1064 nm dispersive Raman spectroscopy of tissues with strong near-infrared autofluorescence.

Raman spectroscopy is an established technique for molecularly specific characterization of tissues. However, even with near-infrared (NIR) excitation, some tissues possess background autofluorescence, which can overwhelm Raman scattering. Here, we report collection of spectra from tissues with strong autofluorescence using a 1064 nm system with a high-throughput dispersive spectrometer and deep-cooled InGaAs array. Spectra collected at 1064 nm were compared with those collected at 785 nm in specimens from human breast, liver, and kidney. The results demonstrate superior performance at 1064 nm in the liver and kidney, where NIR autofluorescence is intense. The results indicate the feasibility of new biomedical applications for Raman spectroscopy at 1064 nm in tissues with strong autofluorescence.

Cross-sectional tracking of particle motion in evaporating drops: flow fields and interfacial accumulation.

The lack of an effective technique for three-dimensional flow visualization has limited experimental exploration of the "coffee ring effect" to the two-dimensional, top-down viewpoint. In this report, high-speed, cross-sectional imaging of the flow fields was obtained using optical coherence tomography to track particle motion in an evaporating colloidal water drop. This approach enables z-dimensional mapping of primary and secondary flow fields and changes in these fields over time. These sectional images show that 1 µm diameter polystyrene particles have a highly nonuniform vertical distribution with particles accumulating at both the air-water interface and the water-glass interface during drop evaporation. Particle density and relative humidity are shown to influence interfacial entrapment, which suggests that both sedimentation rate and evaporation rate affect the dynamic changes in the cross-sectional distribution of particles. Furthermore, entrapment at the air-water interface delays the time at which particles reach the ring structure. These results suggest that the organization of the ring structure can be controlled based on the ratio of different density particles in a colloidal solution.

Students' preparedness and perception toward online learning in dental education - A cross-sectional study.

Background: Amid the health risk posed by coronavirus disease 2019 pandemic, all the educational institutions in India have adopted online teaching as a method of imparting education. Thus, the current study was executed to evaluate the perception and preparedness of dental students toward online learning in dental education. Materials and Methods: The present cross-sectional study was conducted on 286 dental undergraduate (UG) and postgraduate (PG) students employing a pretested questionnaire. The questionnaire focused on technological access, computer and online skills, and motivational skills to use e-learning for educational purpose. The response scale for each domain was classified into low readiness level (mean value = 1-<3), acceptable/moderate readiness level (mean value = 3-<4), and high readiness level (mean value = 4-5). Results: The results showed a high level of computer (4.02 ± 0.73) and online skills (4.05 ± 0.70) and acceptable levels of technology access (3.62 ± 0.73) and motivational level (3.61 ± 0.82). There was a significant difference in computer (P = 0.017) and online skills (P = 0.044) among UG and PG students, with PG students reporting better soft skills. Majority of the students reported participating in online workshops (68.2%) and examinations (76.6%). Around 55.2% of the students reported a positive impact and 8% reported a very positive impact of online methods on their learning. Conclusion: Students showed a high level of readiness toward e-learning methods along with some difficulties faced during e-learning.

Model-based characterization platform of fiber optic extended-wavelength diffuse reflectance spectroscopy for identification of neurovascular bundles.

SIGNIFICANCE: Fiber-optic extended-wavelength diffuse reflectance spectroscopy (EWDRS) using both visible/near-infrared and shortwave-infrared detectors enables improved detection of spectral absorbances arising from lipids, water, and collagen and has demonstrated promise in a variety of applications, including detection of nerves and neurovascular bundles (NVB). Development of future applications of EWDRS for nerve detection could benefit from the use of model-based analyses including Monte Carlo (MC) simulations and evaluation of agreement between model systems and empirical measurements. AIM: The aim of this work is to characterize agreement between EWDRS measurements and simulations and inform future applications of model-based studies of nerve-detecting applications. APPROACH: A model-based platform consisting of an ex vivo microsurgical nerve dissection model, unique two-layer optical phantoms, and MC model simulations of fiber-optic EWDRS spectroscopic measurements were used to characterize EWDRS and compare agreement across models. In addition, MC simulations of an EWDRS measurement scenario are performed to provide a representative example of future analyses. RESULTS: EWDRS studies performed in the common chicken thigh femoral nerve microsurgical dissection model indicate similar spectral features for classification of NVB versus adjacent tissues as reported in porcine models and human subjects. A comparison of measurements from unique EWDRS issue mimicking optical phantoms and MC simulations indicates high agreement between the two in homogeneous and two-layer optical phantoms, as well as in dissected tissues. Finally, MC simulations of measurement over a simulated NVB indicate the potential of future applications for measurement of nerve plexus. CONCLUSIONS: Characterization of agreement between fiber-optic EWDRS measurements and MC simulations demonstrates strong agreement across a variety of tissues and optical phantoms, offering promise for further use to guide the continued development of EWDRS for translational applications.

Development of a mobile phone camera-based transcutaneous bilirubinometer for low-resource settings.

Newborns in high-income countries are routinely screened for neonatal jaundice using transcutaneous bilirubinometery (TcB). In low-and middle-income countries, TcB is not widely used due to a lack of availability; however, mobile-phone approaches for TcB could help expand screening opportunities. We developed a mobile phone-based approach for TcB and validated the method with a 37 patient multi-ethnic pilot study. We include a custom-designed snap-on adapter that is used to create a spatially resolved diffuse reflectance detection configuration with the illumination provided by the mobile-phone LED flash. Monte-Carlo models of reflectance from neonatal skin were used to guide the design of an adapter for filtered red-green-blue (RGB) mobile-phone camera reflectance measurements. We extracted measures of reflectance from multiple optimized spatial-offset regions-of-interest (ROIs) and a linear model was developed and cross-validated. This resulted in a correlation between total serum bilirubin and mobile-phone TcB estimated bilirubin with a R 2= 0.42 and Bland-Altman limits of agreement of +6.4 mg/dL to -7.0 mg/dL. These results indicate that a mobile phone with a modified adapter can be utilized to measure neonatal bilirubin values, thus creating a novel tool for neonatal jaundice screening in low-resource settings.

In Situ Assessment of Porcine Osteochondral Repair Tissue in the Visible-Near Infrared Spectral Region.

Standard assessment of cartilage repair progression by visual arthroscopy can be subjective and may result in suboptimal evaluation. Visible-near infrared (Vis-NIR) fiber optic spectroscopy of joint tissues, including articular cartilage and subchondral bone, provides an objective approach for quantitative assessment of tissue composition. Here, we applied this technique in the 350-2,500 nm spectral region to identify spectral markers of osteochondral tissue during repair with the overarching goal of developing a new approach to monitor repair of cartilage defects in vivo. Full thickness chondral defects were created in Yucatan minipigs using a 5-mm biopsy punch, and microfracture (MFx) was performed as a standard technique to facilitate repair. Tissues were evaluated at 1 month (in adult pigs) and 3 months (in juvenile pigs) post-surgery by spectroscopy and histology. After euthanasia, Vis-NIR spectra were collected in situ from the defect region. Additional spectroscopy experiments were carried out in vitro to aid in spectral interpretation. Osteochondral tissues were dissected from the joint and evaluated using the conventional International Cartilage Repair Society (ICRS) II histological scoring system, which showed lower scores for the 1-month than the 3-month repair tissues. In the visible spectral region, hemoglobin absorbances at 540 and 570 nm were significantly higher in spectra from 1-month repair tissue than 3-month repair tissue, indicating a reduction of blood in the more mature repair tissue. In the NIR region, we observed qualitative differences between the two groups in spectra taken from the defect, but differences did not reach significance. Furthermore, spectral data also indicated that the hydrated environment of the joint tissue may interfere with evaluation of tissue water absorbances in the NIR region. Together, these data provide support for further investigation of the visible spectral region for assessment of longitudinal repair of cartilage defects, which would enable assessment during routine arthroscopy, particularly in a hydrated environment.

Measuring differences in compositional properties of bone tissue by confocal Raman spectroscopy.

The full range of fracture risk determinants arise from each hierarchical level comprising the organization of bone. Raman spectroscopy is one tool capable of characterizing the collagen and mineral phases at a near submicron-length scale, but the ability of Raman spectra to distinguish compositional differences of bone is not well defined. Therefore, we analyzed multiple Raman peak intensities and peak ratios to characterize their ability to distinguish between the typically less mineralized osteonal tissue and the more mineralized interstitial tissue in intracortical human bone. To further assess origins of variance, we collected Raman spectra from embedded specimens and for two orientations of cut. Per specimen, Raman peak intensities or ratios were averaged among multiple sites within five osteons and five neighboring interstitial tissue. The peak ratios of ν(1) phosphate (PO(4)) to proline or amide III detected the highest increases of 15.4 or 12.5%, respectively, in composition from osteonal to interstitial tissue. The coefficient of variance was less than 5% for each as opposed to a value of ~8% for the traditional ν(1)PO(4)/amide I, a peak ratio that varied the most between transverse and longitudinal cuts for each tissue type. Although embedding affected Raman peaks, it did not obscure differences in most peak ratios related to mineralization between the two tissue types. In studies with limited sample size but sufficient number of Raman spectra per specimen for spatial averaging, ν(1)PO(4)/amide III or ν(1)PO(4)/proline is the Raman property that is most likely to detect a compositional difference between experimental groups.

A clinical instrument for combined raman spectroscopy-optical coherence tomography of skin cancers.

BACKGROUND AND OBJECTIVE: The current standard for diagnosis of skin cancers is visual inspection followed by biopsy and histopathology. This process can be invasive, subjective, time consuming, and costly. Optical techniques, including Optical Coherence Tomography (OCT) and Raman Spectroscopy (RS), have been developed to perform non-invasive characterization of skin lesions based on either morphological or biochemical features of disease. The objective of this work is to report a clinical instrument capable of both morphological and biochemical characterization of skin cancers with RS-OCT. MATERIALS AND METHODS: The portable instrument utilizes independent 785 nm RS and 1,310 nm OCT system backbones. The two modalities are integrated in a 4″ (H) × 5″(W) × 8″(L) clinical probe. The probe enables sequential acquisition of co-registered OCT and RS data sets. The axial response of the RS collection in the skin was estimated using scattering phantoms. In addition, RS-OCT data from patients with cancerous and non-cancerous lesions are reported. RESULTS: The RS-OCT instrument is capable of screening areas as large as 15 mm (transverse) by 2.4 mm (in depth) at up to 8 frames/second with OCT, and identifying locations to perform RS. RS signal is collected from a 44 µm transverse spot through a depth of approximately 530 µm. RS-OCT data sets from a superficial scar and a nodular BCC are reported to demonstrate the clinical potential of the instrument. CONCLUSION: The RS-OCT instrument reported here is capable of morphological and biochemical characterization of cancerous skin lesions in a clinical setting. OCT can visualize microstructural irregularities and perform an initial morphological analysis of the lesion. The images can be used to guide acquisition of biochemically specific Raman spectra. The two data sets can then be evaluated with respect to one another to take advantage of the mutually complimentary nature of RS and OCT.

A computationally efficient Monte-Carlo model for biomedical Raman spectroscopy.

Monte Carlo (MC) modeling is a valuable tool to gain fundamental understanding of light-tissue interactions, provide guidance and assessment to optical instrument designs, and help analyze experimental data. It has been a major challenge to efficiently extend MC towards modeling of bulk-tissue Raman spectroscopy (RS) due to the wide spectral range, relatively sharp spectral features, and presence of background autofluorescence. Here, we report a computationally efficient MC approach for RS by adapting the massively-parallel Monte Carlo eXtreme (MCX) simulator. Simulation efficiency is achieved through "isoweight," a novel approach that combines the statistical generation of Raman scattered and Fluorescence emission with a lookup-table-based technique well-suited for parallelization. The MC model uses a graphics processor to produce dense Raman and fluorescence spectra over a range of 800 - 2000 cm-1 with an approximately 100× increase in speed over prior RS Monte Carlo methods. The simulated RS signals are compared against experimentally collected spectra from gelatin phantoms, showing a strong correlation.

Discrimination of malignant and normal kidney tissue with short wave infrared dispersive Raman spectroscopy.

Renal mass biopsy is still controversial due to imperfect accuracy. Raman spectroscopy (RS) demonstrated promise as an in vivo real-time, nondestructive diagnostic tool in many malignancies. Short wave infrared (SWIR) RS has the potential to improve on previous RS systems for renal mass diagnosis. The aim of this study is to evaluate a SWIR RS system in differentiating normal and malignant renal samples. Measurements were acquired using a benchtop RS system with excitation wavelength at 1064 nm and an InGaAs array detector. Processed spectra were classified with a Bayesian machine learning algorithm, sparse multinomial logistic regression. Sensitivity and receiver operating characteristic curve analyses evaluated the classifier accuracy. Accuracy of the classifier was 92.5% with sensitivity and specificity of 95.8% and 88.8%, respectively. For posterior probability of malignant class assignment, the area under the ROC curve is 0.94 (95% confidence interval: 0.89-0.99, P < .001). SWIR RS accurately differentiated normal and malignant kidney tumors. RS has the potential to be used as a diagnostic tool in kidney cancer.

Anterior chamber width measurement by high-speed optical coherence tomography.

OBJECTIVE: To measure anterior chamber (AC) width and other dimensions relevant to the sizing of phakic intraocular lenses (IOLs) with a high-speed optical coherence tomography (OCT) system. DESIGN: Cross-sectional observational study. PARTICIPANTS: Both eyes of 20 normal volunteers. METHODS: A novel high-speed (4000 axial scans/second) OCT prototype was developed for anterior segment scanning. The system uses long wavelength (1310 nm) for deeper angle penetration, rectangular scanning for undistorted imaging, and short image acquisition time (0.125 seconds) to reduce motion error. Three horizontal cross-sectional OCT images (15.5 mm wide and 6 mm deep) of the anterior segment were obtained from each eye with real-time image display to guide centration on the corneal apex. Image processing software was developed to correct for image warping resulting from index transitions. Anterior chamber dimensions were measured using computer calipers by 3 expert raters (ophthalmologists). Analysis of variance was used to determine interrater, interimage, right versus left eye, and intersubject standard deviation (SD) of OCT measurements. MAIN OUTCOME MEASURES: Anterior chamber width (recess to recess), AC depth, and crystalline lens vault as measured by OCT; external white-to-white (WTW) corneal diameter (CD) as measured by Holladay-Godwin gauge. RESULTS: The mean AC width was 12.53+/-0.47 mm (intereye SD), and the mean corneal diameter was 11.78+/-0.57 mm. Optical coherence tomography measurement of AC width has good repeatability from image to image (SD, 0.134 mm), but there was significant difference between raters (SD, 0.215 mm). Estimation of AC width from WTW CD by linear regression was relatively inaccurate (residual SD, 0.41 mm). The mean AC depth was 2.99+/-0.323 mm (intereye SD), with repeatability of less than 0.001 mm (interimage SD), and the mean crystalline lens vault was 0.39+/-0.27 mm with 0.023 mm repeatability. CONCLUSIONS: Reproducible OCT AC biometry was demonstrated using a high-speed OCT prototype. Further improvement in reproducibility may be achieved by automating the measurements with a computer. Direct OCT AC width measurement may improve sizing of angle-supported AC IOLs over conventional estimation by WTW CD. The measurement of AC depth and lens vault also may be useful for other types of phakic AC IOLs.

Discrimination of liver malignancies with 1064 nm dispersive Raman spectroscopy.

Raman spectroscopy has been widely demonstrated for tissue characterization and disease discrimination, however current implementations with either 785 or 830 nm near-infrared (NIR) excitation have been ineffectual in tissues with intense autofluorescence such as the liver. Here we report the use of a dispersive 1064 nm Raman system using a low-noise Indium-Gallium-Arsenide (InGaAs) array to discriminate highly autofluorescent bulk tissue ex vivo specimens from healthy liver, adenocarcinoma, and hepatocellular carcinoma (N = 5 per group). The resulting spectra have been combined with a multivariate discrimination algorithm, sparse multinomial logistic regression (SMLR), to predict class membership of healthy and diseased tissues, and spectral bands selected for robust classification have been extracted. A quantitative metric called feature importance is defined based on classification outputs and is used to guide the association of spectral features with biological indicators of healthy and diseased liver tissue. Spectral bands with high feature importance for healthy and liver tumor specimens include retinol, heme, biliverdin, or quinones (1595 cm(-1)); lactic acid (838 cm(-1)); collagen (873 cm(-1)); and nucleic acids (1485 cm(-1)). Classification performance in both binary (normal versus tumor, 100% sensitivity and 89% specificity) and three-group cases (classification accuracy: normal 89%, adenocarcinoma 74%, hepatocellular carcinoma 64%) indicates the potential for accurately separating healthy and cancerous tissues and suggests implications for utilizing Raman techniques during surgical guidance in liver resection.

Photothermal optical lock-in optical coherence tomography for in vivo imaging.

Photothermal OCT (PTOCT) provides high sensitivity to molecular targets in tissue, and occupies a spatial imaging regime that is attractive for small animal imaging. However, current implementations of PTOCT require extensive temporal sampling, resulting in slow frame rates and a large data burden that limit its in vivo utility. To address these limitations, we have implemented optical lock-in techniques for photothermal optical lock-in OCT (poli-OCT), and demonstrated the in vivo imaging capabilities of this approach. The poli-OCT signal was assessed in tissue-mimicking phantoms containing indocyanine green (ICG), an FDA approved small molecule that has not been previously imaged in vivo with PTOCT. Then, the effects of in vivo blood flow and motion artifact were assessed and attenuated, and in vivo poli-OCT was demonstrated with both ICG and gold nanorods as contrast agents. Experiments revealed that poli-OCT signals agreed with optical lock-in theory and the bio-heat equation, and the system exhibited shot noise limited performance. In phantoms containing biologically relevant concentrations of ICG (1 µg/ml), the poli-OCT signal was significantly greater than control phantoms (p<0.05), demonstrating sensitivity to small molecules. Finally, in vivo poli-OCT of ICG identified the lymphatic vessels in a mouse ear, and also identified low concentrations (200 pM) of gold nanorods in subcutaneous injections at frame rates ten times faster than previously reported. This work illustrates that future in vivo molecular imaging studies could benefit from the improved acquisition and analysis times enabled by poli-OCT.

Quantifying the vascular response to ischemia with speckle variance optical coherence tomography.

Longitudinal monitoring techniques for preclinical models of vascular remodeling are critical to the development of new therapies for pathological conditions such as ischemia and cancer. In models of skeletal muscle ischemia in particular, there is a lack of quantitative, non-invasive and long term assessment of vessel morphology. Here, we have applied speckle variance optical coherence tomography (OCT) methods to quantitatively assess vascular remodeling and growth in a mouse model of peripheral arterial disease. This approach was validated on two different mouse strains known to have disparate rates and abilities of recovering following induction of hind limb ischemia. These results establish the potential for speckle variance OCT as a tool for quantitative, preclinical screening of pro- and anti-angiogenic therapies.

Polarization control of Raman spectroscopy optimizes the assessment of bone tissue.

There is potential for Raman spectroscopy (RS) to complement tools for bone diagnosis due to its ability to assess compositional and organizational characteristics of both collagen and mineral. To aid this potential, the present study assessed specificity of RS peaks to the composition of bone, a birefringent material, for different degrees of instrument polarization. Specifically, relative changes in peaks were quantified as the incident light rotated relative to the orientation of osteonal and interstitial tissue, acquired from cadaveric femurs. In a highly polarized instrument (10(6)∶1 extinction ratio), the most prominent mineral peak (ν1 Phosphate at 961 cm(-1)) displayed phase similarity with the Proline peak at 856 cm(-1). This sensitivity to relative orientation between bone and light observed in the highly polarized regime persisted for certain sensitive peaks (e.g., Amide I at 1666 cm(-1)) in unaltered instrumentation (200∶1 extinction ratio). Though Proline intensity changed with bone rotation, the phase of Proline matched that of ν1 Phosphate. Moreover, when mapping ν1 Phosphate/Proline across osteonal-interstitial borders, the mineralization difference between the tissue types was evident whether using a 20x or 50x objectives. Thus, the polarization bias inherent in commercial RS systems does not preclude the assessment of bone composition when using phase-matched peaks.

A handheld laser scanning confocal reflectance imaging-confocal Raman microspectroscopy system.

Confocal reflectance microscopy and confocal Raman spectroscopy have shown potential for non-destructive analysis of samples at micron-scale resolutions. Current studies utilizing these techniques often employ large bench-top microscopes, and are not suited for use outside of laboratory settings. We have developed a microscope which combines laser scanning confocal reflectance imaging and confocal Raman spectroscopy into a compact handheld probe that is capable of high-resolution imaging and spectroscopy in a variety of settings. The compact size of the probe is largely due to the use of a MEMS mirror for beam scanning. The probe is capable of axial resolutions of up to 4 µm for the confocal imaging channel and 10 µm for the confocal Raman spectroscopy channel. Here, we report instrument design, characterize optical performance, and provide images and spectra from normal skin to demonstrate the instrument's capabilities for clinical diagnostics.

Raman and mechanical properties correlate at whole bone- and tissue-levels in a genetic mouse model.

The fracture resistance of bone arises from the composition, orientation, and distribution of the primary constituents at each hierarchical level of organization. Therefore, to establish the relevance of Raman spectroscopy (RS) in identifying differences between strong or tough bone and weak or brittle bone, we investigated whether Raman-derived properties could explain the variance in biomechanical properties at both the whole bone and the tissue-level, and do so independently of traditional measurements of mineralization. We harvested femurs from wild-type mice and mice lacking matrix metalloproteinase 2 because the mutant mice have a known reduction in mineralization. Next, RS quantified compositional properties directly from the intact diaphysis followed by micro-computed tomography to quantify mineralization density (Ct.TMD). Correlations were then tested for significance between these properties and the biomechanical properties as determined by the three-point bending test on the same femurs. Harvested tibia were embedded in plastic, sectioned transversely, and polished in order to acquire average Raman properties per specimen that were then correlated with average nanoindentation properties per specimen. Dividing the ν(1) phosphate by the proline peak intensity provided the strongest correlation between the mineral-to-collagen ratio and the biomechanical properties (whole bone modulus, strength, and post-yield deflection plus nanoindentation modulus). Moreover, the linear combination of ν(1) phosphate/proline and Ct.TMD provided the best explanation of the variance in strength between the genotypes, and it alone was the best explanatory variable for brittleness. Causal relationships between Raman and fracture resistance need to be investigated, but Raman has the potential to assess fracture risk.

Integrated system for combined Raman spectroscopy-spectral domain optical coherence tomography.

Raman spectroscopy (RS) and optical coherence tomography (OCT) are powerful tools for optical analysis of tissues with mutually complementary strengths and limitations. OCT excels at visualizing tissue microstructure but lacks molecular specificity, while RS can relay tissue biochemical composition but typically cannot relate microstructure. Previous implementations of combined RS-OCT have utilized a common sample arm while maintaining independent RS and OCT detection arms. We present the design and application of an integrated RS-OCT instrument with a common detection arm for both RS and OCT. The detector is a spectrograph capable of sequential detection of the 855-nm OCT signal and the Raman scatter generated by a 785-nm source. The capabilities of the instrument are demonstrated ex vivo in the calvaria and retina of rodents, as well as in vivo in human skin.

Differential effects between the loss of MMP-2 and MMP-9 on structural and tissue-level properties of bone.

Matrix metalloproteinases (MMPs) are capable of processing certain components of bone tissue, including type 1 collagen, a determinant of the biomechanical properties of bone tissue, and they are expressed by osteoclasts and osteoblasts. Therefore, we posit that MMP activity can affect the ability of bone to resist fracture. To explore this possibility, we determined the architectural, compositional, and biomechanical properties of bones from wild-type (WT), Mmp2(-/-) , and Mmp9(-/-) female mice at 16 weeks of age. MMP-2 and MMP-9 have similar substrates but are expressed primarily by osteoblasts and osteoclasts, respectively. Analysis of the trabecular compartment of the tibia metaphysis by micro-computed tomography (µCT) revealed that these MMPs influence trabecular architecture, not volume. Interestingly, the loss of MMP-9 improved the connectivity density of the trabeculae, whereas the loss of MMP-2 reduced this parameter. Similar differential effects in architecture were observed in the L(5) vertebra, but bone volume fraction was lower for both Mmp2(-/-) and Mmp9(-/-) mice than for WT mice. The mineralization density and mineral-to-collagen ratio, as determined by µCT and Raman microspectroscopy, were lower in the Mmp2(-/-) bones than in WT control bones. Whole-bone strength, as determined by three-point bending or compression testing, and tissue-level modulus and hardness, as determined by nanoindentation, were less for Mmp2(-/-) than for WT bones. In contrast, the Mmp9(-/-) femurs were less tough with lower postyield deflection (more brittle) than the WT femurs. Taken together, this information reveals that MMPs play a complex role in maintaining bone integrity, with the cell type that expresses the MMP likely being a contributing factor to how the enzyme affects bone quality.

Inhibition of TGF-ß signaling by 1D11 antibody treatment increases bone mass and quality in vivo.

Transforming growth factor ß (TGF-ß) is an abundant bone matrix protein that influences osteoblast and osteoclast interactions to control bone remodeling. As such, TGF-ß represents an obvious pharmacologic target with the potential to regulate both bone formation and resorption to improve bone volume and strength. To investigate the skeletal effect of TGF-ß inhibition in vivo, we used an antibody (1D11) specifically directed at all three isoforms of TGF-ß. Normal mice were treated with 1D11 or control antibody (4 weeks), and cortical and trabecular bone was assessed by micro-computed tomographic (µCT) scanning. Bone volume and cellular distribution were determined by histomorphometric analysis of vertebrae and long bones. Also, whole-bone strength was assessed biomechanically by three-point bend testing, and tissue-level modulus and composition were analyzed by nanoindentation and Raman microspectroscopy, respectively. TGF-ß blockade by 1D11 increased bone mineral density (BMD), trabecular thickness, and bone volume by up to 54%, accompanied by elevated osteoblast numbers and decreased osteoclasts. Biomechanical properties of bone also were enhanced significantly by 1D11 treatment, with increased bending strength and tissue-level modulus. In addition, Raman microspectroscopy demonstrated that 1D11-mediated TGF-ß inhibition in the bone environment led to an 11% increase in the mineral-to-collagen ratio of trabecular bone. Together these studies demonstrate that neutralizing TGF-ß with 1D11 increases osteoblast numbers while simultaneously decreasing active osteoclasts in the marrow, resulting in a profound increase in bone volume and quality, similar to that seen in parathyroid hormone (PTH)-treated rodent studies.