ir microscopic imaging of pathological states and fracture healing
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Volume 54, Number 8, 2000 APPLIED SPECTROSCOPY 11830003-7028 / 00 / 5408-1183$2.00 / 0q 2000 Society for Applied Spectroscopy
IR Microscopic Imaging of Pathological States and FractureHealing of Bone
RICHARD MENDELSOHN,* ELEFTHERIOS P. PASCHALIS,PAMELA J. SHERMAN, and ADELE L. BOSKEYDepartment of Chemistry, Rutgers University, Newark, New Jersey 07102 (R.M.); and Mineralized Tissues Division,Research Section, Hospital for Special Surgery, New York, New York 10021 (E.P.P., P.J.S., A.L.B.)
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 ; 610 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 size/perfection 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 size/perfection 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 larger/more 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.
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 (310 m m), and excellent-quality spectra may beacquired from tissues. However, for an IR-based methodto provide a biomedically signi cant conclusion (i.e., 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, 110 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 Surgery.During this time, methods of preparation of bone samplesfor IR microscopic examination have been developed andspectrastructure correlations established.1118 In addition,the feasibility of acquiring useful IR images from bio-mineralizing tissues has been reported.5,6 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.
Figure 1 shows, at two distance scales, an optical mi-crograph of the major mineral-containing areas of bone.The 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; i.e., 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 region,which is part of the cylindrical structure forming the outer shell of compact bone, and the inner region containing trabecular bone and marrow.The bottom gure shows a single osteon in which mineral grows in tree-ring 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 spaces.The numbers of trabeculae and their interconnections areboth greatly reduced in osteoporotic bone, as is evidentin Fig. 2B. Generally the trabeculae are anisotropicallyarrangeda possible response to mechanical stresses onthe bone.
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; e.g., CO 32 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 indicated.PMMA 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.
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 elsewhere.18 Brie y, biopsiesare xed in ethanol, dehydrated in a series of washes withincreasing levels of acetone in acetonewater mixtures, xed in polymethylmethacrylate (PMMA), microtomedto the desired thickness (35 m m), and placed on BaF 2windows for IR transmission studies.
Rat Femurs. One hundred and sixty-eight ovariecto-mized rats were split into three groups. One received noestrogen, the second received subcutaneous 17 -estradiolslow-release pellets within three days after ovariectomy,and the third received pellets at the time of fracture.Closed transverse fracture of the mid-shaft right femur ofeach animal was produced six weeks following ovariec-tomy by using a three-point bending device.19 This ani-mal model is an accepted model for the study of bothosteoporosis and fracture healing.2022 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 tre