Download - IR Microscopic Imaging of Pathological States and Fracture Healing

Volume 54 Number 8 2000 APPLIED SPECTROSCOPY 11830003-7028 00 5408-1183$200 0q 2000 Society for Applied Spectroscopy

IR Microscopic Imaging of Pathological States and FractureHealing of Bone

RICHARD MENDELSOHN ELEFTHERIOS P PASCHALISPAMELA J SHERMAN and ADELE L BOSKEYDepartment of Chemistry Rutgers University Newark New Jersey 07102 (RM) and Mineralized Tissues DivisionResearch Section Hospital for Special Surgery New York New York 10021 (EPP PJS ALB)

The application of IR microscopic imaging to the study of bonedisease and fracture healing is demonstrated Samples of normaland osteoporotic human iliac crest biopsies were prepared and ex-amined at 6ndash10 m m spatial resolution and 8 cm 2 1 spectral reso-lution with a 64 3 64 MCT focal plane array detector coupled to aFourier transform infrared (FT-IR) microscope and a step-scanninginterferometer Two spectral parameters one that monitors the ex-tent of mineral (hydroxyapatite) formation in the tissue and anotherthat monitors the sizeperfection of the crystals were compared inthe samples generated from normal and pathological tissues Theaverage mineral levels in the osteoporotic sample were reduced by 40 from the normal In addition the crystal sizeperfection wassubstantially enhanced in the disease state The applicability of IRimaging techniques to the study of therapeutic intervention was alsoinvestigated in a study of the effects of estrogen therapy on fracturehealing in rat femurs Femurs were examined by IR microscopicimaging 4 weeks after fracture IR imaging showed that the minerallevel was enhanced in estrogen-treated samples In addition thecrystals were largermore perfect in the treated specimens Thesedata demonstrate the utility of IR spectroscopic imaging for thestudy of pathological states of hard tissue

Index Headings Infrared microscopic imaging Fracture healing ofbone Estrogen therapy Mineral crystallinity in bone

INTRODUCTION

Two technical issues limit the application of conven-tional infrared (IR) spectroscopy or point-by-point IR mi-croscopy to the study of pathological states of tissues (1)The necessity for sample homogenization in conventionalFourier transform infrared (FT-IR) destroys spatial infor-mation Since spatial heterogeneity is an essential deter-minant of biological function its preservation is an in-herently important element for the successful biomedicalapplication of IR (2) Traditional (point-by-point) IR mi-croscopy can provide useful information at the diffractionlimit (3ndash10 m m) and excellent-quality spectra may beacquired from tissues However for an IR-based methodto provide a biomedically signi cant conclusion (ie areliable diagnosis) thousands of locations in many sam-ples must be examined to allow for statistical variationsin tissue properties within and among individuals Datacollection even from a single sample thus becomes atedious process For example if a single 400 m 3 400m m tissue section is to be examined at 10 m m 3 10 m mspatial resolution then 1600 individual IR spectra wouldbe required The sampling time needed to build up ade-quate signal-to-noise ratios precludes the possibility of

Received 10 February 2000 accepted 10 April 2000 Author to whom correspondence should be sent

examination of many samples and thus limits the appli-cation of traditional IR microscopy for diagnosis In prac-tice either spatial resolution is sacri ced or the exami-nation of only a fraction of sites within a tissue is un-dertaken

Prospects for the ef cient and routine use of IR mi-croscopy in medical applications have greatly improvedthanks to the recent availability of focal plane array de-tectors in the mid-IR spectral region Several applicationsof this technology in both the biomedical and polymerscience areas have recently appeared 1ndash10 but a systematicstudy of disease states has not yet been reported Thepurpose of the current paper is to describe the feasibilityof IR microscopy to study pathological states and fracturehealing of bone The article results from a joint researchprogram that has been underway since 1987 betweenRutgers University and the Hospital for Special SurgeryDuring this time methods of preparation of bone samplesfor IR microscopic examination have been developed andspectrandashstructure correlations established11ndash18 In additionthe feasibility of acquiring useful IR images from bio-mineralizing tissues has been reported56 In the presentarticle we present two examples of the application of themethod to pathological states These include a compari-son of normal and osteoporotic human bone (iliac crestbiopsy) and a discussion of the effect of estrogen therapyon fracture healing in an animal model (rat femur) Theseapplications begin to delineate the range and power of IRmicroscopic imaging for mineralized tissue research Toenable the reader to place the IR spectral results in con-text we summarize some relevant aspects of bone mi-croanatomy below

BONE STRUCTURE

Figure 1 shows at two distance scales an optical mi-crograph of the major mineral-containing areas of boneThe top gure shows the cortical region which is part ofthe cylindrical structure forming the outer shell of com-pact bone Also revealed is the inner region containingtrabecular bone and marrow Normally cortical boneconstitutes 80 of the human skeletal mass and tra-becular bone 20 The latter undergoes the majorchanges during osteoporosis Cor tical bone m ineralgrows by apposition ie mineral layers are depositedaround a central blood vessel known as the Haversiancanal When long bone is sectioned perpendicular to itsmajor axis the system composed of the Haversian canalsurrounded by mineral layers (known as an osteon) ap-

1184 Volume 54 Number 8 2000

FIG 1 Optical micrograph of the major mineral-containing areas of bone The top gure shows the spatial relationship between the cortical regionwhich is part of the cylindrical structure forming the outer shell of compact bone and the inner region containing trabecular bone and marrowThe bottom gure shows a single osteon in which mineral grows in lsquolsquotree-ringrsquorsquo like fashion around the central blood vessel (Haversian canal)

pears as a tree ring-like structure it is illustrated in themore highly magni ed (bottom) panel of Fig 1

Figure 2 provides a micrograph of trabecular bonefrom a 4 mm 3 4 mm section of normal and osteoporotichuman iliac crest biopsies The system is held in placeby a brous tissue network which connects the innerwalls of the cortical bone and the trabeculae Figure 2Ashows normal bone The trabecular structure consists ofplates and struts (occupying darker regions of the micro-graph) while marrow occupies the intervening spacesThe numbers of trabeculae and their interconnections areboth greatly reduced in osteoporotic bone as is evidentin Fig 2B Generally the trabeculae are anisotropicallyarrangedmdasha possible response to mechanical stresses onthe bone

BONE CHEMISTRY

Bone has two main chemical constituents The mineralphase is a poorly crystalline phase of hydroxyapatite(HA) Ca10(PO4)6(OH)2 A variety of substitutions mayoccur in both the anionic and cationic sites eg CO 3

2 2

may substitute either for PO 43 2 or for OH 2 in the lattice

although the former dominates Similar-sized cations mayreplace Ca 2 1 The main organic constituent (protein) inbone collagen consists of three chains intertwined intoa characteristic triple helical structure Type I collagen (atriple helical structure consisting of two a I chains andone a II chain) is the major protein in bone An importantstructural element of the collagen is post-translationalmodi cation of the residues of the amino acids proline

APPLIED SPECTROSCOPY 1185

FIG 2 Optical micrograph of trabecular bone from a 4 mm 3 4 mm section of normal (Fig 2A) and osteoporotic (Fig 2B) human iliac crestbiopsies Note the differences in connectivities and number of the trabeculae (darkened areas)

FIG 3 A series of spectra acquired from a 300 m m wide region ofnormal human trabecular bone Spectral assignments are indicatedPMMA peaks have not been compensated for in this series of spectra

hydroxyproline and lysine to produce cross-links in thestructure which stabilize the triple helix

EXPERIMENTAL

Iliac Crest Biopsies Normal and pathological iliaccrest biopsies are acquired under normal IRB (Institu-

tional Review Board) protocols at the Hospital for Spe-cial Surgery These biopsies are obtained as part of eitherdiagnostic or post-mortem procedures Upon completionof pathological evaluations portions of the biopsies arepreserved for IR evaluation Our sample preparation pro-tocols have been detailed elsewhere18 Brie y biopsiesare xed in ethanol dehydrated in a series of washes withincreasing levels of acetone in acetonendashwater mixtures xed in polymethylmethacrylate (PMMA) microtomedto the desired thickness (3ndash5 m m) and placed on BaF 2

windows for IR transmission studiesRat Femurs One hundred and sixty-eight ovariecto-

mized rats were split into three groups One received noestrogen the second received subcutaneous 17 ucirc -estradiolslow-release pellets within three days after ovariectomyand the third received pellets at the time of fractureClosed transverse fracture of the mid-shaft right femur ofeach animal was produced six weeks following ovariec-tomy by using a three-point bending device19 This ani-mal model is an accepted model for the study of bothosteoporosis and fracture healing20ndash22 Animals werehoused for a minimum of 6 weeks after ovariectomy toensure that an osteoporotic state had been attained Ani-mals were euthanized at 4 6 8 and 12 weeks after bonefracture The preliminary biomechanical data23 suggestthat estrogen treatment prevented the loss of mechanicalintegrity seen in the osteopenic rats Contralateral sec-tions from these bones (right femora) were utilized for


FIG 4 IR images of the spatial distribution of the mineralprotein (matrix) ratio and a histogram of this quantity each given for normal andosteoporotic trabecular bone The parameter presented is a ratio of integrated areas of the PO4

3 2 n 1 n 3 contour to the area of the amide I modeNote the decrease in mineral level in osteoporotic bone

FT-IR imaging The bones were dehydrated by a seriesof washes with increasing acetone proportions embeddedin PMMA and thin sections (4 m m) cut and analyzed bymeans of FT-IR imaging in the areas of compact corticalbone trabecular bone and the fracture callus PMMAfrom sections for FT-IR examination by microscopic im-aging was removed by spectral subtraction with the useof the spectrally isolated C 5 O mode near 1720 cm 2 1 toprovide subtraction factors

Acquisition of IR Images Spectra were acquired onthe BioRad (Cambridge MA) lsquolsquoSting-Rayrsquorsquo system Theinstrument consists of a step-scan interferometer inter-faced to a mercury cadmium telluride (MCT) focal planearray detector imaged to the focal plane of an IR micro-scope Interferograms are simultaneously collected fromeach element of the 64 3 64 array to provide 4096 spec-tra ( 4 min scan time) at a spectral resolution of 8 cm 2 1At each step of the interferometer signals from each el-ement are examined 81 times to provide signal averagingThe (square) sample size imaged (400 m m 3 400 m m)

corresponds to an optimal spatial resolution of about 63m m 3 63 m m The instrumental software permitsstraightforward imaging of IR band areas For images ofpeak height ratios (see below) home-developed softwarewas used based on GRAMS32 (Galactic Software Sa-lem NH) Statistical analysis was accomplished with Mi-crocal Origin For some samples histograms were com-puted for fewer than 4096 data points since the sampleimage did not ll the entire set of pixels

RESULTS

A series of spectra acquired from across a 300 m mwide region of normal human trabecular bone is shownin Fig 3 Band assignments of relevant spectral featuresare included in the gure The phosphate n 1 n 3 contour(900ndash1200 cm 2 1) arises from HA in the tissue while theamide I ( 1630ndash1690 cm 2 1) and II bands (1525ndash1570cm 2 1) arise from collagen As noted above the featurenear 1720 cm 2 1 arises from the PMMA C 5 O stretching


FIG 5 Changes in the PO 43 2 n 1 n 3 contour of hydroxyapatite as crys-

tals are allowed to mature for various lengths of time in a supersaturatedsolution of calcium phosphate The IR data are presented as invertedsecond derivatives The vertical dashed line is drawn at 1020 cm 2 1 Theshift in peak intensity from 1020 to 1030 cm 2 1 as the maturation pro-gresses is evident

FIG 6 IR images of the spatial distribution of the mineral crystallinity index [the peak height ratio I(10301020)] and a histogram of this quantityfor normal and osteoporotic trabecular bone

mode which was not subtracted from the current set ofspectra This peak diminishes greatly in relative intensityfrom the exterior to the interior of the trabeculum Ex-clusion of PMMA from the interior of the bone tissue isthus evident These results are consistent with a prior IRmicroscopy study of rat femurs13 Concomitant with thediminution of intensity of the PMMA spectral features isthe increased intensity of bands arising from the proteinand mineral constituents In addition progressive shapechanges in the phosphate n 1 n 3 contour are evident

Figure 4 presents IR images of the spatial distributionof the mineralprotein (matrix) ratio and a histogram ofthis quantity each given for normal and osteoporotic tra-becular bone The parameter presented is a ratio of in-tegrated areas of the phosphate n 1 n 3 contour to the areaof the amide I mode Images constructed from ratios ofband intensities are preferred to images of a single inten-sity parameter as the use of ratios overcomes problemsarising from sample-to-sample thickness variations andpermits direct comparisons between normal and patho-logical specimens The images of the mineralmatrix ratio


FIG 7 Optical micrographs of two samples of fractured rat femurs examined after four weeks of healingmdashone untreated and one treated withestrogen

are scaled similarly in each case as shown in the gureIt has been demonstrated previously 14 that this parametercorrelates strongly (in macroscopic specimens) with theso-called lsquolsquoash weightrsquorsquo widely used as an index of min-eral quantity

It is evident from Fig 4 that there is substantially moremineral in normal vs osteoporotic bone and that the spa-tial distribution is drastically altered between the two sam-ples Normal bone shows an increase in mineral level inmoving toward the center of the bone from any edgewhile osteoporotic bone mineral levels are consistentlylower toward the center A quantitative evaluation of min-eral distribution is derived from histograms of the mineralmatrix ratio The histogram for the normal sample has anaverage value of 274 (n 5 3164) while osteoporotic bonehas an average value of 191 (n 5 2049) The value of nis the number of pixelsimage that were used in the com-putation Since the sample does not ll the eld of viewsome pixels were excluded from the calculation thus n 4096 The above data con rm and extend the acceptedview that normal bone contains substantially more mineralthan osteoporotic in this instance 40 more IR imagingalso reveals a different spatial distribution of HA betweenthe normal and pathological states

The power of IR microscopic imaging to provide mo-lecular structure information from the tissues is revealedin Figs 5 and 6 As noted above when the HA crystalsmature several changes occur in the n 1 n 3 contour Spec-trandashstructure correlations for this band were derived 1518

from in vitro studies of samples of varying crystal di-

mensions prepared from supersaturated calcium phos-phate solutions that had been allowed to ripen for varioustimes The most obvious spectral change is the growth ofa spectral feature at 1030 cm 2 1 while a band at 1020cm 2 1 loses relative intensity This change is readily evi-dent (Fig 5) in second-derivative spectra of HA samplesthat been permitted to ripen for 0 30 and 60 min re-spectively The peak height ratio of these two compo-nents thus serves as an empirical index of mineral crys-tallinityperfection The 10301020 ratio is imaged fornormal and osteoporotic bone in Fig 6 In addition ahistogram of the parameter is included The quantitativeaspects of the relationship between the intensity param-eter and crystal size are discussed in Ref 18 For ex-ample the 10301020 ratio increases linearly as the crys-tal dimension along the c-axis increases In the currentinstance the HA crystals are much largermore perfectin the osteoporotic bone (average value of the index is104 n 5 1805) compared with normal (average valueof the index is 075 n 5 3098) bone Thus osteoporoticbone possesses less mineral than normal but the averagecrystal sizeperfection is greater in the diseased tissueDiscussions of the biological rami cations of these dataand the results from statistical evaluations of many sam-ples will be deferred to a medical forum

A different type of pathological occurrence is evalu-ated in Figs 7ndash10 Fractures were induced in ovariecto-mized rat femurs Healing at the fracture site after fourweeks in the presence and absence of estrogen was eval-uated by IR microscopic imaging Optical micrographs


FIG 8 IR images of the spatial distribution of the mineralprotein (matrix) ratio and a histogram of this quantity for an estrogen-treated site in afractured rat femur (bottom) and for an untreated site (top)

of two samplesmdashone untreated and one treated with es-trogen after four weeksmdashare shown in Fig 7 The darkerareas correspond to the mineral phase The mineralma-trix ratio derived from the IR data is imaged and histo-grams derived from the images are plotted in Fig 8 Theindex of mineral crystal sizeperfection is imaged andhistograms derived from the images are plotted in Fig 9The general correspondence between the IR- and opti-cally generated images is good Dark regions in the op-tical image (Fig 7) correspond quite well to the presenceof high levels of mineral as shown from an image of themineralmatrix ratio in Fig 8 The ratio is greatly in-creased in the estrogen-treated compared with the untreat-ed femur In addition the distribution of values in thehistogram is much narrower in the treated samples Thecrystalsize perfection (Fig 9) is slightly (15ndash20) in-creased in the treated specimen From a biomedical point

of view these data are consistent with a model in whichestrogen therapy suppresses osteoclastic activity

As a nal illustration of the power of the IR imagingtechnology the distributions of protein (from the amideI integrated area) mineral (from the n 1 n 3 integratedarea) and lipid (from the integrated area of the CH2 sym-metric stretching mode at 2854 cm 2 1) are each plotted inFig 10 for a single fracture callus site (different from thesite studied in Figs 7ndash9) The fracture runs horizontallyas a 300 m m wide swath through the sample There areregions evident at the fracture site where there is sub-stantial protein intensity (light colors) yet little or nomineral is present These locations are the sites predictedfor the next mineral to be formed Also of interest is thespatial distribution of the lipid symmetric methylenestretching mode at 2854 cm 2 1 Although this parameteris weak and the image generated from it is poor the en-


FIG 9 IR images of the index of mineral crystallinityperfection and a histogram of this quantity for an estrogen-treated site in a fractured ratfemur (bottom) and for an untreated site (top)

hanced levels of lipid at the fracture site are representa-tive and reveal the presence of cell membranes ie cel-lular activity

DISCUSSION

In each of the examples cited above IR microscopicimaging has provided spatially resolved informationabout molecular structure and relative concentrations oftissue constituents not available from other methodsMost traditional approaches currently used to study bonetissue (bone density X-ray diffraction etc) focus solelyon the mineral phase None of the traditional approachesprovide information about the protein In addition to FT-IR Raman spectroscopy has that potential and Morrisand his colleagues are beginning to exploit that technol-ogy2425

The crystallinity index I(1030)(1020) provides a directcharacterization of crystal sizeperfection at particularspatial locations in IR images The parameter itself iseasy to extract from the vast data sets acquired from mi-

croscopic imaging as it requires only baseline correctionof the spectra followed by measurement of peak heightratios at the two indicated wavenumber positions Thepoint we wish to emphasize is that development of spec-tral parameters (such as the aforementioned index) foranalysis of imaging data requires considerations beyondthose normally used in IR Traditional analytical ap-proaches such as curve tting and deconvolution inwhich each individual spectrum must be scrutinized areclearly not appropriate for image analysis Thus simplerparameters (frequency shifts halfwidths peak area orpeak height ratios) are required

It seems clear that multivariate methods more sophis-ticated than the univariate approaches used here could beapplied to extract additional diagnostic parameters Suchapproaches have already been reported 25 While usefulthese methods tend to obscure the molecular structureinformation inherent in the individual spectra This lim-itation may or may not be relevant for a particular ap-plication


FIG 10 The distribution of protein (from the amide I mode intensity) mineral (from the n 1 n 3 integrated intensity) and lipid (from the intensityof a band at 2854 cm 2 1) at a fracture site in a rat femur The fracture is a 300 m m 3 m m horizontal swath through the section

CONCLUSION

The current study clearly reveals the ability of IR im-aging to distinguish between normal and pathologicalstates of bone The differences noted above must be test-ed for statistical signi cance by examination of a largenumber of biopsy sections Such studies are currently be-ing prepared for publication This task impossible withconventional technology is feasible with array detectionIf the changes in spectral images and parameters notedabove for the osteoporotic tissues are consistent for a sta-tistically signi cant number of samples then the appli-cation of this method as a biomedical diagnostic willhave been proven In addition the data presented in Figs8ndash10 for the results of estrogen administration on fracturehealing show that the effects of therapeutic interventionsmay be easily evaluated with our current approach

ACKNOWLEDGMENTS

This work was supported by PHS Grants AR-41325 (RM andALB) and AR-46121 (EPP)

1 E N Lewis and I W Levin Appl Spectrosc 49 672 (1995)2 E N Lewis A M Gorbach C Marcott and I W Levin Appl

Spectrosc 50 263 (1996)3 L H Kidder V F Kalasinsky J L Luke I W Levin and E N

Lewis Nature (Medicine) 3 235 (1997)4 L H Kidder P Colarusso S A Stewart I W Levin N M Appel

D S Lester P G Pentchev and E N Lewis J Biomed Opt 47 (1999)

5 C Marcott R C Reeder E P Paschalis D N Tatakis A LBoskey and R Mendelsohn Cell Mol Biol 44 109 (1998)

6 R Mendelsohn E P Paschalis and A L Boskey J Biomed Opt4 14 (1999)

7 R Bhargava S Q Wang and J L Koenig Macromolecules 322748 (1999)

8 C M Snively and J L Koenig J Polymer Sci Part BndashPolymerPhys 37 2261 (1999)


10 C Marcott R C Reeder J A Sweat D D Panzer and D LWetzel Vib Spectrosc 19 123 (1999)

11 R Mendelsohn A Hassankhani A L Boskey and E DiCarloCalci ed Tissue International 44 20 (1989)

12 N L Pleshko A L Boskey and R Mendelsohn Calci ed TissueInternational 51 72 (1992)

13 N L Pleshko A L Boskey and R Mendelsohn J HistochemCytochem 40 1413 (1992)

14 A L Boskey N L Pleshko S B Doty and R Mendelsohn CellsMaterials 2 209 (1992)

15 S J Gadaleta E P Paschalis F Betts R Mendelsohn and A LBoskey Calci ed Tissue International 58 9 (1996)

16 E P Paschalis F Betts E DiCarlo and A L Boskey Calci edTissue International 61 487 (1997)

17 S J Gadaleta N P Camacho and A L Boskey Calci ed TissueInternational 58 17 (1996)

18 E P Paschalis E DiCarlo F Betts P Sherman R Mendelsohnand A L Boskey Calci ed Tissue International 59 480 (1996)

19 F Bonnarens and T Einhorn J Orthop Res 2 97 (1984)20 J M Aitken E Armstrong and J B Anderson J Endocrinol 55

79 (1972)21 P D Saville J Am Geriatr Soc 17 155 (1969)22 T J Wronski P L Lowry C C Walsh and L A Ignaszewski

Calci ed Tissue International 37 324 (1985)23 P J Sherman-Brown E P Paschalis C Rimnac B Robie S B

Doty C Cornell and A L Boskey Trans Orthopaedic Res Soc22 256 (1997)

24 J A Timlin A Carden M D Morris J F Bonadio C E Hof erK M Kozloff and S A Goldstein J Biomed Opt 4 28 (1999)

25 J A Timlin A Carden and M D Morris App Spectrosc 531429 (1999)


FIG 1 Optical micrograph of the major mineral-containing areas of bone The top gure shows the spatial relationship between the cortical regionwhich is part of the cylindrical structure forming the outer shell of compact bone and the inner region containing trabecular bone and marrowThe bottom gure shows a single osteon in which mineral grows in lsquolsquotree-ringrsquorsquo like fashion around the central blood vessel (Haversian canal)

pears as a tree ring-like structure it is illustrated in themore highly magni ed (bottom) panel of Fig 1

Figure 2 provides a micrograph of trabecular bonefrom a 4 mm 3 4 mm section of normal and osteoporotichuman iliac crest biopsies The system is held in placeby a brous tissue network which connects the innerwalls of the cortical bone and the trabeculae Figure 2Ashows normal bone The trabecular structure consists ofplates and struts (occupying darker regions of the micro-graph) while marrow occupies the intervening spacesThe numbers of trabeculae and their interconnections areboth greatly reduced in osteoporotic bone as is evidentin Fig 2B Generally the trabeculae are anisotropicallyarrangedmdasha possible response to mechanical stresses onthe bone

BONE CHEMISTRY

Bone has two main chemical constituents The mineralphase is a poorly crystalline phase of hydroxyapatite(HA) Ca10(PO4)6(OH)2 A variety of substitutions mayoccur in both the anionic and cationic sites eg CO 3

2 2

may substitute either for PO 43 2 or for OH 2 in the lattice

although the former dominates Similar-sized cations mayreplace Ca 2 1 The main organic constituent (protein) inbone collagen consists of three chains intertwined intoa characteristic triple helical structure Type I collagen (atriple helical structure consisting of two a I chains andone a II chain) is the major protein in bone An importantstructural element of the collagen is post-translationalmodi cation of the residues of the amino acids proline





EXPERIMENTAL











RESULTS
























DISCUSSION







CONCLUSION


ACKNOWLEDGMENTS






























EXPERIMENTAL











RESULTS
























DISCUSSION







CONCLUSION


ACKNOWLEDGMENTS
































RESULTS
























DISCUSSION







CONCLUSION


ACKNOWLEDGMENTS
















































DISCUSSION







CONCLUSION


ACKNOWLEDGMENTS




























CONCLUSION


ACKNOWLEDGMENTS


























Download - IR Microscopic Imaging of Pathological States and Fracture Healing

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