Nitric oxide regulates alkaline phosphatase activity in rat fracture callus explant cultures

Nitric oxide (NO) is synthesised by a group of enzymes called nitric oxide synthases (NOS) and oxidizes to its stable end-products nitrite (NO2-) and nitrate (NO3-) We have previously reported in an in vivo rat model that NO is an important regulator for rat bone fracture healing. This study examines the effects of NO on alkaline phosphatase (ALP) activity in a rat fracture callus explant culture system. Explants of rat femoral fracture callus from days 4, 7, 14 and 28 post fracture induced NO2 release and ALP activity in a biphasic temporal manner, with the highest activity on day 7 and the lowest activity on day 14. Inhibition of NOS by co-incubation with an NOS inhibitor, S-(2-aminoethyl) isothiouronium bromide hydrobromide (AETU), inhibited ALP activity by an average of 50% at each time point (P <0.01). Supplementation with NO donor 3-morpholinosydnonomine hydrochloride (SIN-1) at low doses (25 and 0.025 microM) increased ALP activity by 20% (P < 0.01). ALP mRNA and histochemical ALP activity were localised to osteoblast-like and chondrocyte-like cells within fracture callus. The current study provides evidence that NO plays a regulatory role in ALP activity during rat fracture healing.     

骨科损伤控制救治对四肢骨折患者血清骨代谢和炎症反应的影响

为探究骨科损伤控制(damage control orthopaedics, DCO)救治对四肢骨折患者血清骨代谢及炎症反应的影响,本研究选取40例四肢骨折患者,随机分为治疗组和对照组,各20例。治疗组采用骨科损伤控制救治四肢骨折患者,对照组则采用常规骨折治疗方法。酶联免疫法检测试剂盒检测骨代谢指标:骨钙素(bone gla protein, BGP)、Ⅰ型前胶原羧基端前肽(propeptide carboxy-terminal procollagen, PICP)水平、血清碱性磷酸酶(alkaline phosphatase, ALP),骨愈合相关指标:可溶性细胞间粘附分子-1 (soluble intercellular adhesion molecule 1, s ICAM-1)、胰岛素样生长因子-1 (insulin like growth factor-1, IGF-1),以及炎症反应相关因子白细胞介素-1 (interleukin-1, IL-1)、白细胞介素-6 (interleukin-6, IL-6)、肿瘤坏死因子-α(tumor necrosis factor-alpha, TNF-α);统计分析各组患者手术后疼痛程度以及肿胀程度评分。结果表明,骨科损伤控制治疗组的血清骨钙素(BGP)、Ⅰ型前胶原氨基酸末端前肽(PICP)水平、血清碱性磷酸酶(ALP)、可溶性细胞间粘附分子-1 (sICAM-1)和胰岛素样生长因子-1 (IGF-1)水平明显高于对照组;炎症反应因子IL-1、IL-6和TNF-α水平明显低于对照组;骨科损伤控制救治可明显降低四肢骨折患者术后的疼痛程度以及肿胀程度评分。综上所述,骨科损伤控制救治可明显提高四肢骨折患者血清骨代谢和骨愈合水平、降低炎症反应、疼痛以及肿胀程度。     

Nitric oxide regulates alkaline phosphatase activity in rat fracture callus explant cultures

Abstract

Nitric oxide (NO) is synthesised by a group of enzymes called nitric oxide synthases (NOS) and oxidizes to its stable end-products nitrite (NO₂^-) and nitrate (NO₃^-) We have previously reported in an in vivo rat model that NO is an important regulator for rat bone fracture healing.¹ This study examines the effects of NO on alkaline phosphatase (ALP) activity in a rat fracture callus explant culture system. Explants of rat femoral fracture callus from days 4, 7, 14 and 28 post fracture induced NO₂^- release and ALP activity in a biphasic temporal manner, with the highest activity on day 7 and the lowest activity on day 14. Inhibition of NOS by co-incubation with an NOS inhibitor,S-(2-aminoethyl) isothiouronium bromide hydrobromide (AETU), inhibited ALP activity by an average of 50% at each time point (P <0.01). Supplementation with NO donor 3-morpholino-sydnonomine hydrochloride (SIN-1) at low doses (25 and 0.025 µM) increased ALP activity by 20% (P <0.01). ALP mRNA and histochemical ALP activity were localised to osteoblast-like and chondrocyte-like cells within fracture callus. The current study provides evidence that NO plays a regulatory role in ALP activity during rat fracture healing.     

续断对兔骨折愈合过程中相关基因表达和血钙、磷含量的影响

本研究通过检测中药材续断对家兔骨折模型愈合过程中与成骨密切相关基因的表达,以及血清中钙、磷含量变化,探讨其对骨折愈合的促进机制。构建家兔骨折缺损模型,术后按组分别给予续断和蒸馏水灌胃,并分别检测骨保护素(OPG)、骨保护素配体(OPGL)、局部转化生长因子β1(TGF-β1)和骨形态发生蛋白-2(BMP-2)的基因表达以及血清中Ca、P、碱性磷酸酶(ALP)含量。结果表明,续断治疗组中血钙、血磷和ALP的含量在灌胃第2周,第3周和第4周后均有明显升高,且在第三周时达到最大值。同时,OPG、TGF-β1、BMP-2三个基因在用续断治疗的不同时期呈现不同程度的表达上调,OPGL则在治疗早期表达下调。推测续断对骨折的治疗可能是通过调控OPG、OPGL、TGF-β1、BMP-2等基因在骨愈合不同阶段的表达量和血清中Ca、P、ALP的含量来促进骨骼生长。     

Paget's disease of bone: early and late responses to three different modes of treatment with aminohydroxypropylidene bisphosphonate (APD).

Early and late responses to treatment with either oral (600 mg/day) or intravenous (20 mg/day) (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (aminohydroxypropylidene bisphosphonate; APD) were studied in 142 patients with Paget''s disease of bone who had not previously been treated with bisphosphonate. The efficacy of three therapeutic regimens was compared: (a) oral aminohydroxypropylidene bisphosphonate given continuously until six months after the serum alkaline phosphatase activity had returned to normal (long term); (b) oral aminohydroxypropylidene bisphosphonate given until urinary hydroxyproline excretion had returned to normal (short term); (c) intravenous aminohydroxypropylidene bisphosphonate for 10 days. With either oral or intravenous treatment the decrease in urinary hydroxyproline excretion was rapid and always preceded the fall in serum alkaline phosphatase activity. Normal urinary hydroxyproline excretion is essential for return of the serum alkaline phosphatase activity to normal. Complete biochemical remission, defined as return of the serum alkaline phosphatase activity to normal, was obtained in 129 patients (91%). The median duration of remission as assessed by actuarial analysis was 2.7 years. This study found no difference in the long term among the three modes of treatment, suggesting that for most patients with Paget''s disease a short course of intravenous aminohydroxypropylidene bisphosphonate will produce longlasting, complete remission without need for maintenance treatment.     

创伤性骨折血清ALP、OPG表达与Th1/Th2平衡的相关性分析

摘要 目的：探讨创伤性骨折血清碱性磷酸酶（ALP）、骨保护素（OPG）表达与辅助性T细胞1（Th1）/辅助性T细胞2（Th2）平衡的相关性。方法：选取我院2019年2月到2022年12月收治的80例创伤性骨折患者作为研究对象,将其分为观察组,另选取同期来我院体检的80名健康志愿者作为对照组。抽取观察组患者入院后第二天清晨空腹静脉血和对照组清晨空腹静脉血,检测ALP、OPG、Th1、Th2表达水平,并分析血清ALP、OPG表达与Th1/Th2平衡的相关性。术后12个月后对观察组80例患者进行随访,依照患者骨折愈合程度将其分为骨折愈合组（n=54）和延迟愈合组（n=26）,对比两组患者临床一般情况与ALP、OPG、Th1/Th2表达水平,并分析创伤性骨折患者骨折愈合情况影响的多因素。结果：观察组与对照组ALP、OPG表达与辅助性T细胞水平对比差异显著,观察组ALP、OPG、Th2水平明显高于对照组,Th1、Th1/Th2水平明显低于对照组（P＜0.05）;Spearman相关分析结果表明：血清ALP、OPG与Th1/Th2平衡呈负相关（P<0.05）;骨折愈合组和延迟愈合组患者性别、年龄、致伤原因、Th2对比无明显差异（P＞0.05）,两组患者BMI、合并感染ALP、OPG、Th1、Th1/Th2水平对比差异显著（P＜0.05）;对上述单因素分析具有统计学差异指标进行赋值,多因素分析结果表明：ALP、OPG、Th2、Th1/Th2为影响创伤性骨折患者骨折愈合情况的独立影响因素（P＜0.05）。结论：创伤性骨折的发生会导致ALP、OPG水平升高,Th1/Th2平衡改变,患者血清ALP、OPG水平与Th1/Th2平衡关系具有明显相关性。另外,ALP、OPG、Th1、Th1/Th2水平变化可影响创伤性骨折患者术后骨折愈合水平。因此,临床上对于ALP、OPG水平升高,且Th1/Th2失衡患者需警惕骨折愈合不良的发生。     

Ecto-5′-nucleotidase (CD73) regulates bone formation and remodeling during intramembranous bone repair in aging mice

Ecto-5′-nucleotidase (CD73) generates adenosine, an osteoblast activator and key regulator of skeletal growth. It is unknown, however, if CD73 regulates osteogenic differentiation during fracture healing in adulthood, and in particular how CD73 activity regulates intramembranous bone repair in the elderly. Monocortical tibial defects were created in 46–52-week-old wild type (WT) and CD73 knock-out mice (CD73^?/?) mice. Injury repair was analyzed at post-operative days 5, 7, 14 and 21 by micro-computed tomography (micro-CT), histomorphometry, proliferating cell nuclear antigen (PCNA) immunostaining, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) histochemistry. Middle-aged CD73 knock-out mice exhibited delayed bone regeneration and significantly reduced bone matrix deposition detected by histomorphometry and micro-CT. Cell proliferation, ALP activity and osteoclast number were reduced in the CD73^?/? mice, suggesting a combined defect in bone formation and resorption due the absence of CD73 activity in this model of intramembranous bone repair. Results from this study demonstrate that osteoblast activation through CD73 activity is essential during bone repair in aging mice, and it may present a drugable target for future biomimetic therapeutic approaches that aim at enhancing bone formation in the elderly patients.     

The effects of shockwave on bone healing and systemic concentrations of nitric oxide (NO), TGF-β1, VEGF and BMP-2 in long bone non-unions

This study investigated the effects of extracorporeal shockwave treatment (ESWT) on bone healing and the systemic concentrations of nitric oxide (NO), TGF-β1, VEGF and BMP-2 in long bone non-unions. Forty-two patients with 42 established non-unions of the femur and tibia were enrolled in this study. Each long bone non-union was treated with 6000 impulses of shockwave at 28 kV in a single session. Ten milliliters of peripheral blood were obtained for measurements of serum NO level and osteogenic growth factors including TGF-β1, VEGF and BMP-2; serum levels of calcium, alkaline phosphatase, calcitonin and parathyroid hormone before treatment and at 1 day, 1, 3 and 6 months after treatment. The evaluations for bone healing included clinical assessments and serial radiographic examinations. At 6 months, bony union was radiographically confirmed in 78.6%, and persistent non-union in 21.4%. Patients with bony union showed significantly higher serum NO level, TGF-β1, VEGF and BMP-2 at 1 month after treatment as compared to patients with persistent non-union. Shockwave-promoted bone healing was associated with significant increases in serum NO level and osteogenic growth factors. The elevations of systemic concentration of NO level and the osteogenic factors may reflect a local stimulation of shockwave in bone healing in long bone non-unions.     

Assessment of markers of bone formation under controlled environmental factors and their correlation with serum minerals in adult sheep as a model for orthopaedic research

Eighteen healthy skeletally mature (3 years old) ewes, with an average weight of 45 kg, of the Portuguese Churra da Terra Quente breed were used to evaluate the normal values of total and bone-specific isoform of alkaline phosphatase serum activities (ALP and BALP, respectively) and serum osteocalcin (OC) and their correlation with the serum minerals - calcium (Ca), phosphorus (P), magnesium (Mg) and ionized calcium (Ca(2+)). The sheep were maintained under controlled environmental conditions (constant diurnal photoperiod cycle and identical husbandry and feeding) for six weeks before the collection of the blood samples. The measurement of the total ALP and serum minerals was performed with automated biochemistry analysers using the BioMérieux kits, the serum electrolyte Ca(2+) Diametrics Medical, Inc specific cassettes and the BALP and OC METRATM kits from QUIDEL Corporation. The mean +/- standard deviation values obtained were: total ALP 90.17 +/- 85.72 U/L, BALP 15.0 +/- 5.44 U/L, ratio BALP/ total ALP 29.28 +/- 24.22, OC 13.02 +/- 1.87 ng/mL, Ca 2.57 +/- 0.37 mmol/L, P 2.13 +/- 0.42 mmol/L, Mg 1.04 +/- 0.13 mmol/L, Ca(2+) 1.29 +/- 0.04 mmol/L. Significant correlations were observed between the total ALP and Ca (r = 0.5939; P = 0.05) and OC and Ca (r = 0.5706; P = 0.05). Reference to the serum values of bone turnover parameters in sheep could be of great value in research and could provide complementary non-invasive information on the bone healing process, particularly with regard to obtaining an early prognosis of fracture healing.     

Facilitation of human osteoblast apoptosis by sulindac and indomethacin under hypoxic injury

Hypoxic–ischemia injury occurs after trauma causes consequential bone necrosis. Non‐steroid anti‐inflammatory drugs (NSAIDs) are frequently used in orthopedic clinics for pain relief. However, the underlying mechanism and outcome for usage of NSAIDs is poorly understood. To investigate the damage and loss of osteoblast function in hypoxia, two hypoxia mimetics, cobalt chloride (CoCl₂) and desferrioxamine (DFO), were used to create an in vitro hypoxic microenvironment. The cell damage was observed by decreases of cell viability and increases in cyclooxygenase‐2 and cleaved poly(ADP‐ribose) polymerase (PARP). Cell apoptosis was confirmed by WST‐1 cytotoxic assays and flow cytometry. The functional expression of osteoblast in alkaline phosphatase (ALP) activity was significantly decreased by CoCl₂ and inhibited when treated with DFO. To simulate the use of NSAID after hypoxic injury, four types of anti‐inflammatory drugs, sulindac sulfide (SUL), indomethacin (IND), aspirin (Asp), and sodium salicylate (NaS), were applied to osteoblasts after 1 h of hypoxia mimetic treatment. SUL and IND further enhanced cell death after hypoxia. ALP activity was totally abolished in hypoxic osteoblasts under IND treatment. Facilitation of osteoblast apoptosis occurred regardless of IND dosage under hypoxic conditions. To investigate osteoblast in vivo, local hypoxia was created by fracture of tibia and then treated the injured mice with IND by oral feeding. IND‐induced osteoblast apoptosis was confirmed by positive staining of TUNEL assay in fractured mice. Significant delay of fracture healing in bone tissue was also observed with the treatment of IND. These results provide information pertaining to choosing appropriate anti‐inflammatory drugs for orthopedic patients. J. Cell. Biochem. 113: 148–155, 2012. © 2011 Wiley Periodicals, Inc.     

Adult Hypophosphatasia Treated with Teriparatide: Report of 2 Patients and Review of the Literature

Objective: Hypophosphatasia (HPP) is a rare inherited metabolic bone disease from deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Reportedly, teriparatide (parathyroid hormone 1–34) can benefit the adult form of HPP, including fracture healing. We studied 2 women with adult HPP given teripa-ratide and reviewed the reports of 6 additional patients.Methods: A 68-year-old black woman (patient 1) described low-trauma fractures and had subnormal serum alkaline phosphatase (ALP) activity. Biochemical findings were consistent with HPP. Mutation analysis revealed a heterozygous defect in exon 10 of TNSALP (ALPL). Teriparatide was injected daily for 2 years. Four years later, she fractured her right hip. Treatment was resumed for 8 months without further fractures. A 53-year-old white woman (patient 2) reported low-trauma fractures and had subnormal serum ALP. Mutation analysis revealed a heterozygous defect in exon 8 of TNSALP. She injected teriparatide daily for 2 years. One year later, bone mineral density (BMD) declined and treatment was resumed for 3 months. When she sustained a sacral fracture, teriparatide was administered for a further 18 months.Results: Patient 1's serum ALP increased while receiving teriparatide and returned to baseline after its discontinuation. BMD remained unchanged, but no fractures were sustained. Patient 2's serum ALP increased, but the improvement was not sustained. Femoral neck BMD increased significantly during the first cycle, declined significantly afterwards, and was regained during course of teriparatide.Conclusion: Teriparatide shows some benefit for adult HPP.Abbreviations:ALP = alkaline phosphataseBMD = bone mineral densityBSAP = bone-specific alkaline phosphataseCTX = C-telopeptideDXA = dual-energy X-ray absorptiometryFN = femoral neckHPP = hypophosphatasiaLS = lumbar spinePEA = phosphoethanolaminePLP = pyridoxal 5′-phosphatePTH = parathyroid hormoneSQ = subcutaneousTNSALP = tissue-nonspecific isoenzyme of alkaline phosphataseTPTD = teriparatide     

Comparative study of alkaline phosphatase isoenzymes,bone histology,and skeletal radiography in dialysis bone disease.

Liver, intestinal, and bone alkaline phosphatase isoenzymes were measured using heat stability and L-phenylalanine inhibition techniques in 78 patients on intermittent haemodialysis. Fifty-five patients had abnormalities in one or more of the isoenzymes. Changes in bone and intestinal alkaline phosphatase activities seemed to be related and raised liver isoenzyme activity was associated with the development of liver disease. Abnormal histological and radiological findings were better correlated with bone alkaline phosphatase levels than with total alkaline phosphatase, and serial estimations of bone isoenzyme activity were useful in assessing the response of renal osteodystrophy to treatment with a vitamin D analogue. Serum alkaline phosphatase isoenzyme measurement provides another useful and non-invasive index for monitoring metabolic bone disease in patients with chronic renal failure.     

Intramedullary implants coated with cubic boron nitride enhance bone fracture healing in a rat model

BackgroundBoron nitride is a biocompatible and an osteo-inductive material for orthopedic applications. The aim of this study was to evaluate the effects of two different allotrope boron nitride coated implants, cubic boron nitride and hexagonal boron nitride, on fracture healing.MethodsIn this experimental study, a total of 24 rats were divided into three groups. Group A was the control group with Kirschner wire without coating, while the wires were coated dominantly by cubic boron nitride in Group B and hexagonal boron nitride in Group C. Then a mid-third femoral fracture was created. The fracture healing was examined in terms of new bone formation with micro-CT analysis and histopathological examination, quantitative measurement of bone turnover metabolites and scintigraphic examination of osteoblastic activity on 28th day post fracture.ResultsMicro-CT measurement results revealed a statistically significant increase in bone volume/tissue volume ratio and bone surface values in group B compared to group A. Cortex diameter and osteoblast counts were statistically higher in group B compared to group A. Inflammatory response was increased in group C compared to groups A and B. Biochemical test results showed significantly increased alkaline phosphatase levels and decreased osteocalcin levels in group B compared to group A. The increase in serum phosphorus and decrease in serum calcium levels was statistically significant in group C compared to Group A.ConclusionBoth types of boron nitride coating had superior fracture healing features compared to control group. Therefore, c-BN coating can accelerate the fracture healing and could lead to shorten of union time.     

Sustained local delivery of insulin for potential improvement of peri-implant bone formation in diabetes

Dental implantation is an effective standard treatment modality to restore missing teeth and maxillofacial defects. However, in diabetics there is an increased risk for implant failure due to impaired peri-implant osseous healing. Early topical insulin treatment was recently shown to normalize diabetic bone healing by rectifying impairments in osteoblastic activities. In this study, insulin/poly(lactic-co-glycolic acid) (PLGA) microspheres were prepared by a double-emulsion solvent evaporation method. Microspheres were then incorporated in fibrin gel to develop a local drug delivery system for diabetic patients requiring implant treatment. In vitro release of insulin from fibrin gel loaded with these microspheres was assessed, and sustained prolonged insulin release over 21 days ascertained. To assess the bioactivity of released insulin and determine whether slow release might improve impaired diabetic bone formation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, mineralized nodule formation, and ELISA (enzyme-linked immunosorbent assay) assays were performed. The insulin released from the drug delivery system stimulated cell growth in previously inhibited cells, and ameliorated the impaired bone-forming ability of human MG-63 cells under high glucose conditions. Fibrin gel loaded with insulin/PLGA microspheres shows potential for improving peri-implant bone formation in diabetic patients.     

Running inhibits osteoporosis induced by protein-deficient (PD) food intake

Running at 0.7 km/h for 10 min every day inhibited development of osteoporosis caused by protein deficient (PD) food intake. Urine alkaline phosphatase (ALP), a marker of bone formation osteoporosis, was not elevated in rats fed PD, when the osteoporosis was inhibited by running. Estrogen supplementation increased bone-breaking energy (BBE), but did not increase bone mineral density (BMD), and did not decrease urinary ALP levels.     

Characteristics of Bone Turnover in the Long Bone Metaphysis Fractured Patients with Normal or Low Bone Mineral Density (BMD)

The incidence of osteoporotic fractures increases as our population ages. Until now, the exact biochemical processes that occur during the healing of metaphyseal fractures remain unclear. Diagnostic instruments that allow a dynamic insight into the fracture healing process are as yet unavailable. In the present matched pair analysis, we study the time course of the osteoanabolic markers bone specific alkaline phosphatase (BAP) and transforming growth factor β1 (TGFβ1), as well as the osteocatabolic markers crosslinked C-telopeptide of type-I-collagen (β-CTX) and serum band 5 tartrate-resistant acid phosphatase (TRAP5b), during the healing of fractures that have a low level of bone mineral density (BMD) compared with fractures that have a normal BMD. Between March 2007 and February 2009, 30 patients aged older than 50 years who suffered a metaphyseal fracture were included in our study. BMDs were verified by dual energy Xray absorptiometry (DXEA) scans. The levels of BTMs were examined over an 8-week period. Osteoanabolic BAP levels in those with low levels of BMD were significantly different from the BAP levels in those with normal BMD. BAP levels in the former group increased constantly, whereas the latter group showed an initial strong decrease in BAP followed by slowly rising values. Osteocatabolic β-CTX increased in the bone of the normal BMD group constantly, whereas these levels decreased significantly in the bone of the group with low BMD from the first week. TRAP5b was significantly reduced in the low level BMD group. With this work, we conduct first insights into the molecular biology of the fracture healing process in patients with low levels of BMD that explains the mechanism of its fracture healing. The results may be one reason for the reduced healing qualities in bones with low BMD.     

Sequential expression of osteoblast phenotypic genes during medullary bone formation and resorption in estrogen-treated male Japanese quails

Medullary bone is formed reticularly in the bone marrow cavity of the long bones of female birds. Although this bone matrix contains fewer collagen fibers and more acid mucopolysaccharides than cortical bone, it is not clear that the expression pattern of osteoblast phenotypic genes during bone remodeling. Therefore, 17β-estradiol (E2)-treated male Japanese quails were used to examine the temporal expression patterns of osteoblast phenotypic genes, and to simultaneously confirm the morphological changes occurring in the bone marrow cavity during medullary bone formation and resorption. After E2 treatment, bone lining cells proliferated and developed into mature osteoblasts that had intense alkaline phosphatase (ALP) activity. These cells began to form medullary bone that contained acid mucopolysaccharides and tartrate-resistantacid phosphatase. Runt-related gene 2 (Runx2) mRNA was stably expressed throughout the process. The expression of both ALP and type I collagen mRNAs increased initially, and then rapidly decreased after day 7, while osteoclasts began to resorb medullary bone at day 5. The expression of bone matrix-related genes peaked at day 5, and suddenly decreased at day 7, except for osteopontin. Taken together with these results, the expression patterns of bone matrix-related genes during the later stages might be related to osteoclast activity. Additionally, the constant expression of Runx2 during bone formation and resorption suggested that osteoprogenitor cells always exist in the bone marrow cavity. Therefore, the expression patterns of these genes and the characteristics of bone matrix might extremely be related to the quick remodeling of medullary bone.     

Hyperbaric Hyperoxia Accelerates Fracture Healing in Mice

Increased oxygen tension influences bone metabolism. This study comprised two main experiments: one aimed to determine the bone mineral apposition and bone formation rates in vivo under hyperbaric hyperoxia (HBO), and the other aimed to evaluate the effects of exposure to HBO on fracture healing. In experiment 1, male mice were exposed to HBO [90 min/day at 90% O₂ at 2 atmospheres absolute (ATA) for 5 days]. In experiment 2, an open femur fracture model was created in mice, followed by exposure to HBO 5 times/week (90 min/day at 90% O₂ at 2 ATA) for 6 weeks after surgery. In experiment 1, HBO treatment significantly increased the mineral apposition and bone formation rates in the lumbar vertebra and femur and type 1 collagen alpha 1 and alkaline phosphatase mRNA expression in the lumbar vertebra. In experiment 2, at 2 weeks after fracture, the fracture callus was significantly larger in the HBO group than in the non-HBO group. Furthermore, at 4 and 6 weeks after fracture, radiographic findings showed accelerated fracture healing in the HBO group. At 6 weeks after fracture, femur stiffness and maximum load were significantly higher in the HBO group than in the non-HBO group. Urinary 8-hydroxy-2′-deoxyguanosine and plasma calcium concentrations were not significantly different between groups. These results suggest that exposure to HBO enhances bone anabolism and accelerates fracture healing without causing oxidative DNA damage or disruption of plasma calcium homeostasis.     

Obesity regulates miR-467/HoxA10 axis on osteogenic differentiation and fracture healing by BMSC-derived exosome LncRNA H19

This study explored the therapeutic effect of bone marrow mesenchymal stem cell-derived exosomes on the treatment of obesity-induced fracture healing. Quantitative real-time PCR was used to detect the expression of lncRNA H19, miR-467 and Hoxa10 and combined with WB detection to detect osteogenic markers (RUNX2, OPN, OCN). Determine whether exosomes have entered BMSCs by immunofluorescence staining. Alkaline phosphatase (ALP) and alizarin red staining (ARS) staining were used to detect ALP activity and calcium deposition. We found that high-fat treatment can inhibit the secretion of BMSCs-derived exosomes and affect the expression of H19 carried by them. In vivo and in vitro experiments show that high-fat or obesity factors can inhibit the expression of osteogenic markers and reduce the staining activity of ALP and ARS. The treatment of exosomes from normal sources can reverse the phenomenon of osteogenic differentiation and abnormal fracture healing. Further bioinformatics analysis found that miR-467 as a regulatory molecule of lncRNA H19 and Hoxa10, and we verified the targeting relationship of the three through dual luciferase report experiments. Further, we found similar phenomena in ALP and ARS staining. Bone marrow mesenchymal stem cell-derived exosomes improve fracture healing caused by obesity.