momentous correlation between elevated D-dimer values in fresh fractures and venous thrombosis. High values of D-dimer are not a contraindication for surgical treatment of fresh fractures.

Smoking Impairs Hematoma Formation and Dysregulates Angiogenesis as the First Steps of Fracture Healing.

Bone fracture healing is an overly complex process in which inflammation, osteogenesis, and angiogenesis are tightly coupled, and delayed fracture repair is a very common health risk. One of the major causes of delayed healing is the formation of insufficient vasculature. Precise regulation of blood vessels in bone and their interplay with especially osteogenic processes has become an emerging topic within the last years; nevertheless, regulation of angiogenesis in (early) diseased fracture repair is still widely unknown. Here, we aim to develop an in vitro model for the analysis of early fracture healing which also enables the analysis of angiogenesis as a main influencing factor. As smoking is one of the main risk factors for bone fractures and developing a delay in healing, we model smoking and non-smoking conditions in vitro to analyze diverging reactions. Human in vitro fracture hematomas mimicking smokers’ and non-smokers’ hematomas were produced and analyzed regarding cell viability, inflammation, osteogenic and chondrogenic differentiation, and angiogenic potential. We could show that smokers’ blood hematomas were viable and comparable to non-smokers. Smokers’ hematomas showed an increase in inflammation and a decrease in osteogenic and chondrogenic differentiation potential. When analyzing angiogenesis, we could show that the smokers’ hematomas secrete factors that drastically reduced HUVEC proliferation and tube formation. With an angiogenesis array and gene expression analysis, we could identify the main influencing factors: Anpgt1/2, Tie2, and VEGFR2/3. In conclusion, our model is suitable to mimic smoking conditions in vitro showing that smoking negatively impacts early vascularization of newly formed tissue.

A single session of antimicrobial photodynamic therapy does not influence the alveolar repair process in rats.

The aim of this study was to use microscopic and molecular techniques to evaluate the effects of a single session of antimicrobial photodynamic therapy (aPDT) on the alveolar repair process after tooth extraction in rats. The study sample included 84 rats divided into four groups, as follows: a) Control - untreated socket; b) Laser - socket treated using photobiomodulation; c) TBO - socket treated with topic application of the photosensitizer agent, toluidine blue O (TBO); and d) aPDT - socket treated with TBO and laser irradiation. An additional rat was used for thermal mapping during socket irradiation. The animals were euthanatized at 6, 15, and 28 days after unilateral extraction of the upper incisor. Quantitative and qualitative analyses of the connective and bone tissues, blood clot, blood vessel, and inflammatory infiltrate were performed, and real-time polymerase chain reaction was used to study the expression of genes (collagen type I, osteocalcin, alkaline phosphatase [ALP], runt-related transcription factor 2 [RUNX2], and vascular endothelial growth factor [VEGF]) involved in the bone healing process. No statistically significant differences in microscopic and molecular outcomes were observed between the groups (p > 0.05). A positive correlation was seen to exist between blood clot and VEGF (p = 0.000), and a negative correlation was observed between bone tissue and ALP (p = 0.028) and blood vessel and VEGF (p = 0.018). A single session of aPDT in the dental extraction site did not influence the alveolar repair process in rats.

Diversity of Vascular Niches in Bones and Joints During Homeostasis, Ageing, and Diseases.

The bones and joints in the skeletal system are composed of diverse cell types, including vascular niches, bone cells, connective tissue cells and mineral deposits and regulate whole-body homeostasis. The capacity of maintaining strength and generation of blood lineages lies within the skeletal system. Bone harbours blood and immune cells and their progenitors, and vascular cells provide several immune cell type niches. Blood vessels in bone are phenotypically and functionally diverse, with distinct capillary subtypes exhibiting striking changes with age. The bone vasculature has a special impact on osteogenesis and haematopoiesis, and dysregulation of the vasculature is associated with diverse blood and bone diseases. Ageing is associated with perturbed haematopoiesis, loss of osteogenesis, increased adipogenesis and diminished immune response and immune cell production. Endothelial and perivascular cells impact immune cell production and play a crucial role during inflammation. Here, we discuss normal and maladapted vascular niches in bone during development, homeostasis, ageing and bone diseases such as rheumatoid arthritis and osteoarthritis. Further, we discuss the role of vascular niches during bone malignancy.

Reorganization of the osteocyte lacuno-canalicular network characteristics in tumor sites of an immunocompetent murine model of osteotropic cancers.

Mechanosensitive osteocytes are central regulators of bone resorption and formation. However, during the formation of bone metastases, which arise as consequences of breast and prostate cancer and skew homeostatic bone remodeling to favor osteolytic, osteosclerotic or mixed lesions, only a paucity of data exists on tumor-associated osteocyte interaction. Herein, we used a suite of high-resolution imaging and histological techniques to evaluate the effect of osteotropic cancer on cortical bone microarchitecture. Confocal imaging highlighted a direct contact between tumor cells residing in the bone marrow and osteocytes. High-resolution microcomputed tomography revealed a 10-12% larger osteocyte lacuna volume in the presence of tumor cells at day 21 after intratibial injection of EO771-Luc breast and RM1-Luc prostate cancer cells. The 3D representative of the spatial distribution of cortical bone microporosity showed i) a regional accumulation of vascular canals and large lacunae with low connectivity in osteosclerotic regions of interest and ii) an absence of vascular canals and large lacunae in osteolytic regions. These findings pinpoint the relationship between the presence of tumor cells in the bone marrow microenvironment and osteocyte lacunar characteristics and cortical bone blood vessel structure.

The changes of bone vessels and their role in bone loss in tail-suspended rats

Bone loss is one of the highest risk factors during long spaceflight for astronauts which can lead to health problems and increase fracture risk, but the mechanisms are still unclear. Bone is highly vascularized tissue, the vascular system in bone plays an important role in the development, regeneration and remodeling of bone. However, little is known about how the blood vessels in bones change and its effect on microgravity-induced osteoporosis, we investigated the above issues with tail-suspended rat model in this study. The result showed that the blood vessel volume and vessel number in long bones increased and H-type blood vessels (CD31hiEmcnhi vessels) reduced; moreover, exercise could alleviate the decrease of H-type blood vessels and osteogenesis in tail-suspended rats. These results revealed that changes of bone blood vessels may be a main reason for microgravity-induced bone mass rapid loss.

Magic-TT (Magnetism-induced cell target transplantation) Improves the Effect of Intra-Bone Marrow Transplantation Resulting in Rapid Hematopoietic Reconstitution in Allogenic Murine Transplantation Model

Background: Intra-bone marrow transplantation has been proven to be efficient in improving the homing of transplanted cells, resulting in faster hematopoietic reconstitution and a reduction in severity of GVHD. However, because of staggered blood vessels in bone, many transplanted cells leak out to peripheral circulation. In our previous study (Blood 2015 126:5404), it has been initially proven that MagIC-TT could enhance target transplantation to femurs by flow cytometry and pathological examination. After that, by bioluminescent imagine system, we further proved that most intra-bone marrow transplanted cells moved to lung immediately without MagIC-TT (Figure 1 A); while the cells were retained in the bone marrow with MagIC-TT (Figure 1 B), but if the magnetic field withdrawal 1 hour after, the cells also moved to the lung quickly(Figure 1 C). In this study, the therapeutic effect of intra-bone marrow transplantation combined with MagIC-TT was explored in allogenic murine transplantation model.MethodsMice and cell preparation: Sixty female BALB/c mice with specific pathogen free (SPF) were randomly separated in 4 groups (16 and 14 mice in 5×10e6M and 1×10e7 M group respectively; 16 and 14 mice in 5×10e6W and 1×10e7 W group respectively, M/W referring to MagIC-TT/non-MagIC-TT). The recipients received 7.5Gy irradiation before transplantation. Twelve SPF C57BL/6 CAG-eGFP transgenic mice were used as donors. The bone marrow were magnetized by nanoparticles and then adjusted to the concentration of 5×10e6/20μL and 1×10e7/20μl for use. Every mouse was injected with 20μL, 5×10e6 or 1×10e7 cells.Intra-bone marrow injection and fixed magnetic field After anesthetizing the mice and disinfecting the knee joint, the femoral puncture was operated with the medulla-puncture needle invented by our team (Chinese patent No.201620090904.2). In M groups, special magnetic fields were applied to the femurs of mice during and after injection. The effects of intra-bone marrow injection had been proven by X-ray and bioluminescent imaging system, etc .The therapeutic effects The therapeutic effects were determined by the survival of mice, the percentage of GFP+ cells in peripheral blood, blood routine test etc. to evaluate the hematopoietic reconstitution.ResultsSurvival: The survival rate was 66.7% and 33.3% in M groups and W groups on the 40th day after transplantation (P=0.096) (Figure 2 A), which was 62.5% and 71.4% in 5×10e6M and 1×10e7M, 37.5% and 28.6% in 5×10e6W and 1×10e7W, respectively(Figure 2 B).Donor cells percentage in peripheral blood (Figure 3 A): Flow cytometry analysis was performed on the 5th, 13rd, 17th, and 25thday after transplantation. The percentage of donor GFP+ cells grew much faster on d5~d13 in M groups than that in W groups (P<0.05). Pairwise comparisons showed that 10e7M group grew faster than 10e7W (P<0.05), donor GFP+ cells in 5×10e6M grew even faster than those in both 5×10e6W and 1×10e7W (P<0.05). No significant difference was found between 5×10e6M and 1×10e7M.Blood Routine Test: Generally, WBC in M groups reached growth peak on d9-13 vs d13-17 in W groups, and the WBC number of M groups are higher than those in W group (P<0.05) (Figure 3B). This trend also existed for HGB and PLT, but not as significant as WBC (Figure 3 C, 3 D) .ConclusionMagIC-TT can improve the effect of intra-bone marrow transplantation by avoiding transplanted cells leakage to peripheral circulation, resulting in faster hematopoietic reconstitution in allo-murine transplantation model. The mechanism of MagIC-TT and its relation with reduction of complication are under further study. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis

Synthetic glucocorticoids (GCs), one of the most effective treatments for chronic inflammatory and autoimmune conditions in children, have adverse effects on the growing skeleton. GCs inhibit angiogenesis in growing bone, but the underlying mechanisms remain unclear. Here, we show that GC treatment in young mice induces vascular endothelial cell senescence in metaphysis of long bone, and that inhibition of endothelial cell senescence improves GC-impaired bone angiogenesis with coupled osteogenesis. We identify angiogenin (ANG), a ribonuclease with pro-angiogenic activity, secreted by osteoclasts as a key factor for protecting the neighboring vascular cells against senescence. ANG maintains the proliferative activity of endothelial cells through plexin-B2 (PLXNB2)-mediated transcription of ribosomal RNA (rRNA). GC treatment inhibits ANG production by suppressing osteoclast formation in metaphysis, resulting in impaired endothelial cell rRNA transcription and subsequent cellular senescence. These findings reveal the role of metaphyseal blood vessel senescence in mediating the action of GCs on growing skeleton and establish the ANG/PLXNB2 axis as a molecular basis for the osteoclast-vascular interplay in skeletal angiogenesis.

SPTBN1 Prevents Primary Osteoporosis by Modulating Osteoblasts Proliferation and Differentiation and Blood Vessels Formation in Bone.

Osteoporosis is a common systemic skeletal disorder that leads to increased bone fragility and increased risk of fracture. Although βII-Spectrin (SPTBN1) has been reported to be involved in the development of various human cancers, the function and underlying molecular mechanisms of SPTBN1 in primary osteoporosis remain unclear. In this study, we first established a primary osteoporosis mouse model of senile osteoporosis and postmenopausal osteoporosis. The results showed that the expression of SPTBN1 was significantly downregulated in primary osteoporosis mice model compared with the control group. Furthermore, silencing of SPTBN1 led to a decrease in bone density, a small number of trabecular bones, wider gap, decreased blood volume fraction and number of blood vessels, as well as downregulation of runt-related transcription factor 2 (Runx2), Osterix (Osx), Osteocalcin (Ocn), and vascular endothelial growth factor (VEGF) in primary osteoporosis mice model compared with the control group. Besides, the silencing of SPTBN1 inhibited the growth and induced apoptosis of mouse pre-osteoblast MC3T3-E1 cells compared with the negative control group. Moreover, the silencing of SPTBN1 significantly increased the expression of TGF-β, Cxcl9, and the phosphorylation level STAT1 and Smad3 in MC3T3-E1 cells compared with the control group. As expected, overexpression of SPTBN1 reversed the effect of SPTBN1 silencing in the progression of primary osteoporosis both in vitro and in vivo. Taken together, these results suggested that SPTBN1 suppressed primary osteoporosis by facilitating the proliferation, differentiation, and inhibition of apoptosis in osteoblasts via the TGF-β/Smad3 and STAT1/Cxcl9 pathways. Besides, overexpression of SPTBN1 promoted the formation of blood vessels in bone by regulating the expression of VEGF. This study, therefore, provided SPTBN1 as a novel therapeutic target for osteoporosis.

Update on Acute Bone and Joint Infections in Paediatrics: A Narrative Review on the Most Recent Evidence-Based Recommendations and Appropriate Antinfective Therapy.

Acute bone and joint infections (BJIs) in children may clinically occur as osteomyelitis (OM) or septic arthritis (SA). In clinical practice, one-third of cases present a combination of both conditions. BJIs are usually caused by the haematogenous dissemination of septic emboli carried to the terminal blood vessels of bone and joints from distant infectious processes during transient bacteraemia. Early diagnosis is the cornerstone for the successful management of BJI, but it is still a challenge for paediatricians, particularly due to its nonspecific clinical presentation and to the poor specificity of the laboratory and imaging first-line tests that are available in emergency departments. Moreover, microbiological diagnosis is often difficult to achieve with common blood cultures, and further investigations require invasive procedures. The aim of this narrative review is to provide the most recent evidence-based recommendations on appropriate antinfective therapy in BJI in children. We conducted a review of recent literature by examining the MEDLINE (Medical Literature Analysis and Retrieval System Online) database using the search engines PubMed and Google Scholar. The keywords used were “osteomyelitis”, OR “bone infection”, OR “septic arthritis”, AND “p(a)ediatric” OR “children”. When BJI diagnosis is clinically suspected or radiologically confirmed, empiric antibiotic therapy should be started as soon as possible. The choice of empiric antimicrobial therapy is based on the most likely causative pathogens according to patient age, immunisation status, underlying disease, and other clinical and epidemiological considerations, including the local prevalence of virulent pathogens, antibiotic bioavailability and bone penetration. Empiric antibiotic treatment consists of a short intravenous cycle based on anti-staphylococcal penicillin or a cephalosporin in children aged over 3 months with the addition of gentamicin in infants aged under 3 months. An oral regimen may be an option depending on the bioavailability of antibiotic chosen and clinical and laboratory data. Strict clinical and laboratory follow-up should be scheduled for the following 3–5 weeks. Further studies on the optimal therapeutic approach are needed in order to understand the best first-line regimen, the utility of biomarkers for the definition of therapy duration and treatment of complications.

Bone Marrow Microvasculature.

The skeleton is highly vascularized due to the various roles blood vessels play in the homeostasis of bone and marrow. For example, blood vessels provide nutrients, remove metabolic by-products, deliver systemic hormones, and circulate precursor cells to bone and marrow. In addition to these roles, bone blood vessels participate in a variety of other functions. This article provides an overview of the afferent, exchange and efferent vessels in bone and marrow and presents the morphological layout of these blood vessels regarding blood flow dynamics. In addition, this article discusses how bone blood vessels participate in bone development, maintenance, and repair. Further, mechanical loading-induced bone adaptation is presented regarding interstitial fluid flow and pressure, as regulated by the vascular system. The role of the sympathetic nervous system is discussed in relation to blood vessels and bone. Finally, vascular participation in bone accrual with intermittent parathyroid hormone administration, a medication prescribed to combat age-related bone loss, is described and age- and disease-related impairments in blood vessels are discussed in relation to bone and marrow dysfunction. © 2020 American Physiological Society. Compr Physiol 10:1009-1046, 2020.

Experimental Acute Sepsis Reduced Number of Osteocalcin Immunolabeled Cells in Periodontal Ligament.

The aim of this study was to evaluate the effect of acute sepsis in the periodontal ligament, alveolar and furcation bone in absence of periodontitis induction through histological and immunohistochemical analyses. A septic rat model was established by cecal ligation and puncture (CLP). Twelve rats were randomly divided into CLP (n=6) and Sham (n=6) groups. The animals were euthanized at 24 h and hemimandibles were submitted to histomorfometric (bone matrix, collagenous fibers, fibroblasts, osteocytes, inflammatory cells, and blood vessels) and immunohistochemical (BMP-2/4, RANKL and osteocalcin) evaluation in alveolar bone, furcation bone and periodontal ligament. Our results demonstrated that histomorphometric parameters were similar in alveolar bone, furcation bone and periodontal ligament of Sham and CLP rats. Regarding to immunohistochemical analyses, the number of BMP-2/4 and RANKL immunolabeled cells was also similar in both groups. Furthermore, it was detected a reduction in the osteocalcin immunolabeled cells in periodontal ligaments of CLP compared to Sham rats (p=0.0014). In conclusion, the acute sepsis induction resulted in reduced number of osteocalcin labelled cells in periodontal ligament region. Moreover, no significant histological differences were observed in the periodontium of rats under acute sepsis. Considering the role of osteocalcin in bone remodeling, the study contributes to revealing the importance of careful periodontal evaluation in the presence of sepsis.

Влияние на регенерацию костной ткани локальных изменений ионного и ферментативного гомеостаза скаффолдами из поликапролакона минерализованными ватеритом

Цель исследования - оценка влияния на регенерацию костной ткани локальных изменений ферментативного и ионного гомеостаза минерализованными ватеритом скаффолдами из поликапролакона. Методика. Эксперимент выполнен на 47 нелинейных крысах-самцах. Животные были разделены на 4 экспериментальные группы: контроль (интактные животные), группа сравнения (ложнооперированные крысы), группа отрицательного контроля (крысы с имплантацией матрицы, на которой сорбирован нативный овальбумин) и экспериментальная группа - крысы, которым имплантировали поликапролактоновый скаффолд, минерализованный ватеритом (ПКЛ/СаСО3-скаффолд) с адсорбированной щелочной фосфатазой в дефект бедренной кости. На 7-е и 28-е сут в крови определяли содержание щелочной фосфатазы, а также концентрацию остеокальцина. Количественное определение остеокальцина производили методом иммуноферментного анализа с использованием набора реактивов фирмы «Immunodiagnostic systems» (Франция) и полуавтоматического ИФА-анализатора «Anthos Reader 2020» («Biochrom», Великобритания). Для выявления изменений ферментативной активности в сыворотке крови определяли кинетическим методом активность щелочной фосфатазы на биохимическом анализаторе «Сапфир-400» («Hirose Electronic System», Япония) с использованием набора реактивов фирмы «ДиаС» (Россия). Морфологическую верификацию процессов остеогенеза осуществляли при изучении гистологических препаратов на 28-е сут эксперимента. Поперечные срезы диафиза бедренной кости изучали при помощи микроскопа AxioImager Z2 («Carl Zeiss», Германия) и микровизора проходящего света серии μVizo-103 (ООО «ЛОМО ФОТОНИКА», Россия). Оценивали интеграцию матриц с краями пропила, изменения кости в области имплантации матриц, преобладающий тип клеточных элементов матриц, наличие в них костных балок и сосудов. Результаты. Установлено, что изменение локального ионного гомеостаза (СаСО3) и ферментативного гомеостаза щелочной фосфатазы, вызывает в матрице имплантированной в область дефекта формирование костных балок и биохимические изменения в крови, характеризующиеся повышением активности щелочной фосфатазы и увеличением концентрации остеоакальцина. Заключение. При имплантации ПКЛ/СаСО3-матриц с щелочной фосфатазой локальные изменения ферментативного и ионного гомеостаза, вызванные введением в состав кальцийсодержащих скаффолдов щелочной фосфатазы, в значительной степени стимулируют остеогенез. Биосовместимость и остеоиндуктивные свойства ПКЛ/СаСО3-матриц с щелочной фосфатазой позволяют говорить о перспективности их клинической апробации для стимуляции процессов регенерации костной ткани. Aim. To study the effect of local changes in enzymatic and ionic homeostasis induced by vaterite-mineralized polycaprolactone scaffolds with adsorbed alkaline phosphatase on bone tissue regeneration. The experiment was conducted on 47 mongrel male rats divided into four experimental groups: control (intact animals), comparison group (sham-operated rats), negative control (rats implanted with a matrix with adsorbed native ovabumin), and experimental group (rats implanted with PLC-CaCO3-scaffold with adsorbed alkaline phosphatase into the femoral bone defect). Alkaline phosphatase and osteocalcin contents were measured in blood of all animals on days 7 and 28. Osteocalcin was measured using the immune enzyme assay with an Immunodiagnostic Systems (France) kit and semi-automatic immune Anthos Reader 2020 (Biochrom, Great Britain) enzyme analyzer. Changes in serum alkaline phosphatase activity were detected using the kinetic method with a Sapphire-400 biochemical analyzer (Hirose Electronic Systems, Japan) and a DiaC (Russia) kit. Osteogenesis processes were verified morphologically on day 28 of the experiment by a histological assay. Transversal sections of the femoral diaphysis were examined with an AxioImager Z2 (Carl Zeiss, Germany) microscope and a µVizo-103 transmitted-light microvisor (OOO LOMO FOTONIKA, Russia). The evaluated indexes included matrix integration with saw-cut edges; bone changes in the area of matrix implantation; predominating type of matrix cell elements and the presence there of bone trabecula and blood vessels. Results. Changes in the local ionic homeostasis and homeostatic changes of alkaline phosphatase induced formation of bone trabecula in the matrix implanted into the defect area and biochemical changes in blood evident as upregulation of serum alkaline phosphatase associated with increased osteocalcin. Conclusion. In implantation of PLC-CaCO3 matrices with alkaline phosphatase, local changes in the enzymatic and ionic homeostasis induced by supplementation of calcium-containing scaffolds with alkaline phosphatase considerably stimulate osteogenesis. The biocompatibility and osteoinductive properties of PLC-CaCO3 matrices with alkaline phosphatase suggested that clinical testing of the use of the matrices for stimulation of bone tissue regeneration would be promising.

Enhanced in vivo biocompatibility of magnesia-contained bioactive glasses

Human blood compatibility of previously prepared magnesia-doped bioactive glass samples was evaluated by hemolysis assay. The in vivo evaluation was carried out using a rat model in which a hole was made in the rat femur bone by drilling and the bioactive glass samples were implanted in the cavity. Histological examination had demonstrated the better bone regeneration and blood vessel formation as compared to the reference one. The in vivo complete blood count (CBC) was also analyzed at different time periods. The in vivo results of magnesium-containing bioactive glasses had shown improved bioactivity as well as better biocompatibility.

Intermittent PTH 1-34 administration improves the marrow microenvironment and endothelium-dependent vasodilation in bone arteries of aged rats.

Inflammation coincides with diminished marrow function, vasodilation of blood vessels, and bone mass. Intermittent parathyroid hormone (PTH) administration independently improves marrow and vascular function, potentially impacting bone accrual. Currently, the influence of marrow and intermittent PTH administration on aged bone blood vessels has not been examined. Vasodilation of the femoral principal nutrient artery (PNA) was assessed in the presence and absence of marrow. Furthermore, we determined the influence of PTH 1-34 on 1) endothelium-dependent vasodilation and signaling pathways [i.e., nitric oxide (NO) and prostacyclin (PGI2)], 2) endothelium-independent vasodilation, 3) cytokine production by marrow cells, and 4) bone microarchitecture and bone static and dynamic properties. Young (4-6 mo) and old (22-24 mo) male Fischer-344 rats were treated with PTH 1-34 or a vehicle for 2 wk. In the absence and presence of marrow, femoral PNAs were given cumulative doses of acetylcholine, with and without the NO and PGI2 blockers, and diethylamine NONOate. Marrow-derived cytokines and bone parameters in the distal femur were assessed. Exposure to marrow diminished endothelium-dependent vasodilation in young rats. Reduced bone volume and NO-mediated vasodilation occurred with old age and were partially reversed with PTH. Additionally, PTH treatment in old rats restored endothelium-dependent vasodilation in the presence of marrow and augmented IL-10, an anti-inflammatory cytokine. Endothelium-independent vasodilation was unaltered, and PTH treatment reduced osteoid surfaces in old rats. In conclusion, the marrow microenvironment reduced vascular function in young rats, and PTH treatment improved the marrow microenvironment and vasodilation with age. NEW & NOTEWORTHY This study investigated the influence of the marrow microenvironment on bone vascular function in young and old rats. An inflamed marrow microenvironment may reduce vasodilator capacity of bone blood vessels, diminishing delivery of blood flow to the skeleton. In young rats, the presence of the marrow reduced vasodilation in the femoral principal nutrient artery (PNA). However, intermittent parathyroid hormone administration (i.e., a treatment for osteoporosis) improved the marrow microenvironment and vasodilator capacity in old PNAs.

Simultaneous visualisation of calcified bone microstructure and intracortical vasculature using synchrotron X-ray phase contrast-enhanced tomography

3D imaging of the bone vasculature is of key importance in the understanding of skeletal disease. As blood vessels in bone are deeply encased in the calcified matrix, imaging techniques that are applicable to soft tissues are generally difficult or impossible to apply to the skeleton. While canals in cortical bone can readily be identified and characterised in X-ray computed tomographic data in 3D, the soft tissue comprising blood vessels that are putatively contained within the canal structures does not provide sufficient image contrast necessary for image segmentation. Here, we report an approach that allows for rapid, simultaneous visualisation of calcified bone tissue and the vasculature within the calcified bone matrix. Using synchrotron X-ray phase contrast-enhanced tomography we show exemplar data with intracortical capillaries uncovered at sub-micrometre level without the need for any staining or contrast agent. Using the tibiofibular junction of 15 week-old C57BL/6 mice post mortem, we show the bone cortical porosity simultaneously along with the soft tissue comprising the vasculature. Validation with histology confirms that we can resolve individual capillaries. This imaging approach could be easily applied to other skeletal sites and transgenic models, and could improve our understanding of the role the vasculature plays in bone disease.

Mechanical, hormonal and metabolic influences on blood vessels, blood flow and bone.

Bone tissue is highly vascularized due to the various roles bone blood vessels play in bone and bone marrow function. For example, the vascular system is critical for bone development, maintenance and repair and provides O2, nutrients, waste elimination, systemic hormones and precursor cells for bone remodeling. Further, bone blood vessels serve as egress and ingress routes for blood and immune cells to and from the bone marrow. It is becoming increasingly clear that the vascular and skeletal systems are intimately linked in metabolic regulation and physiological and pathological processes. This review examines how agents such as mechanical loading, parathyroid hormone, estrogen, vitamin D and calcitonin, all considered anabolic for bone, have tremendous impacts on the bone vasculature. In fact, these agents influence bone blood vessels prior to influencing bone. Further, data reveal strong associations between vasodilator capacity of bone blood vessels and trabecular bone volume, and poor associations between estrogen status and uterine mass and trabecular bone volume. Additionally, this review highlights the importance of the bone microcirculation, particularly the vascular endothelium and NO-mediated signaling, in the regulation of bone blood flow, bone interstitial fluid flow and pressure and the paracrine signaling of bone cells. Finally, the vascular endothelium as a mediator of bone health and disease is considered.