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.

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.

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.

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.

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.

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.

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.

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.

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.

ZIP4 silencing improves bone loss in pancreatic cancer.

Metabolic bone disorders are associated with several types of human cancers. Pancreatic cancer patients usually suffer from severe nutrition deficiency, muscle wasting, and loss of bone mass. We have previously found that silencing of a zinc transporter ZIP4 prolongs the survival and reduces the severity of the cachexia in vivo. However, the role of ZIP4 in the pancreatic cancer related bone loss remains unknown. In this study we investigated the effect of ZIP4 knockdown on the bone structure, composition and mechanical properties of femurs in an orthotopic xenograft mouse model. Our data showed that silencing of ZIP4 resulted in increased bone tissue mineral density, decreased bone crystallinity and restoration of bone strength through the RANK/RANKL pathway. The results further support the impact of ZIP4 on the progression of pancreatic cancer, and suggest its potential significance as a therapeutic target for treating patients with such devastating disease and cancer related disorders.

Clinical Evaluation of High-Volume Hemofiltration with Hemoperfusion Followed by Intermittent Hemodialysis in the Treatment of Acute Wasp Stings Complicated by Multiple Organ Dysfunction Syndrome.

Multiple organ dysfunction syndrome (MODS) is a rare complication of wasp stings. Currently, there is no standardized treatment for MODS secondary to multiple wasp stings, although blood purification techniques are often used. This study aimed to analyze our experiences of using intermittent hemodialysis (IHD) with or without high-volume hemofiltration (HVHF) for treating acute wasp stings complicated by MODS. In this retrospective study, 36 patients with wasp stings complicated by MODS received either IHD combined with hemoperfusion, or HVHF (ultrafiltration flow rate, 70 mL/kg/h) combined with hemoperfusion for 5 days followed by IHD. Clinical symptoms, blood biochemical parameters, duration of mechanical ventilation, use of vasoactive agents, duration of hospital stay and survival rate were recorded, and Acute Physiology and Chronic Health Evaluation II (APACHE II) and multiple organ dysfunction (MOD) scores estimated. Patients treated with HVHF followed by IHD appeared to exhibit a faster recovery than those receiving IHD alone, as evidenced by superior improvements in MOD (4.29±1.08 vs. 2.27±1.07) and APACHE II (7.09±2.62 vs. 4.20±1.69) scores (P < 0.05). Patients treated with HVHF had significantly lower myoglobin, creatine kinase-MB, lactate dehydrogenase, bilirubin and creatinine levels than patients treated with IHD alone. In addition, the durations of hospital stay (13.15±2.77 vs. 27.92±3.18 days), vasopressor use (1.76±0.24 vs. 3.43 ± 1.01 days), mechanical ventilation (3.02±1.63 vs. 5.94 ± 2.11 days) and oliguria (6.57±2.45 vs. 15.29 ± 3.51 days) were reduced, and renal function more often recovered (85.1% vs. 53.1%), in the HVHF group compared with the IHD group (P < 0.05). These results raise the possibility that HVHF plus IHD may be superior to IHD alone for the treatment of acute wasp stings complicated by MODS; additional prospective studies are merited to explore this further.

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.

Synergistic acceleration in the osteogenesis of human mesenchymal stem cells by graphene oxide-calcium phosphate nanocomposites.

Nanocomposites consisting of oblong ultrathin plate shaped calcium phosphate nanoparticles and graphene oxide microflakes were synthesized and have demonstrated markedly synergistic effect in accelerating stem cell differentiation to osteoblasts.

Development of Raman spectral markers to assess metastatic bone in breast cancer.

Bone is the most common site for breast cancer metastases. One of the major complications of bone metastasis is pathological bone fracture caused by chronic bone loss and degeneration. Current guidelines for the prediction of pathological fracture mainly rely on radiographs or computed tomography, which are limited in their ability to predict fracture risk. The present study explored the feasibility of using Raman spectroscopy to estimate pathological fracture risk by characterizing the alterations in the compositional properties of metastatic bones. Tibiae with evident bone destruction were investigated using Raman spectroscopy. The carbonation level calculated by the ratio of carbonate/phosphate ν1 significantly increased in the tumor-bearing bone at all the sampling regions at the proximal metaphysis and diaphysis, while tumor-induced elevation in mineralization and crystallinity was more pronounced in the metaphysis. Furthermore, the increased carbonation level is positively correlated to bone lesion size, indicating that this parameter could serve as a unique spectral marker for tumor progression and bone loss. With the promising advances in the development of spatially offset Raman spectroscopy for deep tissue measurement, this spectral marker can potentially be used for future noninvasive evaluation of metastatic bone and prediction of pathological fracture risk.

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.

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

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.