U.S. patent application number 12/785933 was filed with the patent office on 2010-09-16 for method and apparatus for evaluating ocular tissue conditions.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Karen A. Dehring, Michael D. Morris, Blake J. Roessler.
Application Number | 20100234740 12/785933 |
Document ID | / |
Family ID | 37805246 |
Filed Date | 2010-09-16 |
United States Patent
Application |
20100234740 |
Kind Code |
A1 |
Roessler; Blake J. ; et
al. |
September 16, 2010 |
Method and Apparatus for Evaluating Ocular Tissue Conditions
Abstract
Methods and apparatus are provided for evaluating an ocular
tissue condition of a patient (e.g., a disease, a risk of
developing a disease, etc.). For example, an indicator associated
with the ocular tissue condition may be generated. A portion of
ocular tissue of the patient is irradiated using a light source.
Light from the portion of the ocular tissue is received. Raman
spectra information associated with the received light is
determined. An indicator of an ocular tissue condition is generated
based at least on the Raman spectra information. The indicator may,
for example, assist a physician in diagnosing or ruling out the
ocular tissue condition.
Inventors: |
Roessler; Blake J.; (Ann
Arbor, MI) ; Morris; Michael D.; (Ann Arbor, MI)
; Dehring; Karen A.; (Ypsilanti, MI) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 SOUTH WACKER DRIVE, 6300 WILLIS TOWER
CHICAGO
IL
60606-6357
US
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
MICHIGAN
Ann Arbor
MI
|
Family ID: |
37805246 |
Appl. No.: |
12/785933 |
Filed: |
May 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11217755 |
Sep 1, 2005 |
7729749 |
|
|
12785933 |
|
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Current U.S.
Class: |
600/476 ;
356/301 |
Current CPC
Class: |
G01N 21/359 20130101;
A61B 5/413 20130101; A61B 5/4528 20130101; G01N 21/35 20130101;
A61B 5/0059 20130101; A61B 5/4514 20130101; G01N 2021/656
20130101 |
Class at
Publication: |
600/476 ;
356/301 |
International
Class: |
A61B 6/00 20060101
A61B006/00; G01J 3/44 20060101 G01J003/44 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant
numbers P30 AR46024, R01 AR34399, and R01 AR47969 awarded by the
Public Health Service division of the Department of Health and
Human Services, and Grant number T32 AR07080, awarded by the
National Institute of Arthritis and Musculoskeletal and Skin
Disease, National Institutes of Health. The Government may own
certain rights in this invention.
Claims
1. A method for evaluating ocular tissue of a patient, the method
comprising: irradiating a portion of ocular tissue of the patient
using a light source; receiving light from the portion of the
ocular tissue; determining Raman spectra information associated
with the received light; and generating, based at least on the
Raman spectra information, an indicator of an ocular tissue
condition.
2. A method as defined in claim 1, wherein the Raman spectra
information includes a plurality of bands corresponding to received
light at one or more wavelengths; wherein generating the indicator
of the ocular tissue condition comprises determining at least one
intensity of at least one band.
3. A method as defined in claim 2, wherein determining the at least
one intensity of the at least one band comprises at least one of:
fitting a curve to at least one band; calculating an area of at
least one band; or calculating a height of at least one band.
4. A method as defined in claim 2, wherein generating the indicator
of the connective tissue condition comprises: determining a first
intensity of at least a first band; determining a second intensity
of at least a second band; and determining a first ratio of the
first intensity and the second intensity.
5. A method as defined in claim 4, wherein generating the indicator
of the connective tissue condition further comprises generating the
indicator based at least in part on the first ratio.
6. A method as defined in claim 1, wherein the ocular tissue
condition is at least one of an acquired disorder or a genetic
disorder including macular degeneration, cataracts, glaucoma,
keratitis, scleritis, or episcleritis.
7. A method as defined in claim 1, wherein the ocular tissue
condition is an increased risk of developing an ocular disease.
8. A method as defined in claim 1, wherein the ocular tissue
condition comprises at least one of compatibility of donated ocular
tissue, integrity of a cornea after a corneal transplant, or an
indication of whether cataract surgery is needed.
9. A method as defined in claim 1, wherein the indicator is for
monitoring the progression of the ocular tissue condition and/or
monitoring a response to treatment of the ocular tissue
condition.
10. A method as defined in claim 1, wherein generating the
indicator is further based on at least one of an age of the
patient, a medical history of the patient, or a family history of
the patient.
11. A method as defined in claim 1, further comprising repeating
the acts of irradiating, receiving, and determining over a period
of time to generate spectral content information that reflects the
ocular tissue condition of the patient over the period of time.
12. An apparatus for evaluating ocular tissue of a patient,
comprising: a light source; a Raman probe to receive light
scattered from a portion of ocular tissue of a patient irradiated
by the light source; a spectrum analyzer coupled to receive light
received by the light receiver and to determine Raman spectra
information for the received light; and a computing device coupled
to the spectrum analyzer, the computing device configured to
generate diagnostic information indicative of an ocular tissue
condition based at least in part on the Raman spectra
information.
13. An apparatus as defined in claim 12, wherein the Raman spectra
information includes a plurality of bands corresponding to received
light at one or more wavelengths; wherein generating the indicator
of the ocular tissue condition comprises determining at least one
intensity of at least one band.
14. An apparatus as defined in claim 13, wherein determining the at
least one intensity of the at least one band comprises at least one
of: fitting a curve to at least one band; calculating an area of at
least one band; or calculating a height of at least one band.
15. An apparatus as defined in claim 13, wherein generating the
indicator of the connective tissue condition comprises: determining
a first intensity of at least a first band; determining a second
intensity of at least a second band; and determining a first ratio
of the first intensity and the second intensity.
16. An apparatus as defined in claim 15, wherein generating the
indicator of the connective tissue condition further comprises
generating the indicator based at least in part on the first
ratio.
17. An apparatus as defined in claim 12, wherein the diagnostic
information corresponds to an indicator of at least one of an
acquired disorder or a genetic disorder including macular
degeneration, cataracts, glaucoma, keratitis, scleritis, or
episcleritis.
18. An apparatus as defined in claim 12, wherein the diagnostic
information corresponds to whether an increased risk of developing
an ocular disease exists.
19. An apparatus as defined in claim 12, wherein the diagnostic
information corresponds to an indicator of at least one of
compatibility of donated ocular tissue, integrity of a cornea after
a corneal transplant, or an indication of whether cataract surgery
is needed.
20. An apparatus as defined in claim 12, wherein the diagnostic
information is further based on at least one of an age of the
patient, a medical history of the patient, or a family history of
the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present disclosure is a divisional of U.S. patent
application Ser. No. 11/217,755, filed on Sep. 1, 2005, and
entitled "METHOD AND APPARATUS FOR EVALUATING CONNECTIVE TISSUE
CONDITIONS." U.S. patent application Ser. No. 11/217,755 is hereby
incorporated by reference herein in its entirety.
FIELD OF THE DISCLOSURE
[0003] The present disclosure generally relates to medical
diagnostic apparatus and methods, and more particularly to
apparatus and methods that may be used to help diagnose conditions
of connective tissue.
BACKGROUND
[0004] Osteoporosis is an important healthcare problem. It is
estimated that 24 million Americans are affected by osteoporosis
and that osteoporosis led to $13.8 billion in healthcare costs in
1995. The risk of dying from hip fracture complications is the same
as the risk of dying from breast cancer. For Caucasian females over
50, the risk of hip, spine, or distal forearm fractures is 40%.
Osteoporosis is currently defined as a condition in which bone
mineral density is greater than two standard deviations below the
mean of a young healthy population.
[0005] Current techniques for screening individuals for fracture
susceptibility are relatively inaccurate and/or pose risks to the
patient. For example, the present preferred technique for diagnosis
of osteoporosis is dual X-ray absorption (DXA), which measures the
amount of mineral in the bone. In some patients, however, a low
mineral content does not appear to lead to an increased risk of
fracture. Additionally, DXA requires that the patient is exposed to
ionizing radiation.
[0006] Osteoarthritis is another important health care problem. It
has been estimated that 40 million Americans and 70 to 90 percent
of persons older than 75 years are affected by osteoarthritis. The
prevalence of osteoarthritis among men and women is equal, though
its symptoms occur earlier in women. Risk factors include age,
joint injury, obesity, and mechanical stress.
[0007] Studies suggest physio-chemical alteration of the articular
cartilage surface is an early event in the pathogenesis of
osteoarthritis. The changes involve physical damage to structural
matrix proteins, mediated by physical forces and degradative
enzymes.
[0008] Current techniques for diagnosing or ruling out
osteoarthritis include taking an X-ray image of a joint, analyzing
blood samples, and analyzing synovial fluid withdrawn from the
joint with a needle. The diagnosis is largely clinical because
radiographic findings do not always correlate with symptoms. An
X-ray image of a joint may indicate osteoarthritis if a normal
space between the bones in a joint is narrowed, an abnormal
increase in bone density is evident, or if bony projections or
erosions are evident. A blood sample may indicate osteoarthritis if
byproducts of hyaluronic acid are present. Hyaluronic acid is a
joint lubricant and the presence of its byproducts in the blood may
indicate the lubricant's breakdown, a sign of osteoarthritis. Also,
elevated levels of a factor called C-reactive protein, which is
produced by the liver in response to inflammation, may indicate
osteoarthritis. On the other hand, elevated levels of rheumatoid
factor and so-called erythrocyte sedimentation rates may indicate
rheumatoid arthritis rather than osteoarthritis. An analysis of
synovial fluid withdrawn from the joint may indicate osteoarthritis
if cartilage cells are present in the fluid. On the other hand, a
high white blood cell count in the synovial fluid is an indication
of infection, and high uric acid in the synovial fluid is an
indication of gout.
SUMMARY
[0009] Methods and apparatus are provided for evaluating an ocular
tissue condition of a patient (e.g., a disease, a risk of
developing a disease, etc.). For example, an indicator associated
with the ocular tissue condition may be generated. First, a portion
of ocular tissue of the patient is irradiated using a light source.
Then, spectral content information for light scattered, reflected,
or transmitted by the ocular tissue is determined. The spectral
content information is used, at least in part, to generate the
indicator. The indicator may assist a physician in diagnosing or
ruling out the ocular tissue condition. Also, the indicator may
assist in estimating a risk of developing a disease, monitoring the
progression of a disease, monitoring a response to treatment of a
disease, determining whether surgery is needed, determining the
integrity of ocular tissue before or after transplant, etc.
[0010] In another embodiment, a method for evaluating ocular tissue
of a patient is disclosed. The method may comprise irradiating a
portion of ocular tissue of the patient using a light source, and
receiving light from the portion of the ocular tissue. The method
may also comprise determining Raman spectra information associated
with the received light, and generating, based at least on the
Raman spectra information, an indicator of an ocular tissue
condition.
[0011] In another aspect, an apparatus for evaluating ocular tissue
of a patient is provided. The apparatus may comprise a light
source, and a Raman probe to receive light scattered from a portion
of ocular tissue of a patient irradiated by the light source. Also,
the apparatus may comprise a spectrum analyzer coupled to receive
light received by the light receiver and to determine Raman spectra
information for the received light, and a computing device coupled
to the spectrum analyzer, the computing device configured to
generate diagnostic information indicative of an ocular tissue
condition based at least in part on the Raman spectra
information.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The features and advantages of the apparatus and methods
described herein will be best appreciated upon reference to the
following detailed description and the accompanying drawings, in
which:
[0013] FIG. 1 is a block diagram of one embodiment of an apparatus
for determining susceptibility to fracture;
[0014] FIG. 2 is a flow diagram of one embodiment of a method for
determining a susceptibility to fracture;
[0015] FIG. 3 is a flow diagram of one embodiment of a method for
determining a susceptibility to fracture based on spectral content
information;
[0016] FIG. 4 is a flow diagram of another embodiment of a method
for determining a susceptibility to fracture based on spectral
content information;
[0017] FIG. 5 is a chart showing measured spectral content
information for a group of patients that suffered fractures and for
a control group;
[0018] FIG. 6 is a block diagram of a computer that can be used
with the apparatus of FIG. 1;
[0019] FIG. 7 is a flow diagram of one embodiment of a method for
determining a cartilage tissue condition;
[0020] FIG. 8 is a flow diagram of one embodiment of a method for
determining a cartilage tissue condition based on spectral content
information;
[0021] FIG. 9 is a flow diagram of another embodiment of a method
for determining a cartilage tissue condition based on spectral
content information;
[0022] FIG. 10 is a flow diagram of another embodiment of a method
for determining a cartilage tissue condition based on spectral
content information;
[0023] FIG. 11 is a flow diagram of another embodiment of a method
for determining a connective tissue condition;
[0024] FIG. 12 is a flow diagram of another embodiment of a method
for determining a connective tissue condition based on spectral
content information;
[0025] FIG. 13 is a flow diagram of one embodiment of a method for
determining an ocular tissue condition;
[0026] FIG. 14A is a chart showing measured spectral content
information associated with cartilage tissue for wildtype mice;
[0027] FIG. 14B is a chart showing measured spectral content
information associated with cartilage tissue for transgenic
mice;
[0028] FIG. 15 is a graph of a Raman spectrum of ocular tissue;
[0029] FIG. 16 is a portion of the graph of FIG. 15;
[0030] FIG. 17 is another graph of a Raman spectrum of ocular
tissue;
[0031] FIG. 18 is a graph showing an example of peak fitting in a
region of the graph of FIG. 17; and
[0032] FIG. 19 is a Raman spectrum of articular cartilage (upper
plot) and a Raman spectrum of eye tissue (lower plot) taken from a
single mouse specimen.
DETAILED DESCRIPTION
[0033] Diagnostic Apparatus
[0034] FIG. 1 is a block diagram of an example apparatus 100 that
may be used to help diagnose a condition of the bone tissue of a
patient. For example, the apparatus 100 may be used to help
diagnose osteoporosis, help estimate a susceptibility to fracture
of the bone tissue, help diagnose a defect (e.g., osteogenesis
imperfecta), help diagnose a nutritional disorder, or help diagnose
other disorders related to bone tissue. The apparatus 100 may be
used on a patient once, for example, or may be used multiple times
over time to help track changes in the bone tissue.
[0035] The apparatus 100, which may be used for a Raman
spectrometry analysis of a bone tissue or an infrared (IR) analysis
of the bone tissue, includes a light source 104 optically coupled
to at least one optical fiber 108. For Raman spectrometry, the
light source 104 may comprise a laser, for example, that generates
substantially monochromatic light. The optical fiber 108 is
optically coupled to an optical probe 116. The optical probe 116
may be positioned proximate to a portion of bone tissue 120 from a
patient, and may be used to irradiate the bone tissue 120 with the
light generated by the light source 104.
[0036] In one embodiment, the optical probe 116 is also optically
coupled to at least another optical fiber 124. In this embodiment,
the optical probe 116 may be used to collect light scattered or
reflected by the bone tissue 120 and to transmit the scattered
light through the optical fiber 124. This embodiment may be used
for Raman spectrometry or for "attenuated total reflection" IR
spectrometry.
[0037] In another embodiment, another optical probe 128 may be
positioned proximate to the portion of the bone tissue 120 such
that the optical probe 128 can collect light transmitted by the
bone tissue 120. The optical probe 128 may be optically coupled to
the optical fiber 124 and can transmit the light transmitted by the
bone tissue 120 through the optical fiber 124. This embodiment may
be used for "line of sight" IR spectrometry.
[0038] The optical fiber 124 is optically coupled to a spectrum
analyzer 132 via an optical processor 140 which may include one or
more lenses and/or one or more filters. The spectrum analyzer 132
may include, for example, a spectrograph optically coupled to an
array of optical detectors, and is communicatively coupled to a
computing device 144.
[0039] FIG. 2 is a flow diagram of a method for determining a
condition related to the bone tissue of a patient. The method 170
may be implemented by an apparatus such as the apparatus 100 of
FIG. 1, and will be described with reference to FIG. 1. At a block
174, a portion of bone tissue of a patient is irradiated with
light. For example, the optical probe 116 may be used to irradiate
the bone tissue 120 with light generated by the light source 104.
In one embodiment, the bone tissue 120 may be irradiated
non-invasively through the skin of the patient. In other
embodiments, bone tissue 120 exposed by an incision, or removed as
a biopsy, may be irradiated.
[0040] In some embodiments, bone tissue at or near a site presumed
at risk for fracture (e.g., the hip) may be irradiated.
Alternatively, bone tissue not at or near a site of presumed risk
may be measured. For in vivo measurements, irradiation may occur at
a site at which bone tissue is close to the skin. For example, the
proximal diaphysis of the tibia may be irradiated. As biopsy
measurements, an iliac crest biopsy could be irradiated as just one
example.
[0041] At a block 178, light scattered, reflected, or transmitted
by the bone tissue may be collected. For example, the optical probe
116 may collect light scattered by the bone tissue 120 (Raman
spectrometry). As another example, the optical probe 116 may
collect light reflected by the bone tissue 120 ("attenuated total
reflection" IR spectrometry). Alternatively, the optical probe 128
may collect light transmitted by the bone tissue 120 ("line of
sight" IR spectrometry). As with the optical probe 116, the optical
probe 128 may collect light non-invasively through the skin of the
patient. In other embodiments, the light may be collected via an
incision or collected from an irradiated biopsy.
[0042] At a block 182, spectral content information associated with
the collected light is generated. For example, the light collected
by the optical probe 116 or the optical probe 128 may be provided
to the spectrum analyzer 132 via the optical processor 140. The
spectrum analyzer 132 may then generate spectral content
information associated with the light received by the spectrum
analyzer 132.
[0043] In Raman spectrometry, the collected light may include light
at wavelengths shifted from the wavelength of the incident light.
The spectrum of the collected light scattered from bone tissue
(referred to hereinafter as the "Raman spectrum of the bone
tissue") is indicative of the physico-chemical state of the bone
tissue. The Raman spectrum of the bone tissue includes bands
indicative of various components of the bone tissue including
phosphate of bone mineral, carbonate of bone mineral, interstial
water, residual water, hydroxide of the bone mineral, etc. Also
included are bands indicative of various components of the collagen
matrix of the bone tissue including amide I, hydroxyproline,
proline, cross-links, etc. The wavelength at which a band is
located is indicative of the component of the bone mineral or
matrix to which it corresponds. The height and/or intensity of a
band are indicative of the amount of the corresponding component of
the bone tissue.
[0044] In IR spectrometry, the light generated by the light source
104 includes light at a variety of IR wavelengths. Some of the
light at various wavelengths is absorbed by components of the bone
tissue, and different components absorb different wavelengths.
Thus, similar to the Raman spectrum of the bone tissue, in IR
spectrometry, the spectrum of the collected light transmitted by
the bone tissue (referred to hereinafter as the "IR spectrum of the
bone tissue") includes bands indicative of components and structure
of the bone tissue. Unlike in Raman spectrometry, however, the
bands in the IR spectrum of the bone tissue are indicative of light
absorbed by the bone tissue, rather than light scattered by the
bone tissue. Nevertheless, the IR spectrum of the bone tissue is
also indicative of the physico-chemical state of the bone tissue.
As is known to those of ordinary skill in the art, the Raman
spectrum of a bone tissue and an IR spectrum of the same bone
tissue may provide indications of different components and/or
different structure of the bone tissue.
[0045] At a block 186, it is determined whether the patient has a
bone tissue disorder based on the spectral content information
generated at block 182. For example, the computing device 144 may
receive spectral content information from the spectrum analyzer
132. The computing device 144 may then generate an indication of
whether the patient has a bone tissue disorder. As another example,
the computing device 144 may generate an indication, based on the
spectral content information generated at block the 182, that may
be used by a physician to determine whether the patient has a bone
tissue disorder. For example, the indication may be indicative of a
susceptibility of the bone tissue of the patient to fracture. The
bone tissue disorder may be, for example, osteoporosis, a genetic
disorder (e.g., osteogenesis imperfecta), an acquired disorder,
etc.
[0046] The determination of the block 186 may be based on
additional factors. For example, the determination may be further
based on one or more of an age of the patient, a height of the
patient, a weight of the patient, a bone mineral density of the
patient (e.g., determined using DXA), a family history of the
patient, etc. Determining the estimate of susceptibility to
fracture will be described in more detail below.
[0047] Blocks 174, 178, and 182 may optionally be repeated over a
period of time (e.g., weeks, months, years) to generate spectral
content information that reflects the condition of the bone tissue
of the patient over the period of time. This spectral content
information over the period of time may be used in the
determination of block 186.
[0048] Estimating Susceptibility to Fracture
[0049] In one embodiment, the determination of block 186 comprises
estimating a susceptibility of the bone tissue of the patient to
fracture. Examples of techniques for estimating a susceptibility to
fracture based on spectral content information are provided below.
Many other techniques may be employed as well. In general,
embodiments of methods for estimating susceptibility to fracture
may vary according to the environment in which they are to be used.
For example, different embodiments may be used in a clinical
setting as compared to a laboratory setting because signal-to-noise
ratios likely will be higher in the laboratory setting as compared
to the clinical setting.
[0050] In some embodiments in which Raman spectrometry is employed,
the area under a band or height of particular bands in the Raman
spectrum of the bone tissue may be used to determine a
susceptibility to fracture.
[0051] Amide I and amide III are observable in both IR and Raman
spectrometry. Amide I and amide III spectra include information
similarly indicative of the structure of collagen in the bone
tissue, although amide I appears to produce more intense bands as
compared to amide III. In Raman spectrometry, amide I of bone
tissue is associated with a plurality of bands that can extend over
much of the 1600 cm.sup.-1 to 1700 cm.sup.-1 region. For example,
amide I of bone tissue is associated with a band approximately at
1650 cm.sup.-1 and a band approximately at 1680 cm.sup.-1 to 1690
cm.sup.-1.
[0052] It is believed that the absence of collagen intrafibral
cross-links weakens bone tissue. The disruption or absence of
collagen cross-links can result in changes to the relative
intensities of the bands associated with amide I. For example,
denaturing collagen to gelatin causes the high frequency shoulder
associated with amide I to become more prominent. Additionally, the
intrafibral cross-links in bone matrix collagen cause shifts in the
proline bands (proline-2 and proline-3) from 1660 cm.sup.-1 to 1663
cm.sup.-1 and from 1670 cm.sup.-1 to 1690 cm.sup.-1 respectively.
Research has shown that the 1690 cm.sup.-1 band intensity in bone
matrix increases relative to the intensity of the 1663 cm.sup.-1
band when dehydrodihydroxylysinonorleucine,
dehydrohydroxylysinonorleucine or
dehydrohistindohydroxymerodesmosine cross-links are chemically
reduced. Further research with fetal murine calvarial tissue has
shown that the matrix amide I band in newly deposited tissue has a
prominent shoulder at approximately 1690 cm.sup.-1 that becomes
smaller as the tissue ages and cross-links are formed.
[0053] FIG. 3 is a flow diagram illustrating one embodiment of a
method for determining susceptibility to fracture based on areas of
particular bands in a Raman spectrum of bone tissue. A similar
technique may be employed for use with an IR spectrum of bone
tissue.
[0054] At a block 204, an area of the amide I bands substantially
between 1680 cm.sup.-1 and 1690 cm.sup.-1 is determined.
Determining the area of these amide I bands may include curve
fitting using a function such as a mixed Gaussian-Lorentzian
function. Determining the area of the bands may also include
measuring the area without curve fitting. For example, the area
could be measured based on the raw data. As another example, the
raw data could be filtered (e.g., with a smoothing filter), and the
area could be measured based on the filtered data. In general, the
areas under one or more bands may be determined using any of a
variety of techniques, including known techniques. At a block 208,
an area of the amide I band approximately at 1665 cm.sup.-1 is
determined. Determining the area of this amide I band may be
performed in the same or similar manner as described with reference
to block 204.
[0055] At a block 212, a ratio of the area determined at the block
204 with the area determined at the block 208 may be determined.
Then, at a block 216, an estimate of the susceptibility to fracture
of the bone tissue is determined based on the ratio determined at
the block 212. Determining the estimate of the susceptibility to
fracture may comprise determining in which of one or more sets of
values the ratio falls. In one embodiment, the estimate of the
susceptibility to fracture may comprise an indication of whether or
not the bone tissue is susceptible to fracture. In other
embodiments, the estimate of the susceptibility to fracture may
additionally comprise an indication of one of a plurality of risk
levels (e.g., high risk, increased risk, normal risk).
[0056] As described previously, the estimate of the susceptibility
to fracture determined at the block 216 may be based on additional
factors such as one or more of an age of the patient, a height of
the patient, a weight of the patient, a bone mineral density of the
patient, a family history of the patient, etc.
[0057] FIG. 4 is a flow diagram illustrating another embodiment of
a method for determining susceptibility to fracture based on areas
of particular bands. At a block 254, an area of a band associated
with phosphate .nu..sub.1 and having a peak at approximately 957
cm.sup.-1 and having a shoulder at approximately 945 cm.sup.-1 is
determined. Other phosphate bands could be used, although it is
believed that the .nu..sub.1 band is more intense than other
phosphate bands. Determining the area of this phosphate .nu..sub.1
band may include curve fitting to resolve the phosphate .nu..sub.1
band into two components using a function such as a mixed
Gaussian-Lorentzian function or some other suitable function. In
general, the area of this band may be performed using any of a
variety of techniques, including known techniques such as those
described previously. .nu.1
[0058] At a block 258, the area of the collagen amide I envelope
(the plurality of bands between approximately 1600 cm.sup.-1 to
1700 cm.sup.-1) is determined. Other matrix bands could be used,
for example bands indicative of hydroxyproline (853 cm.sup.-1),
proline (919 cm.sup.-1), etc. Determining the area of the collagen
amide I band may be performed in the same or similar manner as
described previously. At a block 262, the area of the carbonate
.nu..sub.1 band (circa 1070 cm.sup.-1) is determined. Determining
the area of the carbonate .nu..sub.1 band may be performed in the
same or similar manner as described previously. Additionally, other
carbonate bands could be used, although it is believed that the
.nu..sub.1 band is more intense than other carbonate bands.
[0059] At a block 266, a ratio of the area of the phosphate
.nu..sub.1 band to the area of the collagen amide I bands is
determined. At a block 270, a ratio of the area of the carbonate
.nu..sub.1 band to the area of phosphate .nu..sub.1 band is
determined. It is believed that this ratio is a rough measure of
the size and crystallinity of mineral crystals.
[0060] FIG. 5 is a plot of the above-described ratios determined
from bone tissue taken from the proximal femur in the same location
for each individual in a matched set of females. A control group
included eleven individuals who had died without having a hip
fracture. A fracture group included eighteen individuals who had
sustained a hip fracture and were treated with arthroplasty. In the
fracture group, those who had sustained fracture due to trauma such
as automobile accidents or falls from a ladder were excluded. The
control group and the fracture group were selected such that the
age of the individuals and the bone volume fractions were similar
between the two groups.
[0061] As can be seen in FIG. 5, a relationship exists between the
carbonate/phosphate ratio and the phosphate/amide I ratio. As the
phosphate/amide I ratio decreases, the carbonate/phosphate ratio at
first generally remains approximately constant. As the
phosphate/amide I ratio continues to decrease, the
carbonate/phosphate ratio then tends to increase considerably. The
fracture specimens tend to be concentrated at the low end of the
phosphate/amide I ratio range, while the control specimens tend to
be concentrated at the upper end of the phosphate/amide I ratio
range. A low phosphate/amide I ratio and a high carbonate/phosphate
ratio appear strongly associated with hip fracture. Student t-tests
were conducted on the data illustrated graphically in FIG. 4. A
comparison of the carbonate/phosphate ratios between the two groups
(the fracture group and the control group) resulted in a p-value of
0.08. A comparison of the phosphate/collagen ratios between the two
groups resulted in a p-value of 0.28.
[0062] Referring again to FIG. 4, at a block 274, an estimate of
the susceptibility to fracture of the bone tissue is determined
based on the ratios determined at the blocks 266 and 270.
Determining an estimate of the susceptibility to fracture may
comprise determining whether the ratios determined at the blocks
266 and 270 fall within one or more sets of values. Additionally,
in one embodiment, the estimate of the susceptibility to fracture
may comprise an indication of whether or not the bone tissue is
susceptible to fracture. In other embodiments, the estimate of the
susceptibility to fracture may additionally comprise an indication
of one of a plurality of risk levels (e.g., high risk, increased
risk, normal risk).
[0063] The estimate of the susceptibility to fracture determined at
the block 274 may be based on additional factors such as one or
more of an age of the patient, a height of the patient, a weight of
the patient, a bone mineral density of the patient, a family
history of the patient, etc. Additionally, the estimate of the
susceptibility to fracture determined at block 274 may be based on
spectral content information taken over a period of time (e.g.,
weeks, months, years).
[0064] Other information in the IR spectrum or the Raman spectrum
of the bone tissue can be used in addition to, or as an
alternative, the information described above. For example,
information related to bands other than those described above could
be used. Additionally, information related to the width, shape
(e.g., whether or not a band has "shoulders"), height, etc. of
particular bands could be used in determining susceptibility to
fracture. Additionally, more sophisticated analyses could be
employed such as a cluster analysis.
[0065] In a study separate from the study associated with the data
of FIG. 5, iliac crest biopsies were analyzed from ten subjects
without fractures (mean age 56 years, range 43-70 years) and five
subjects with osteoporotic fractures (mean age 63 years, range
50-72 years). In particular, for each specimen, trabecular and
cortical regions were scanned using Raman spectroscopy and average
carbonate/phosphate and phosphate/amide I band area rations were
obtained for the trabecular and cortical regions. No corrections
were made for multiple comparisons.
[0066] Both carbonate .nu..sub.1/phosphate .nu..sub.1 ratio and
phosphate .nu..sub.1/amide I ratio were higher in cortical than
trabecular bone for all specimens (p=0.005 and p=0.01,
respectively, paired t-tests). This may suggest that mineralized
matrix chemistry differs between bone types due to, for example, a
fundamental difference or a result of differing average tissue age.
Chemical composition of cortical bone mineralized matrix appears to
change with age, as demonstrated by a decrease in phosphate/amide I
ratio (p=0.005, linear regression model). Neither carbonate
.nu..sub.1/phosphate .nu..sub.1 ratio in cortical bone nor any
measure in trabecular bone showed significant change with age. The
phosphate .nu..sub.1/amide I ratio in patients without fractures
was greater in cortical than trabecular bone until age 55 (in all 6
subjects), but greater in trabecular bone in those 55 y or older
(in all 4 subjects). In all 5 patients with fractures, the
phosphate .nu..sub.1/amide I ratio was greater in cortical bone.
Thus, patients with fractures demonstrated the pattern seen in
younger (under 55) non-fractured subjects, as opposed to the
pattern of patients of similar age without fractures. It is
possible that failure to alter mineralized matrix chemistry results
in increased fracture risk. Another possibility is that the greater
phosphate .nu..sub.1/amide I ratio in cortical bone for patients
with fractures, as compared to phosphate .nu..sub.1/amide I ratio
in the trabecular bone, was a result of the fracture. There may be
other explanations as well for the differences in the relationship
between phosphate .nu..sub.1/amide I ratio in cortical bone and
trabecular bone between patients with fractures and patients
without fractures.
[0067] Comparing patients with fractures to patients without
fractures, trabecular bone from patients with fractures had a lower
phosphate .nu..sub.1/amide I ratio (p=0.03, t-test). No differences
appeared to be found in cortical bone or in carbonate
.nu..sub.1/phosphate .nu..sub.1 ratio in trabecular bone. This
lower mineral/matrix ratio (decreased mineral) in trabecular bone
with patients with fractures may suggest a systemic increase in
remodeling prior to or following fracture, and is likely
demonstrated more clearly in trabecular bone because of its more
rapid turnover. If this increase in remodeling occurs prior to
fracture, chemical composition from iliac crest biopsy specimens
may improve fracture risk assessment. The lower phosphate
.nu..sub.1/amide I ratio in trabecular bone for patients with
fractures, however, could be a result of the fracture. There may be
other explanations as well for the lower phosphate .nu..sub.1/amide
I ratio in trabecular bone for patients with fractures.
[0068] Yet another study was conducted that was designed to help
understand whether, and how, the chemical composition of the bone
extracellular matrix changes immediately after fracture. Raman
spectroscopy was used to compare chemical composition between the
fracture site and a location away from the fracture site. With this
experimental model, it was assumed that there was originally no
difference along the length of the bone. It was also assumed that
there was little change far from the fracture site as a result of
the fracture. Thus, differences in chemical composition found in
this study between the fracture site and far from it may model
changes in the chemical composition of the bone as a result of the
fractures.
[0069] In this study, the tibiae of five mice were fractured in a
controlled manner. One day later, the tibiae were dissected out and
Raman spectra were obtained for cortical bone at/near the fracture
site and approximately 2 mm from the fracture site (no trabecular
bone was analyzed). Data from both locations were available for 4
limbs, each from separate animals.
[0070] The results indicated a decreased phosphate .nu..sub.1/amide
I ratio and increased carbonate .nu..sub.1/phosphate .nu..sub.1
ratio at the fracture site as compared to the site 2 mm away from
the fracture. This data may suggest there is some change in the
chemical composition of the bone extracellular matrix following
fracture. It is important to note, however, that this assumes that
there was no difference in chemical composition existed prior to
the fracture between the two sites. It also assumes that there was
little change at the site 2 mm away from the fracture site as a
result of the fracture. There may be other explanations for why the
study indicates decreased phosphate .nu..sub.1/amide I ratio and
increased carbonate .nu..sub.1/phosphate .nu..sub.1 ratio at the
fracture site as compared to the site 2 mm away from the
fracture.
[0071] Further Description of the Diagnosis Apparatus
[0072] In general, embodiments of apparatus for determining a bone
tissue disorder may vary in design according to the environment in
which they are to be used. For example, an apparatus to be used in
a clinical setting may be designed to obtain spectrum information
more quickly as compared to an apparatus to be used in a laboratory
setting.
[0073] Referring again to FIG. 1, many types of light sources 104
may be employed. With regard to Raman spectrometry, a substantially
monochromatic light source can be used. In general, near-infrared
wavelengths provide better depth of penetration into tissue. On the
other hand, as wavelengths increase, they begin to fall outside the
response range of silicon photo detectors (which have much better
signal-to-noise ratios than other currently available detectors).
One example of a light source that can be used is the widely
available 830 nanometer diode laser. This wavelength can penetrate
at least 1 to 2 millimeters into tissue. Additionally, this
wavelength is not absorbed by blood hemoglobin and is only weakly
absorbed by melanin. If the bone tissue is to be exposed by
incision, or if biopsied bone tissue is to be examined, other
wavelengths may be employed. For example, a 785 nanometer diode
laser could be used.
[0074] Many other wavelengths may be used as well. In general, a
wavelength of a light source may be chosen based on various factors
including one or more of a desired depth of penetration,
availability of photo detectors capable of detecting light at and
near the wavelength, efficiency of photo detectors, cost,
manufacturability, lifetime, stability, scattering efficiency,
penetration depth, etc. Any of a variety of substantially
monochromatic light sources can be used, including commercially
available light sources. For example, the article "Near-infrared
multichannel Raman spectroscopy toward real-time in vivo cancer
diagnosis," by S. Kaminaka, et al. (Journal of Raman Spectroscopy,
vol. 33, pp. 498-502, 2002) describes using a 1064 nanometer
wavelength light source with an InP/InGaAsP photomultiplier.
[0075] With regard to IR spectrometry, any of a variety of types of
light sources can be used, including commercially available light
sources. For example, light sources known to those of ordinary
skill in the art as being suitable for analysis of bone tissues can
be used.
[0076] With regard to the optical probe 116, any of variety optical
probes can be used, including commercially available optical
probes. For instance, the Handbook of Vibrational Spectroscopy,
Volume 2: Sampling Techniques, 1587-1597 (J. Chalmers et al. eds.,
John Wiley & Sons Ltd. 2002) describes examples of fiber optic
probes that can be used. For Raman spectrometry, optical probes
designed for Raman spectrometry may be used. For example, any of a
variety of commercially available fiber optic probes can be used.
Some commercially available fiber optic probes include filters to
reject Raman scatter generated within the excitation fiber and/or
to attenuate laser light entering the collection fiber or fibers.
Loosely focused light may help eliminate or minimize patient
discomfort as compared to tightly focused light. As is known to
those of ordinary skill in the art, loosely focused light may be
achieved by a variety of techniques including multimode delivery
fibers and a long focal length excitation/collection lens(es).
[0077] Existing commercially available fiber optic probes may be
modified, or new probes developed, to maximize collection
efficiency of light originating at depths of 1 millimeter or more
below the surface of a highly scattering medium, such as tissue.
Such modified, or newly developed probes, may offer better
signal-to-noise ratios and/or faster data collection. The probe may
be modified or may be coupled to another device to help maintain a
constant probe-to-tissue distance, which may help to keep the
system in focus and help maximize the collected signal.
[0078] If the bone is to be irradiated via an incision (and/or the
light is to be collected via an incision), relay optics may be
coupled to, or incorporated in, a needle. For example, two optical
fibers or an "n-around-one" array could be used. In general, the
size and the number of fibers should be appropriate to fit into a
needle. The diameter of the excitation/collection lens or lenses
used in such an embodiment could be small to help minimize the size
of the incision. For example, lenses of diameters between 0.3 and 1
millimeter could be used. Lenses having larger or smaller diameters
could be used as well. The lens(es) and or optical fibers could be
incorporated into a hypodermic needle such as a #12 French type
needle.
[0079] Additionally, a microprobe or microscope (e.g., a modified
epi-fluorescence microscope) may be used instead of the optical
probe 116 of FIG. 1. In this embodiment, the optical fiber 108
and/or the optical fiber 124 may be omitted.
[0080] The optical processor 140 may include one or more lenses for
focusing the collected light. The optical processor 140 may also
include one or more filters to attenuate laser light. Although
shown separate from the spectrum analyzer 132, some or all of the
optical processor 140 may optionally be a component of the spectrum
analyzer 132.
[0081] The spectrum analyzer 132 may comprise a spectrograph
optically coupled with a photo detector array. The photo detector
array may comprise a charge coupled device, or some other photo
detection device. For example, the article "Near-infrared
multichannel Raman spectroscopy toward real-time in vivo cancer
diagnosis," by S. Kaminaka, et al. (Journal of Raman Spectroscopy,
vol. 33, pp. 498-502, 2002) describes using a 1064 nanometer
wavelength light source with an InP/InGaAsP photomultiplier.
[0082] In another embodiment, the spectrum analyzer 132 may
comprise one or more filters to isolate a plurality of wavelengths
of interest. Then, one or more photo detectors (e.g., a CCD, an
avalanche photodiode, photomultiplier tube, etc.) could be
optically coupled to the output of each filter. A single detector
could be used with a tunable filter (e.g., an interferometer,
liquid crystal tunable filter, acousto-optic tunable filter, etc.)
or if fixed passband filters (e.g., dielectric filters, holographic
filters, etc.) are placed in front of the detector one at a time
using, for example, a slider, filter wheel, etc. In general, any of
a variety of spectrum analyzers could be used such as a Raman
analyzer, an IR spectrum analyzer, an interferometer, etc.
[0083] The computing device 144 may comprise, for example, an
analog circuit, a digital circuit, a mixed analog and digital
circuit, a processor with associated memory, a desktop computer, a
laptop computer, a tablet PC, a personal digital assistant, a
workstation, a server, a mainframe, etc. The computing device 144
may be communicatively coupled to the spectrum analyzer 132 via a
wired connection (e.g., wires, a cable, a wired local area network
(LAN), etc.) or a wireless connection (a BLUETOOTH.TM. link, a
wireless LAN, an IR link, etc.). In some embodiments, the spectral
content information generated by the spectrum analyzer 132 may be
stored on a disk (e.g., a floppy disk, a compact disk (CD), etc.),
and then transferred to the computing device 144 via the disk.
Although the spectrum analyzer 132 and the computer 144 are
illustrated in FIG. 1 as separate devices, in some embodiments the
spectrum analyzer 132 and the computing device 144 may be part of a
single device. For example, the computing device 144 (e.g., a
circuit, a processor and memory, etc.) may be a component of the
spectrum analyzer 132.
[0084] FIG. 5 is a block diagram of an example computing device 144
that may be employed. It is to be understood that the computer 340
illustrated in FIG. 5 is merely one example of a computing device
144 that may be employed. As described above, many other types of
computing devices 144 may be used as well. The computer 340 may
include at least one processor 350, a volatile memory 354, and a
non-volatile memory 358. The volatile memory 354 may include, for
example, a random access memory (RAM). The non-volatile memory 358
may include, for example, one or more of a hard disk, a read-only
memory (ROM), a CD-ROM, an erasable programmable ROM (EPROM), an
electrically erasable programmable ROM (EEPROM), a digital
versatile disk (DVD), a flash memory, etc. The computer 340 may
also include an I/O device 362. The processor 350, volatile memory
354, non-volatile memory 358, and the I/O device 362 may be
interconnected via one or more address/data buses 366. The computer
340 may also include at least one display 370 and at least one user
input device 374. The user input device 374 may include, for
example, one or more of a keyboard, a keypad, a mouse, a touch
screen, etc. In some embodiments, one or more of the volatile
memory 354, non-volatile memory 358, and the I/O device 362 may be
coupled to the processor 350 via one or more separate address/data
buses (not shown) and/or separate interface devices (not shown),
coupled directly to the processor 350, etc.
[0085] The display 370 and the user input device 374 are coupled
with the I/O device 362. The computer 340 may be coupled to the
spectrum analyzer 132 (FIG. 1) via the I/O device 362. Although the
I/O device 362 is illustrated in FIG. 5 as one device, it may
comprise several devices. Additionally, in some embodiments, one or
more of the display 370, the user input device 374, and the
spectrum analyzer 132 may be coupled directly to the address/data
bus 366 or the processor 350. Additionally, as described
previously, in some embodiments the spectrum analyzer 132 and the
computer 340 may be incorporated into a single device.
[0086] The previously described additional factors that may be used
for diagnosing a bone tissue disorder (e.g., one or more of an age
of the patient, a height of the patient, a weight of the patient, a
bone mineral density of the patient, a family history of the
patient, etc.) may be entered via the user input device 374, loaded
from a disk, received via a network (not shown), etc. These
additional factors may be stored in one or more of the memories 354
and 358. Additionally, previously measured spectral content
information may be loaded from a disk, received via a network (not
shown), etc., and stored in one or more of the memories 354 and
358.
[0087] A routine, for example, for estimating a susceptibility to
fracture based on spectral content information may be stored, for
example, in whole or in part, in the non-volatile memory 358 and
executed, in whole or in part, by the processor 350. For example,
the method 200 of FIG. 3 and/or the method 250 of FIG. 4 could be
implemented in whole or in part via a software program for
execution by the processor 350. The program may be embodied in
software stored on a tangible medium such as CD-ROM, a floppy disk,
a hard drive, a DVD, or a memory associated with the processor 350,
but persons of ordinary skill in the art will readily appreciate
that the entire program or parts thereof could alternatively be
executed by a device other than a processor, and/or embodied in
firmware and/or dedicated hardware in a well known manner. With
regard to the method 200 of FIG. 3 and the method 250 of FIG. 4,
one of ordinary skill in the art will recognize that the order of
execution of the blocks may be changed, and/or the blocks may be
changed, eliminated, or combined.
[0088] Although the method 200 of FIG. 3 and the method 250 of FIG.
4 were described above as being implemented by the computer 340,
one or more of the blocks of FIGS. 3 and 4 may be implemented by
other types of devices such as an analog circuit, a digital
circuit, a mixed analog and digital circuit, a processor with
associated memory, etc.
[0089] Determining Cartilage Conditions
[0090] Although the example apparatus described above were
described in the context of analyzing bone tissue, these apparatus
or similar apparatus can be used to determine conditions associated
with other connective tissues. Generally, connective tissue
comprises a biological tissue having an extensive extracellular
matrix. Connective tissue helps form a framework and/or support
structure for body tissues, organs, etc. Examples of connective
tissue that can be analyzed include supporting connective tissue
(e.g., bone, cartilage, etc.), fibrous connective tissue (e.g.,
cartilage, tendons, ligaments, etc.), loose connective tissue,
adipose tissue, etc.
[0091] As described above, connective tissues such as cartilage may
be analyzed. At least some spectral information associated with
cartilage can be distinguished from spectral information associated
with bone, and thus techniques for determining cartilage conditions
based on spectral information may be performed in vivo.
[0092] FIG. 7 is a flow diagram of an example method for
determining a condition related to cartilage tissue of a patient.
The method 400 may be implemented by an apparatus such as the
apparatus 100 of FIG. 1, and will be described with reference to
FIG. 1. At a block 404, a portion of cartilage tissue of a patient
is irradiated with light. For example, the optical probe 116 may be
used to irradiate the cartilage tissue with light generated by the
light source 104. In one embodiment, the cartilage tissue may be
irradiated non-invasively through the skin of the patient. In other
embodiments, cartilage tissue exposed by an incision, or removed as
a biopsy, may be irradiated.
[0093] At a block 408, light scattered, reflected, or transmitted
by the cartilage tissue may be collected. For example, the optical
probe 116 may collect light scattered by the cartilage tissue
(Raman spectrometry). As another example, the optical probe 116 may
collect light reflected by the cartilage tissue ("attenuated total
reflection" IR spectrometry). Alternatively, the optical probe 128
may collect light transmitted by the cartilage tissue ("line of
sight" IR spectrometry). As with the optical probe 116, the optical
probe 128 may collect light non-invasively through the skin of the
patient. In other embodiments, the light may be collected via an
incision or collected from an irradiated biopsy.
[0094] At a block 412, spectral content information associated with
the collected light is generated. For example, the light collected
by the optical probe 116 or the optical probe 128 may be provided
to the spectrum analyzer 132 via the optical processor 140. The
spectrum analyzer 132 may then generate spectral content
information associated with the light received by the spectrum
analyzer 132.
[0095] In Raman spectrometry, the cartilage spectrum of the
collected light scattered from cartilage tissue (referred to
hereinafter as the "Raman spectrum of the cartilage tissue") is
indicative of the physico-chemical state of the cartilage tissue.
The Raman spectrum of the cartilage tissue includes bands
indicative of various components present in cartilage tissue
including phosphate, carbonate, etc. Also included are bands
indicative of various components of the collagen matrix of the
cartilage tissue including amide I, amide III, etc. The wavelength
at which a band is located is indicative of the component of the
mineral or matrix to which it corresponds. The height and/or
intensity of a band are indicative of the amount of the
corresponding component.
[0096] Similar to the Raman spectrum of the cartilage tissue, in IR
spectrometry, the spectrum of the collected light transmitted by
the cartilage tissue (referred to hereinafter as the "IR spectrum
of the cartilage tissue") includes bands indicative of components
and structure of the cartilage tissue. Unlike in Raman
spectrometry, however, the bands in the IR spectrum of the
cartilage tissue are indicative of light absorbed by the cartilage
tissue, rather than light scattered by the cartilage tissue.
Nevertheless, the IR spectrum of the cartilage tissue is also
indicative of the physico-chemical state of the cartilage tissue.
As is known to those of ordinary skill in the art, the Raman
spectrum of a cartilage tissue and an IR spectrum of the same
cartilage tissue may provide indications of different components
and/or different structure of the cartilage tissue.
[0097] At a block 416, it is determined whether the patient has a
cartilage tissue condition based on the spectral content
information generated at block 412. For example, the computing
device 144 may receive spectral content information from the
spectrum analyzer 132. The computing device 144 may then generate
an indication, based at least in part on the spectral content
information, of whether the patient has a cartilage tissue
condition. The cartilage tissue condition may be, for example,
osteoarthritis, rheumatoid arthritis, chondromalacia,
polychondritis, relapsing polychondritis, a genetic disorder, an
acquired disorder, etc. Also, the cartilage tissue condition may be
an increased risk of developing a disease such as osteoarthritis,
rheumatoid arthritis, chondromalacia, polychondritis, etc. A
computing device such as the computing device 340 of FIG. 6 may be
used to generate the indication. In some embodiments, the computing
device 144 may generate, based on the spectral content information
generated at block the 412, an indicator associated with the
cartilage tissue condition. Such an indicator may be used by a
physician to determine whether the patient has a cartilage tissue
condition, to monitor the progression of a cartilage tissue
condition, to monitor a response to treatment of a cartilage tissue
condition, etc.
[0098] The determination of the block 416 may be based on
additional factors. For example, the determination may be further
based on one or more of an age of the patient, a weight of the
patient, a history of weight of the patient, a blood test, an
analysis of synovial fluid, a medical history of the patient (e.g.,
past joint injuries), an X-ray, a family history of the patient,
etc. Determining whether the patient has a cartilage tissue
condition will be described in more detail below.
[0099] Blocks 404, 408, and 412 may optionally be repeated over a
period of time (e.g., weeks, months, years) to generate spectral
content information that reflects the cartilage tissue condition of
the patient over the period of time. This spectral content
information over the period of time may be used in the
determination of block 416.
[0100] Evaluating Osteoarthritis Conditions
[0101] Examples of techniques for generating an indicator, based on
spectral content information, of osteoarthritis are provided below.
Many other techniques may be employed as well. In general,
embodiments of methods for generating such an indicator may vary
according to the environment in which they are to be used. For
example, different embodiments may be used in a clinical setting as
compared to a laboratory setting because signal-to-noise ratios
likely will be higher in the laboratory setting as compared to the
clinical setting.
[0102] In some embodiments in which Raman spectrometry is employed,
the intensity of particular bands in the Raman spectrum of the
cartilage tissue may be used to generate an indicator of
osteoarthritis. The intensity of a band may be determined by, for
example, determining an area under the band or determining a height
of the band.
[0103] Amide I and amide III are observable in both IR and Raman
spectrometry. Amide I and amide III spectra include information
similarly indicative of the structure of collagen in the cartilage
tissue. In Raman spectrometry, amide III of cartilage tissue is
associated with a plurality of bands that can extend over much of
the 1240 cm.sup.-1 to 1270 cm.sup.-1 region. Also observable are
bands associated with minerals present in the cartilage tissue. For
example, bands associated with carbonate .nu..sub.1 and phosphate
.nu..sub.1 are observable.
[0104] FIG. 8 is a flow diagram illustrating one embodiment of a
method 430 for generating an indicator of a cartilage tissue
condition based on intensities of particular bands in a Raman
spectrum of cartilage tissue. A similar technique may be employed
for use with an IR spectrum of cartilage tissue.
[0105] At a block 434, an intensity of a carbonate .nu..sub.1 band
(nominally located at approximately 1070 cm.sup.-1) associated with
the cartilage tissue is determined. Determining the intensity of
this band may include measuring the height of a peak of the band.
Also, determining the intensity of this band may include
determining the area under the band by curve fitting using a
function such as a mixed Gaussian-Lorentzian function. Determining
the area of the band may also include measuring the area without
curve fitting. For example, the area could be measured based on the
raw data. As another example, the raw data could be filtered (e.g.,
with a smoothing filter), and the height or area could be measured
based on the filtered data. In general, the intensity of one or
more bands may be determined using any of a variety of techniques,
including known techniques. At a block 438, an intensity of a
phosphate .nu..sub.1 band (nominally located at approximately 959
cm.sup.-1) associated with the cartilage tissue is determined.
Determining the intensity of this band may be performed in the same
or similar manner as described with reference to block 434.
[0106] At a block 442, a ratio of the intensity determined at the
block 434 with the intensity determined at the block 438 may be
determined. Then, at a block 446, an indicator of osteoarthritis is
determined based on the ratio determined at the block 446. It is
believed that cartilage tissue affected by osteoarthritis has a
higher carbonate/phosphate ratio as compared with cartilage not
affected by osteoarthritis.
[0107] Determining the indicator may comprise determining in which
of one or more sets of values the ratio falls by, for example,
comparing the ratio to one or more thresholds. In one embodiment,
the indicator may comprise an indication of whether or not
osteoarthritis is present. In other embodiments, the indicator may
comprise one of a plurality of levels indicating a probability or
confidence level that osteoarthritis is present. In still other
embodiments, the indicator may comprise one of a plurality of
levels indicating a risk of developing osteoarthritis. In yet other
embodiments, the indicator may comprise one of a plurality of
levels indicating a level of severity, a level of progression,
etc., of osteoarthritis.
[0108] As described previously, the indicator determined at the
block 446 may be based on additional factors such as one or more of
an age of the patient, a weight of the patient, a prior weight of
the patient, blood test data, synovial fluid test data, medical
history data (e.g., past joint injuries), X-ray data, family
history data, etc.
[0109] FIG. 9 is a flow diagram illustrating another embodiment of
a method 460 for generating an indicator of osteoarthritis based on
intensities of particular bands in a Raman spectrum of cartilage
tissue. A similar technique may be employed for use with an IR
spectrum of cartilage tissue.
[0110] At a block 464, an intensity of a band associated with the
cartilage tissue having peak nominally at approximately 1240
cm.sup.-1 is determined. Determining the intensity of this band may
be performed in the same or similar manner as described above. At a
block 468, an intensity of a band associated with the cartilage
tissue having peak nominally at approximately 1270 cm.sup.-1 is
determined. Determining the intensity of this band may be performed
in the same or similar manner as described above.
[0111] At a block 472, a ratio of the intensity determined at the
block 464 with the intensity determined at the block 468 may be
determined. Then, at a block 476, an indicator of osteoarthritis is
determined based on the ratio determined at the block 472. It is
believed that cartilage tissue affected by osteoarthritis has a
higher 1240 cm.sup.-1 band/1270 cm.sup.-1 band ratio as compared
with cartilage not affected by osteoarthritis.
[0112] Determining the indicator may comprise determining in which
of one or more sets of values the ratio falls. In one embodiment,
the indicator may comprise an indication of whether or not
osteoarthritis is present. In other embodiments, the indicator may
comprise one of a plurality of levels indicating a probability or
confidence level that osteoarthritis is present. In still other
embodiments, the indicator may comprise one of a plurality of
levels indicating a risk of developing osteoarthritis. In yet other
embodiments, the indicator may comprise one of a plurality of
levels indicating a level of severity, a level of progression,
etc., of osteoarthritis. As described previously, the indicator
determined at the block 476 may be based on additional factors.
[0113] FIG. 10 is a flow diagram illustrating yet another
embodiment of a method 480 for generating an indicator of a
cartilage tissue condition based on intensities of particular bands
in a Raman spectrum of cartilage tissue. A similar technique may be
employed for use with an IR spectrum of cartilage tissue.
[0114] At a block 484, an intensity of one or more mineral bands
associated with the cartilage tissue is determined. For example,
the intensity of the carbonate .nu..sub.1 band and the phosphate
.nu..sub.1 band may be determined by adding their individual
intensities together. Determining the intensity of each individual
band in the one or more mineral bands may be performed in the same
or similar manner as described above. At a block 486, an intensity
of a CH.sub.2 wag band associated with the cartilage tissue having
peak nominally at approximately 1446 cm.sup.-1 is determined.
Determining the intensity of this band may be performed in the same
or similar manner as described above.
[0115] At a block 488, a ratio of the intensity determined at the
block 484 with the intensity determined at the block 486 may be
determined. Then, at a block 490, an indicator of osteoarthritis is
determined based on the ratio determined at the block 488. It is
believed that cartilage tissue affected by osteoarthritis has a
lower mineral/CH.sub.2 wag ratio as compared with cartilage not
affected by osteoarthritis. With regard to the block 486, other
bands associated with the collagen matrix of the cartilage tissue
may be used in place of the CH.sub.2 wag band such as amide I (1665
cm.sup.-1), amide III (1240 cm.sup.-1-1270 cm.sup.-1), 855 cm-1,
880 cm.sup.-1, 919 cm.sup.-1, etc. Generally, the ratio determined
at the block 486 indicates the amount of mineral per collagen.
[0116] Determining the indicator may comprise determining in which
of one or more sets of values the ratio falls. In one embodiment,
the indicator may comprise an indication of whether or not
osteoarthritis is present. In other embodiments, the indicator may
comprise one of a plurality of levels indicating a probability or
confidence level that osteoarthritis is present. In still other
embodiments, the indicator may comprise one of a plurality of
levels indicating a risk of developing osteoarthritis. In yet other
embodiments, the indicator may comprise one of a plurality of
levels indicating a level of severity, a level of progression,
etc., of osteoarthritis. As described previously, the indicator
determined at the block 490 may be based on additional factors.
[0117] In other embodiments, one or more of the methods described
above with respect to FIGS. 8-10 may be combined. For example, an
indicator of osteoarthritis could be determined based on the ratio
determined at the block 442 of FIG. 8 and the ratio determined at
the block 472 of FIG. 9. As another example, the indicator of
osteoarthritis could be determined based on the ratio determined at
the block 442 of FIG. 8 and the ratio determined at the block 488
of FIG. 10. As yet another example, the indicator of osteoarthritis
could be determined based on the ratio determined at the block 472
of FIG. 9 and the ratio determined at the block 488 of FIG. 10. As
still another example, the indicator of osteoarthritis could be
determined based on the ratio determined at the block 442 of FIG.
8, the ratio determined at the block 472 of FIG. 9, and the ratio
determined at the block 488 of FIG. 10. Multiple ratios may be used
to determine an indicator of osteroarthritis using any of a variety
of techniques. As one example, the multiple ratios may be
mathematically combined and then the result could be compared to
one or more thresholds. As another example, multiple indicators
determined based on the multiple ratios could be mathematically
combined.
[0118] Other information in the IR spectrum or the Raman spectrum
of the cartilage tissue can be used in addition to, or as an
alternative, the information described above. For example,
information related to bands other than those described above could
be used. Additionally, information related to the width, shape
(e.g., whether or not a band has "shoulders"), height, etc. of
particular bands could be used in determining a cartilage tissue
condition. Additionally, more sophisticated analyses could be
employed such as a cluster analysis, pattern matching, etc.
[0119] The locations of particular Raman bands described above with
reference to FIGS. 8-10 were determined based on experiments with
mouse tissues. One of ordinary skill in the art will recognize that
the locations of bands may vary based on, for example, testing
error, age, species, etc. For instance, the locations may vary by
up to plus or minus 3 cm.sup.-1, or even more.
[0120] Evaluating a Tissue Condition Based on Spectral Analysis of
Another Tissue
[0121] Spectral content information obtained from one tissue in a
first portion of the body may be used to determine a condition
associated with tissue in a second portion of the body remote from
the first portion. As an example, spectral content information
obtained from an eyeball, a nose, an ear, etc., may be used to
generate an indicator of osteoarthritis in a hand (including
fingers), a foot (including toes), a knee, a hip, a back, a neck,
etc. Examples of techniques for generating, based on spectral
content information obtained one portion of the body, an indicator
of osteoarthritis in another portion of the body are provided
below. Many other techniques may be employed as well. In general,
embodiments of methods for generating such an indicator may vary
according to the environment in which they are to be used. For
example, different embodiments may be used in a clinical setting as
compared to a laboratory setting because signal-to-noise ratios
likely will be higher in the laboratory setting as compared to the
clinical setting.
[0122] It has been observed that some spectral content obtained
from a first tissue of the body may exhibit similarities with a
second tissue remote from the first tissue. For example, ocular
tissue includes collagen structures, and spectral content
information associated with collagen in ocular tissue may be used
to determine if a condition exists with respect to cartilage tissue
in a joint of a hand, a foot, a back, a neck, a knee joint, a hip
joint, etc. As another example, spectral content information
associated with cartilage in nose and/or ear may be used to
determine if a condition exists with respect to cartilage tissue in
a joint of a hand, a foot, a back, a neck, a knee joint, a hip
joint, etc.
[0123] FIG. 11 is a flow diagram of an example method for
determining a connective tissue condition. The method 500 may be
implemented by an apparatus such as the apparatus 100 of FIG. 1,
and will be described with reference to FIG. 1. At a block 504, a
portion of connective tissue of a patient is irradiated with light,
the connective tissue being at a first location of the patient's
body. For example, the optical probe 116 may be used to irradiate
the connective tissue with light generated by the light source 104.
In one embodiment, the connective tissue may be irradiated
non-invasively. For example, ocular tissue may be irradiated. As
another example, nasal or ear cartilage may be irradiated through
skin or mucus membrane. In other embodiments, connective tissue
exposed by an incision, or removed as a biopsy, may be
irradiated.
[0124] At a block 508, light scattered, reflected, or transmitted
by the connective tissue may be collected. For example, the optical
probe 116 may collect light scattered by the connective tissue
(Raman spectrometry). As another example, the optical probe 116 may
collect light reflected by the connective tissue ("attenuated total
reflection" IR spectrometry). Alternatively, the optical probe 128
may collect light transmitted by the connective tissue ("line of
sight" IR spectrometry). As with the optical probe 116, the optical
probe 128 may collect light non-invasively (e.g., through the skin
of the patient). In other embodiments, the light may be collected
via an incision or collected from an irradiated biopsy, for
example.
[0125] At a block 512, spectral content information associated with
the collected light is generated. For example, the light collected
by the optical probe 116 or the optical probe 128 may be provided
to the spectrum analyzer 132 via the optical processor 140. The
spectrum analyzer 132 may then generate spectral content
information associated with the light received by the spectrum
analyzer 132.
[0126] At a block 516, it is determined whether the patient has a
condition related to connective tissue at a second location in the
body, remote from the first location, based on the spectral content
information generated at block 512. For example, the computing
device 144 may receive spectral content information from the
spectrum analyzer 132. The computing device 144 may then generate
an indication, based at least in part on the spectral content
information, of whether the patient has a connective tissue
condition regarding connective tissue at the second location. The
condition may be a cartilage condition such as osteoarthritis,
rheumatoid arthritis, chondromalacia, polychondritis, relapsing
polychondritis, a genetic disorder, an acquired disorder, etc.
Also, the connective tissue condition may be an increased risk of
developing a disease such as osteoarthritis, rheumatoid arthritis,
chondromalacia, polychondritis, etc. A computing device such as the
computing device 340 of FIG. 6 may be used to generate the
indication. In some embodiments, the computing device 144 may
generate, based on the spectral content information generated at
block the 412, an indicator associated with the connective tissue
condition. Such an indicator may be used by a physician to
determine whether the patient has a connective tissue condition, to
monitor the progression of a connective tissue condition, to
monitor a response to treatment of a connective tissue condition,
etc.
[0127] The determination of the block 516 may be based on
additional factors. For example, the determination may be further
based on one or more of an age of the patient, a weight of the
patient, a history of weight of the patient, a blood test, an
analysis of synovial fluid, a medical history of the patient (e.g.,
past joint injuries), an X-ray, a family history of the patient,
etc. Determining whether the patient has a connective tissue
condition associated with connective tissue at the second location
will be described in more detail below.
[0128] Blocks 504, 508, and 512 may optionally be repeated over a
period of time (e.g., weeks, months, years) to generate spectral
content information that reflects the connective tissue condition
of the patient over the period of time. This spectral content
information over the period of time may be used in the
determination of block 516.
[0129] FIG. 12 is a flow diagram illustrating another embodiment of
a method 550 for generating an indicator of osteoarthritis in a
cartilage tissue at a first location in the body based on
intensities of particular bands in a Raman spectrum of tissue at a
second location in the body remote from the first location. A
similar technique may be employed for use with an IR spectrum of
tissue.
[0130] At a block 554, an intensity of a band associated with the
tissue having peak nominally at approximately 1230 cm.sup.-1 is
determined. Determining the intensity of this band may be performed
in the same or similar manner as described above. At a block 558,
an intensity of a band associated with the tissue having peak
nominally at approximately 1260 cm.sup.-1 is determined.
Determining the intensity of this band may be performed in the same
or similar manner as described above.
[0131] At a block 562, a ratio of the intensity determined at the
block 554 with the intensity determined at the block 558 may be
determined. Then, at a block 566, an indicator of osteoarthritis is
determined based on the ratio determined at the block 562. It is
believed that ocular tissue, nasal cartilage tissue, ear cartilage
tissue, etc., in a person affected by osteoarthritis may have a
higher 1230 cm.sup.-1 band/1260 cm.sup.-1 band ratio as compared
with tissues in a person not affected by osteoarthritis.
[0132] Determining the indicator may comprise determining in which
of one or more sets of values the ratio falls. In one embodiment,
the indicator may comprise an indication of whether or not
osteoarthritis is present. In other embodiments, the indicator may
comprise one of a plurality of levels indicating a probability or
confidence level that osteoarthritis is present. In still other
embodiments, the indicator may comprise one of a plurality of
levels indicating a risk of developing osteoarthritis. In yet other
embodiments, the indicator may comprise one of a plurality of
levels indicating a level of severity, a level of progression,
etc., of osteoarthritis. As described previously, the indicator
determined at the block 566 may be based on additional factors.
[0133] Generating the indicator of osteoarthritis may additionally
or alternatively comprise other types of analysis of the spectral
content information. For example, different ratios such as the
ratios described in FIGS. 8, 9, and 10 may be used additionally or
alternatively in generating the indicator. In general, the spectral
content information obtained from tissue at a first location in the
body an indicator of a tissue condition in a connective tissue at a
first location in the body and used to generate an indicator of a
condition of connective tissue at a second location in the body
remote from the first location may depend on the type of tissue at
the first location, the type of tissue at the second location, the
type of condition, etc.
[0134] Evaluating Ocular Tissue
[0135] Although examples described above include apparatus and
techniques for analyzing bone tissue and cartilage tissue, similar
techniques and these apparatus or similar apparatus can be used to
determine conditions associated with ocular tissues as well. For
example, collagen in ocular tissues may become damaged as a result
of natural aging, genetic defects, disease, etc. Alterations to eye
collagen may occur before clinical symptoms are evident.
[0136] FIG. 13 is a flow diagram of an example method for
determining a condition related to ocular tissue of a patient. The
method 600 may be implemented by an apparatus such as the apparatus
100 of FIG. 1, and will be described with reference to FIG. 1. At a
block 604, a portion of ocular tissue of a patient is irradiated
with light. For example, the optical probe 116 may be used to
irradiate the ocular tissue with light generated by the light
source 104. In one embodiment, the ocular tissue may be irradiated
non-invasively.
[0137] At a block 608, light scattered, reflected, or transmitted
by the ocular tissue may be collected. For example, the optical
probe 116 may collect light scattered by the ocular tissue (Raman
spectrometry). As another example, the optical probe 116 may
collect light reflected by the ocular tissue ("attenuated total
reflection" IR spectrometry). Alternatively, the optical probe 128
may collect light transmitted by the ocular tissue ("line of sight"
IR spectrometry). As with the optical probe 116, the optical probe
128 may collect light non-invasively, in one embodiment.
[0138] At a block 612, spectral content information associated with
the collected light is generated. For example, the light collected
by the optical probe 116 or the optical probe 128 may be provided
to the spectrum analyzer 132 via the optical processor 140. The
spectrum analyzer 132 may then generate spectral content
information associated with the light received by the spectrum
analyzer 132.
[0139] In Raman spectrometry, the spectrum of the collected light
scattered from ocular tissue (referred to hereinafter as the "Raman
spectrum of the ocular tissue") is indicative of the
physico-chemical state of the ocular tissue. The Raman spectrum of
the ocular tissue includes bands indicative of various components
present in ocular tissue including bands indicative of various
components of the collagen matrix of the ocular tissue. The
wavelength at which a band is located is indicative of the
component, and the height and/or intensity of a band is indicative
of the amount of the corresponding component.
[0140] Similar to the Raman spectrum of the ocular tissue, in IR
spectrometry, the spectrum of the collected light transmitted by
the ocular tissue (referred to hereinafter as the "IR spectrum of
the ocular tissue") includes bands indicative of components and
structure of the ocular tissue. Unlike in Raman spectrometry,
however, the bands in the IR spectrum of the ocular tissue are
indicative of light absorbed by the ocular tissue, rather than
light scattered by the ocular tissue. Nevertheless, the IR spectrum
of the ocular tissue is also indicative of the physico-chemical
state of the ocular tissue. The Raman spectrum of an ocular tissue
and an IR spectrum of the same ocular tissue may provide
indications of different components and/or different structure of
the ocular tissue.
[0141] At a block 616, it is determined whether the patient has an
ocular tissue condition based on the spectral content information
generated at block 412. For example, the computing device 144 may
receive spectral content information from the spectrum analyzer
132. The computing device 144 may then generate an indication,
based at least in part on the spectral content information, of
whether the patient has an ocular tissue condition. The ocular
tissue condition may be, for example, macular degeneration,
cataracts, glaucoma, keratitis, scleritis, episcleritis, a genetic
disorder, an acquired disorder, etc. Also, the ocular tissue
condition may be an increased risk of developing an ocular disease.
As further examples, the indication may be of the compatibility of
donated ocular tissue, integrity of a cornea after a corneal
transplant, an indication of whether cataract surgery is needed,
etc.
[0142] A computing device such as the computing device 340 of FIG.
6 may be used to generate the indication. In some embodiments, the
computing device 144 may generate, based on the spectral content
information generated at block the 612, an indicator associated
with the ocular tissue condition. Such an indicator may be used by
a physician to determine whether the patient has an ocular tissue
condition, to monitor the progression of an ocular tissue
condition, to monitor a response to treatment of an ocular tissue
condition, etc.
[0143] The determination of the block 616 may be based on
additional factors. For example, the determination may be further
based on one or more of an age of the patient, a medical history of
the patient (e.g., past ocular tissue conditions), a family history
of the patient, etc.
[0144] Blocks 604, 608, and 612 may optionally be repeated over a
period of time (e.g., weeks, months, years) to generate spectral
content information that reflects the ocular tissue condition of
the patient over the period of time. This spectral content
information over the period of time may be used in the
determination of block 616.
[0145] Experiments
[0146] In one experiment, Del 1 (+/-) transgenic mice containing 6
copies of a transgene with a small deletion mutation in the type II
collagen gene and that were predisposed to early osteoarthritis
were analyzed. The femoral articular cartilage was obtained from
Del 1 (+/-) mice at 8 ages (2.5, 3, 5, 7, 9, 10, 13, and 16
months), with age-matched wildtype (wt) controls. The femoral
articular cartilage was isolated en bloc and subject to Raman
spectroscopy with 785 nm laser excitation and an Olympus BH-2
microscope equipped with a 20.times./0.75 NA Zeiss Fluar objective.
At each time point, the articular surfaces of three to four
transects per femur were examined. Raw spectra were baselined with
a polynomial, and curve fitted with GRAMS/AI.COPYRGT. software.
Bands associated with cartilage matrix proteins were analyzed using
the Students t-test. All p values less than 0.05 were considered
statistically significant.
[0147] No statistically significant difference was observed between
the 2.5 and 3 month old Del1 (+/-) and wt mice. At 5 months of age,
however, some differences in the composition and structure of the
tissue were detected between the Del 1 (+/-) and wt mice. In
general, the Del 1 (+/-) mice had larger carbonate
.nu..sub.1:phosphate .nu..sub.1 (CO.sub.3:PO.sub.4) ratios than wt
mice, and this difference increased with age. The higher
CO.sub.3:PO.sub.4 ratio reflects a more carbonated mineral, which
is more crystalline and has the potential to compensate for tissue
weaknesses. In addition, the Del 1 (+/-) mice exhibited higher 1240
cm.sup.-1:1270 cm.sup.-1 band (1240:1270 band) area ratios than
their age-matched controls, which indicates a more disordered
secondary structure of collagen. The correlation between age and
the 1240:1270 ratio was best fit with a second-degree polynomial.
FIG. 14A is a graph showing 1240:1270 ratios of wt mice, the
second-degree polynomial to which it was fit, and the R.sup.2 value
associated with the fit. FIG. 14B is a graph showing 1240:1270
ratios of Del 1 (+/-) mice, the second-degree polynomial to which
it was fit, and the R.sup.2 value associated with the fit. In both
of FIGS. 11A and 11B, the vertical axes are in arbitrary units
(A.U.).
[0148] Differences between Del 1 (+/-) and wt mice were discerned
at as early as 5 months of age. The non-linear relationship between
age and the 1240:1270 ratio suggests that at a particular age, the
extracellular matrix accumulates changes in the tertiary structure
of the collagen fibrils, as is evidenced by the progressive
decrease in overall order. This change occurred for the Del 1 (+/-)
mice at approximately 6 months of age, and for the wt mice at
approximately 11 months of age. It is possible that the 1240:1270
band area ratio indicates the age at which irreversible damage
begins to occur within the femoral articular cartilage.
[0149] In another experiment, Del 1 (+/-) transgenic mice
containing 6 copies of a transgene with a small deletion mutation
in the type II collagen gene and that were predisposed to early
osteoarthritis were analyzed. Murine femoral articular cartilage
was obtained from Del 1 (+/-) mice at 8 ages (2, 2.5, 3, 5, 7, 9,
13, and 16 months), with age-matched wildtype (wt) controls. The
Del 1 (+/-) mice had early onset of flattening of femoral condyles,
erosion of articular cartilage, sclerosis of subchondral bone,
degeneration of the menisci, pyknotic chondrocyte nuclei, with
clusters of reactive chondrocytes at the margins of the
defects.
[0150] Raman spectra were obtained with 785 nm laser excitation. To
improve signal-to-noise ratio images were acquired and component
spectra were extracted using multivariate analysis allowing the
separation of cartilage spectra from mineral spectra. Although
there are similarities between the spectra of cartilage and bone
matrix, the Raman spectra patterns are distinct because type II
collagen is not chemically identical to type I collagen.
Additionally, the Raman spectra information includes bands
associated with specific proteoglycans in cartilage.
[0151] Differences between the Raman spectra of cartilage of Del 1
(+/-) and wt mice were observed. Also, differences in the Raman
spectra of cartilage were observed with differences in age.
Differences were particularly notable at the 1685 cm.sup.-1 band,
comparing the ages 13 months to 16 months, and comparing the ages 2
months and 7 months. It is believed that differences at the 1685
cm.sup.-1 band possibly reflect immature crosslinks or ruptured
crosslinks in collagen. At each point, the Del 1 (+/-) mice had a
higher carbonate/phosphate ratio. This may indicate that Del 1
(+/-) mice cartilage had a more crystalline mineral content. Also,
at each point, the Del 1 (+/-) mice had a higher 1240:1270 ratio.
This may indicate that Del 1 (+/-) mice cartilage had a more
disordered structure of collagen. Further, at each point, the Del 1
(+/-) mice had a lower mineral/matrix ratio, where the
mineral/matrix ratio was calculated based on bands associated with
carbonate and phosphate (mineral) and a band located at
approximately 1446 cm-1 (matrix). This may indicate that Del 1
(+/-) mice cartilage had less mineral per collagen.
[0152] In yet another experiment, Del 1 (+/-) transgenic mice were
analyzed. For each animal, one or both femurs, with articular
cartilage of the knee joints intact, were analyzed from mice at ten
ages (2, 2.5, 3, 5, 7, 9, 10, 12, 13, and 16 months), with
age-matched wildtype (wt) controls.
[0153] Three to 10 transects were taken across the entire femoral
articular surface, with a majority of the transects taken along the
interior edges of the medial and lateral condyles. Transects taken
from the posterior or anterior cruciate ligaments, located between
the femoral condyles, were easily distinguishable from cartilage in
both white-light images and Raman spectra (data not shown), and
were not used in band area calculations. The near-IR excitation
laser, 785 nm (Kaiser Optical Systems Invictus), was line-focused
through an Olympus BH-2 microscope equipped with a 20.times./0.75
NA Zeiss Fluar objective. Incident power on the specimen was
approximately 100 mW with a 0.25 neutral density filter. The light
was focused to a line approximately 100 micrometers long and 1
micrometer wide. Raman-scattered light was collected through the
objective and analyzed by an axial transmissive spectrograph
(Kaiser Optical Systems Holospec f/1.8i) operated at 4 cm.sup.-1
resolution. The scattered light was integrated for 5 minutes on a
back-illuminated deep depletion CCD camera (Andor Technology DU
420-BR-DD).
[0154] Because line-focusing was used, transects consisting of 254
Raman spectra each, spaced at intervals of 0.5 micrometers along
the line, were taken at each sampling area on the femur. From each
transect, a normalized mean spectrum was obtained to offset the
effect of sample heterogeneity. For each femur, three to ten
transects were obtained. Transects were spectra were pre-processed
and averaged into a normalized spectrum using MATLAB.RTM. software
from The MathWorks. The normalized spectra were baselined with a
user-defined multi-point baseline, and curve fitted with GRAMS.RTM.
software from Thermo Electron Corporation. The band envelopes in
the baselined spectra were fitted to mixed Gaussian/Lorentzian
bands. The constraints for the curve fitting were that all band
intensities were non-negative and that the fit yielded an R.sup.2
value of 0.99 or greater. The curve fitting procedure generally
yielded bands that were readily identified based on Raman band
assignments from previous experiments on bone matrix as summarized
in Table 1.
TABLE-US-00001 TABLE 1 Raman Shift (cm.sup.-1) Assignment Component
850 .sub..nu.CC hydroxproline matrix 876 .sub..nu.CC hydroxproline
matrix 945 disordered apatite mineral 958 .sub..nu.1
PO.sub.4.sup.3- mineral 1001 ring breathing of phenylalanine matrix
1070 .sub..nu.2 CO.sub.3.sup.2- mineral 1240 amide III (disordered)
matrix 1270 amide III (ordered) matrix 1450 CH.sub.2 wag matrix
1667 amide I matrix
[0155] Band area ratios were used to measure carbonate:phosphate
ratios (carbonate 1070 cm.sup.-1:phosphate 958 cm.sup.-1 and 945
cm.sup.-1), disordered:ordered phosphate (disordered phosphate 945
cm.sup.-1:ordered phosphate 958 cm.sup.-1) and cartilage collagen
disorder (amide III components, 1240 cm.sup.-1:1270 cm.sup.-1, or
amide I, 1670 cm.sup.-1:1685 cm.sup.-1).
[0156] Three to 10 Raman transects were taken on each femur, and a
normalized mean spectrum from each transect was used to calculate
band areas. All band areas ratios from specimens corresponding to a
single age and genotype were grouped to yield a single mean ratio.
P-values were determined using Student's T-test (two-tailed with
unequal variances).
[0157] To elucidate spectral components of cartilage, Raman spectra
of solid samples of type I collagen, type II collagen, proteoglycan
and chondroitin-6-sulfate were obtained. Spectra from proteoglycan
and chondroitin-6-sulfate are not presented because contributions
from these two components were not observed in articular cartilage
spectra. Previous literature showed that polysaccharide components
(chondroitin sulfate, proteoglycan, and keratin sulfate) of
non-mineralized cartilage also made an insignificant contribution
to the overall Raman spectrum. Raman spectra from types I and II
collagen were compared. Spectral features from hydroxyproline,
aromatic ring breathing, amide III and amide I are observed (880
cm.sup.-1, 1001 cm.sup.-1, 1220-1280 cm.sup.-1 and 1600-1720
cm.sup.-1, respectively). An obvious difference in the spectra of
solid type I and type II collagen is the location of local peak
maxima in the Amide III region. The amide III band corresponding to
disordered collagen in type I collagen is located around 1244
cm.sup.-1, while it is located closer to 1230 cm.sup.-1 in type II
collagen. A comparison of the articular cartilage spectra with
these dry reference spectra indicates that major spectral
contributions in articular cartilage arose from mineral components
and the collagen proteins.
[0158] Raw and baselined spectra were compared. The phosphate band
at 958 cm.sup.-1 is strong in the raw spectrum but individual peaks
are difficult to identify. Raman spectra of articular cartilage for
both wild-type and transgenic mice showed strong mineral signatures
at 958 cm.sup.-1 and 1070 cm.sup.-1 in addition peaks attributed to
type II collagen. Raman bands corresponding to phosphate and
carbonate species (958 cm.sup.-1 and 1070 cm.sup.-1 respectively)
were readily observed and the mean band positions for bands in the
amide III region represented type II collagen. Given a variable
cartilage depth, the weak Raman scatter of polysaccharide
components, and a lower concentration of collagen in the surface
layers, this observation was expected. It is believed that a mean
normalized spectrum representative of the Raman transect minimized
the effects of cartilage depth and sample heterogeneity on band
areas.
[0159] Raman spectroscopic values for Del1 (+/-) and wt mice are
listed in Table 2. Raman spectroscopic analysis revealed no
significant spectroscopic difference between the age-matched 2.5
and 3 month old wt and Del1 (+/-) mice. However, by age 6 months,
differences in the composition and structure of the tissue between
the wt and transgenic mice were identified and these trends
continued as the mice aged. With respect to the mineral and matrix
components of the Raman spectrum, these data suggest a mechanism of
early and rapid type II collagen deterioration in the Del1 (+/-)
mice, followed by a biochemical change in the mineral.
TABLE-US-00002 TABLE 2 Specimen Age and Transgenic
Disordered:Ordered status CO.sub.3:PO.sub.4 ratio Amide III ratio
Phosphate ratio 2 months wt 0.05 (n = 2) 1.04 (n = 2) 0.28 (n = 2)
2 months tg 0.09 (n = 2) 0.84 (n = 2) 0.25 (n = 2) 2.5 months wt
0.16 .+-. 0.06 7.9 .+-. 6.8 0.33 .+-. 0.15 2.5 months tg 0.14 .+-.
0.04 3.6 .+-. 1.6 0.25 .+-. 0.15 3 months wt 0.10 .+-. 0.03 1.18
.+-. 0.47 0.11 .+-. 0.05 .sup.++ 3 months tg 0.09 .+-. 0.02 0.75
.+-. 0.22 0.36 .+-. 0.14 .sup.++ 5 months wt 0.08 .+-. 0.009 .sup.+
1.01 .+-. 0.25 0.48 .+-. 0.12 5 months tg 0.1 .+-. 0.01 .sup.+ 1.03
.+-. 0.21 0.33 .+-. 0.09 .sup.+ 7 months wt 0.13 .+-. 0.01 .sup.+
1.99 .+-. 0.91 .sup.+ 0.42 .+-. 0.10 7 months tg 0.17 .+-. 0.02
.sup.+ 5.89 .+-. 2.23 .sup.+ 0.46 .+-. 0.07 9 months wt 0.07 .+-.
0.008 .sup.+ 0.81 .+-. 0.21 .sup.+ 0.37 .+-. 0.04 9 months tg 0.09
.+-. 0.01 .sup.+ 1.15 .+-. 0.15 .sup.+ 0.32 .+-. 0.07 10 months wt
0.08 .+-. 0.02 0.71 .+-. 0.19 .sup.+ 0.17 .+-. 0.11 .sup.++ 10
months tg 0.07 .+-. 0.02 1.25 .+-. 0.48 .sup.+ 0.40 .+-. 0.13
.sup.++ 12 months wt 0.15 .+-. 0.02 0.81 .+-. 0.21 0.33 .+-. 0.03
12 months tg 0.15 .+-. 0.02 0.69 .+-. 0.15 0.31 .+-. 0.04 13 months
wt 0.13 .+-. 0.01 .sup.++ 4.99 .+-. 1.43 .sup.++ 0.32 .+-. 0.05 13
months tg 0.17 .+-. 0.008 .sup.++ 1.88 .+-. 0.60 .sup.++ 0.36 .+-.
0.05 16 months wt 0.07 .+-. 0.03 .sup.+ 1.25 .+-. 0.57 .sup.+ 0.38
.+-. 0.11 16 months tg 0.17 .+-. 0.06 .sup.+ 2.51 .+-. 0.57 .sup.+
0.32 .+-. 0.08 20 months wt 0.065 .+-. 0.14 1.22 .+-. 0.17 0.39
.+-. 0.08
[0160] With regard to the data in Table 2, mean band area ratios
are presented for the wt and Del1 (+/-) mice. The number of
transects used in calculating the mean ranged from 2-20, depending
on the age and type of mouse. Raman spectra band area ratios are
presented as means from all of the specimens per transgenic status
and age. The .+-.value is the 95% confidence interval, p-values are
denoted: .sup.+p<0.05, .sup.++p<0.01. The carbonate:phosphate
ratio is the ratio of band areas corresponding to the 1070
cm.sup.-1 band area divided by the sum of the 958 cm.sup.-1 and 945
cm.sup.-1 band areas. The amide III ratio is the ratio of band
areas in the amide III envelope corresponding to disordered
collagen (1240 cm.sup.-1) to ordered collagen (1270 cm.sup.-1). The
disordered: ordered phosphate ratio is the ratio of band areas
corresponding to disordered apatite (945 cm.sup.-1) to normal
apatite (958 cm.sup.-1).
[0161] Within the Amide III envelope (1220-1280 cm.sup.-1), the
1240 cm.sup.-1 band corresponds to a disordered secondary structure
of collagen and the 1270 cm.sup.-1 band corresponds to an ordered
secondary structure. The ratio of these two bands may be used to
gauge the degree of disorder in collagen's secondary structure. In
addition to matrix information, Raman spectra yielded information
from peaks that are ascribed to apatite phosphate and carbonate
moieties. The carbonate (CO.sub.3):phosphate (PO.sub.4) band area
ratio, 1070 cm.sup.-1: (958 cm.sup.-1+945 cm.sup.-1), may be an
indication of the quality of the mineral, since as carbonation of
the apatite increases, the mineral crystals become larger. The
disordered:ordered phosphate ratio, 945 cm.sup.-1:958 cm.sup.-1,
may be used to gauge the relative order of the phosphate within the
apatite.
[0162] Time-based trends in both the mineral and matrix components
were analyzed. Data outliers in the 1240 cm.sup.-1:1270 cm.sup.-1
ratio were seen at 2.5 months, 7 months (both Del1 (+/-) and wt),
and 13 months (wt), and an outlier in the 958 cm.sup.-1:945
cm.sup.-1 ratio was seen at 10 months (wt). These outliers probably
result from a low signal-to-noise ratio that affected band area
calculations.
[0163] A Mankin score, as modified by Turner, et al, was used to
evaluate histopathologic changes of OA on the femoral articular
cartilage of Del1 (+/-) and wt mice. In contrast to other studies
using the Del1 model, the scores did not consistently increase as a
function of age. We observed two significant differences in the
Mankin scores: an increase between the 5 and 12 month old wild-type
mice (p=0.045) and a decrease in Mankin score between the 2 and 3
month old Del1 (+/-) mice (p=0.020). Mankin scores were not
consistent between observers at any of the time points.
[0164] The matrix of articular cartilage is comprised of a network
of collagen fibers and entrapped proteoglycans. The collagen fibers
consist of types II, IX, and XI collagens, but is predominately
type II collagen, a homotrimer of three identical
.alpha.1(II)-chains. In adults, type II collagen is found mainly in
hyaline articular cartilage and in the nucleus propulsus of
intervertebral discs, and it is transiently expressed in other
areas during embryonic development.
[0165] In humans, mutations in Col2a1 have been linked to familial
osteoarthritis, as well as mild chondrodysplasia, Stickler and
Wagner syndromes, and more lethal syndromes such as type II
achondrogenesis (Langer-Saldino syndrome). Similarly, the Del1
(+/-) transgenic mouse develops a progressive articular
degeneration phenotype. Studies have shown an increased expression
of cartilage oligomeric matrix protein in the Del1 (+/-) articular
cartilage, particularly along the tidemark, at the border of
calcified and uncalcified cartilage. This increase parallels
cartilage degeneration. The chondrocytes at early degenerative
lesions of Del1 (+/-) articular cartilage are rich in Sox9, an
indicator of chondrocyte activation. The Sox9 staining co-localizes
with type Ha procollagen, likely reflecting an attempt at repair.
Matrix metalloproteinase-13 mRNA and its inhibitor TIMP-1 are also
expressed during early cartilage degeneration in this OA model, and
are localized in the synovial tissue, deep calcified cartilage, and
subchondral bone. These may be involved in tissue remodeling in
response to cartilage damage.
[0166] Mankin scoring is a frequently used system for scoring
cartilage degradation in animal models of osteoarthritis (Hausser,
Osteoarthritis and Cartilage 12: 870-877. 2004; Chu, American
Journal of Sports Medicine 32: 699-709, 2004; Lahm, Acta Orthop
Scand 75: 762-767, 2004; Tibesku, Arthritis & Rheumatism 52:
810-817, 2005). Within the Mankin system, sub-scores are given for
surface integrity, hyper- and hypo-cellularity, and matrix quality
using hematoxylin/eosin and Safranin-O staining. The final score
ranges from 0 (no osteoarthritis) to 20 (severe osteoarthritis).
Murine models of OA are more often graded with a less descriptive 0
through 3 score (de Hooge, Osteoarthritis and Cartilage, 13: 66-73,
2005; Zemmyo Arthritis & Rheumatism 48: 2873-2880, 2003), a 0
through 4 score (Helminen. Journal of Clinical Investigation 92:
582-595, 1993; Saamanen, Osteoarthritis and Cartilage, 8: 248-257,
2000) a 0 through 5 score (Lapvetelainen, Annals of Rheum Dis 61:
810-817, 2002), or a 0 through 6 score (Mistry, Osteoarthritis and
Cartilage 12: 131-141, 2004; Glasson, Arthritis & Rheumatism
50: 2547-2558, 2004). A review of the literature reveals one other
murine study using a modified Mankin scoring system (Xu, Arthritis
& Rheumatism 48: 2509-2518, 2003).
[0167] The modified Mankin score has been validated as an adequate
measure of histological damage in at least one animal model of OA.
The authors of this validation study examined rabbit articular
cartilage, comparing wild-type to experimental OA (either medial
meniscectomy or plaster immobilization extension), with each sample
being evaluated by two blinded observers. They found that most
scores were less than 6, with reasonable intra-observer agreement
(standard deviation varying between 1.5 and 2.6 points). Other
researchers, however, have questioned the validity of this scoring
system and have identified inherent problems associated with both
inter- and intra-observer variation. In the present experiment,
significant inter-observer variation were observed and the mean
score did not consistently increase as a function of age.
Statistical analyses showed no significant differences between Del1
(+/-) and wild-types in total modified Mankin scores at ages 2, 3,
10, or 13 months of age (FIG. 7c). In contrast to the high degree
of variability using conventional histology, the spectra obtained
using Raman spectra were consistent from animal to animal. Raman
spectra were normalized to minimize sample heterogeneity. Thus, any
changes observed in band area ratios was more likely due to
biochemical changes in the mineral or matrix than to decreased
scatter from the collagen matrix.
[0168] Previous studies have shown that compared to wt mice, Del1
(+/-) mice have collagen with greater fiber disorder. Raman spectra
results of the present experiment are consistent with these prior
reports, as seen by an increased 1240 cm.sup.-1:1270 cm.sup.-1 band
area ratio for the age-matched Del1 (+/-) mice. This increased
ratio indicates a more disordered secondary structure for collagen,
suggesting the Del1 (+/-) mice had an altered molecular response to
the truncated type II collagen fiber. Del1 (+/-) mice showed an
increasing 1240 cm.sup.-1:1270 cm.sup.-1 ratio starting at
approximately 6 months of age. The wt mice maintained a more
ordered collagen secondary structure for a longer period of time,
with a slight gradual upward trend starting at approximately 12
months of age. The increasing disorder of collagen in the Del1
(+/-) articular cartilage is consistent with the abnormal light
microscopy seen in previous studies.
[0169] The 1685 cm.sup.-1:1667 cm.sup.-1 ratio (ratio of the 2
bands in the amide I envelope) in Del1 (+/-) mice increased at a
rate about 1.5 times greater than the wt 1685 cm.sup.-1:1667
cm.sup.-1 ratio (data not shown). As observed in bone, and also
expected for cartilage, the 1685 cm.sup.-1: 1667 cm.sup.-1 ratio
increases as tissue damage progresses. While the differences in the
amide I envelope may be reflective of contributions from both types
I and II collagen, these data add weight to the argument that Raman
spectra can readily detect increased fragility of collagen
crosslinks in Del1 (+/-) mice. Due to the intricate ultrastructure
of tissues such as cartilage, it is expected that structural
changes in collagen (in this case truncation) will also cause
changes in the mineral content of the mineralized layer of
cartilage.
[0170] Data from the present experiment tend to show that Del1
(+/-) mice have an increased CO.sub.3:PO.sub.4 ratio when compared
to wt mice. A more carbonated mineral is more crystalline and thus
has the potential to compensate for other tissue weaknesses or
disorder. Other studies of mineralized tissues have shown an
increase in CO.sub.3:PO.sub.4 ratio for osteoarthritic joints. The
CO.sub.3:PO.sub.4 ratio increased with age in the Del1 (+/-) and wt
mice until approximately 7 months of age, and increased again after
10 months for the Del1 (+/-) mice. The elevated carbonate content
in the older transgenic mice may be indicative of an increased
mineralization to compensate for a degraded collagen matrix or as a
chemical response to the decreasing pH of the synovial fluid. Both
mechanisms are plausible, as lower pH and increased mineralization
of cartilage in OA patients has been reported. It is possible that,
in the Del1 (+/-) model, the mineral becomes more carbonated as a
response to the inherent instability of matrix composed of
truncated type II collagen fibers.
[0171] Further analysis of the mineralized species revealed that
while there was an increase in carbonated apatite in the Del1 (+/-)
mice, there was no significant change in the relative order of the
phosphate species. In previous studies, the Raman band at 945
cm.sup.-1 generally corresponded to a more disordered phosphate
species. The ratio of the 945 cm.sup.-1 to the 958 cm.sup.-1 bands
can be used to further assess the mineralization process. The
relative amount of disordered phosphate was observed to plateau at
approximately 9 months for both the Del1 (+/-) and wt mice. There
was no statistically significant difference (p>0.15) between the
wt and Del1 (+/-) mice after 5 months, except at age 10 months. The
data for the wt and Del1 (+/-) mice can be fitted into a parabolic
shaped curve with an apparent plateau of this ratio after 9 months.
Thus, the increased carbonation, as seen in older Del1 (+/-) mice,
may be either a result of the substitution from both the ordered
and disordered phosphate mineral species or as an increased
carbonate secretion as a chemical response to acidic synovial
fluid.
[0172] The Del1 transgenic mouse is a model of human OA
characterized by altered cartilage matrix structure. It was
hypothesized that RS of Del1 transgenic femoral articular cartilage
would identify age-dependent patterns that would correlate with
histomorphometric changes characteristic of OA. Consistent changes
in Del1 (+/-) articular cartilage Raman spectral patterns over time
were observed, which were not seen in wt samples. The increasing
amide III ratio and the increasing CO.sub.3:PO.sub.4 ratio
distinguish the Del1 (+/-) samples from the wt samples at an early
age. Moreover, time-based trends for early collagen deformation and
subsequent mineral changes that may be reacting to the disordered
collagen were observed.
[0173] Previous studies show that the weight difference between
Del1 (+/-) and wild-type mice is greater for male than female mice.
In the present experiment, due to insufficient numbers of mice,
they were not matched for gender. This random gender pairing could
have affected the Raman spectra. To address this issue multiple
mice at each age were evaluated. The data for male and female mice
were graphed separately, and no significant differences were
observed. Thus, it appears that the data analysis was not biased by
the genders of the mice evaluated.
[0174] In still another experiment, Del 1 (+/-) ocular tissue from
a wildtype mouse, aged 20 months, and younger mice but of unknown
ages (a transgenic mouse and a wildtype mouse) were analyzed. At
least seven transects were taken from each eye, although not all
transects produced a usable signal either due to fluorescence or
sample movement. The transects were preprocessed to remove
curvature and spikes, and dark-subtract spectra. The spectra were
baselined and peak-fitted using GRAMS/AI spectroscopy software
available from Thermo Electron Corporation. Peak areas were
recorded if the curve-fit showed an R.sup.2 greater than 0.990 and
had no negative peaks. The averaged peak areas were from both eyes.
A 5X/0.25 NA objective and a 0.3 neutral density filter (laser
intensity at objective approximately 105 mW) was used to help keep
the entire specimen in focus despite the large radius of curvature
of the eye, and to help avoid specimens burning.
[0175] The Del1 mice are well-characterized animal models for
early-onset osteoarthritis because they have collagen fibrils
truncated by 15 amino acids, the result of a 150 basepair deletion
mutation. Expression of this truncated collagen gene is also seen
in ocular tissue, and it is postulated that the Del1 mice may also
be used to model age-related degeneration in the eye. It was
observed that the amide III envelope (1220-1320 cm.sup.-1) had the
most consistent signal. The 1230:1260 band ratios were looked at
when comparing transgenic and wildtype. The ratio of 1230:1260 area
bands are used to estimate disorder in collagen, with contributions
from cross-linking and degree of hydration. A Raman spectrum of the
ocular globe is presented in FIG. 15. FIG. 16 is a portion of the
Raman spectrum of FIG. 15. Contributions from ocular tissue, amino
acids and collagen are distinguishable, and band assignments are
provided in Table 3. The amide III envelope in ocular tissue
appears wider than the amide III envelope in articular cartilage,
likely due to several types of collagen structure in the ocular
tissue.
TABLE-US-00003 TABLE 3 Sample ID 1230:1260 Ratio (Average) St. Dev.
Del1 mouse, 0.9080 (+/-0.159) 0.1812 unknown age Wildtype mouse,
0.5587 (+/-0.136) 0.2298 unknown age Wildtype mouse, 0.9857
(+/-0.127) 0.2143 20 months
[0176] With respect to articular cartilage of mice, an increased
1230:1260 ratio as a function of age was observed in Del1 mice,
whereas there was no observable difference in wildtype mice. In the
analysis of ocular tissue, there was a statistically significant
(p=0.008) discrepancy of ratios between the Del1 and wildtype for
the mice of unknown age. As seen in Table 3, the Del1 mouse of
unknown age has a higher 1230:1260 ratio, as expected. The
1230:1260 ratio of the 20-month wildtype mouse is comparable but
not statistically significant (p=0.4) to that of the Del1 mouse,
indicating that collagen deformation caused by the transgene is
comparable to months of the normal "wear and tear" as seen in an
older wildtype mouse. Upon comparison between the two wildtype
mice, there is a significant statistical difference in the values
(p<0.01).
[0177] A statistically significant difference in the 1230:1260 band
ratios when comparing Del 1 and wildtype mice was seen. The
transgenic mice show increased collagen disorder, as expected,
compared to the wildtype mice. Moreover, increased collagen
disorder as a function of mouse age was seen.
[0178] In yet another experiment, eyes were excised from Del1 (+/-)
and wild-type (wt) mice including a 12-14 month transgenic mouse, a
12-14 month wild-type mouse, and a 20 month old wild-type
mouse.
[0179] An eye mount was constructed and included a disposable tube
cap secured onto a microscope slide and filled with pH 7.4
phosphate buffered saline (PBS). A "blank" spectrum of the eye
mount filled with PBS (spectrum not shown) confirmed that the
spectral features of ocular tissue arose from the eye tissue, with
minimal spectral interference from the mount. Minor contributions
from the phosphate buffer/polypropylene cap were observed at 1883
cm.sup.-1 and 1151 cm.sup.-1 in spectra taken from eye tissue. Both
eyes from each specimen were analyzed.
[0180] A 5X/0.25 NA objective and a 0.3 neutral density filter
(laser intensity at objective .about.105 mW) was used to avoid
specimen burning and keep the entire specimen in focus, despite the
small radius of curvature of the eye. The Raman signal was
integrated for 600 seconds on a charged coupled device (CCD)
detector to maximize the signal-to-noise ratio (SNR). Specimens
were analyzed with no coverslip over the mount. The levels of PBS
in the mount were continually monitored and refreshed as needed.
The outer edges of the sclera were examined. Raman transects, point
spectroscopy at intervals along a line, were taken along these
outer sclera edges. Spectra from transects with excessive
fluorescence interference or specimen movement were discarded.
[0181] Transects were pre-processed using MATLAB.RTM. software from
The MathWorks to remove curvature and spikes, and to remove the
dark signal from the spectra. To minimize the effect of sample
inhomogeneity on band areas, a single normalized spectrum
(representative of the entire transect) was exported to the
GRAMS.RTM. software after the initial processing. The spectra were
baselined and the amide III envelope was peak-fitted using GRAMS/AI
software. Peak areas were recorded if the best-fit curve showed an
R.sup.2 greater than 0.990 and had no negative peaks. The peak
areas for both eyes were grouped together to form a single average
value for each specimen.
[0182] Depending on the specimen, spectra from five to twelve Raman
transects were used to calculate an average band area ratio. All
band areas ratios from specimens corresponding to a single age and
transgenic status were grouped to yield a single averaged ratio.
Student's T-tests (two-tailed with unequal variances) were used to
compare band ratios between the transgenic and wild-type (wt) mice
and between ageing wt mice. P-values below 0.05 are denoted in
Table 4 by +, p-values below 0.01 are denoted in Table 4 by ++.
TABLE-US-00004 TABLE 4 Specimen transgenic status and age 1235
cm.sup.-1:1265 cm.sup.-1 Ratio Std. Dev. Wild-type, age 0.5587
(.+-.0.136) 0.2298 12-14 months N = 12 Wild-type, age 0.9857
(.+-.0.127) 0.2143 20 months N = 11 Del1 (+/-), 0.9080 (.+-.0.159)
0.1812 age 12-14 months N = 5
[0183] A Raman spectrum of eye tissue is presented in FIG. 17, and
an example of peak fitting in the amide III region of FIG. 17 is
presented in FIG. 18. An amide III envelope analysis was used to
estimate disorder in collagen. The maximum at a lower Raman shift
(range: 1230-1240 cm.sup.-1) may be associated with disordered
collagen fiber, and the maxima at a higher Raman shift (range: 1260
1270 cm.sup.-1) may be associated with ordered collagen. For eye
tissue, the maxima were 1235 cm.sup.-1 and 1265 cm.sup.-1 and an
amide III ratio was determined as the ratio of the 1235 cm.sup.-1
and 1265 cm.sup.-1 band areas. Increases in the amide III ratio may
indicate an increase in the disorder of the collagen fiber. Amide
III band ratios may also be used to gauge collagen disorder in
comparisons in articular cartilage from Del1 (+/-) transgenic and
wild-type mice. Spectral contributions from ocular tissue, amino
acids and collagen are distinguishable in FIG. 17. Band assignments
are provided in Table 5 and are based on previous assignments of
ocular tissue from the literature. Bands at 1235, 1240, 1265 and
1270 cm.sup.-1 are visible in the amide III envelope, likely due to
several types of collagen structure in the ocular tissue. FIG. 19
illustrates the Raman spectra of articular cartilage (upper plot)
and Raman spectra of eye tissue (lower plot) taken from the same
specimen. As can be seen in FIG. 19, local maxima in the amide III
envelope are less defined in ocular tissue than in articular
cartilage, as seen in FIG. 19. Contributions from phosphate and
carbonate mineral components at 959 cm.sup.-1 and 1070 cm.sup.-1,
as observed in Raman spectra of articular cartilage, are not
observed in eye tissue.
TABLE-US-00005 TABLE 5 Raman Shift (cm.sup.-1) Band Assignment
Component 642 Aromatic Collagen Tyrosine 757 Aromatic Tryptophan
877, 853, 827 Aromatic Tryptophan 1003 Aromatic ring breathing
Phenylalanine 1220-1280 Amide III envelope Collagen 1450 C--H
scissor Ocular Tissue 1650 O--H bend Water/Ocular Tissue 1667 Amide
I Collagen 1685 Amide I Collagen
[0184] Raman spectra of eye tissue from the Del1 (+/-) mouse showed
a deformed collagen fiber that was also observed in the aged
wild-type mouse. In the analysis of ocular tissue, there is a
statistically significant (p=0.008) difference of ratios between
the 12-14 month transgenic and wild-type specimens. As seen in
Table 4, the transgenic mouse has a higher 1235 cm.sup.-1:1265
cm.sup.-1 ratio, as expected. The amide III ratio of the 20 month
wild-type mouse is comparable to that of a transgenic mouse,
indicating that collagen deformation caused by the transgene may be
comparable to 20 months of the normal ageing as seen in the older
wild-type mouse. The wild-type mice also show a significant
statistical difference in the measured ratios (p<0.01), which
may indicate that collagen deformation in older wild-type mice
occurs due to the natural effect of ageing and the low turnover
rate of ocular collagen. An increase in collagen deformation as a
function of age in wild-type mice is expected and a slight increase
in the amide III ratio was also observed in Raman analysis of
femurs from ageing wild type mice.
[0185] In addition to a higher band area ratio, the average peak
position in the amide III envelope may provide another marker of
collagen damage in the transgenic and 20 month specimens. Raman
spectroscopy of proteins has shown that changes in the secondary
structure, due to environmental, mechanical, or chemical stresses,
will give rise to changes in band position, band area or both.
Unlike articular cartilage, eye collagen is not subject to damage
from mechanical loads and the observed shifts in band position are
plausibly the result of chemical modifications, such as an altered
cross-link content, that may affect the elasticity of the collagen
fiber. The shift to higher wavenumbers in the amide III region may
be another indication of collagen fiber deformation that is due to
either the truncated collagen or long-term normal use, depending on
the transgenic status of the mouse. There is a shift to a higher
wavenumber for the 1235 cm.sup.-1 peak for the 20 month wild-type
and transgenic mouse. Spectra from the 12-14 month transgenic and
20 month wild-type mouse have an average peak position of 1234
cm.sup.-1 (.+-.1.7) and 1236 cm.sup.-1 (.+-.2.0) respectively,
while spectra from the 12-14 month wild-type mouse have an average
peak position of 1231 cm.sup.-1 (.+-.1.4). The difference in band
position seen in the 12-14 month wild type mouse is statistically
different (p<0.05) from both the 12-14 month transgenic and 20
month wild type mice.
[0186] There is a statistically significant difference in the amide
III band ratios between the 12-14 month transgenic and wild-type
mice. The transgenic mouse showed a higher collagen disorder, as
expected, compared to its age-matched wild-type littermate.
Moreover, the increased collagen disorder in the 20 month wild-type
mouse may indicate that Raman can also identify changes to collagen
that are a result of normal ageing. Truncation of the collagen
fiber caused by the transgene or loss of collagen fiber elasticity
from normal ageing causes deformation that is observed in both the
amide III band area ratio and band position associated with
disordered collagen in the amide III region. Raman spectroscopy
yields useful markers to identify increased collagen disorder in
both transgenic mice and older wild type mice. Raman bands in the
amide III envelope can be used as markers for collagen fiber
disorders in eye tissue. Because it is non invasively accessible,
eye tissue may serve as a good site for study of collagen damage
due to genetic defects or the natural ageing process.
[0187] At least portions of the techniques described above,
including the blocks described with reference to FIGS. 2-4 and
7-13, may be implemented using software comprising computer program
instructions. Such computer program instructions may control the
operation of a computing device such as a desktop computer, a
laptop computer, a tablet computer, a workstation, a server, a
mainframe, etc. The computing device may have a memory in which the
computer program instructions may be stored. The computer program
instructions may be written in any high level language such as C,
C++, C#, Java or the like or any low-level assembly or machine
language. By storing computer program instructions in a memory of
the computing device, the computing device is physically and/or
structurally configured in accordance with the computer program
instructions.
[0188] While many methods and systems have been described herein as
being implementable in software, they may be implemented in
hardware, firmware, etc., and may be implemented by a variety of
computing systems and devices. Thus, at least some method blocks
and system blocks described herein may be implemented in a standard
multi-purpose central processing unit (CPU), a special purpose CPU,
or on specifically designed hardware or firmware such as an
application-specific integrated circuit (ASIC) or other hard-wired
device as desired. When implemented in software, the software
routine may be stored in any computer readable memory such as on a
magnetic disk, a laser disk (such as a compact disk (CD), a digital
versatile disk (DVD)), a flash memory, a memory card, a memory
stick, etc., or other storage medium, in a RAM or ROM of a computer
or processor, in any database, etc. Likewise, this software may be
delivered via any known or desired delivery method including, for
example, on a computer readable memory or other transportable
computer storage mechanism or over a communication channel such as
a telephone line, the internet, etc. (which are viewed as being the
same as or interchangeable with providing such software via a
transportable storage medium).
[0189] While the invention is susceptible to various modifications
and alternative constructions, certain illustrative embodiments
thereof have been shown in the drawings and are described in detail
herein. It should be understood, however, that there is no
intention to limit the disclosure to the specific forms disclosed,
but on the contrary, the intention is to cover all modifications,
alternative constructions and equivalents falling within the spirit
and scope of the disclosure as defined by the appended claims.
* * * * *