Osteocyte morphology and orientation in relation to strain in the jaw bone

INTRODUCTION

Bone quality at the patient’s implant site is an important local factor for the success of dental implants1,2.It influences the dental implant stability at the time of surgery3,4and osseointegration at the second stage5,6.Implant success rates are different at various implant sites in the jaw,with the highest failure rates occurring in the maxillary posterior region7.Bone quality is determined by mechanical properties as well as the bone mineral density,bone architecture,and extracellular matrix composition1,8.

Bone structure is continuously remodelled by bone-resorbing osteoclasts and bone-forming osteoblasts,which are regulated by osteocytes9.Osteocytes act as mechanosensors of bone producing signalling molecules that affect osteoblastic and/or osteoclastic activities.A prominent theory is that mechanosensing by osteocytes occurs via strain-induced fluid flow through the lacuno-canalicular network10.

Osteocyte morphology varies in different typesof bone.Elongated osteocytes are found in load-bearing long bones that are predominantly loaded parallel to their longitudinal direction.On the other hand,round osteocytes are found in flat bones,such as calvariae,loaded with much lower amplitudes,radially and/or tangentially,due to intracranial pressure and/or mastication11.Osteocyte morphology and orientation thus seem to be affected by the mechanical loading direction.Osteocyte lacunae are aligned to the collagen fibre orientation12,13,which may correspond to the orientation of the tensile strain in the bone14.External mechanical forces on cells are known to affect the cytoskeletal structure and thus the cell morphology9,15.Moreover,round osteocytes are much more mechanosensitive than elongated cells16.Round osteocytes in calvarial bone experience much lower mechanical loads than long bones,which might indicate that their morphologies maintain their physiological functions even in the presence of low mechanical loads and hence are more mechanosensitive than elongated osteocytes in long bones that are exposed to higher mechanical loads11.Therefore,the osteocyte morphology at the implant location may predict the success of dental implants.

Bone quality is assessed by bone density in bone biopsies using histomorphometry and densitometry,but the cellular parameters for bone quality have not been determined17.To date,not much is known about the osteocyte morphology and orientation in the human jaw bone.

In this study,it is hypothesised that 1)tensile strains in the maxillary bone are larger and more uniformly directed in a single gap compared to free-ending locations and 2)osteocytes are larger,more elongated,and more uniformly oriented in a single gap versus free-ending locations.Therefore,this study aimed to investigate the relationship between the levels and orientation of tensile strain and the morphology and orientation of osteocytes in single gap versus free-ending dental implant positions in the maxillarybonethrough afinite element(FE)modeland histomorphometric analyses.

RESULTS

The maxillary bone biopsy details and histomorphometrical analysis results of the residual native bone(RNB)data in the eight patients are shown in Table 1.The evaluation results of two patients,a single gap location(Patient#1)and free-ending locations(Patient#5),are shown in detail in Figs 1 and 2.

Patients

The period between the tooth extraction and biopsy retrieval at the single gap location was eight to nine months(Table 1:Patient#1 and#2).While this period at the multiple gap locations varied from nine to＞36 months(Table 1:Patient#3 and#4),this period spanned three to＞36 months in the free ending locations(Table 1:Patient#5–#8).

Preoperatively,the clinical bone quality was classified as type II in all of the single gap locations and as type III in all of the freeending locations(Table 1:Bone class)18.Clinical bone quality did not seem to be directly correlated with the period between the tooth extraction and biopsy retrieval.No clinical signs of inflammation were observed during biopsy retrieval.

Radiological evaluation

Panoramic radiographs were made preoperatively(not shown)and six months after maxillary sinus floor elevation(MSFE)prior to biopsy retrieval.A single gap location in patient#1(Fig.1a)and free-ending locations in patient#5(Fig.1c)are shown.The mean height gain of the maxillary sinus floor at the biopsy positions was similar for all patients(mean±standard deviations:7.5mm±1.7mm).

为了尽可能降低在房屋建筑过程中发生冷桥现象，目前国内很多建筑商在进行房屋构建层面，一般会采取保温层建设的方式进行该现象的规避。常用的材料为聚苯板，这种材料不仅能够更好的满足房屋保温需求，而且在使用过程中起成本相对较低，使用范畴非常广泛。

A limitation of our study was that we only used twodimensional sections to analyse the orientation of the threedimensional osteocytes.However,this did not affect our conclusion regarding any possible differences in the morphology between the different implant locations since histomorphometry is based on the principle that statistical information of threedimensional structures can be obtained from two-dimensional cross-sections if a sufficient number of cross sections is measured.Information regarding surfaces can be obtained from crosssections of these surfaces,i.e.,lines.Another limitation of this study is the small number of patients.

The FE model predicted that the tensile strains were the largest close to the natural tooth and decreased in magnitude with increasing distance from the tooth.This strain profile was caused by the bending of the sinus floor between the mesial and distal teeth.To better understand the strain profile,it should be kept in mind that the respective figures show a 2D section from a 3D model(Fig.3).In the 3D model,the maxillary sinus floor was also supported by the buccal and lingual sides of the sinus cavity.

The tensile strain magnitude and direction were different in the single gap and free-ending implant locations.In the single gap locations,the tensile strain was large and uniformly directed in the cranial-caudal direction(Fig.1b).In the free-ending locations,the tensile strain magnitude decreased＞2-fold by one tooth distance from the natural tooth(Fig.1d).The tensile strain was less uniformly oriented than in the single gap locations.

Histology

Allofthe intactbiopsies and a few broken butwell evaluable biopsies were included in the study.Newly formed mineralised bone tissue, containing lacunae with live osteocytes,unmineralized osteoid areas,and connective tissue were observed around the β-tricalcium phosphate(β-TCP)particles cranial to the native residual bone in the biopsies.Approximately 10%of the observed lacunae were empty in all of the regions.Bone ingrowth was determined from the border between the RNB and the grafted maxillary sinus floor(GMSF)towards the cranial side.The newly formed bone was in close contact with the bone substitute granules(Fig.2a,e,l,m).Some biopsies showed no newly formed mineralised bone tissue,but only unmineralized osteoid areas and connective tissue in the most cranially located region of interests(ROIs)(Fig.2i).

Histomorphometrical evaluation of the mineralised residual native bone tissue

发展较为成熟粉丝社群会成为其他粉丝的学习对象，促进个体实现个人价值，最终使整个粉丝群体的素质和形象都有所提升。

No differences were observed in the mean percentage of the mineralised native residual bone tissue volume between the single gap and free-ending implant locations(mean±standard deviations(%);single gap:33.4±14.1;free-ending:32.8±20.4,27.3±13.9,37.4±19.7).

The osteocyte morphology and orientation were analysed in each ROI(Fig.4).In a random bone area in each ROI,a digital image was acquired at×400 magnification(Fig.4b1).Only sharp and clearly displayed lacunae with live osteocytes were analysed.The osteocyte number,surface area,morphology,and orientation were determined “blindly”by two researchers.

Osteocyte orientation

The osteocyte orientation in the single gap locationsof two patients was similar(Table 1:Patient#1 and#2).As a reference value,the average osteocyte orientation of patient#1 and#2 was taken for the other multiple gap and free-ending locations.The osteocyte orientations of a single gap location patient#1 and a free-ending location patient#5 are shown in detail(Fig.2).

The osteocyte orientation was similar in the single gap(26;patient#1)RNB(median:27.4°)and GMSF(median:27.4°)(Fig.2b).In the RNB,54.8%of osteocytes had a cranial-caudal orientation(Fig.2c),and in the GMSF,53.1%of osteocytes had a cranialcaudal orientation(Fig.2d).

The osteocyte orientation was similar in the free-ending(25;patient#5)RNB(median:32.9°)and GMSF(median:30.3°)(Fig.2f).A total of 47.8%of the osteocytes in the RNB had a cranial-caudal orientation(Fig.2g),and 45.5%of the osteocytes in the GMSF had a cranial-caudal orientation(Fig.2h).Moreover,the osteocyte orientation was different in the free-ending location(26)between the RNB(median 58.0°)and GMSF(median:25.8°)(Fig.2j)(P ＜ 0.05).A total of 22.5%of the osteocytes in the RNB had a cranial-caudal orientation(Fig.2k),and 52.9%of the osteocytes in the GMSF had a cranial-caudal orientation(Fig.2l).The osteocyte orientation in the free-ending location(27)was only measured in the RNB(median 43.2)since the GMSF was lacking(Fig.2n).A total of 33.3%of the osteocytes had a cranial-caudal orientation (Fig.2o).The osteocyte orientation in the RNB in the free-ending location(26)was different from the single gap reference value(Table 1:Patient#5)(P＜0.05).

Moreover,the osteocyte orientations in the free-ending(25;patient#8)and(26;patient#8)RNB were significantly different from the reference values of patient#1 and#2.

DISCUSSION

The aim of this study was to investigate the relationship between the tensile strain and osteocyte morphology and orientation in human maxillary bone.An FE analysis and histological and histomorphometrical data were used to predict the possible differences in the maxillary bone quality between the single gap versus free-ending locations.The FE model predicted larger and differently oriented tensile strains in the single gap compared to free-ending implant locations.Histomorphometrically,no differences were observed for the mineralised RNB volume and the number and morphology of the osteocytes between single gap and free-ending locations.The osteocytes in the single gap locations had a more cranial-caudal orientation and a larger surface area than in the free-ending locations.These results suggestpossible differences in the dental implant success related to the osteocyte mechanosensitivity in the single gap and free-ending implant positions in the maxilla.

此外，11月USDA将阿根廷大豆预计进口量由上月222万吨激增至420万吨，略低于2017/2018年度的进口量477万吨，但远高于2016/2017年度进口167万吨。除却阿根廷本国大豆消费量外，不排除未来阿根廷通过消费进口大豆的方式，增加本国对华大豆出口量的可能性。

Although FE modelling is used extensively to predict the biomechanicalstress directionsin dental implantsand its surrounding bone in relationship to implant success19,it has never been used in relationship to osteocyte morphology and orientation.The presence of the remaining teeth near the implant position keeps the bone mechanically strained.The tensile strains were directed from the natural tooth to the biopsy location(s),resulting in a difference in the tensile strain orientation between the single gap and free-ending locations.

In the FE model,individual teeth were removed to simulate patient-specific cases.Whereas the biopsies showed a heterogeneous patchwork of cortical bone,trabecular bone,β-TCP granules,and connective tissue,the sinus floor was modelled as a homogeneous tissue of intermediate stiffness.This was done for two reasons:(i)there were not sufficient 3D data present for each patient to model the actual heterogeneity of the whole sinus floor and(ii)the focus was on the broad stress trajectories resulting from the remaining dentition,which was best investigated by leaving the other factors equal.

Even though the time of extraction appeared to be comparable(8–13 months)for most of the retrieved biopsies,there was a clinical difference in the bone quality between the single gap versus free-ending locations,which was class II in most of the gap locations and class III in the free-ending locations.This suggests a higher amount of cortical bone and lower amount of cancellous bone in the single gap than in the free-ending locations.However,no differences were observed between the mineralised bone tissue volumes between the different locations,suggesting no changes in bone formation at the time of biopsy retrieval.Since osteocytes fulfil a role as mechanosensors of bone,it is plausible that bone formation is affected differently in the single gap than in the free-ending implant positions in the long term due to differences in the tensile strain magnitude and orientation.

果不其然，第二天王施凯“华丽丽”地感冒了。可惜“病假”这种借口用过太多次，老师根本不信，“狼孩子”王施凯只能“挺着病躯”去学校。

Nearly all osteocytes were elongated in both the single gap and free-ending locations,implying a dominant loading direction in these bone regions.Osteocytesin the gap locations showed significantly larger surface areas than those in the freeending locations, suggesting differences in osteocyte mechanosensitivity.Since the osteocyte cell body likely plays a role in direct mechanosensing of the matrix stiffness,this might relate to differences in bone architecture16,20.Moreover,it has been shown in vitro that different mechanical stimuli cause different cellular deformations21.This would suggest that differences in tensile strain result in changes in the osteocyte cytoskeleton and different morphologies9.Since differences in osteocyte morphology were not observed,the differences in the tensile strain might have been too small to cause substantial cytoskeletal changes.

Osteocytes in the single gap locations and free-ending locations directly neighbouring a natural tooth on one side had a cranialcaudal orientation,resulting from large and uniformly directed tensile strain.The osteocytes in the various free-ending locations of one patient had different orientations from each other and the single gap location,resulting from a decrease in the tensile strain magnitude from the natural tooth to the most distal free-ending location.These data are in line with previous observations showing elongated osteocytes aligned in the principal loading direction and osteocytes aligned to the collagen fibre orientation12,13,which corresponds to the orientation of the tensile strain in the bone14.

1990年春，见廉小花对儿子特别好，徐河开始向廉小花套近乎。廉小花觉得徐河肯干，心地也不坏，自己和小云天又天生有缘，于是廉小花不顾家人的反对，于1990年冬天，执意嫁给了徐河。

FE model

房间的角落里，电视机静静地嗡嗡响着。大多数电视频道都不再播放内容，但BBC一直播送着新闻，中间穿插无厘头的动画片。电视画面上的突发新闻字幕上宣布，英国首相和其他世界领导人已经上了外星人的母舰，与格拉斯克帝国协商。格拉斯克帝国先前突然出现，要“邀请”地球加入他们的帝国。

此次研究中所检出22例患者反流程度差异明显，可见不完全闭合状动脉瓣且呈现为线性反流并部分占据左室流出道，4例患者存在喷泉状严重反流现象并完全占据左室流出道。

In conclusion,these data show significant differences in the surface area and orientation of osteocytes,in particular,in the areas of the maxillary bone that are related to the tensile strain magnitude and orientation.The exact implication of the osteocyte orientation on the dental implant success,however,is complex and deserves further study.This exploratory study provides,for the first time,a view on the relationship between tensile strain and the osteocyte morphology and orientation in the maxillary bone,which might contribute to a better understanding of the cellular processes that lead to different bone qualities in various dental implant positions and,eventually,to the success of dental implants in the maxilla.

MATERIALS AND METHODS

Patient selection

Eight patients,six men and two women,who were partially edentulous in the posterior maxilla,were selected.All patients required a MSFE due to insufficient maxillary bone height,and the vertical bone height before MSFE was 4–10 mm.

All material behaviour was assumed to be linear-elastic and isotropic.The maxillary bone was modelled with a Young’s modulus of 10GPa and Poisson’s ratio of 0.3.The cortical and cancellous bone were not distinguished,since the demarcation between these two bone types varied greatly in MSFE patients.The teeth were given a Young’s modulus of 24GPa,which was based on the stiffness of dentin.The focus was on the strains at the implant positions rather than within the remaining teeth,and therefore,the enamel and pulp were not modelled as separate materials.

The study was performed in accordance with the principles of the Declaration of Helsinki.Since the study involved CE-marked calcium phosphates being used for their intended purpose(carrier material for bone augmentation in MSFE procedures),no specific regulatory approval from a medical ethical committee was required.Patients provided written informed consent before inclusion in the study.

Clinical bone quality classification

The bone quality was pre-operatively assessed and was classified based on the amount of cortical bone versus cancellous bone18.

Maxillary sinus floor elevation surgery

A preoperative panoramic radiograph was made from each patient to calculate the alveolar bone height at each planned implant position.The MSFE procedure was performed with Ceros®β-TCP granules with 60%porosity/0.7–1.4mm grain size(Thommen Medical AG,Grenchen,Switzerland)as previously described22.The oral mucoperiosteal flap was closed using Gore-Tex sutures(W.L.Gore and Associates,Newark,DE,USA),which were removed 10–14 dayspost-operatively.Allpatientsreceived antibiotic prophylaxis consisting of 500mg of amoxicillin 4-times daily,starting one day preoperatively and continuing for one week postoperatively.After a healing period of six months,prior to dental implant placement,a panoramic radiograph was made to measure the available tissue height for the dental implant placement.The dental implants were placed as previously described23.

建设工程项目一般都是在露天环境中施工，因此工程项目的质量总与自然环境、施工条件和各级管理机构的状况以及各种社会因素密切相关，但是有些项目管理者只将注意力集中在工程项目的实体本身，往往忽视环境因素对工程项目质量的影响。同时，由于环境因素复杂多变，管理者很难进行准确的估计和把握，进而造成环境因素对项目质量管理的深刻干预和影响。

Biopsy retrieval

Bone biopsies were collected using a trephine burr(outer and inner diameter 3.5mm and 2.5mm,respectively)and were fixed in 4%phosphate-buffered formaldehyde(Klinipath BV,Duiven,The Netherlands).Subsequently,the biopsies were transferred to 70%ethanoland were stored untiluse forhistomorphometry.One midsagittal section per biopsy was evaluated as described below.

Seventeen biopsies from gap or free-ending locations were evaluated.The following dental implant location definitions were used:1)single gap location:a natural tooth is present at both sides of the implant location;2)multiple gap location:a natural tooth is present on either side of at least two implant locations next to each other,and multiple bone biopsies can be retrieved in this gap;and 3)free-ending location:there is only one natural tooth present at one side(mesially)of the implant location(s),and multiple bone biopsies can be retrieved in this situation.

FE model

A three-dimensional model of the maxillary sinus floor(Fig.3)was designed with the FE software Abaqus/CAE(version 6.12,Dassault Systemes Simulia,Providence,RI,USA)to predict the tensile strain in the maxillary bone.Individual teeth were removed from this model to simulate patient-specific cases.While the dimensions of the model approximate the anatomical dimensions,the dental arch was straightened to simplify the visualisation and for comparison with the dental radiographs.

The mean age of patients was 58 years(range:40–73 years).All patients were non-smokers or smoked＜10 cigarettes per day.Patients with systemic diseases,drug abuse,and/or pregnancy were excluded from participation,as well as patients requiring horizontal bone augmentation.

输尿管结石当前的主要治疗方式就是体外冲击波碎石(ESWL)和输尿管镜下取石(URSL),临床中能够获得比较好的效果[1]。可是因为治疗失败和大结石嵌顿情况会导致患者转为开放手术治疗,手术对患者的创伤比较大,恢复速度缓慢,术后对患者的腹腔引流管通畅情况进行观察,记录引流量,如果存在异常情况,需要及时的通知医生来为患者进行处理。如果患者的引流液体比较多,则可能是漏尿的结果,临床中需要对患者的导尿管进行监控,让其保持通畅,定时挤压导尿管,防止漏尿。此次我院就后腹腔镜技术治疗输尿管结石的效果进行了分析,有以下报道。

Teeth were loaded with an occlusal(vertical)load of 100 N,which is comparable to the human bite force24.As a boundary condition,the bone section was fixed in the mesio-distal direction at its mesial surface and was fixed in all directions at its cranial surface.

Histology and histomorphometrical analysis

After dehydration in an ascending alcohol series,the bone specimens were embedded without prior decalcification in low temperaturepolymerising methylmethacrylate(MMA,Merck Schuchardt OHG, Hohenbrunn, Germany) as previously described25.Longitudinalsectionsof5μm thicknesswere prepared,and the midsagittal sections were stained with Goldner’s Trichome to distinguish mineralised bone tissue(green)and unmineralized osteoid(red)26.

从销售数据上看，茅台酱香系列酒大单品打造成效显著，数据最为抢眼的是茅台王子酒，完成销售额超32亿元，成为茅台集团仅次于茅台酒的第二大单品。茅台迎宾酒、赖茅酒、汉酱酒、贵州大曲以稳定的发展态势，即将形成10亿元级大单品群。

The histological sections were divided into ROI of 1mm2.Each ROI was analysed separately using a Leica DC 200 digital camera and Leica QWin©software(Leica Microsystems Image Solutions,Rijswijk,The Netherlands)as well as ImageJ(US National Institutes of Health,Bethesda,MD,1997–2014).A demarcation line indicated the transition from the RNB floor to the regenerated GMSF bone.

Mineralised bone tissue was calculated as the mean percentage of the mineralised volume in each ROI as previously described22,27.

“2005年实行垂直管理后，医院整体护理工作发生了根本性的变化。”主管医疗，自2004年开始主抓护理工作，盛京医院副院长张成普告诉记者，医院在国内较早实行了垂直管理，而且“非常彻底”，人、财、物与责、权、利全部划归护理部统一管理。

Osteocyte morphology and orientation

Osteocyte number and morphology in the residual native bone The single gap,multiple gap and free-ending implant locations showed similar numbers of osteocytes per ROI(mean±standard deviations:97±40)(Table 1).In all locations,most of the osteocytes(～90%)were elongated,while only ～10%of cells were round(Table 1).One free-ending location(24)had significantly more round cells than other locations(Table 1;Patient#7).In a single gap location(26),the osteocyte surface area was～1.5 times larger than in the multiple gap and free-ending locations(Table 1;Patient#1–6)(P＜0.05).

For the osteocyte surface area calculation,the ellipse formula“π×½ length×½ width”was used(Fig.4c1).Osteocyte morphologywasdefined with theformula “width/length”; “＜0.8”distinguishing elongated and “≥0.8” distinguishing round(Fig.4c1).The osteocyte orientation was described by an angle from 0°–90°measured from the caudal-cranial axis.The cranialcaudal axis side was labelled 0°,and the transversal axis was labelled 90°.The osteocyte orientation was measured by the angle between the length axis of the osteocyte(Fig.4c2:yellow line)and cranial-caudal axis(Fig.4c2:white line).

Statistical analysis

The data are presented as the mean±standard deviation.Statistical analyses were performed using SPSS version 20 software.The Mann–Whitney test and Pearson Chi-square test were performed to compare the results obtained from the different volumes of interest between the biopsies in a gap and free-ending implant position.Statistical significance was considered when P＜0.05.

通过能源的综合利用与智慧管控，可以有效提高能源利用效率，降低用户用能成本；利用不同类别能源之间的特性差异和相互转换，可以促进可再生能源的消纳；结合多能源的耦合互补，可以提高用户用能的可靠性，为电网运行提供更多柔性资源；同时，综合能源系统的协同规划亦可减少基础设施的重复建设。

ACKNOWLEDGEMENTS

The work of R.F.M.v.O.was supported by a grant from the University of Amsterdam for the stimulation of a research priority area in Oral Regenerative Medicine.R.G.B.received logistic support from the Research Office of the University of San Carlos,Cebu City.We thank Marion A.van Duin,Jan Harm Koolstra,and Noortje de Boer for their technical support and advice.

ADDITIONAL INFORMATION

Conflict of interest:The authors declare that they have no conflict of interest.

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