Biomedical subjects

Michael D Morris

Publications and source records attributed to Michael D Morris.

At least 19 recordsLinked to original sources

PubMed2006

Bone tissue compositional differences in women with and without osteoporotic fracture.

It is generally accepted that the hallmark of osteoporosis is a reduction in bone mass. There is significant overlap, however, in bone mineral density between osteoporotic and normal individuals. This study examined the chemical composition of bone tissue obtained from women who had sustained a fracture and women without fracture to determine if there are differences between the two groups. Nineteen fractured and eleven non-fractured proximal femurs were obtained, matched for age and bone volume fraction obtained from micro-computed tomography. Trabecular bone specimens were examined by Raman spectroscopy to determine measures of chemical composition. A subset of the specimens was utilized to compare locations at the fracture and regions at least 2 mm away from apparent tissue damage using Raman spectroscopy. In addition, fifteen iliac crest biopsies each were obtained from women who had sustained a fracture and from normal controls. Raman spectroscopy was used to determine measures of chemical composition of trabecular and cortical bone. The results demonstrated that femoral bone tissue in the region of visible damage had a trend towards differences compared to regions at least 2 mm from visible damage. Femoral trabecular bone in fractured women had a higher carbonate/amide I area ratio than in unfractured women. Iliac crest biopsies revealed a higher carbonate/phosphate ratio in cortical bone from women who had sustained a fracture. Results suggest that the chemical composition of bone tissue may be an additional risk factor for osteoporotic fracture.

Aged↗

PubMed2006

Raman spectroscopic evidence for octacalcium phosphate and other transient mineral species deposited during intramembranous mineralization.

UNLABELLED: To understand early mineralization events, we studied living murine calvarial tissue by Raman spectroscopy using fibroblast growth factor 2 (FGF2)-soaked porous beads. We detected increased levels of a transient phase resembling octacalcium phosphate in sutures undergoing premature suture closure. INTRODUCTION: Several calcium phosphates have been postulated as the earliest inorganic precursors to bone mineral. They are unstable and have not been previously detected in tissue specimens. Whether the same intermediates are formed in sutures undergoing premature closure is also unknown. METHODS: Six coronal suture tissue specimens from fetal day 18.5 B6CBA F1/J wild-type mice were studied. Three sutures specimens were treated with FGF2-soaked heparin acrylic beads to induce accelerated mineralization and premature suture closure. Three control specimens were treated with empty heparin acrylic beads. All sutures were maintained as organ cultures to permit repeated spectral analyses at 12-24 h intervals over a 72-h period. RESULTS: During the first 24 h, the spectra contained bands of octacalcium phosphate (OCP) or an OCP-like mineral. The main phosphorus-oxygen stretch was at 955 cm(-1), instead of the 957-959 cm(-1) seen in bone mineral, and there was an additional band at 1010-1014 cm(-1), as expected for OCP. A broad band was found at 945 cm(-1), characteristic of a highly disordered or amorphous calcium phosphate. An increased amount of mineral was observed in FGF2-treated sutures, but no qualitative differences in Raman spectra were observed between experimental and control specimens. CONCLUSIONS: Inorganic mineral deposition proceeds through transient intermediates, including an OCP-like phase. Although this transient phase has been observed in purely inorganic model systems, this study is the first to report OCP or an OCP-like intermediate in living tissue. Raman microspectroscopy allows observation of this transient mineral and may allow observation of other precursors as well.

Animals↗

PubMed2006

Reexamination of hexafluorosilicate hydrolysis by 19F NMR and pH measurement.

The dissociation of hexafluorosilicate has been reinvestigated due to recent suggestions that fluorosilicate intermediates may be present in appreciable concentrations in drinking water. 19F NMR spectroscopy has been used to search for intermediates in the hydrolysis of hexafluorosilicate. No intermediates were observable at 10(-5) M concentrations under excess fluoride forcing conditions over the pH range of 3.5-5. A single intermediate species, assigned as SiF5(-) or its hydrate, was detected below pH 3.5. At moderate pH values of 4 and 5 silica oligomerization in the solutions studied made it difficult to directly determine the hexafluorosilicate equilibrium constant. Under more acidic conditions the average pKd, or negative log of the dissociation constant Kd, determined by 19F NMR measurements, was 30.6. We also investigated the behavior of hexafluorosilicate in common biological buffer reagents including phosphate/citrate, veronal/HCI buffers, and Ringer's solution. The buffer capacity of all of these systems was found to be insufficient to prevent acidic shifts in pH when hexafluorosilicate was added. The pH change is sufficient explanation for the observed inhibition of acetylcholinesterase that was previously attributed to hexafluorosilicate hydrolysis intermediates.

Barbital↗

PubMed2006

Is photobleaching necessary for Raman imaging of bone tissue using a green laser?

Raman microspectroscopy is widely used for musculoskeletal tissues studies. But the fluorescence background obscures prominent Raman bands of mineral and matrix components of bone tissue. A 532-nm laser irradiation has been used efficiently to remove the fluorescence background from Raman spectra of cortical bone. Photochemical bleaching reduces over 80% of the fluorescence background after 2 h and is found to be nondestructive within 40 min. The use of electron multiplying couple charge detector (EMCCD) enables to acquire Raman spectra of bone tissues within 1-5 s range and to obtain Raman images less than in 10 min.

Animals↗

PubMed2006

Three structural roles for water in bone observed by solid-state NMR.

Hydrogen-bearing species in the bone mineral environment were investigated using solid-state NMR spectroscopy of powdered bone, deproteinated bone, and B-type carbonated apatite. Using magic-angle spinning and cross-polarization techniques three types of structurally-bound water were observed in these materials. Two of these water types occupy vacancies within the apatitic mineral crystal in synthetic carbonated apatite and deproteinated bone and serve to stabilize these defect-containing crystals. The third water was observed at the mineral surface in unmodified bone but not in deproteinated bone, suggesting a role for this water in mediating mineral-organic matrix interactions. Direct evidence of monohydrogen phosphate in a (1)H NMR spectrum of unmodified bone is presented for the first time. We obtained clear evidence for the presence of hydroxide ion in deproteinated bone by (1)H MAS NMR. A (1)H-(31)P heteronuclear correlation experiment provided unambiguous evidence for hydroxide ion in unmodified bone as well. Hydroxide ion in both unmodified and deproteinated bone mineral was found to participate in hydrogen bonding with neighboring water molecules and ions. In unmodified bone mineral hydroxide ion was found, through a (1)H-(31)P heteronuclear correlation experiment, to be confined to a small portion of the mineral crystal, probably the internal portion.

Animals↗

PubMed2006

Subsurface Raman spectroscopy and mapping using a globally illuminated non-confocal fiber-optic array probe in the presence of Raman photon migration.

We report the use of a fiber-optic probe with global illumination and an array of 50 collection fibers (PhAT probe, Kaiser Optical Systems, Inc.) to obtain Raman spectra and 50 spatial element maps of polymers through overlayers of other polymers that are highly scattering. Band target entropy minimization (BTEM) is used to recover the spectra of the subsurface components and generate maps of their distributions. This approach to subsurface mapping is tested with model systems consisting of two or three layers of polyethylene, polytetrafluoroethylene (Teflon), and polyoxymethylene (Delrin) arranged in different geometries. Raman spectra and maps were obtained through overlayer thicknesses of up to 13 mm. Subsurface spatial resolution is achieved because each fiber views an asymmetric distribution of Raman scattered light from surface and subsurface components that depends on the position of the fiber relative to the depth and position of a component and the extent of photon diffusion through the system.

Equipment Design↗

PubMed2006

Correlating changes in collagen secondary structure with aging and defective type II collagen by Raman spectroscopy.

A novel application of Raman spectroscopy for monitoring damage to ocular collagen in wild-type mice and Del1 (+/-) transgenic mice, a murine animal model of osteoarthritis, is described. In order to understand the progression of diseases of collagen, it is necessary to use methods that can recognize alterations in affected tissue due to chemical and/or genetic modifications. The heterozygous Del1 (+/-) transgenic mouse is established as a model for early-onset osteoarthritis caused by modifications to the type II collagen gene (COL2A1) that result in a truncated collagen fiber. We expect that abnormal type II collagen is expressed in articular cartilage and eye tissue of the Del1 (+/-) mouse. Eyes excised from a subset of specimens from another study using Del1 (+/-) mice were examined by Raman spectroscopy for evidence of defective collagen. Spectral contributions from the collagen protein were readily observed. The amide III envelope (1220-1280 cm-1) was used to characterize changes in collagen secondary structure. Raman spectra of the sclera component of eyes taken from transgenic and older wild-type mice show an increased collagen disorder, as expected. These preliminary results suggest that Raman is capable of recognizing and measuring abnormality in eye collagen and may have potential as a diagnostic tool for ocular collagen damage.

Aging↗

PubMed2006

Identifying chemical changes in subchondral bone taken from murine knee joints using Raman spectroscopy.

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.

Animals↗

PubMed2005

Novel assessment of bone using time-resolved transcutaneous Raman spectroscopy.

UNLABELLED: With fragility fractures increasing as the population ages, there is a need for improved means to estimate risk of fracture. We recorded Raman spectra of both the mineral and organic phases of bone transcutaneously, a technology with potential to enhance bone quality and fracture risk assessment. INTRODUCTION: The current "gold standard" assessment of bone quality is BMD determined by DXA. However, this accounts for only 60-70% of bone strength. X-rays are absorbed by the mineral phase of bone, whereas the organic phase remains essentially invisible; however, bone strength is critically dependent on both phases. We report, for the first time, a Raman spectroscopic technique that analyses both phases of bone beneath unbroken skin by eliminating spectral components of overlying tissues. MATERIALS AND METHODS: We used an 800-nm laser (1-kHz, 1-ps pulses) with a synchronized 4-ps Kerr gate with variable picosecond delay that effectively shuttered out photons from overlying tissues. We measured bone Raman spectra at a point 2 mm above the carpus from two mouse genotypes with extreme differences in bone matrix quality: wildtype and oim/oim (matched for age, sex, and weight). Typical depth was 1.1 mm. We repeated the measurements with overlying tissues removed down to bone. Oim/oim mice produce only homotrimeric collagen, which results in poorly mineralized bone tissue. RESULTS: The main spectral features were present from both bone phases. The spectral bands were in similar ratios when measured through the skin or directly from bone (in both genotypes). The band of the mineral phase (phosphate nu1) was smaller in oim/oim mice when measured directly from bone and through skin. The band associated with a particular vibrational mode of organic phase collagen (CH2 wag) showed a frequency shift between the genotypes. CONCLUSIONS: This novel technique allowed us, for the first time, to make objective transcutaneous spectral measurements of both the mineral and the organic phases of bones and distinguish between normal and unhealthy bone tissue. After further optimization, this technology may help improve fracture risk assessments and open opportunities for screening in anticipation of the predicted increase in fragility fractures.

Animals↗

PubMed2005

Anabolic actions of PTH (1-34): use of a novel tissue engineering model to investigate temporal effects on bone.

PTH is in clinical use for the treatment of osteoporosis and is under intensive investigation for its potential in applications of tissue engineering, fracture healing, and implant integration. However, the mechanisms of its action to stimulate bone formation are still unclear. A novel bone tissue engineering model was used to elucidate basic mechanisms of PTH anabolic actions. Ectopic ossicles containing cortical bone, trabecular bone, and a hematopoietic marrow were generated from implanted bone marrow stromal cells (BMSC). One week after implantation, nude mice were administered PTH or vehicle for 1 week (group 1), 3 weeks (group 2), or 7 weeks (group 3). Another group was also treated for 3 weeks, initiated 12 weeks after implantation (group 4). Micro-radiography and histomorphometry revealed increased marrow cellularity in group 1 PTH-treated ossicles, increased bone in group 2 PTH-treated ossicles, and similar amounts of bone in both group 3 and 4 ossicles regardless of treatment. Incidence of phosphate mineral and phosphate mineral to hydroxyproline ratio via Raman spectroscopy were significantly higher after 3 weeks versus 1 week of PTH treatment, but there was no difference between PTH- and vehicle-treated ossicles. Early events of PTH action in group 1 ossicles and the effects of a single injection of PTH on 1- and 2-week-old ossicles were evaluated by Northern blot analysis. Osteocalcin (OC) mRNA was increased after 1 week of intermittent PTH treatment in ossicles and calvaria but an acute injection did not alter OC mRNA. In contrast, a single injection of PTH increased matrix gamma-carboxyglutamic acid protein (MGP) mRNA in 2-week-old ossicles. Differential and temporal-dependent effects of PTH on OC and MGP suggest at the molecular level, that PTH acts to inhibit osteoblast mineralization. However, this does not translate into tissue level alterations. These data indicate that anabolic actions of PTH in ectopic ossicles are temporally dependent on the BMSC implanted and suggest that cell implantation strategies are particularly responsive to PTH.

Adipocytes↗

PubMed2004

Highly ordered interstitial water observed in bone by nuclear magnetic resonance.

UNLABELLED: NMR was used to study the nanostructure of bone tissue. Distance measurements show that the first water layer at the surface of the mineral in cortical bone is structured. This water may serve to couple the mineral to the organic matrix and may play a role in deformation. INTRODUCTION: The unique mechanical characteristics of bone tissue have not yet been satisfactorily connected to the exact molecular architecture of this complex composite material. Recently developed solid-state nuclear magnetic resonance (NMR) techniques are applied here to the mineral component to provide new structural distance constraints at the subnanometer scale. MATERIALS AND METHODS: NMR dipolar couplings between structural protons (OH(-) and H(2)O) and phosphorus (PO(4)) or carbon (CO(3)) were measured using the 2D Lee-Goldburg Cross-Polarization under Magic-Angle Spinning (2D LG-CPMAS) pulse sequence, which simultaneously suppresses the much stronger proton-proton dipolar interactions. The NMR dipolar couplings measured provide accurate distances between atoms, e.g., OH and PO(4) in apatites. Excised and powdered femoral cortical bone was used for these experiments. Synthetic carbonate ( approximately 2-4 wt%)-substituted hydroxyapatite was also studied for structural comparison. RESULTS: In synthetic apatite, the hydroxide ions are strongly hydrogen bonded to adjacent carbonate or phosphate ions, with hydrogen bond (O-H) distances of approximately 1.96 A observed. The bone tissue sample, in contrast, shows little evidence of ordered hydroxide. Instead, a very ordered (structural) layer of water molecules is identified, which hydrates the small bioapatite crystallites through very close arrangements. Water protons are approximately 2.3-2.55 A from surface phosphorus atoms. CONCLUSIONS: In synthetic carbonated apatite, strong hydrogen bonds were observed between the hydroxide ions and structural phosphate and carbonate units in the apatite crystal lattice. These hydrogen bonding interactions may contribute to the long-range stability of this mineral structure. The biological apatite in cortical bone tissue shows evidence of hydrogen bonding with an ordered surface water layer at the faces of the mineral particles. This structural water layer has been inferred, but direct spectroscopic evidence of this interstitial water is given here. An ordered structural water layer sandwiched between the mineral and the organic collagen fibers may affect the biomechanical properties of this complex composite material.

Animals↗

PubMed2004

Brittle IV mouse model for osteogenesis imperfecta IV demonstrates postpubertal adaptations to improve whole bone strength.

UNLABELLED: The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties. INTRODUCTION: The Brittle IV (Brtl) mouse was developed as a knock-in model for osteogenesis imperfecta (OI) type IV. A Gly349Cys substitution was introduced into one col1a1 allele, resulting in a phenotype representative of the disease. In this study, we investigate the effect of the Brtl mutation on whole bone architecture, strength, and composition across a range of age groups. MATERIALS AND METHODS: One-, 2-, 6-, and 12-month-old Brtl and wildtype (WT) mice were analyzed. Femurs were assessed at the central diaphysis for cortical geometric parameters using microCT and were subsequently mechanically tested to failure by four-point bending. Matrix material properties were predicted using microCT data to normalize data from mechanical tests. Raman spectroscopy and DXA were used to assess matrix composition. RESULTS: Our findings show a postpubertal adaptation in which Brtl femoral strength and stiffness increase through a mechanism independent of changes in whole bone geometry. These findings suggest an improvement in the material properties of the bone matrix itself, rather than improvements in whole bone geometry, as seen in previous mouse models of OI. Raman spectroscopic results suggest these findings may be caused by changes in mineral/matrix balance rather than improvements in mineral crystallinity. CONCLUSIONS: Our findings parallel the currently unexplained clinical observation of decreased fractures in human OI patients after puberty. The Brtl mouse remains an important tool for investigating therapeutic interventions for OI.

Adaptation, Physiological↗

PubMed2004

Distribution of single DNA molecule electrophoretic mobilities in semidilute and dilute hydroxyethylcellulose solutions.

The distribution of center of mass electrophoretic mobility mobilities and normalized migration time of up to 1080 lambda DNA molecules per experiment were measured in both semidilute hydroxyethylcellulose HEC/0.5 x Tris-borate-EDTA (TBE) solutions and dilute HEC/0.5 x TBE solution by high-speed video microscopy. Measurements were made microscopically over a short migration distance in homogeneous DNA HEC/0.5 x TBE solution and after electrophoretic migration of a plug of DNA through 7 cm. Video at 120 frames/s (semidilute HEC solution) and 236 frames/s (dilute HEC solution) allowed visualization with adequate resolution for single molecule mobility measurements. The electrophoretic migration times and band shapes predicted from the measurements corresponded well with those measured by conventional capillary electrophoresis (CE) in both semidilute and dilute HEC. In semidilute solution, the band width predicted by a square root of time scaling is in good agreement with the results of conventional CE. However, in dilute solution the precision of the measurements was not good enough to allow scaled estimates of band widths.

Cellulose↗

PubMed2004

Bone tissue ultrastructural response to elastic deformation probed by Raman spectroscopy.

Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components in real time of murine cortical bone as it responds to elastic deformation. Because bone is ia composite material, its mechanical properties are dependent on the structure and composition at a variety of dimensional scales. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed as perturbations to tissue spectra. We load murine femora in a custom-made mechanical tester that fits on the stage of a Raman microprobe and can accept hydrated tissue specimens. As the specimen is loaded in tension, the shifts in mineral P-O4 v1 are followed with the microprobe. Average load and strain are measured using a load cell. These devices ensure that specimens are not loaded to or beyond the yield point. Changes occur in the mineral component of bone as a response to loading in the elastic regime. We propose that the mineral apatitic crystal lattice is deformed by movement of calcium and other ions. Raman microspectroscopy shows that bone mineral is not a passive contributor to tissue strength. The mineral active response to loading may function as a local energy storage and dissipation mechanism, thus helping to protect tissue from catastrophic damage.

Algorithms↗

PubMed2004

Earliest mineral and matrix changes in force-induced musculoskeletal disease as revealed by Raman microspectroscopic imaging.

UNLABELLED: Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common human birth defect in the skull. Raman microspectroscopy was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Raman imaging revealed decreased relative mineral content in skulls undergoing craniosynostosis compared with unloaded specimens. INTRODUCTION: Raman microspectroscopy, a nondestructive vibrational spectroscopic technique, was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common birth defect in the face and skull. The calvaria, or flat bones that comprise the top of the skull, are most often affected, and craniosynostosis is a feature of over 100 human syndromes and conditions. MATERIALS AND METHODS: Raman images of the suture, the tips immediately adjacent to the suture (osteogenic fronts), and mature parietal bones of loaded and unloaded calvaria were acquired. Images were acquired at 2.6 x 2.6 microm spatial resolution and ranged in a field of view from 180 x 210 microm to 180 x 325 microm. RESULTS AND CONCLUSIONS: This study found that osteogenic fronts subjected to uniaxial compression had decreased relative mineral content compared with unloaded osteogenic fronts, presumably because of new and incomplete mineral deposition. Increased matrix production in osteogenic fronts undergoing craniosynostosis was observed. Understanding how force affects the composition, relative amounts, and location of the mineral and matrix provides insight into musculoskeletal disease in general and craniosynostosis in particular. This is the first report in which Raman microspectroscopy was used to study musculoskeletal disease. These data show how Raman microspectroscopy can be used to study subtle changes that occur in disease.

Animals↗

PubMed2003

Effect of urea on the polymer buffer solutions used for the electrophoretic separations of nucleic acids.

The present study describes the effect of urea on the properties of hydroxyethyl cellulose (HEC) polymer solutions used for the separation of nucleic acids. Solution properties were investigated by viscosity measurements and supplemented by Raman spectroscopy of the solution components. By using viscosimetry, it was possible to identify that borate, urea, and HEC participate in an interaction causing an increase in the viscosity of dilute solutions. In addition, short polymer chains exhibited a 4-fold decrease in the entanglement threshold in the presence of 4 M urea and 0.356 M total borate concentration. These interactions were found to be specific to HEC. Raman spectroscopy monitored serial addition of 8 M urea to HEC sieving solutions combined with factor analysis indicated formation of minor urea species. Lack of change in the Raman spectrum and relative amount of borate suggested that there is no direct interaction between borate and urea. These effects on HEC sieving solution properties lead to the use of low HEC concentrations that are beneficial for the separation of nucleic acids under denaturing conditions.

Buffers↗

PubMed2003

Band-target entropy minimization (BTEM) applied to hyperspectral Raman image data.

Band-target entropy minimization (BTEM) has been applied to extraction of component spectra from hyperspectral Raman images. In this method singular value decomposition is used to calculate the eigenvectors of the spectroscopic image data set. Bands in non-noise eigenvectors that would normally be used for recovery of spectra are examined for localized spectral features. For a targeted (identified) band, information entropy minimization or a closely related algorithm is used to recover the spectrum containing this feature from the non-noise eigenvectors, plus the next 5-30 eigenvectors, in which noise predominates. Tests for which eigenvectors to include are described. The method is demonstrated on one synthesized Raman image data set and two bone tissue specimens. By inclusion of small amounts of signal that would be unused in other methods, BTEM enables the extraction of a larger number of component spectra than are otherwise obtainable. An improvement in signal/noise ratio of the recovered spectra is also obtained.

Aged↗

PubMed2002

Comparison of RNA, single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions.

We present a study of the separation of RNA, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in semidilute linear hydroxyethylcellulose (HEC) solution. Our results strive to provide a better understanding of the mechanisms of nucleic acid migration during electrophoresis in polymer solutions under native and denaturing conditions. From a study of the dependence of mobility on chain length and applied electric field, we found that RNA and ssDNA show better separation and higher resolution over a larger range of sizes compared to dsDNA. In addition, RNA reptation without orientation extends to longer chain lengths in comparison to ssDNA, possibly as a result of different type of short-lived secondary structure formations. Such a comparative study between nucleic acid capillary electrophoresis helps to optimize RNA separation and provides better understanding of RNA migration mechanisms in semidilute polymer solutions under denaturing conditions.

DNA↗