Introduction

Leptin is a peptide hormone which is secreted especially by white adipose tissue and encoded by the obese gene.It is produced primarily by adipocytes and acts on the hypothalamus.Moreover,salient nonadipocytic,extra-hypothalamic pathways also exist[1].Recently,the role of leptin in bone metabolism has been identified[2-3].Therole of leptin on the skeletal system is complex.Present information on leptin indicates that it is involved in at least 2 different bone-controlling mechanisms,a direct stimulatory effect on bone growth,and/or an indirect suppressive effect on bone formation through the hypothalamus via the sympathetic nervous system[4].By acting on the hypothalamus and increasing both sympathetic output,and β2-adrenergic receptors on the surface of osteoblasts,leptin exerts an antiosteogenic effect on the central nervous system[5].Recent findings suggested that blocking the central effects of leptin would increase bone mass by stimulating bone formation[6,7]and hypothalamic leptin gene therapy actually increased bone length and total bone mass in growing obese(ob/ob)mice.Overall,the aforementioned findings are congruous with the hypothesis that the central nervous system-mediated actions of leptin are anti-osteogenic.

However,peripherally speaking,some hypothesis demonstrated that leptin has an opposite effect by promoting bone mineralization[8]and osteoblast to osteocyte differentiation in vitro[9-10].Despite this,questions still remain about the concept that peripheral leptin is responsible for the putative bone anabolic effects of the hormone in vivo.Consequently,in this study,we aimed to explore possible effects of leptin on bone growth and fracture healing.

术前所有患者均行裸眼视力、矫正视力及裂隙灯显微镜检查，非接触眼压（TX-F，日本Canon公司）及矫正眼压检查（ORA，美国Richard公司），电脑验光（kr-8800，日本Topcon公司）及散瞳后综合验光（RT-2100，日本Niderk公司），眼前节分析仪（Allegro Oculyzer，德国Oculus公司）测量角膜厚度及角膜形态，三面镜检查眼底，A超（AVISO，法国光太公司）测量眼轴长度。

Materials and methods

Adult male SD rats(n=72,250-290 g,purchased from Shanghai Slaccas Laboratory Animal Co,Shanghai,China)were maintained at 23±1°C on a 12-h light/12-h dark cycle.They were all housed individually in specific pathogen-free conditions with unlimited access to water and laboratory food and were treated strictly in accordance with institutional ethical guidelines.These rats were randomly and equally divided into three groups.Group A was treated with 1 mL normal saline(NS)intraperitoneally and used as the operative control group.Group B was treated with 0.3 μg/kg leptin(Leptin Rat,Recombinant,Linco Corp.,USA)in 1 mL NS intraperitoneally.Group C was treated with 0.5 μg/kg leptin in 1 mL NS intraperitoneally.Treatments were started on the day of surgery and lasted for 2 weeks.The average body weights of the rats at the 2,4 and 8 weeks were 262±12.2 g,255±16.5 g,and 265±13.7 g,respectively,and analysis of variance(ANOVA)did not detect a significant difference in average weights.All three groups were divided into three subgroups(n=8 rats)for evaluation at 2,4 and 8 weeks after the operation.These rats were sacrificed by sodium pentobarbital(100 mg/kg,i.p.)overdose,perfused transcardically with NS and 10%neutral formalin.Approval for this study was granted by the Ethics Board of the First Affiliated Hospital with Nanjing Medical University,China.

Femoral fracture model

Femoral osteotomy and fixation were performed in the same manner as previously reported[11].Brie fly,a transverse osteotomy was made at the midshaft of the femur and intramedullary fixation was performed using a stainless steel wire(diameter,1.5 mm).The fracture fragments were reduced and stabilized.Wires were cut on the surface of the intercondylar groove to avoid restriction of knee joint motion.Unrestricted activity was allowed after recovery from anesthesia.

Tissue preparation and histologic analysis

实施最严格的耕地保护制度。全省耕地保有量1.14亿亩，划定永久基本农田9587万亩，连续18年实现耕地占补平衡，保障粮食生产能力稳步提升。建立生态红线管理和生态补偿制度，对重要生态区域实施红线管理，对生态公益林、重要湿地和海域海岛保护给予生态补偿。建立自然保护地管理制度。在资源集中、区位重要、特色鲜明区域，建立各类省级以上自然保护区45处、地质矿山公园67处、森林公园119处、湿地公园204处、海洋公园12处，典型自然生态系统和生物多样性得到有效保护。持续推进矿产资源整顿规范，取缔关闭非法采矿和一批露天开采矿山，建成国家级绿色矿山37处，数量居全国第二位。

Radiological evaluation

The bone formation part of the Lane-Sandhu histopathological scoring system was used for radiological evaluation of fracture healing[13].The standard lateral　and　AP　radiographs　that　were　taken after euthanasia were evaluated by a double blinded observer.

Table 1　Radiological and histological results of the groups*

*Data are expressed as median(range).In radiological results:at 8 weeks,the formation of callus in group B and C were significantly better than group A(P=0.04,P=0.013,respectively);In histological results:there was a statistically significant difference between group B,C,and group A at 4weeks(P=0.01,P=0.002,respectively)and 8 weeks(P=0.008,P=0.003,respectively).

Microcomputerized tomography(micro-CT)analysis

随着现代人生活节奏加快，生活压力不断加大，心血管疾病人群不断扩大，逐渐向低龄化方向发展，不稳定性心绞痛发病率呈逐年上升的趋势。本文将在我院治疗不稳定型心绞痛的患者作为研究对象，实践组采用复方丹参滴丸与曲美他嗪联合应用治疗。结果实践组疗效、每搏量（SV）及射血分数（EF）明显优于对照组，且差异明显有效。

The explanted femoral bones were scanned by micro-CT using SkyScan 1172 scanner with a voxel size of 20 mm.The data was collected at 100 kVand 100 mA and reconstructed using the cone-beam algorithm.Each femoral bone sample scanning was performed over a 180°rotation with an exposure time of 105 ms.A cylindrical volume of interest with a diameter of 20 mm and a height of 27 mm was selected,which displayed the microstructure of the rat femoral bones(comprising the cortical and cancellous bone).We scanned femoral samples every 1 mm and all the samples were done under the same experimental condition,utilizing the same radiographical machine,the same position and the same people for experimenting and another for measuring.Data analysis was performed using CT Analyzer software(BrukermicroCT,Kontich,Belgium).The region of interest was set at the area of fracture healing,de fined by the fracture area filled with new bone,and the structural indices of the femoral fracture areas were calculated using this software.In three-dimensional(3D)analysis,bone callus volume(BV)and fibrous callus volume were measured.

Statistical analysis

All statistical analyses were performed using SPSS 12.0(SPSS,Inc.,Chicago,IL,USA).The clinical data was expressed as mean±standard deviation.All variables were normally distributed(Shapiro-Wilk test).Differences between body weights of the three groups before operation were detected using one-way analysis of variance(ANOVA),and after the operation we used ANOVA with a post hoc test to analyze data of the three groups.P value less than 0.05 was considered statistically significant.

After the rats were sacrificed,the fractured femurs were dissected and carefully cleaned of muscle around the fracture callus to preserve callus integrity.Calluses from weeks 2,4 and 8 after fracture were then fixed in 4% paraformaldehyde,the intramedullary wire was removed,and the specimen was decalcified before paraffin embedding.Sagittal sections,which were chosen blindly and randomly from the middle of each sampled callus and the distance between each section,were 5µm through the fracture site and mounted on Fisher brand Superfrost Plus Slides(Fisher scientific,Pittsburgh,PA,USA).Sections were stained with hematoxylin-eosin for histological evaluation of healing at 2,4 and 8 weeks after fracture.The criteria described by Huo et al.[12]was used for histological evaluation of the specimens.

Results

At the 4 week stage,fracture callus from group A showed a well formed callus with areas of newly formed fibrous callus,calcified cartilage,chondrocytes and hypertrophic chondrocytes,group B and C demonstrated more mature trabecular bone formation and larger external callus compared to group A(P=0.01,P=0.002,respectively).

Radiological analysis

In all rats different Lane-Sandhu scores of fracture formation were calculated in radiographs(Table1,Fig.1).At 2 weeks after operation,evidence of callus formation was found at the fracture site in these groups,but there was no statistically significant difference.At 4 weeks after operation,callus formation in the three groups was more rapid and the average evaluation scores were higher than 2 weeks;however,we did not detect significant difference in these groups.

This study has demonstrated the influence of leptin on femoral fracture healing in rats.The results showed that leptin enhanced fracture healing,as characterized by a significant increase in the parameters regarding callus volume and fracture healing of the fractured femora compared with those in the control group.

Histological results

There was no statistically significant difference between the groups at 2 weeks(P=0.20).

云计算资源调度本质上来看其实是一个多目标优化(MOOP)问题[5].在云计算环境下，由于资源使用者和资源提供者的目标各不相同，前者较多地考虑工作流的执行时间和资源的租赁成本，而后者则更关注于资源的利用率.由于资源有限且要满足不同用户多样性需求，资源竞争现象频繁出现，因此，需要合理高效的资源调度算法对资源-任务进行分配，尽量避免多个任务争抢一个资源的现象的发生[6]．

There were no operation-related complications after anesthesia and intraperitoneal interventions.There were no wound infections and no complications related to surgery.No rats died in the experiment.

Fig.1　Radiological images at 8 weeks.The formation of callus(white arrow)in group B(B)and group C(C)continued to be significantly better than group A(A).

Fig.2　Histological cross-section of the fractured rat femurs obtained 8 weeks after surgery.In group A,predominantly cartilage with some woven bone was observed(A)(black arrows).In group B,predominantly woven bone occurred in the fracture line(B).In group C,predominantly woven bone and lamellar bone were observed(C)(black arrows).Magnification×100.

At the 8 week phase,group A displayed pronounced cartilage with some woven bone.In group B,predominantly woven bone occurred in the fracture line.In group C,predominantly woven and lamellar bone was observed(Fig.2).There was a statistically significant difference between group A and B,and between group A and C(P=0.008,P=0.003,respectively).There was no statistically significant difference between group B and C(P=0.235).

Micro-CT analysis

Computer analysis of the Micro-CT images revealed the volume of new bone and the quality of the femoral fracture area in all rats(Table 2,Fig.3).At 2 weeks,fracture lines were clear and some fibrous calluses were observed in all 3 groups.There was no statistically significant　difference　among　the　three　groups(P=0.35).At 4 weeks,in group A,the mineralized callus bridging was deemed insufficient.More mature mineralized callus bridging was detected in group B and C compared with group A(P=0.02,P=0.04,respectively).At 8 weeks,some mineralized callus and newly formed bone were seen in group A.A large amount of newly formed bone was seen in group B,greater quantities of bony callus and evidence of bone fusion were observed in group C.Quantitatively,the bone callus volume was higher in group B and C than in group A(P=0.005,P=0.001,respectively).Group C also had better fracture healing than group B at 8 weeks(P=0.01).

数学教育在中国有很长的历史，近现代的数学教学理论至少可以追溯到大学者王国维翻译日本学者藤泽利喜太郎的《算术条目及教授法》[6]．但中国的数学教育作为一个科学理论体系的提出及方法论的引入，则是从20世纪80年代开始．

Discussion

At 8 weeks,complete fracture healing was found in both group B and group C.The fracture line became nearly invisible compared to 2 and 4 weeks.The formation of callus in group B and C continued to be significantly better than group A(P=0.04,P=0.013,respectively).There was no significant difference between group B and group C(P=0.197).

Research shows that fracture healing is a varied and complex process[14].It involves an orderly and intricate succession of events,beginning with inflammation,followed by fibrous tissue and cartilage differentiation,and finally,endochondral and intramembranous ossification.Multiple factors are involved in this process,such as hormonal factors(growth hormone and transforming growth factor)and an extracellular matrix comprised of a wound healing process which summarizes skeletal growth and development[15].Therefore,bone union is attributed to two major factors,bone callus formation across the fracture site and mineralization to stabilize the fracture site.

Leptin,the circulating protein product of the obesity(Ob)gene,was initially discovered as a satiety factorregulating food intake and energy expenditure,and it is synthesized and secreted by adipocytes[16-17].As an important hormonal regulatory factor,leptin not only influences lipid metabolism but also has positive effects on peripheral bone metabolism by regulating bone mass and promoting bone mineralization[18-19].

Table 2　Volume of fracture callus in three groups

Unit:mm3 ▲P＜0.01 compared with group A at 4 weeks and 8 weeks ★P＜0.01 compared with group B at 8 weeks

Fig.3　Micro-CT images of the fractured rat femurs at 8 weeks after surgery.Some mineralized callus and newly formed bone are seen in group A(A).Large volume of newly formed bone is seen in group B(B),greater amounts of bony callus and evidence of bone fusion are observed in group C(C).Quantitatively,the bone callus volume is higher in group B,C than in group A.

Experimental researches showed that the elevation of peripheral serum leptin levels starts immediately after trauma,such as spinal cord injury or traumatic brain injury,by activation of endogenous leptin secretion.These higher serum leptin levels are associated with increased callus formation in the fracture site[20-21].Furthermore,elevated serum leptin may act peripherally to induce myeloid precursor cell differentiation and osteoblast proliferation,and accelerate the mineralization of bone at the fracture site[22-23].Similarly,in our study,because daily application of exogenous leptin might provide consistently higher concentrations of serum leptin levels,better fracture healing was observed in the groups given exogenous leptin(fracture healing of group B,and C were better than group A).We also found that callus volume was higher in the group where more exogenous leptin was administered(group C also had larger bone formation than group B at the 8 week stage)In a study by Wang and colleagues[24],it was reported that elevated leptin expression might also enhance alkaline phosphatase activity,secretion of osteocalcin,and expression of type I collagen mRNA.Turner et al.suggested that leptin acts on growth plate cartilage cells,osteoblasts and osteoclasts to enhance their number and/or activity.Leptin deficiency results in an overall decrease in bone turnover.Therefore,as found with other major regulators of bone metabolism[25],regional changes in bone mass and structure depend upon peripheral serum leptin levels.

Our study,in which leptin was administered intraperitoneally,distinguishes between direct peripheral and indirect central actions of leptin on bone formation because peripheral and central leptin have opposing actions on fracture healing.In all groups,the experimental data obtained continuously without interruption,indicated the suitability of the applied method.The experiments described above suggests that a dose dependent positive effect of leptin on fracture healing when we evaluated radiological,histological and especially micro-CT results.

观察两组患儿并发症的情况，其中包括：肝内胆管扩张、术后胆管炎、门脉高压症，总发生率=发生例数/总例数×100%。

Several weaknesses exist with respect to paucity in technical explanations of our observations.In addition,a limited number of time points prevented a more detailed view of the stages of healing and the optimal time for administration of leptin.Furthermore,in vivo leptin distribution and local concentration present within the bone were not explored.Future work should investigate the molecular mechanism of action of leptin upon bone healing.In addition,use of impaired fracture healing models,including rats with increased age,diabetes mellitus,or undergoing steroid treatment,could lead to a more complete understanding of the effect of leptin.

References

[1]　Thomas T.The complex effects of leptin on bone metabolism through multiple pathways[J].Curr Opin Pharmacol,2004,4(3):295-300.

[2]　Rodrigues L,Mouta R,Costa AR,et al.Effects of high fat diet on salivary a-amylase,serum parameters and food consumption in rats[J].Arch Oral Biol.2015,27;60(6):854-862.

[3]　Bertoni L,Ferretti M,Cavani F,et al.Leptin increases growth of primary ossification centers in fetal mice[J].J Anat,2009,215(5):577-583.

[4]　Upadhyay J,Farr OM,Mantzoros CS.The role of leptin in regulating bone metabolism[J].Metabolism,2015,64(1):105-113.

[5]　Takeda S,Elefteriou F,Levasseur R,et al.Leptin regulates bone formation via the sympathetic nervous system[J].Cell,2002,111(3):305-317.

[6]　Bertoni L,Ferretti M,Cavani F,et al.Leptin increases growth of primary ossification centers in fetal mice[J].J Anat,2009,215(5):577-583.

[7]　Kume K,Satomura K,Nishisho S,et al.Potential role of leptin in endochondral ossification[J].J Histochem Cytochem,2002,50(2):159-169.

[8]Gaspar AP,Brandão CM,Lazaretti-Castro M.Bone mass and hormone analysis in patients with spinal cord injury:evidence for agonadal axis disruption[J].JClin Endocrinol Metab,2014,99(12):4649-4655.

[9]　Bonnet N,Gadois C,McCloskey E,et al.Protective effect of beta blockers in post menopausal women:influence on fractures,bone density,micro and macroarchitecture[J].Bone,2007,40(5):1209-1216.

[10]Li J,Mori S,Kaji Y,et al.Concentration of bisphosphonate(incadronate)in callus area and its effects on fracture healing in rats[J].J Bone Miner Res,2000,15(10):2042-2051.

[11]Li J,Mori S,Kaji Y,et al.Concentration of bisphosphonate(incadronate)in callus area and its effects on fracture healing in rats[J].J Bone Miner Res,2000,15(10):2042-2051.

[12]Huo MH,Troiano NW,Pelker RR,et al.The influence of ibuprofen on fracture repair:biomechanical,biochemical,histologic,and histomorphometric parameters in rats[J].J Orthop Res,1991,9(3):383-390.

[13]Lane JM,Sandhu HS.Current approaches to experimental bone grafting[J].Orthop Clin North Am,1987,18(2):213-225.

[14]Schindeler A,McDonald MM,Bokko P,et al.Bone remodeling during fracture repair:The cellular picture[J].Semin Cell Dev Biol,2008,19(5):459-466.

[15]Park AG,Paglia DN,Al-Zube L,et al.Local insulin therapy affects fracture healing in a rat model[J].J Orthop Res,2013,31(5):776-782.

[16]Mantzoros CS,Moschos S,Avramopoulos I,et al.Leptin concentrations in relation to body mass index and the tumor necrosis factor-alpha system in humans[J].J Clin Endocrinol Metab,1997,82(10):3408-3413.

[17]Mantzoros CS,Qu D,Frederich RC,et al.Activation of beta(3)adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice[J].Diabetes,1996,45(7):909-914.

[18]Cadosch D,Gautschi OP,Thyer M,et al.Humoral factors enhance fracture-healing and callus formation in patients with traumatic brain injury[J].J Bone Joint Surg Am,2009,91(2):282-288.

[19]Zhang J,Li T,Xu L,et al.Leptin promotes ossification through multiple ways of bone metabolism in osteoblast:a pilot study[J].Gynecol Endocrinol,2013,29(8):758-762.

[20]Hamrick MW,Ferrari SL.Leptin and the sympathetic connection of fat to bone[J].Osteoporos Int,2008,19(7):905-912.

[21]Bigford GE,Bracchi-Ricard VC,Nash MS,et al.Alterations in mouse hypothalamic adipokine gene expression and leptin signaling following chronic spinal cord injury and with advanced age[J].PLoS One,2012,7(7):e41073.

[22]Brock JH,Rosenzweig ES,Blesch A,et al.Local and remote growth factor effects after primate spinal cord injury[J].J Neurosci,2010,30(29):9728-9737.

[23]Aloe L,Bianchi P,De Bellis A,et al.Intranasal nerve growth factor bypasses the blood-brain barrier and affects spinal cord neurons in spinal cord injury[J].Neural RegenRes,2014,9(10):1025-1030.

[24]Wang L,Tang X,Zhang H,et al.Elevated leptin expression in rat model of traumatic spinal cord injury and femoral fracture[J].J Spinal Cord Med,2011,34(5):501-509.

[25]Turner RT,Kalra SP,Wong CP,et al.Peripheral leptin regulates bone formation[J].J Bone Miner Res,2013,28(1):22-34.