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.

Use of a mechanical iris-based fiber optic probe for spatially offset Raman spectroscopy.

We demonstrated a mechanical iris-based fiber optic probe with adjustable spatial offsets for spatially offset Raman spectroscopy (SORS). In the fiber probe, the excitation fiber was fixed at the center of the iris, and the collection fibers were movable with blades of the iris. Moreover, we studied the gap effect between the probe and the sample and demonstrated this fiber optic probe can be used as a platform for surface-enhanced SORS applications. This fiber probe design could potentially provide a design-efficient and cost-effective solution for various Raman applications.

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.

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

BACKGROUND: Ulcerative colitis and Crohn's 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 tissue's 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 Crohn's disease and ulcerative colitis. METHODS: Fresh frozen colon tissue biopsies from patients with ulcerative colitis (n = 12) and with Crohn's 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 Crohn's 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 Crohn's disease has been demonstrated, indicating the potential to improve diagnostic accuracy of inflammatory bowel disease.

Osteoclast Differentiation Assay.

Osteoclasts are highly specialized multinucleated cells derived from the monocyte/macrophage hematopoietic lineage that are uniquely capable of adhering to bone matrix and resorbing bone. The tartrate-resistant acid phosphatase (TRAP) assay is the most common method to detect osteoclasts population in vitro. Here we described a general protocol of inducing osteoclast differentiation from the murine macrophage cell line, RAW264.7, and identification of osteoclasts with the classical TRAP assay.

An eight-year epidemiologic study of head and neck tuberculosis in Texas, USA.

BACKGROUND: Head and neck tuberculosis (HNTB) especially cervical lymphadenopathy are the most common extrapulmonary indications of TB, but remain a diagnostic challenge. In this study, we describe and analyze the epidemiologic characteristics of HNTB on a population-level. MATERIALS AND METHODS: We retrospectively assessed 547 HNTB cases reported to the Centers for Disease Control and Prevention's TB Genotyping Information Management System in Texas from 2009 to 2016 and compare and contrast differences between diagnosed exclusively HNTB and HNTB with concurrent pulmonary tuberculosis (PTB). RESULTS: The majority of patients with HNTB were diagnosed with cervical lymphadenopathy (96.9%), age 25-44 (47.3%) and female (52.7%). Co-infection with human immunodeficiency virus, being homeless, excessive alcohol use within the past 12 months and drug use were more frequently seen for HNTB with concurrent pulmonary involvement compared to reported patients with exclusively HNTB. The highest prevalence of Mycobacterium tuberculosis lineage in exclusively HNTB was Euro-American L4 (52.3%), followed by Indo-Oceanic L1 (21.5%) and East-Asian L2 (13.1%). One multidrug resistant TB case was identified. Seven deaths were reported during treatment. CONCLUSION: Our findings provide a better understanding of the epidemiology of HNTB and characteristics associated with the disease at the population-level, which is important in managing HNTB patients.

A comparison of BMP2 delivery by coacervate and gene therapy for promoting human muscle-derived stem cell-mediated articular cartilage repair.

BACKGROUND: Osteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study. METHOD: In this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair. RESULTS: We observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining. CONCLUSIONS: We conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

Head and neck tuberculosis: Literature review and meta-analysis.

BACKGROUND: Head and neck tuberculosis (HNTB), including cervical lymphadenopathy, is the most common extrapulmonary manifestation of TB. The proposed study investigated the epidemiologic and clinical characteristics of HNTB. MATERIALS AND METHODS: A literature search was conducted via PubMed, Embase, Cochrane Library and Wanfang for keywords (tuberculosis, head and neck, laryngeal, pharyngeal, tongue, oropharyngeal, nasopharyngeal, and oral cavity). Scientific articles published from January 1990 through July 2017 were selected and reviewed to assess the epidemiology, presentation, diagnosis and treatment of HNTB disease. RESULTS: Results from the included 57 studies revealed that the majority of HNTB cases were age<40 years and female. The most common HNTB sites were cervical lymph nodes (87.9%), followed by larynx (8.7%). Involvement of other HN-regions was rare (3.4%). Multidrug resistant TB was not common among the majority of studies. Given the paucibacillary nature of HNTB, sputum tests did not have a good performance on HNTB diagnosis. Most of HNTB cases were diagnosed by fine-needle aspiration, cytology and excision biopsies in combination with clinical presentations. CONCLUSION: HNTB disease is an important manifestation in the diagnostic process in an otolaryngologist practice. The developments of rapid, ultrasensitive, simple and cost-effective high-throughput methods for early diagnosis of HNTB are urgently needed.

TRIM44 promotes quiescent multiple myeloma cell occupancy and survival in the osteoblastic niche via HIF-1α stabilization.

Despite progress in the treatment of MM, including the use of high-dose chemotherapy and autologous stem cell transplantation, a considerable proportion of patients are refractory to all therapies. This resistance is related to the molecular genetic heterogeneity in MM cells as well as to the contributions from the BM, which is one of the key determinants of treatment outcome. Our previous studies using fluorescent tracers revealed that MM heterogeneity is correlated with the presence of quiescent stem-like cancer cells, which prefer to reside within the osteoblastic niche of the BM. In this report, we identified a novel protein, tripartite motif containing 44 (TRIM44), which is overexpressed in the osteoblastic niche of the BM, enabling MM cells to compete with HSCs for niche support. TRIM44 expression in MM cells promoted cell quiescence but increased bone destruction in xenograft mice, similar to what is observed in MM patients. TRIM44 functions as a deubiquitinase for hypoxia inducible factor-1α (HIF-1α), which stabilizes HIF-1α expression during hypoxia and normoxia. Stabilized HIF-1α stimulates MM cell growth and survival during hypoxia. Our work is the first report to reveal signaling in quiescent MM cells and the functions of TRIM44.

Risk factors for extrapulmonary dissemination of tuberculosis and associated mortality during treatment for extrapulmonary tuberculosis.

Many environmental, host, and microbial characteristics have been recognized as risk factors for dissemination of extrapulmonary tuberculosis (EPTB). However, there are few population-based studies investigating the association between the primary sites of tuberculosis (TB) infection and mortality during TB treatment. De-identified population-based surveillance data of confirmed TB patients reported from 2009 to 2015 in Texas, USA, were analyzed. Regression analyses were used to determine the risk factors for EPTB, as well as its subsite distribution and mortality. We analyzed 7007 patients with exclusively pulmonary TB, 1259 patients with exclusively EPTB, and 894 EPTB patients with reported concomitant pulmonary involvement. Age ≥45 years, female gender, human immunodeficiency virus (HIV)-positive status, and end-stage renal disease (ESRD) were associated with EPTB. ESRD was associated with the most clinical presentations of EPTB other than meningeal and genitourinary TB. Patients age ≥45 years had a disproportionately high rate of bone TB, while foreign-born patients had increased pleural TB and HIV+ patients had increased meningeal TB. Age ≥45 years, HIV+ status, excessive alcohol use within the past 12 months, ESRD, and abnormal chest radiographs were independent risk factors for EPTB mortality during TB treatment. The epidemiologic risk factors identified by multivariate analyses provide new information that may be useful to health professionals in managing patients with EPTB.

Mechanical properties of infant bone.

Although an understanding of bone material properties is crucial for interpreting and predicting fracture patterns due to injury or defining the effects of disease on bone strength, information about infant bone properties is scant in the literature. In this study we present the mechanical testing results from 47 tibia and 52 rib specimens taken from 53 infant decedents in order to further our understanding of infant bone strength. Bone specimens were imaged using microCT and tested in three-point bending until failure. Extrinsic and intrinsic properties demonstrated an increase in strength and stiffness over the first year of life, while ductility measures remained largely unchanged. Donor race had no effect on the material properties, but tibia bone specimens showed significant sex differences, with the elastic modulus from females being larger than males. When compared to properties from adolescent and adult donors, infant bone is less strong, less stiff, and more ductile.

Mutant cartilage oligomeric matrix protein (COMP) compromises bone integrity, joint function and the balance between adipogenesis and osteogenesis.

Mutations in COMP (cartilage oligomeric matrix protein) cause severe long bone shortening in mice and humans. Previously, we showed that massive accumulation of misfolded COMP in the ER of growth plate chondrocytes in our MT-COMP mouse model of pseudoachondroplasia (PSACH) causes premature chondrocyte death and loss of linear growth. Premature chondrocyte death results from activation of oxidative stress and inflammation through the CHOP-ER pathway and is reduced by removing CHOP or by anti-inflammatory or antioxidant therapies. Although the mutant COMP chondrocyte pathologic mechanism is now recognized, the effect of mutant COMP on bone quality and joint health (laxity) is largely unknown. Applying multiple analytic approaches, we describe a novel mechanism by which the deleterious consequences of mutant COMP retention results in upregulation of miR-223 disturbing the adipogenesis - osteogenesis balance. This results in reduction in bone mineral density, bone quality, mechanical strength and subchondral bone thickness. These, in addition to abnormal patterns of ossification at the ends of the femoral bones likely contribute to precocious osteoarthritis (OA) of the hips and knees in the MT-COMP mouse and PSACH. Moreover, joint laxity is compromised by abnormally thin ligaments. Altogether, these novel findings align with the PSACH phenotype of delayed ossification and bone age, extreme joint laxity and joint erosion, and extend our understanding of the underlying processes that affect bone in PSACH. These results introduce a novel finding that miR-223 is involved in the ossification defect in MT-COMP mice making it a therapeutic target.

Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage.

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of approximately 6.25 microm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm(-1)/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.

In vivo analysis of mucosal lipids reveals histological disease activity in ulcerative colitis using endoscope-coupled Raman spectroscopy.

The goal of this study is to evaluate endoscopic Raman spectroscopy as a noninvasive technique to determine histological inflammatory status of colitis. Colon mucosal composition was investigated in vivo from patients with ulcerative colitis (UC) and from age- and body mass index (BMI) matched controls using endoscope-coupled Raman spectroscopy. The results were co-registered with histological assessment of inflammatory status at the same locations. Substantial decreases (50-60%) in the content of phosphotidylcholines (PCs) and total lipids were observed in inflamed colon tissue (histology grade 1, 2 and 3) compared to those from the quiescent (histology grade 0) and from the controls. No significant difference was observed in lipids or PC contents between control and grade 0, or among grades 1 - 3. The degree of lipid unsaturation increased in the inflamed tissue regardless of disease severity. The inflammation-associated alterations in lipids and PC are observed independent of BMI or the anatomical locations for data collection. Multivariate analysis using support vector machine (SVM) algorithm classified the spectra of the controls or the inactive colitis from those of inflamed tissue with a sensitivity of 83.5% and 97.1% respectively. Our results showed that mucosal lipid content is related to the microscopic disease activity, and thus could serve as a valuable spectral marker to differentiate active colitis from the quiescent.

Clinical characterization of in vivo inflammatory bowel disease with Raman spectroscopy.

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), affects over 1 million Americans and 2 million Europeans, and the incidence is increasing worldwide. While these diseases require unique medical care, the differentiation between UC and CD lacks a gold standard, and therefore relies on long term follow up, success or failure of existing treatment, and recurrence of the disease. Here, we present colonoscopy-coupled fiber optic probe-based Raman spectroscopy as a minimally-invasive diagnostic tool for IBD of the colon (UC and Crohn's colitis). This pilot in vivo study of subjects with existing IBD diagnoses of UC (n = 8), CD (n = 15), and normal control (n = 8) aimed to characterize spectral signatures of UC and CD. Samples were correlated with tissue pathology markers and endoscopic evaluation. The collected spectra were processed and analyzed using multivariate statistical techniques to identify spectral markers and discriminate IBD and disease classes. Confounding factors including the presence of active inflammation and the particular colon segment measured were investigated and integrated into the devised prediction algorithm, reaching 90% sensitivity and 75% specificity to CD from this in vivo data set. These results represent significant progress towards improved real-time classification for accurate and automated in vivo detection and discrimination of IBD during colonoscopy procedures.

Correlations Between Bone Mechanical Properties and Bone Composition Parameters in Mouse Models of Dominant and Recessive Osteogenesis Imperfecta and the Response to Anti-TGF-ß Treatment.

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by brittle bones that are prone to fracture. Although previous studies in animal models investigated the mechanical properties and material composition of OI bone, little work has been conducted to statistically correlate these parameters to identify key compositional contributors to the impaired bone mechanical behaviors in OI. Further, although increased TGF-ß signaling has been demonstrated as a contributing mechanism to the bone pathology in OI models, the relationship between mechanical properties and bone composition after anti-TGF-ß treatment in OI has not been studied. Here, we performed follow-up analyses of femurs collected in an earlier study from OI mice with and without anti-TGF-ß treatment from both recessive (Crtap-/- ) and dominant (Col1a2+/P.G610C ) OI mouse models and WT mice. Mechanical properties were determined using three-point bending tests and evaluated for statistical correlation with molecular composition in bone tissue assessed by Raman spectroscopy. Statistical regression analysis was conducted to determine significant compositional determinants of mechanical integrity. Interestingly, we found differences in the relationships between bone composition and mechanical properties and in the response to anti-TGF-ß treatment. Femurs of both OI models exhibited increased brittleness, which was associated with reduced collagen content and carbonate substitution. In the Col1a2+/P.G610C femurs, reduced hydroxyapatite crystallinity was also found to be associated with increased brittleness, and increased mineral-to-collagen ratio was correlated with increased ultimate strength, elastic modulus, and bone brittleness. In both models of OI, regression analysis demonstrated that collagen content was an important predictor of the increased brittleness. In summary, this work provides new insights into the relationships between bone composition and material properties in models of OI, identifies key bone compositional parameters that correlate with the impaired mechanical integrity of OI bone, and explores the effects of anti-TGF-ß treatment on bone-quality parameters in these models. © 2016 American Society for Bone and Mineral Research.

Fkbp10 Deletion in Osteoblasts Leads to Qualitative Defects in Bone.

Osteogenesis imperfecta (OI), also known as brittle bone disease, displays a spectrum of clinical severity from mild (OI type I) to severe early lethality (OI type II), with clinical features including low bone mass, fractures, and deformities. Mutations in the FK506 Binding Protein 10 (FKBP10), gene encoding the 65-kDa protein FKBP65, cause a recessive form of OI and Bruck syndrome, the latter being characterized by joint contractures in addition to low bone mass. We previously showed that Fkbp10 expression is limited to bone, tendon, and ligaments in postnatal tissues. Furthermore, in both patients and Fkbp10 knockout mice, collagen telopeptide hydroxylysine crosslinking is dramatically reduced. To further characterize the bone specific contributions of Fkbp10, we conditionally ablated FKBP65 in Fkbp10fl/fl mice (Mus musculus; C57BL/6) using the osteoblast-specific Col1a1 2.3-kb Cre recombinase. Using µCT, histomorphometry and quantitative backscattered electron imaging, we found minimal alterations in the quantity of bone and no differences in the degree of bone matrix mineralization in this model. However, mass spectroscopy (MS) of bone collagen demonstrated a decrease in mature, hydroxylysine-aldehyde crosslinking. Furthermore, bone of mutant mice exhibits a reduction in mineral-to-matrix ratio and in crystal size as shown by Raman spectroscopy and small-angle X-ray scattering, respectively. Importantly, abnormalities in bone quality were associated with impaired bone biomechanical strength in mutant femurs compared with those of wild-type littermates. Taken together, these data suggest that the altered collagen crosslinking through Fkbp10 ablation in osteoblasts primarily leads to a qualitative defect in the skeleton. © 2017 American Society for Bone and Mineral Research.

Nature of phosphate substrate as a major determinant of mineral type formed in matrix vesicle-mediated in vitro mineralization: An FTIR imaging study.

Membrane-bound extracellular matrix vesicles play an important role in the de novo initiation and propagation of calcium-phosphate mineral formation in calcifying cartilage, bone, dentin, and in pathologic calcification. Characterization of the phase, composition, crystal size, and perfection provides valuable insight into the mechanism of the mineral deposition. In the present study, Fourier transform infrared imaging spectroscopy (FT-IRIS) was used to characterize the mineral phase generated during MV-mediated in vitro mineralization. FT-IRIS studies revealed that the mineral phase associated with MVs calcified in the presence of AMP and beta-GP was always found to be crystalline hydroxyapatite while with ATP only a small amount of immature mineral, most likely an amorphous or poorly crystalline hydroxyapatite, was observed. Low concentrations of pyrophosphate (PPi) (< or = 0.01 mM) showed apatitic mineral while high concentrations showed immature calcium pyrophosphate dihydrate (CPPD). The implications of these findings are that (a) hydrolysis of AMP or beta-GP, monophosphoester substrates of MV-5' AMPase (substrate: AMP) and TNAP (substrates: AMP, beta-GP), yields orthophosphate (Pi) which leads to the formation of mature crystalline, apatite mineral, while the hydrolysis of ATP, substrate for MV-TNAP or ATPase or NPP1, inhibits the formation of mature hydroxyapatite, and (b) pyrophosphate (PPi) has a bimodal effect on mineralization, i.e., at low PPi concentrations, alkaline phosphatase activity of matrix vesicles is able to hydrolyze PPi to orthophosphate and thus facilitates the formation of basic calcium phosphate mineral which subsequently transforms into apatitic mineral. We hypothesize that, at high PPi concentrations, PPi by itself or Pi released by partial PPi hydrolysis could act as inhibitors of alkaline phosphatase activity, thereby preventing complete hydrolysis of PPi to Pi, and thus resulting in the accumulation of calcium pyrophosphate dihydrate. Therefore, in order for physiological mineralization to proceed, a balance is required between levels of Pi and PPi.

Reverse-Time Migration Based Optical Imaging.

We theoretically demonstrated a new optical imaging technique based on reverse-time migration (RTM) for reconstructing optical structures in homogeneous media for the first time. RTM is a powerful wave-equation-based method to reconstruct the image of the structure by modeling the wave propagation inside the media with both forward modeling and reverse-time extrapolation. While RTM is commonly used with acoustic seismic waves, this paper represents the first effort to develop optical RTM imaging method for biomedical research. To refine the image quality, we further developed new methods to suppress the low-wavenumber artifact (LWA). When compared with the conventional means for LWA suppression such as Laplacian filtering, illumination normalization, and the ratio method, our new derivative-based and power-image methods are able to significantly reduce LWA, resulting in high-quality reconstructed images with sufficient contrasts and spatial resolutions for structure identification. The optical RTM imaging technique may provide a new platform for non-invasive optical imaging of structures in deep layers of tissues for biomedical applications.

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.