Xiaohong Bi

Sustained Osteomalacia of Long Bones Despite Major Improvement in Other Hypophosphatasia- Related Mineral Deficits in Tissue Nonspecific Alkaline Phosphatase/Nucleotide Pyrophosphatase Phosphodiesterase 1 Double-Deficient Mice

We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2-/-) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp1-/- mice is corrected in [Akp2-/-; Enpp1-/-] double-knockout mice. Here we have examined the appendicular skeletons of Akp2-/-, Enpp1-/-, and [Akp2-/-; Enpp1-/-] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2-/-, and [Akp2-/-; Enpp1-/-] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2-/- but also in Enpp1-/- and [Akp2-/-; Enpp1-/-] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1-/- calvarial osteoblasts but decreased mineral deposition by Enpp1-/- long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2-/- mice and the hypomineralized phenotype of the long bones of Enpp1-/- mice are not rescued by simultaneous deletion of TNAP and NPP1 functions.

Fourier transform infrared imaging spectroscopic analysis of tissue engineered cartilage: histologic and biochemical correlations

The composition of cartilage is predictive of its in vivo performance. Therefore, the ability to assess its primary macromolecular components, proteoglycan (PG) and collagen, is of great importance. In the current study, we hypothesized that PG content and distribution in tissue engineered cartilage could be determined using Fourier-transform infrared imaging spectroscopy (FT-IRIS). The cartilage was grown from chondrocytes within a hollow fiber bioreactor (HFBR) system previously used extensively to study cartilage development. FT-IRIS analysis showed a gradient of PG content, with the highest content in the center near the nutritive fibers and the lowest near the interior surface of the HFBR. Further, we found significantly greater PG content in the region near culture medium inflow (45.0%) as compared to the outflow region (24.7%) (p<0.001). This difference paralleled the biochemically determined glycosaminoglycan difference of 42.6% versus 27.8%. In addition, FT-IRIS-determined PG content at specific positions within the tissue sections correlated with histologically determined PG content (R=0.73, p=0.007). In summary, FT-IRIS determination of PG correlates with histological determination of PG and yields quantitatively similar results to biochemical determination of glycosaminoglycan in developing cartilage.

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.

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 L5 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.

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 ν1 phosphate (PO4) 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 ν1PO4/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, ν1PO4/amide III or ν1PO4/proline is the Raman property that is most likely to detect a compositional difference between experimental groups.

Development of spectral markers for the discrimination of ulcerative colitis and Crohn's disease using Raman spectroscopy.

BACKGROUND: Ulcerative colitis and Crohns disease are 2 distinct forms of IBD that can overlap radiologically, endoscopically, and pathologically. This difficulty complicates surgical options. The development of new technologies providing accurate diagnosis of IBD is needed. Raman spectroscopy is a noninvasive method that uses the intrinsic properties of tissue and that tissues vibrational energy in reaction to light. PURPOSE: We hypothesize that Raman spectroscopy can detect the structural and compositional changes that occur in the tissue during the development of inflammatory bowel disease, and thus may offer increased diagnostic certainty in the differentiation between Crohns disease and ulcerative colitis. METHODS: Fresh frozen colon tissue biopsies from patients with ulcerative colitis (n = 12) and with Crohns disease (n = 9) were measured in vitro using a custom-designed Raman fiber-optic probe. For spectra collection, the probe was placed in gentle contact with the mucosa surface for 3 seconds, with excitation power at 150 mW. Five spectra were acquired from each biopsy to increase the signal-to-noise ratio and to ensure repeatability of data collection. Mean spectra were analyzed for peak difference and molecular origin. RESULTS: Significant difference was observed between the spectra from each disease in the spectral regions assigned to nucleic acid, phenylalanine, and lipids. Tissue samples from patients with ulcerative colitis demonstrated higher content of lipid and lower amount of phenylalanine and nucleic acid. These characteristic Raman features could serve as spectral markers that can potentially be applied to distinguish ulcerative colitis and Crohns disease. CONCLUSIONS: This study presents the only application of Raman spectroscopy in the diagnosis of inflammatory bowel disease. The feasibility of this technique in differentially detecting molecular alterations in ulcerative colitis and Crohns disease has been demonstrated, indicating the potential to improve diagnostic accuracy of inflammatory bowel disease.

The swaying mouse as a model of osteogenesis imperfecta caused by WNT1 mutations

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue characterized by bone fragility and low bone mass. Recently, our group and others reported that WNT1 recessive mutations cause OI, whereas WNT1 heterozygous mutations cause early onset osteoporosis. These findings support the hypothesis that WNT1 is an important WNT ligand regulating bone formation and bone homeostasis. While these studies provided strong human genetic and in vitro functional data, an in vivo animal model to study the mechanism of WNT1 function in bone is lacking. Here, we show that Swaying (Wnt1(sw/sw)) mice previously reported to carry a spontaneous mutation in Wnt1 share major features of OI including propensity to fractures and severe osteopenia. In addition, biomechanical and biochemical analyses showed that Wnt1(sw/sw) mice exhibit reduced bone strength with altered levels of mineral and collagen in the bone matrix that is also distinct from the type I collagen-related form of OI. Further histomorphometric analyses and gene expression studies demonstrate that the bone phenotype is associated with defects in osteoblast activity and function. Our study thus provides in vivo evidence that WNT1 mutations contribute to bone fragility in OI patients and demonstrates that the Wnt1(sw/sw) mouse is a murine model of OI caused by WNT1 mutations.

Prostate cancer metastases alter bone mineral and matrix composition independent of effects on bone architecture in mice — A quantitative study using microCT and Raman spectroscopy

Prostate cancer is the most common primary tumor and the second leading cause of cancer-related deaths in men in the United States. Prostate cancer bone metastases are characterized by abnormal bone remodeling processes and result in a variety of skeletal morbidities. Prevention of skeletal complications is a crucial element in prostate cancer management. This study investigated prostate cancer-induced alterations in the molecular composition and morphological structure of metastasis-bearing bones in a mouse model of prostate cancer using Raman spectroscopy and micro-computed tomography (microCT). LNCaP C4-2B prostate cancer cells were injected into the right tibiae of 5-week old male SCID mice. Upon sacrifice at 8weeks post tumor inoculation, two out of the ten tumor-bearing tibiae showed only osteoblastic lesions in the radiographs, 4 osteolytic lesions only and 4 mixed with osteoblastic and osteolytic lesions. Carbonate substitution was significantly increased while there was a marked reduction in the level of collagen mineralization, mineral crystallinity, and carbonate:matrix ratio in the cortex of the intact tumor-bearing tibiae compared to contralateral controls. MicroCT analysis revealed a significant reduction in bone volume/total volume, trabecular number and trabecular thickness, as well as significant increase in bone surface/volume ratio in tibiae with osteolytic lesions, suggesting active bone remodeling and bone loss. None of the changes in bone compositional properties were correlated with lesion area from radiographs or the changes in bone architecture from microCT. This study indicates that LNCaP C4-2B prostate cancer metastases alter bone tissue composition independent of changes in architecture, and altered bone quality may be an important contributor to fracture risk in these patients. Raman spectroscopy may provide a new avenue of investigation into interactions between tumor and bone microenvironment.

Infrared Fiber Optic Probe Evaluation of Degenerative Cartilage Correlates to Histological Grading

Background: Osteoarthritis (OA), a degenerative cartilage disease, results in alterations of the chemical and structural properties of tissue. Arthroscopic evaluation of full-depth tissue composition is limited and would require tissue harvesting, which is inappropriate in daily routine. Fourier transform infrared (FT-IR) spectroscopy is a modality based on molecular vibrations of matrix components that can be used in conjunction with fiber optics to acquire quantitative compositional data from the cartilage matrix. Purpose: To develop a model based on infrared spectra of articular cartilage to predict the histological Mankin score as an indicator of tissue quality. Study Design: Comparative laboratory study. Methods: Infrared fiber optic probe (IFOP) spectra were collected from nearly normal and more degraded regions of tibial plateau articular cartilage harvested during knee arthroplasty (N = 61). Each region was graded using a modified Mankin score. A multivariate partial least squares algorithm using second-derivative spectra was developed to predict the histological modified Mankin score. Results: The partial least squares model derived from IFOP spectra predicted the modified Mankin score with a prediction error of approximately 1.4, which resulted in approximately 72% of the Mankin-scored tissues being predicted correctly and 96% being predicted within 1 grade of their true score. Conclusion: These data demonstrate that IFOP spectral parameters correlate with histological tissue grade and can be used to provide information on tissue composition. Clinical Relevance: Infrared fiber optic probe studies have significant potential for the evaluation of cartilage tissue quality without the need for tissue harvest. Combined with arthroscopy, IFOP analysis could facilitate the definition of tissue margins in debridement procedures.

Evaluating HER2 amplification status and acquired drug resistance in breast cancer cells using Raman spectroscopy

Abstract. The overexpression of human epidermal growth factor receptor 2 (HER2) is associated with increased breast cancer recurrence and worse prognosis. Effective treatments such as trastuzumab and lapatinib for patients with HER2 overexpression target the blockade of HER2 signaling activities but are often limited by the emergence of acquired drug resistance. This study applied Raman spectroscopy to differentially identify the amplification status of HER2 in cells and to characterize the biochemical composition of lapatinib resistant and sensitive HER2+ breast cancer cells in response to the drug. Raman spectra from BT474 (HER2+ breast cancer cell), MCF-10A (HER2− control), and HER2+ MCF-10A (HER2+ control) were analyzed using lasso and elastic-net regularized generalized linear models (glmnet) for multivariate statistical analysis and were discriminated to groups of different HER2 expression status with an overall 99% sensitivity and specificity. Enhanced lipid content and decreased proteome were observed in HER2+ cells. With lapatinib treatment, lapatinib-resistant breast cancer cells demonstrated sustained lipogenesis compared with the sensitive cells.