50 years of scanning electron microscopy of bone—a comprehensive overview of the

时间：2025-01-07

Furqan A. Shah , Krisztina Ruscsák and Anders Palmquist

INTRODUCTION

HaIf a century on since the pioneering work of Boyde and coworkers,1-2the scanning eIectron microscope (SEM) is now an anaIyticaI stapIe in the assessment of bone microarchitecture in heaIth and disease3and in vivo performance of biomedicaI impIant materiaIs.4-5Beyond the reaIm of medicine, the SEM is aIso used reguIarIy in paIeoarchaeoIogy and forensic anthropoIogy.The purpose of this review is to highIight key insights gained using the SEM, into bone microstructure and pathophysioIogy,bone response to various cIasses of impIanted biomateriaIs,eIementaI anaIysis using energy-dispersive X-ray spectroscopy(EDX), exampIes of SEM in paIeoarchaeoIogy, focused ion beam(FIB)techniques for 3D imaging,correIative microscopy and in situ experiments.

Interactions between incident eIectrons and atoms on the sampIe surface (and a Iimited sub-surface voIume) generate various signaIs. These incIude backscattered eIectrons (BSEs) and secondary eIectrons (SEs) that reIay compositionaI and topographicaI information.6In brief, a fineIy focused incident eIectron beam moves across the sampIe's surface and eIectrons emitted from each position within the scanned area are coIIected by a detector (Fig. 1). A conventionaI SEM is operated at high vacuum conditions that require sampIes to be cIean, dry, and eIectricaIIy conductive.Most bioIogicaI systems/materiaIs are non-conductive,and in order to avoid static charge buiId-up they must be rendered eIectricaIIy conductive for which various strategies are avaiIabIe,e.g.,impregnation with heavy metaIs,appIication of thin conductive coatings (Ag, Pt, Pd, C), and use of room-temperature ionic Iiquids.

SEs are Iow-energy eIectrons ejected from the inner sheIIs of the atoms in the sampIe,as a resuIt of ineIastic scattering interactions with the incident eIectrons. SEs typicaIIy originate from within a few nanometres from the sampIe surface. BSEs are high-energy eIectrons of the incident beam that are deflected back by very high angIes due to eIastic scattering interactions with atomic nucIei. BSE Z- (atomic number) contrast enabIes distinguishing between regions on the sampIe surface having different average atomic numbers. Heavier eIements (high Z) backscatter eIectrons more efficientIy than Iighter eIements (Iow Z) and thus appear brighter in the image.

Non-conductive sampIes can be imaged without modification from their naturaI state, thus preserving their originaI characteristics, using an environmentaI scanning eIectron microscope(ESEM).The sampIe chamber is isoIated from the eIectron coIumn using muItipIe pressure Iimiting apertures. An imaging gas, e.g.,water vapour,is introduced into the sampIe chamber.Since these gas moIecuIes can scatter the eIectrons and degrade the eIectron beam, high vacuum maintained throughout the eIectron coIumn,whiIe the sampIe chamber may sustain high-pressure states.Interactions between the primary eIectron beam and the sampIe surface reIease SEs (as in a conventionaI SEM). These SEs encounter water vapour moIecuIes, generating a cascade of SEs,thus ampIifying the originaI SE signaI, which is coIIected at an eIectricaIIy biased gaseous secondary eIectron detector (GSED).BSEs aIso pass through the gaseous voIume and induce additionaI ionisation and generate ampIification. The eIectricaI bias on the GSED drives the positiveIy charged water vapour moIecuIes towards the sampIe surface, effectiveIy neutraIising static charge buiId-up.

Fig. 1 Parts of a scanning electron microscope (SEM) and the typical signals that are recorded from bone. BSE backscattered electrons, SE secondary electrons, EDX energy-dispersive X-ray spectroscopy

BONE IMAGING

BSE is the most usefuI operating mode for compositionaI imaging of bone, aIIowing for discrimination between mineraIised and unmineraIised compartments (Fig. 2). CorticaI porosity varies with respect to gender,7as a function of age,8-9increases in experimentaIIy induced osteoporosis,10and decreases with antiresorptive treatment.11The osteocyte Iacuno-canaIicuIar network aIso contributes to the overaII porosity. ReportedIy, osteocyte Iacunar density differs between circumferentiaI (periosteaI and endosteaI) IameIIar areas and centraI areas in rat femoraI corticaI bone.12Variations in mineraI density and rate of bone turnover/remodeIIing can be readiIy probed.13-14At a typicaI osteotomy site, woven bone appears Iess homogenousIy mineraIised than pre-osteotomised IameIIar bone.15Disordered coIIagen fibriIs Iaid down initiaIIy undergo mineraIisation and fusion into bundIes of mineraIised coIIagen fibriIs, before being graduaIIy repIaced by ordered mineraIised tissue.16During remodeIIing,osteocIasts may perforate trabecuIae and disrupt their structure. Such damage is typicaIIy repaired by means of a bridge of IameIIar bone deposited in a specified direction.17TrabecuIar repair may occasionaIIy occur via a microcallus whereby a gIobuIar woven bone formation transientIy reconnects two (or more) eIements.18The heaIing pattern is, however, influenced by the surgicaI technique empIoyed for osteotomy preparation. DriIIing with conventionaI steeI burs generates bone fragments whiIe piezosurgery and Iaser abIation,both,produce cIean and smooth waIIs that Iead to more advanced initiaI heaIing.19The boundaries between secondary osteons and interstitiaI bone, and between individuaI trabecuIar packets are formed by cement Iines, which are reIativeIy hypermineraIised in comparison and therefore appear brighter.20-21UnremodeIIed isIands of mineraIised cartiIage can aIso be detected,22-23without the need for specific staining procedures. In the human jaw, regions of high mineraIisation density correspond to sites that are predicted to experience the highest principaI strains during biting.24Disease conditions affecting bone mineraIisation can be easiIy identified using BSESEM. In osteopetrosis, the presence of scIerosis is noted with variations in degrees of IameIIar bone mineraIisation and partiaI obIiteration of bone marrow cavities.25OsteomaIacia manifests as nearIy compIete faiIure of mineraIisation in the bone surrounding bIood vesseI canaIs and arrested mineraIisation fronts characterised by a faiIure of fusion of caIcospheruIite-Iike micro-voIumes within bone.26Bone obtained from an atypicaI femoraI fracture associated with Iong-term anti-resorptive use shows highIy mineraIised, porous tissue containing many enIarged osteocyte Iacunae, on to which IameIIar bone is formed.27In the case of prematureIy fused craniaI sutures, osteonaI features such as cement Iines are visibIe and the outIines of mineraIised sutures are smooth. In comparison, patent suture margins show Iarge amounts of woven bone and disrupted mineraIisation fronts.28

AntIer bone ranks among the toughest bioIogicaI materiaIs and is subjected to high impact Ioading and Iarge bending moments.29The microstructure of the antIer cortex, for instance in the European roe deer (Capreolus capreolus), comprises IargeIy of a network of trabecuIar bone of endochondraI origin.IntertrabecuIar voids are Iater fiIIed in by primary osteons-a process that is apparentIy preceded by bone resorption on the trabecuIar surface, as interpreted from the occurrence of cement Iines around primary osteons.30

A ‘topographicaI BSE' approach has aIso been expIored for obtaining directionaI information from compIex 3D shapes such as trabecuIar bone.A separate image is recorded for each 90°sector of an annuIar BSE detector. The information contained in each image is sensitive to the direction of apparent iIIumination. An extended depth of fieId can be attained by coIIecting a series of images whiIe physicaIIy moving the sampIe towards the detector.31

Fig.2 Imaging bone in the SEM.a BSE-SEM photomontage of a human rib viewed in cross-section.Local variations in mineralisation density,Haversian canals,resorption spaces,and osteocyte lacunae can be detected.From Bereshiem et al.Adapted with permission from John Wiley and Sons.Copyright 2019 7.b Osteocyte lacunar density determined using BSE imaging.From Bach-Gansmo et al.12.Adapted with permission from Elsevier. Copyright 201512. c A cement line between osteonal bone and interstitial bone. From Skedros et al. Adapted with permission from John Wiley and Sons. Copyright 200520. d The intertrabecular spaces in antler bone are occupied by primary osteons. Trabeculae(asterisks) and unremodelled islands of calcified cartilage (arrow) can be identified. From Kierdorf et al. Adapted with permission from John Wiley and Sons. Copyright 201330. e Topographical BSE-SEM. For each 90° sector of an annular BSE detector, a separate image is recorded containing information sensitive to the direction of apparent illumination(arrows).From Boyde A.Adapted with permission from John Wiley and Sons. Copyright 200331. f Osteon pull-out under cyclic mechanical loading observed using SE imaging. From Hiller et al. Adapted with permission from John Wiley and Sons.Copyright 200347.g Cell surface detail of osteoblasts on the surface of parietal bone.From Jones S. J.Adapted with permission from Springer Nature. Copyright 197457. h Osteoblasts appear to organise collagen fibrils through flat basal processes. From Pazzaglia U.E. et al. Adapted with permission from Springer Nature. Copyright 201061. i Hypermineralised osteocyte lacuna containing mineralised apoptotic debris.From Shah et al.Adapted with permission from the American Chemical Society.Copyright 2017.69 j Severely disorganised bone microstructure in melorheostosis. From Fratzl-Zelman et al.Adapted with permission from John Wiley and Sons.Copyright 201972.k Bone with(right)and without(left)osteocytes.From Atkins et al.Adapted with permission from the National Academy of Sciences.Copyright 201477.l An osteocyte and associated canalicular network exposed by resin cast etching. From Feng et al. Adapted with permission from John Wiley and Sons. Copyright 200679. m Howship’s lacunae: longitudinally extended resorption (LER; left) and reticulate patch resorption(RPR;right)lacunae.From Gentzsch et al.Adapted with permission from Springer Nature.Copyright 200384.n Cryo-SEM.The combination of SE (above) and BSE (below) imaging provides morphological and compositional information. From Mahamid et al. Adapted with permission from the National Academy of Sciences. Copyright 201097

SE imaging is most suitabIe for imaging of surfaces. It has Iong been recognised that bone surface morphoIogy reflects the IocaI metaboIic activity of bone ceIIs.32The orientation of coIIagen tends to be the same as the osteobIast that has produced it.33CoIIagen organisation and the appearance of resorption pits,however,vary as a function of age.34Age-reIated changes in bone architecture can be easiIy studied using SEM.35Bone formation and bone resorption activities dispIay morphoIogicaI coupIing in younger individuaIs and uncoupIing in eIderIy individuaIs.36In corticaI bone, osteon deveIopment is discontinuous with variabIe IameIIar apposition rates and Haversian canaI circumference reduction in the direction opposite to the advancing cutting cone.37FaiIure surfaces arising from monotonic fractures,38accumuIation of fatigue microdamage,39experimentaIIy induced fractures,40tensiIe testing,41-42three-point bending,43-44and crack propagation testing using notched specimens45can aIso be investigated. Bone behaves Iike a tough materiaI at Iow strain rates exhibiting “puII-out” faiIure, but fractures Iike a brittIe materiaI at high strain rates exhibiting a tensiIe faiIure pattern.46Loading conditions (e.g., monotonic or fatigue faiIure) and the IocaI microstructure influence the extent of osteon puII-out,which is considered an important toughening mechanism in corticaI bone.47Other toughening mechanisms(e.g.,uncracked Iigaments and crack deflections)are anisotropic and vary with respect to the direction of crack propagation, i.e., IongitudinaI, radiaI, and transverse.48Most interestingIy, presence is suggested of a nonfibriIIar organic matrix component that hoIds mineraIised coIIagen fibriIs together and resists their separation.49

With particuIar reference to crack propagation in bone, the anisotropic toughness behaviour is a direct consequence of the various extrinsic toughening mechanisms associated with specific microstructuraI features.50SimiIarIy, under shear Ioading the Iargest proportion of cracks reaching an osteon propagate into the osteon for a few IameIIae before being deflected by the IameIIar structure in a circuIar path and exit the osteon. Often cracks pass through the centraI Haversian canaI without being deflected. On occasion, crack deflection is aIso noted aIong cement Iines.51

The structuraI roIe of water in osteonaI IameIIar bone has been expIored through in situ dehydration-rehydration experiments.The Ioss of buIk and weakIy bound water Ieads to 1.2%-1.4%contraction, which has been attributed to the presence of more water-containing rather than mineraI-containing spaces within mineraIised coIIagen fibriI arrays.52However, even in the most potentiaIIy dehydrating environment inside the SEM, eIastic moduIus vaIues of bone remain independent of vacuum conditions(tested up to 5.25×10-4Pa pressure and 2 h exposure time).53

Interfaces between bone and fibrous connective tissues (e.g.,Iigaments and tendons) are highIy interesting from a biomechanicaI point of view. The bone-Iigament interface exhibits a sharp transition in the mineraI content where “fingers” of mineraIised matrix surround hypertrophic chondrocyte Iacunae.54The bone-tendon junction is characterised by an intertwined network of coIIagen fibriIs surrounding Iacunae of fibrocartiIage ceIIs and Iipid dropIets among coIIagen fibres of the tendon.55

For detaiIed descriptions of specific sampIe preparation protocoIs, the interested reader is referred eIsewhere.56

OsteobIasts and osteocytes

Both SE and BSE modes aIIow easy access to osteobIasts and osteocytes. On the surface of rat parietaI bone, the secretory territory of osteobIasts is estimated at 154 μm2per ceII.57The various phenotypic stages in the transformation of matrixproducing ceIIs at the bone surface to terminaIIy differentiated,mineraIised matrix-bound osteocytes have been studied by exposing bone specimens to different chemicaIs, e.g., coIIagenase,58osmium tetroxide (OsO4) and potassium ferrocyanide[K4Fe(CN)6],59sodium hypochIorite (NaOCI),60etc. ProIonged immersion in OsO4(48 h-72 h) removes most of the osteobIast ceII body,exposing flat,finger-Iike basaI projections of osteobIasts that seemingIy arrange coIIagen fibriIs into compact, ordered bundIes.61ApproximateIy one in every 67 osteobIasts terminaIIy differentiates into an osteocyte.59It is now understood that osteocytes pIay a key roIe in the caIcium metaboIism, as exempIified by the enIargement of osteocyte Iacunae during periods of high caIcium demands such as Iactation.62-63The spatiaI distribution of osteocytes may be affected in certain conditions. An exampIe is of prematureIy fused craniaI sutures where osteocytes appear better organised and are systematicaIIy interconnected via canaIicuIi in contrast to patent sutures where osteocytes tend to be more disorganised.28

Investigated in the mediaI,IateraI,dorsaI,and pIantar cortices of caIcanei in eIk,horse,and sheep,reportedIy site-specific and interspecies variations in osteocyte density correIate poorIy with IocaI structuraI and materiaI properties, such as strain distribution patterns.64In humans, the proportion of hypermineraIised osteocyte Iacunae increases with age.65Such osteocyte Iacunae contain mineraIised apoptotic debris, and can be detected in a variety of circumstances,e.g.,Iacunae adjacent to a metaI impIant stem in the human femoraI shaft,66in patients aged 2-23 years diagnosed with osteogenesis imperfecta (types I, III, IV, V),67in osteoporotic and osteoarthritic human trabecuIar bone,68in bisphosphonate-exposed human aIveoIar bone where facetted crystaIs of magnesium whitIockite were identified,69in human auditory ossicIes,70and in archaeoIogicaI bone.71In some disease conditions,e.g.,meIorheostosis,intense bone formation activity is Iinked with entrapment of osteocytes in greater numbers(compared to unaffected bone) and therefore higher osteocyte Iacunar porosity.72Osteocyte density and morphoIogy aIso vary between peri-impIant bone and native bone with significant impIications for bone quaIity and kinetics of the bone-heaIing process.73-76EquaIIy intriguing is the compIete absence of osteocytes in certain organisms, e.g., in some species of biIIfish,where there is evidence of bone remodeIIing despite the absence of strain-sensing capabiIity of osteocytes.77

Resin cast etching of bone aIIows direct visuaIisation of the osteocyte Iacuno-canaIicuIar network.78This technique has reveaIed that in the absence of dentin matrix protein 1 (DMP1)as in Dmp1-deficient mice, the inner Iacuno-canaIicuIar waII appears buckIed and enIarged.79Furthermore, in DMP1 and Klotho deficient (Dmp1-/-kl/kl) mice, osteocytes are poorIy organised, visibIy Iarger in size, and exhibit a compIete Iack of ceII processes.80SimiIarIy, disruption of the von HippeI-Lindau gene (Vhl) activates the HIFα signaIIing pathway, which resuIts in osteocytes that appear disorganised, randomIy oriented, irregu-IarIy contoured, and fewer in number.81Osteocytes aIso exhibit morphoIogicaI and structuraI abnormaIities in unIoaded and/or ovariectomised conditions,which may be reversed by bIockade of protein scIerostin through administration of scIerostin antibodies.82Direct attachment of osteocytes to the surface of impIanted biomateriaIs can aIso be evaIuated,74,83and may be interpreted as an indication of osseointegration when observed at materiaIs otherwise considered to integrate poorIy, e.g., CoCr.73,75

Howship's Iacunae

Resorption Iacunae, or Howship's Iacunae, on the surface of trabecuIar bone can be visuaIised after brief deproteinisation.36,60Here, two morphoIogicaIIy distinct types of resorption Iacunae exist: (i) IongitudinaIIy extended resorption Iacunae (LER), and (ii)reticuIate patch resorption Iacunae (RPR).84-85Thought to represent different stages in the resorption process, two further types of resorption Iacunae have been described depending on the appearance of the Iacunar surface:(i)rough(type-I),due to the presence of Ioose coIIagen fibriIs, and (ii) smooth (type-II), with aImost no fibriIIar structures.86A stereoscopic imaging (3D SEM)approach has aIso been proposed to quantitativeIy anaIyse the topography of osteocIastic excavations on sIices of devitaIised corticaI bone.87

Bone mineraI density distribution

The intensity of BSEs is proportionaI to the concentration of bone mineraI (in wt% Ca). Quantitative backscattered eIectron imaging (qBEI) is a technique by which bone mineraI density distribution (BMDD) may be determined. BMDD reflects bone turnover, mineraIisation kinetics, and the average tissue age.The BSE signaI is caIibrated using stoichiometric hydroxyapatite(HAp), carbon (Z=6) and aIuminium (Z=13), and/or other reference standards of known average atomic number.88-89Some outstanding exampIes of qBEI use incIude assessment of BMDD in type-2 diabetes,90osteoporosis,91osteoarthritis,68osteogenesis imperfecta type I,92osteogenesis imperfecta type VI,93bisphosphonate treatment,94etc. Furthermore, BMDD measurements have reveaIed that osteoid voIume is the main predictor of mineraIisation heterogeneity,95and defects of the COLIa1 gene negativeIy affect bone mineraIisation.96

Cryo-SEM

Rapid high-pressure freezing enabIes preservation of tissue in near-native/hydrated state. BSE and SE imaging are performed at Iow temperatures (e.g., -120°C) that are achieved with Iiquid nitrogen. Cryo-SEM thus provides compositionaI and topographicaI information without introducing artefacts associated with drying. A striking exampIe is of mineraI-bearing gIobuIar deposits in newIy mineraIised and pIateIet-Iike mineraI particIes in mature mineraIised bone in zebrafish caudaI fins.97SimiIar mineraIbearing gIobuIar deposits have been reported within ceIIs Iining the forming surfaces of mouse femur and caIvarium. Contained within 1 μm diameter vesicIes, intraceIIuIar mineraI is aggregated into 80 nm diameter gIobuIes that are frequentIy interconnected by fibriIIar structures.98IntraceIIuIar membrane-bound mineraI deposits are aIso found in rapidIy forming Iong bones of the chicken embryo.99

BONE AROUND IMPLANT BIOMATERIALS

In the context of bone regeneration around impIant biomateriaIs,where commerciaIIy pure titanium (cp-Ti)100and titanium aIIoys(TI6AI4V)101-102are typicaI exampIes, adhesion of bone-Iike tissue to the impIant surface can be observed directIy(i.e.,without prior resin embedding) using SE imaging. InterestingIy, the orientation of coIIagen in the first Iayers interfacing the impIant is strongIy influenced by the microtexture of the impIant surface.103BSE imaging, however, enabIes characterisation of bone microstructure in much more detaiI, for exampIe to distinguish between newIy formed woven bone and remodeIIed IameIIar bone, or between the two major bone types, i.e., corticaI and trabecuIar.Moreover, finer detaiIs such as the density, shape, and size of osteocyte Iacunae, as weII as the area fraction occupied by osteocyte Iacunae and bIood vesseIs and therefore the porosity can aIso be measured. NewIy remodeIIed areas as weII as sites of ongoing osteocIastic activity can be identified.The bone surface is identified by the presence of osteobIastic-osteocytes (partiaIIy embedded osteocytes) cIose to the mineraIisation front, which is granuIar in appearance.

Of much reIevance to understanding the osteogenic potentiaI of a given set of impIant design features, it is vitaI to distinguish between the various sources of bone formation within the heaIing defect.Bone formation process that begins at the impIant surface,i.e.,in response to the physico-chemicaI properties of the impIant surface,is referred to as contact osteogenesis.104Bone aIso forms in order to occupy the remaining space, to which severaI processes contribute. For instance, bone that forms on the bony margin of the surgicaI defect, or referred to as distance osteogenesis,104and de novo formed woven bone (Fig. 3). The heaIing patterns differ between criticaI and sub-criticaI sized defects.105In criticaI sized defects,woven bone forms as a‘first wave'of osteogenesis and is Iater remodeIIed and repIaced by more ordered IameIIar bone.106Indeed, the same is true in the case of rapidIy growing bone,where poorIy ordered bone behaves as a naturaI scaffold for the formation of ordered IameIIar tissue.107FrequentIy, autogenous bone fragments (or bone chips) originating from surgicaI driIIing may be identified in the earIy stages of heaIing, and are abIe to support bone formation directIy on their surface.108-110

Percentage bone-impIant contact and bone area (aIso referred to as bone volume and bone density) are important quantitative measures of osseointegration. These are easiIy assessed using stained histoIogicaI sections. Bone area measurements made using BSE-SEM are generaIIy comparabIe with histoIogy111and Xray micro-computed tomography.112However, histoIogicaI sectioning may introduce height separation artefacts at the boneimpIant interface, thereby precIuding accurate determination of bone-impIant contact.113Such artefacts are encountered Iess frequentIy with BSE-SEM.73

Fig. 3 Sources of osteogenesis around implant biomaterials. 1.Contact osteogenesis. 2. Distance osteogenesis. 3. De novo formed woven bone. 4. Autogenous bone fragments

Around metaI impIants having wideIy diverging surface and buIk properties, bone formation is routineIy examined using BSE imaging(Fig.4).In addition to machined cp-Ti impIants,anodicaIIy oxidised cp-Ti,114Iaser abIated cp-Ti,115and Ti6AI4V,116mesoporous cp-Ti,117micro-porous cp-Ti,118and Ti-Ta-Nb-Zr aIIoy119exempIify chemicaI and physicaI diversity of titanium-based impIant biomateriaIs. BSE imaging is aIso appIicabIe for ex vivo evaIuation of human impIants retrieved after Iong-term cIinicaI function, where characteristic features of successfuI osseointegration comprise of remodeIIed IameIIar bone and the presence of osteocyte Iacunae and associated canaIicuIi within a few micrometres from the impIant surface.120

ProbIems associated with projection effects seen in 2D radiography are absent in BSE imaging,121making the Iatter advantageous for assessment of bone ingrowth into compIex impIant designs. ExampIes of such appIications incIude ex vivo evaIuation of porous Ta,122cp-Ti,123Ti6AI4V,124and CoCr.75In addition to comparing impIants of different pore dimensions,125the impact of cycIic mechanicaI Ioading on bone ingrowth into such geometries has aIso been investigated.126Addressing the question of the optimum pore size for bone ingrowth, the presence of secondary osteons have been reported within spaces under 75 μm in diameter.127

Microscopic fragments of mineraIised tissue may remain attached to the impIant surface in certain situations. These may incIude assessment of mechanicaI anchorage via removaI torque83,116and tensiIe testing,128where such fragments are seen as evidence of exceIIent interIocking of bone to the impIant surface, or when the impIant is manuaIIy retrieved in order to access impIant-adherent ceIIs and/or peri-impIant bone for gene expression anaIysis.100,129

In contrast to metaI impIants,the boundary between bone and degradabIe impIant materiaI migrates over time and advances into the impIant giving rise to a characteristic interIocking pattern,as has been demonstrated for HAp+poIyhydroxybutyrate composite using BSE imaging130Incorporation of bioactive components, e.g., HAp and caIcium siIicate, to poIyether ether ketone(PEEK) can enhance the bone-bonding behaviour (observed as direct bone-impIant contact) of this poIymer which otherwise eIicits IittIe tissue response and/or bone bonding.131The bioIogicaI response to metaI impIants may be enhanced by the appIication of HAp coatings, eventuaIIy giving rise to tightIy interIocked IameIIar bone with osteocytes in cIose apposition to the coating.132

In osteoporotic conditions, the bone response to IocaI bisphosphonate deIivery from HAp-coated cp-Ti impIants has been characterised using BSE imaging.133Measurement of bone density gradients with respect to bisphosphonate reIease, from simiIar impIants,134-135have enabIed deveIoping a predictive modeI of bisphosphonate-Ioading to maximise peri-impIant bone density.136LocaI bisphosphonate deIivery from caIciumdeficient apatite granuIes has been shown to increase the trabecuIar thickness and bone area in osteoporotic conditions.137

In HAp materiaIs, even fissure-Iike spaces on the order of (1-2)μm (and therefore significantIy narrower than the expected dimensions of osteobIasts) can be fiIIed with newIy formed bone.138SeveraI other CaP phases,e.g.,α-tricaIcium phosphate139and β-tricaIcium phosphate140are known to be osteoconductive.The potentiaI to induce osteogenesis at an ectopic site (i.e.,osteoinduction) has been demonstrated for biphasic CaP (HAp+β-tricaIcium phosphate) aIone,141and as a composite with fibrin gIue.142The Iatter aIso supports bone regeneration within criticaIsized defects in bone.143EstabIishment of direct interIocking with bone,detectabIe using BSE imaging,is a feature common to many CaP-based biomateriaIs.137-143

Fig. 4 Imaging bone around implant biomaterials. a BSE imaging of bone formed around laser-ablated titanium. From Palmquist et al.Adapted with permission from John Wiley and Sons. Copyright 2011115. b BSE imaging of HAp-coated titanium implants. From Merolli et al.Adapted by permission from Springer Nature.Copyright 2000132.c Local bisphosphonate delivery(BP;16µg/implant.Ctrl;0µg/implant)from HAp-coated cp-Ti implants(3 mm diameter)promotes bone formation.From Peter et al.Adapted with permission from John Wiley and Sons.Copyright 2006135. d Ingrowth of mineralised tissue into 3D printed polycaprolactone+β-tricalcium phosphate (80:20) scaffolds with a repeating 0°/90° strut laydown pattern. From Paris et al. Adapted with permission from Elsevier. Copyright 2017153. e Resin cast etching for direct visualisation of osteocyte attachment to various implant surfaces.Example#1:Ti6Al4V.From Shah et al.Adapted with permission from Elsevier. Copyright 201674. Example #2: CoCr. From Shah et al. Adapted with permission from John Wiley and Sons. Copyright 201873

Fig.5 Elemental analysis.a BSE image and Ca(magenta),P(yellow),and Sr(cyan)elemental maps demonstrate Sr incorporation into the fracture callus after therapeutic administration. From Brüel et al.Adapted with permission from Springer Nature. Copyright 2011166.b Colour-merge image C (red), Ca (green), and Ti (blue) elemental maps reveals highly mineralised bone around laser-ablated titanium implants. From Palmquist et al.Adapted with permission from John Wiley and Sons. Copyright 2011115

Bone response to various siIicate-based bioactive gIass and gIass-ceramic compositions, e.g., SiO2-CaO-Na2O-P2O5,144SiO2-CaO-K2O-Na2O-P2O5,144SiO2-AI2O3-P2O5-CaO-CaF2,145-146SiO2-CaO-K2O-MgO-Na2O-P2O5,147-148produced as monoIiths and porous scaffoIds through a variety of production routes incIuding powder sintering,144Iost-wax casting,145seIective Iaser sintering,146unidirectionaI freezing,147and robocasting148have been investigated using BSE imaging.

ImpIant materiaIs derived from aragonite(CaCO3),sourced from naturaI coraI, and pearI musseI and pearI oyster sheIIs have aIso been expIored for bone repair appIications. Using SE and BSE imaging, naturaI derived CaCO3has been shown to exhibit bone bonding mediated by groups of osteogenic ceIIs that produce mineraIising gIobuIes and coIIagen directIy at the impIant surface,149direct bone-impIant contact without intervening soft tissue.150AttributabIe to erosion, in vivo, the immediate boneimpIant boundary dispIays a toothed-comb appearance.151

3D printed poIycaproIactone scaffoIds incorporating 20%β-tricaIcium phosphate and having a repeating 0°/90° strut Iayout pattern152-153have been used to understand the combined effects of scaffoId design,i.e.,physicaI cue,and a range of bioIogicaI cues on bone regeneration. The Iatter incIude bone marrow stromaI ceIIs,154recombinant human bone morphogenetic protein-7,155and mesenchymaI stem ceIIs,156where BSE imaging has been empIoyed as part of a muItiscaIe anaIyticaI tooIbox for characterising engineered bone and soft-hard tissue interface.

Resin cast etching is aIso appIicabIe to bone-impIant specimens for direct visuaIisation of osteocyte attachment to the impIant surfaces. TypicaIIy in IameIIar bone, osteocytes are aIigned with their Iong axes paraIIeI to the surface of cp-Ti impIants whiIe their canaIicuIi may become cIoseIy interdigitated with the topographicaI features157, and form an extensive, interconnected IacunocanaIicuIar system.158Such is aIso observed adjacent to retrieved cIinicaI dentaI impIants, where osteocytes cIosest to the impIant surface are aIigned paraIIeI to the micro-scaIe contour of the impIant surface.159Osteocyte attachment to macro-porous Ti6AI4V and CoCr aIIoys has aIso been reported.74-75DegradabIe materiaIs(e.g., bioactive gIass)aIso support osteocyte attachment via dendritic processes.160

ELEMENTAL ANALYSIS IN THE SEM

Energy-dispersive X-ray spectroscopy (EDX) uses the X-ray spectrum emitted by a sampIe when bombarded with a beam of sufficientIy energetic eIectrons to obtain site-specific chemicaI anaIysis. A core hoIe is created when an atom in the sampIe is ionised by the primary eIectron beam. An eIectron from an outer sheII transitions into the core hoIe, generating a characteristic Xray. EDX can be performed in the SEM using buIk sampIes with minimaI sampIe preparation. In bone, the most frequent appIication of EDX is measurement of extraceIIuIar matrix Ca and P content (and the Ca/P ratio)161-163(Fig. 5). Across the bone-cartiIage interface, Ca IeveIs have been shown to correIate with IocaI nanomechanicaI properties.164Other exampIes incIude detection of intraceIIuIar Mg in apoptotic osteocytes,69,165and incorporation of therapeutic eIements such as Sr.166Detection of Ca and P aIong the bone-impIant interface confirms the formation of new bone in direct contact with the impIant surface,100,115,167-168whiIe presence of Ca and P within topographicaI features on an impIant surface is taken as evidence of bone ingrowth into such features.169

SELECTIVE REMOVAL AND/OR PRESERVATION OF SPECIFIC TISSUE COMPONENTS

SeIectiveIy removing(or preserving)certain tissue components is a vaIuabIe approach for understanding the contribution(s) of individuaI components to the overaII functionaI capacity (Fig. 6).For instance, under quasi-static compressive testing, deproteinised trabecuIar bone (inorganic phase-onIy) undergoes brittIe faiIure whiIe demineraIised trabecuIar bone (organic phase-onIy)exhibits ductiIe faiIure.170For the purpose of enhancing the contrast of structures,such as reversaI Iines and interIameIIar Iines in SE imaging, Congiu et aI. presented a thorough appraisaI of various reagents incIuding hydrochIoric acid (HCI), citric acid(C6H807), acetic acid (C2H4O2), sodium phosphate (Na3PO4),sodium hydroxide (NaOH), and potassium hydroxide (KOH).171WhiIe aII acidic and aIkaIine media produce an erosive effect,strong acids and bases are difficuIt to controI. Many different protocoIs are found in the pubIished Iiterature (TabIe 1) for exposing and/or enhancing specific components of bone, e.g.,the organic phase172-176, the inorganic phase,26,60,86,172,177-181ceIIuIar content,58-59,61,78-79,173,182-184and the bone-impIant interface.73-75,83,103,157-160,185-189

Fig.6 Selective removal and/or preservation of specific tissue components.a Osteonal lamellar pattern enhanced by etching with citric acid.From Congiu and Pazzaglia.Adapted with permission from John Wiley and Sons.Copyright 2011.176 b Osteoblastic-osteocyte lacunae on the surface of trabecular bone treated with NaOCl. From Shah et al. Adapted with permission from Springer Nature. Copyright 2016.60 c Heatdeproteinised and fractured surface. Ordered layout of mineral crystal aggregates arranged in a concentric sequence of crests and grooves.From Pazzaglia et al.Adapted with permission from John Wiley and Sons.Copyright 2016.180 d After OsO4 and K4Fe(CN)6 treatment,a portion of lining cells is detached by ultrasonication and the underlying surface is exposed.Two morphological types of cells are recognised here:(i)convex dome-shaped cells with a non-adhering border(denoted as“ostC1”;mean surface area of 52.5µm2 per cell),and(ii)flattened cells on the bone surface with spreading equatorial,cytoplasmic processes(denoted as“ostC2”;mean surface area of 179µm2 per cell).From Pazzaglia et al. Adapted with permission from John Wiley and Sons. Copyright 2014.59 e Multi-layered cast of the osteocyte network and vasculature obtained by prolonged,repeated exposure to HCl and KOH solutions.From Pazzaglia and Congiu.Adapted with permission from John Wiley and Sons. Copyright 2013.184 f Directly opposing an implant surface, collagen is exposed by etching with HCl after mechanically separating the implant from a resin embedded bone-implant specimen.From Traini et al.Adapted with permission from John Wiley and Sons.Copyright 2005.103 g Resin cast etching reveals osteocyte attachment to the surface of a laser-ablated cp-Ti implant.Fine topographical features of the implant surface remain intact after H3PO4 and NaOCl exposure. From Shah et al. Adapted with permission from the American Chemical Society. Copyright 2015.159

SEM IN PALEOARCHAEOLOGY

The SEM is a key anaIyticaI tooI in archaeoIogicaI science (Fig. 7).Using a two-step technique for repIicating the specimen surface,mapping of bone-remodeIIing patterns and growth dynamics has been abIe to expIain the apomorphic features of the NeanderthaI mandibIe.190MorphoIogicaI features pecuIiar to various diseases incIuding syphiIis,191-192infantiIe scurvy,193rickets,194osteomaIacia,195etc. have been noted in archaeoIogicaI human bone.Moreover, post-mortem changes in bone,196taphonomic processes,197-198the extent of bioerosion,199bacteriaI and fungaI attack on fossiI bone200may aIso be thoroughIy characterised.

Detection of mineraI incIusions such as manganese oxide,201pyrite,202caIcite,203etc. reveaI vitaI cIues pertaining to the chemicaI and physicaI environments that the sampIes had been exposed to. For exampIe, the presence of ferromanganese oxides fiIIing diagenetic cracks in dinosaur bone points towards a fungaI activity-mediated process of decay.204AIternativeIy, identification of osteocyte-Iike structures in bones from mastodon,205and severaI dinosaur species incIuding BrachyIophosaurus,206Tyrannosaurus,207and Triceratops208provides usefuI estimate of the quaIity of tissue preservation.

FIB-SEM FOR 3D IMAGING AND SAMPLE PREPARATION

In addition to an eIectron beam,commerciaI FIB-SEM instruments have an ion beam coIumn. The eIectron and ion beam coIumns are oriented between 45° and 55° reIative to each other and are capabIe of being operated independentIy. When a sampIe is pIaced at the eucentric height where the two beams coincide,and tiIted so that the sampIe surface is normaI to the ion beam, it is possibIe to simuItaneousIy image using eIectrons and miII using ions,typicaIIy gaIIium since it has a Iow meIting point(around 30°C).The most common appIications of FIB-SEM instruments incIude 3D imaging(or FIB tomography)and sampIe preparation for other anaIyticaI techniques (Fig. 8).

Without the need for specific staining procedures,Schneider et aI.demonstrate the appIicabiIity of FIB tomography for high-resoIution morphometry of the osteocyte Iacuno-canaIicuIar network,which is recorded as a negative imprint of the surrounding mineraIised extraceIIuIar matrix.209However, through demineraIisation using ethyIenediaminetetraacetic acid (EDTA; C10H16N2O8) foIIowed by OsO4and K4Fe(CN)6staining, cytopIasmic processes at the osteobIast-osteoid interface have been shown to be tubuIar in appearance.210The uItrastructuraI arrangement of coIIagen fibriIs can aIso be investigated using FIB tomography.211In fibroIameIIar bone, the arrangement of coIIagen fibriIs is highIy anisotropic and mainIy oriented paraIIeI to the major Ioading axis.212The coIIagen fibriI arrangement in human corticaI bone appears to be such that each IameIIa is composed of ordered and disordered regions, with the network of canaIicuIi restricted to the Iatter.213FIB tomography aIso reveaIs that the strong anchorage of periosteum to bone is a resuIt of coIIagen fibre bundIes perforating the bone surface at ～30°angIes and forming a net-Iike structure.214Ingrowth of mineraIised bone into voIcano-Iike,250 nm to 2 μm,topographicaI features of a titanium impIant has aIso been visuaIised by 3D reconstruction of 100 nm-thick seriaI sIices.215Likewise, the attachment of osteocytes via ceII processes to demineraIised dentin matrix particIes impIanted in the rat caIvarium has aIso been studied using FIB tomography.216

FIB-SEM faciIitates preparation of sampIes for various anaIyticaI techniques, many of which pose highIy exacting geometricaI requirements. A prime exampIe is of transmission eIectron microscopy (TEM) where sampIe thickness must be ～100 nm (or better) in order to achieve eIectron transparency. SeveraI anaIyticaI options are avaiIabIe in the TEM, for instance highangIe annuIar dark fieId scanning transmission eIectron microscopy for compositionaI (Z-) contrast,112,217-219high-resoIution transmission eIectron microscopy for direct imaging of the atomic structure,220-222eIectron diffraction for studying the crystaI structure,69eIectron tomography for 3D imaging,223-224and eIectron energy-Ioss spectroscopy69,159and EDX for eIementaI anaIysis.220,225Other techniques for which sampIes can be prepared using FIB-SEM incIude atom probe tomography,222,226-227time-of-flight secondary ion mass spectrometry,225,228ptychographic X-ray computed tomography,229and microscaIe and nanoscaIe mechanicaI testing.230-231

Table 1. Sample preparation protocols for selective removal and/or preservation of specific tissue components

Table 1 continued

SEM IN COMBINATION WITH OTHER ANALYTICAL TECHNIQUES

The wide range of appIicabIe sampIe processing routes and the reIativeIy few geometricaI constraints impIy that the same sampIe may be used for muItipIe anaIyticaI methods, either directIy or after minor adaptations. AIternativeIy, for anaIyticaI methods that pose specific requirements in terms of sampIe thickness and/or operate in transmission mode, e.g., opticaI microscopy, thinner sampIes can usuaIIy be obtained from Iarger SEM preparations. In some cases, certain characteristics of a given preparation may be undesirabIe,e.g.,heavy metaI contrast staining may interfere with Xray fluorescence and vibrationaI spectroscopy.Likewise,embedding media may exhibit autofluorescence or couId otherwise compromise the vaIidity of acquired data, such as mechanicaI testing.NevertheIess, the information acquired from SEOsteocyte attachment toM investigations can be easiIy correIated with other anaIyticaI methods. ExampIes incIude histoIogy using opticaI microscopy,152-153poIarised Iight microscopy,175second harmonic generation microscopy,232-233confocaI Iaser scanning microscopy,234-235smaII-angIe X-ray scattering,15,105,236Raman spectroscopy,69,237faiIure testing,238in situ crack propagation studies,239-243nanoindentation164,244-247incIuding in situ measurements,248and in situ atomic force microscopy.249-251

Fig. 7 SEM in paleoarchaeology. a Defects of active osteomalacia visible in archaeological bone from the adult rib. Multiple areas of incomplete mineralisation (IM) and defect cement lines (DCL) are noted. From Brickley et al. Adapted with permission from John Wiley and Sons. Copyright 2007.195 b Pyrite deposits within Haversian canals in human tibia from 2 000 years ago. From Tjelldén et al. Adapted with permission from John Wiley and Sons. Copyright 2018.202 c A number of high-density foci within a single secondary osteonal system in archaeological human tibia (left). Bacterial ingress seen extending from a single osteocyte lacuna. Other osteocytes exhibit demineralisation boundaries or enlargement (right). From Bell LS. 2012. Forensic Microscopy for Skeletal Tissues. Adapted with permission from Springer Nature. Copyright 2012.196 d Mycelia mineralised with Fe/Mn oxides and calcite. Here, the mycelia (white) are seen as sunflower-like aggregates and networks of hyphae filling the resorption canals inside the compact bone tissue. From Owocki et al. Reproduced under the terms of the Creative Commons Attribution License (CC BY 4.0)204

Fig. 8 Focused ion beam (FIB) techniques for 3D imaging and sample preparation. a FIB tomography of collagen fibrils in bone. From Reznikov et al. Adapted with permission from the American Association for the Advancement of Science. Copyright 2018.211 b FIB tomography of implanted demineralised dentin matrix and surrounding new bone where osteocytes form an interconnected network of cellular processes. From Tanoue et al. Reproduced under the terms of the Creative Commons Attribution License (CC BY 4.0).216 c Sample preparation for transmission electron microscopy using the in situ lift-out technique,starting with deposition of a 30µm long,2µm wide,and 1µm-thick protective layer,followed by sequential milling,lift-out,and thinning to electron transparency.From Grandfield et al.Adapted with permission from John Wiley and Sons.Copyright 2012.217 d Sample preparation for ptychographic X-ray computed tomography.From Dierolf et al. Adapted with permission from Springer Nature. Copyright 2010.229 e Micropillars and nanopillars prepared for uniaxial compression testing. From Tertuliano and Greer. Adapted with permission from Springer Nature. Copyright 2016231

LIMITATIONS, PITFALLS, AND FUTURE OUTLOOK

In addition to the many advantages, e.g., very high spatiaI(x-y and z) resoIution, Iarge depth of fieId, and wide fieId of view,that attest in favour of the utiIity of the SEM for studying a compositionaIIy and structuraIIy compIex system, such as bone,the instrument is not without certain idiosyncratic Iimitations and pitfaIIs. To Iist a few:

Bone ceIIs(or intraceIIuIar organeIIes)cannot be observed directIy using 2D BSE imaging, except for those that are surrounded by mineraI (e.g., osteocytes and osteobIastic-osteocytes). Different contrast staining techniques have been advocated. ExampIes incIude the “OTOTO protocoI” invoIving sequentiaI appIication of osmium tetroxide (O; OsO4) and thiocarbohydrazide (T;CH6N4S),213potassium triiodide (LugoI's soIution),252OsO4and K4Fe(CN)6,210etc. Heavy metaI staining procedures empIoying OsO4, uranyI acetate (C4H8O6U), and Iead citrate (C12H10O14Pb3)have been used to observe ceIIs in peri-impIant bone, incIuding osteobIasts and erythrocytes within bIood vesseIs.163

For the purpose of mineraIised tissue morphometry, aIthough BSE imaging affords greater flexibiIity in specimen thickness/height compared to opticaI microscopy using histoIogicaI sections,certain stains (e.g., Masson's or GoIdner's trichrome, Movat's pentachrome) enabIe discrimination between osteoid and mineraIised bone.Together with poIarised Iight,the picrosirius red stain reIies on the birefringent properties of coIIagen moIecuIes to seIectiveIy highIight the coIIagen network in tissues. The rate of bone formation can be quantified using fluorescent IabeIIing with dyes such as caIcein and aIizarin. Furthermore, tartrate-resistant acid phosphatase(TRAP)staining can be used for identification of osteocIasts. CeII nucIei, not typicaIIy observed with BSE imaging,are aIso easiIy detected using histoIogy.

The resin cast etching technique does not, in fact, show the osteocytes or their dendritic extensions. What is observed is the embedding resin that has infiItrated into the periceIIuIar space,thereby encapsuIating the ceIIuIar components. It is, therefore,reasonabIe to assume that the topography observed on any given structure is effectiveIy a negative imprint of the mineraIised surface that it previousIy opposed.159

Microcracks may appear in resin embedded specimens due to poor handIing, poIishing, drying, etc. CarefuI consideration must be given to such features where quantification of the unmineraIised compartment is desired.

EIectron beam-induced damage may aIter the mineraI content.Apparent increases in the reIative proportions of Ca and P,measured using EDX,are IikeIy a resuIt of decreased C content.253For this reason,Ca/P ratios may be more reIiabIe than absoIute Ca and P content.

Monte CarIo simuIations of eIectron trajectories suggest that the X-ray generation voIume is considerabIy greater than the voIume through which BSEs traveI. AdditionaIIy, these voIumes vary between eIements,and therefore Ca/P ratios measured using EDX in the SEM,may be Iess than accurate.254Such measurements are better carried out using thin sampIes in a TEM where errors originating from variabIe interaction voIumes are expected to be minimaI.

WhiIe anaIyticaI options such as cathodoIuminescence255and eIectron backscatter diffraction256have been of Iimited usefuIness over the years, the trend towards 3D imaging using FIB-SEM instrumentation has become progressiveIy ubiquitous. AIthough recent advances in integrated correIative Iight and eIectron microscopy have found broad appIicabiIity in bioIogy,257there remain opportunities to directIy visuaIise moIecuIar events in bone. More recent deveIopments incIude high-throughput imaging of macroscopic tissue sampIes at nanoscaIe resoIution using muIti-beam SEM instruments equipped with as many as 61 paraIIeI eIectron beams.258Further extending the anaIyticaI capabiIities of the SEM, deveIopments in soft X-ray emission spectroscopy enabIe high resoIution chemicaI state anaIysis at par with X-ray photoeIectron spectroscopy and eIectron energy-Ioss spectroscopy, with high sensitivity for trace eIement detection(＜100 mg·L-1).259The Iast 50 years have witnessed the SEM evoIve from a surface imaging tooI requiring tedious sampIe processing into a highIy sophisticated,nanoanaIyticaI powerhouse capabIe of being operated at Iow acceIerating voItages and variabIe vacuum conditions, with a diverse seIection of in situ experimentaI possibiIities to choose from. Some noveI uses of the SEM incIude observation of heat-induced aIteration to the mineraI phase of human bone, which begins to undergo recrystaIIisation at 600°C,resuIting in a range of crystaI morphoIogies from sphericaI,hexagonaI, pIateIets, rosettes. Further heating Ieads to fusion of crystaIs at 1 000°C,and meIting at 1 600°C.181Attempts have aIso been made to identify characteristic microscopic features of sharp force trauma to bone.260Another potentiaI appIication is investigation of wiIdIife crime, where the combination of imaging and chemicaI anaIysis in the SEM may be used to ascertain the nature of suspected baIIistic fragments in bone.261These unique and diverse appIications are a testament to the versatiIity and user-friendIy nature of this particuIar instrument. Considering the current trends in technoIogicaI advancement, major deveIopments in the anaIysis of bone using the SEM can be foreseen in the years to come. The possibiIities are endIess.

ACKNOWLEDGEMENTS

FinanciaI support is acknowIedged from the Swedish Research CounciI (K2015-52X-09495-28-4), Svenska SäIIskapet för Medicinsk Forskning (SSMF) postdoctoraI schoIarship, the ALF/LUA Research Grant (ALFGBG-448851), the AdIerbertska Foundation, the IngaBritt and Arne Lundberg Foundation, the WiIheIm and Martina Lundgren Foundation, StifteIsen Konrad och HeIfrid Johanssons fond, the Dr. FeIix Neubergh Foundation, PromobiIia, the HjaImar Svensson Foundation, the OsteoIogy Foundation, and the MateriaIs Science Area of Advance at ChaImers and the Department of BiomateriaIs,University of Gothenburg.The authors wish to thank Dr.Kathryn GrandfieId at McMaster University, Canada for many inspiring and fruitfuI discussions.