Publications by authors named "Nancy Pleshko"

68 Publications

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo.

Int J Mol Sci 2021 Mar 22;22(6). Epub 2021 Mar 22.

Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, FL 32611, USA.

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin's influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.
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http://dx.doi.org/10.3390/ijms22063223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004833PMC
March 2021

Characterization of Collagen Structure in Normal, Wooden Breast and Spaghetti Meat Chicken Fillets by FTIR Microspectroscopy and Histology.

Foods 2021 Mar 6;10(3). Epub 2021 Mar 6.

Department of Bioengineering, Temple University, Philadelphia, PA 19122, USA.

Recently, two chicken breast fillet abnormalities, termed Wooden Breast (WB) and Spaghetti Meat (SM), have become a challenge for the chicken meat industry. The two abnormalities share some overlapping morphological features, including myofiber necrosis, intramuscular fat deposition, and collagen fibrosis, but display very different textural properties. WB has a hard, rigid surface, while the SM has a soft and stringy surface. Connective tissue is affected in both WB and SM, and accordingly, this study's objective was to investigate the major component of connective tissue, collagen. The collagen structure was compared with normal (NO) fillets using histological methods and Fourier transform infrared (FTIR) microspectroscopy and imaging. The histology analysis demonstrated an increase in the amount of connective tissue in the chicken abnormalities, particularly in the perimysium. The WB displayed a mixture of thin and thick collagen fibers, whereas the collagen fibers in SM were thinner, fewer, and shorter. For both, the collagen fibers were oriented in multiple directions. The FTIR data showed that WB contained more β-sheets than the NO and the SM fillets, whereas SM fillets expressed the lowest mature collagen fibers. This insight into the molecular changes can help to explain the underlying causes of the abnormalities.
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http://dx.doi.org/10.3390/foods10030548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998852PMC
March 2021

Applications of Vibrational Spectroscopy for Analysis of Connective Tissues.

Molecules 2021 Feb 9;26(4). Epub 2021 Feb 9.

Department of Bioengineering, Temple University, Philadelphia, PA 19122, USA.

Advances in vibrational spectroscopy have propelled new insights into the molecular composition and structure of biological tissues. In this review, we discuss common modalities and techniques of vibrational spectroscopy, and present key examples to illustrate how they have been applied to enrich the assessment of connective tissues. In particular, we focus on applications of Fourier transform infrared (FTIR), near infrared (NIR) and Raman spectroscopy to assess cartilage and bone properties. We present strengths and limitations of each approach and discuss how the combination of spectrometers with microscopes (hyperspectral imaging) and fiber optic probes have greatly advanced their biomedical applications. We show how these modalities may be used to evaluate virtually any type of sample (ex vivo, in situ or in vivo) and how "spectral fingerprints" can be interpreted to quantify outcomes related to tissue composition and quality. We highlight the unparalleled advantage of vibrational spectroscopy as a label-free and often nondestructive approach to assess properties of the extracellular matrix (ECM) associated with normal, developing, aging, pathological and treated tissues. We believe this review will assist readers not only in better understanding applications of FTIR, NIR and Raman spectroscopy, but also in implementing these approaches for their own research projects.
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http://dx.doi.org/10.3390/molecules26040922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916244PMC
February 2021

Characterization of connective tissues using near-infrared spectroscopy and imaging.

Nat Protoc 2021 02 18;16(2):1297-1329. Epub 2021 Jan 18.

Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.

Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and label-free assessment of biological materials. Compared to mid-infrared spectroscopy, NIR spectroscopy excels in penetration depth, allowing intact biological tissue assessment, albeit at the cost of reduced molecular specificity. Furthermore, it is relatively safe compared to Raman spectroscopy, with no risk of laser-induced photothermal damage. A typical NIR spectroscopy workflow for biological tissue characterization involves sample preparation, spectral acquisition, pre-processing and analysis. The resulting spectrum embeds intrinsic information on the tissue's biomolecular, structural and functional properties. Here we demonstrate the analytical power of NIR spectroscopy for exploratory and diagnostic applications by providing instructions for acquiring NIR spectra, maps and images in biological tissues. By adapting and extending this protocol from the demonstrated application in connective tissues to other biological tissues, we expect that a typical NIR spectroscopic study can be performed by a non-specialist user to characterize biological tissues in basic research or clinical settings. We also describe how to use this protocol for exploratory study on connective tissues, including differentiating among ligament types, non-destructively monitoring changes in matrix formation during engineered cartilage development, mapping articular cartilage proteoglycan content across bovine patella and spectral imaging across the depth-wise zones of articular cartilage and subchondral bone. Depending on acquisition mode and experiment objectives, a typical exploratory study can be completed within 6 h, including sample preparation and data analysis.
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http://dx.doi.org/10.1038/s41596-020-00468-zDOI Listing
February 2021

MRI-derived porosity index is associated with whole-bone stiffness and mineral density in human cadaveric femora.

Bone 2021 02 30;143:115774. Epub 2020 Nov 30.

Department of Radiology, University of Pennsylvania, United States of America; Department of Orthopaedic Surgery, University of Pennsylvania, United States of America.

Ultrashort echo time (UTE) magnetic resonance imaging (MRI) measures proton signals in cortical bone from two distinct water pools, bound water, or water that is tightly bound to bone matrix, and pore water, or water that is freely moving in the pore spaces in bone. By isolating the signal contribution from the pore water pool, UTE biomarkers can directly quantify cortical bone porosity in vivo. The Porosity Index (PI) is one non-invasive, clinically viable UTE-derived technique that has shown strong associations in the tibia with μCT porosity and other UTE measures of bone water. However, the efficacy of the PI biomarker has never been examined in the proximal femur, which is the site of the most catastrophic osteoporotic fractures. Additionally, the loads experienced during a sideways fall are complex and the femoral neck is difficult to image with UTE, so the usefulness of the PI in the femur was unknown. Therefore, the aim of this study was to examine the relationships between the PI measure in the proximal cortical shaft of human cadaveric femora specimens compared to (1) QCT-derived bone mineral density (BMD) and (2) whole bone stiffness obtained from mechanical testing mimicking a sideways fall. Fifteen fresh, frozen whole cadaveric femora specimens (age 72.1 ± 15.0 years old, 10 male, 5 female) were scanned on a clinical 3-T MRI using a dual-echo UTE sequence. Specimens were then scanned on a clinical CT scanner to measure volumetric BMD (vBMD) and then non-destructively mechanically tested in a sideways fall configuration. The PI in the cortical shaft demonstrated strong correlations with bone stiffness (r = -0.82, P = 0.0014), CT-derived vBMD (r = -0.64, P = 0.0149), and with average cortical thickness (r = -0.60, P = 0.0180). Furthermore, a hierarchical regression showed that PI was a strong predictor of bone stiffness which was independent of the other parameters. The findings from this study validate the MRI-derived porosity index as a useful measure of whole-bone mechanical integrity and stiffness.
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http://dx.doi.org/10.1016/j.bone.2020.115774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769997PMC
February 2021

DMOG Negatively Impacts Tissue Engineered Cartilage Development.

Cartilage 2020 Oct 26:1947603520967060. Epub 2020 Oct 26.

Tissue Imaging and Spectroscopy Lab, Department of Bioengineering, Temple University, Philadelphia, PA, USA.

Objective: Articular cartilage exists in a hypoxic environment, which motivates the use of hypoxia-simulating chemical agents to improve matrix production in cartilage tissue engineering. The aim of this study was to investigate whether dimethyloxalylglycine (DMOG), a HIF-1α stabilizer, would improve matrix production in 3-dimensional (3D) porcine synovial-derived mesenchymal stem cell (SYN-MSC) co-culture with chondrocytes.

Design: Pellet cultures and scaffold-based engineered cartilage were grown to determine the impact of chemically simulated hypoxia on 2 types of 3D cell culture. DMOG-treated groups were exposed to DMOG from day 14 to day 21 and grown up to 6 weeks with = 3 per condition and time point.

Results: The addition of DMOG resulted in HIF-1α stabilization in the exterior of the engineered constructs, which resulted in increased regional type II collagen deposition, but the stabilization did not translate to overall increased extracellular matrix deposition. There was no increase in HIF-1α stabilization in the pellet cultures. DMOG treatment also negatively affected the mechanical competency of the engineered cartilage.

Conclusions: Despite previous studies that demonstrated the efficacy of DMOG, here, short-term treatment with DMOG did not have a uniformly positive impact on the chondrogenic capacity of SYN-MSCs in either pellet culture or in scaffold-based engineered cartilage, as evidenced by reduced matrix production. Such 3D constructs generally have a naturally occurring hypoxic center, which allows for the stabilization of HIF-1α in the interior tissue. Thus, short-term addition of DMOG may not further improve this in cartilage tissue engineered constructs.
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http://dx.doi.org/10.1177/1947603520967060DOI Listing
October 2020

Near infrared spectroscopic assessment of loosely and tightly bound cortical bone water.

Analyst 2020 May 28;145(10):3713-3724. Epub 2020 Apr 28.

Department of Bioengineering, Temple University, USA.

Water is an important component of bone and plays a key role in its mechanical and structural integrity. Water molecules in bone are present in different locations, including loosely or tightly bound to the matrix and/or mineral (biological apatite) phases. Identification of water location and interactions with matrix components impact bone function but have been challenging to assess. Here, we used near infrared (NIR) spectroscopy to identify loosely and tightly bound water present in cortical bone. In hydrated samples, NIR spectra have two primary water absorption bands at frequencies of ∼5200 and 7000 cm. Using lyophilization and hydrogen-deuterium exchange assays, we showed that these absorption bands are primarily associated with loosely bound bone water. Using further demineralization assays, thermal denaturation, and comparison to standards, we found that these absorption bands have underlying components associated with water molecules tightly bound to bone. In dehydrated samples, the peak at ∼5200 cm was assigned to a combination of water tightly bound to collagen and to mineral, whereas the peak at 7000 cm was exclusively associated with tightly bound mineral water. We also found significant positive correlations between the NIR mineral absorption bands and the mineral content as determined by an established mid infrared spectroscopic parameter, phosphate/amide I. Moreover, the NIR water data showed correlation trends with tissue mineral density (TMD) in cortical bone tissues. These observations reveal the ability of NIR spectroscopy to non-destructively identify loosely and tightly bound water in bone, which could have further applications in biomineralization and biomedical studies.
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http://dx.doi.org/10.1039/c9an02491cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301914PMC
May 2020

Approaches for Monitoring of Matrix Development in Hydrogel-Based Engineered Cartilage.

Tissue Eng Part C Methods 2020 04 3;26(4):225-238. Epub 2020 Apr 3.

Tissue Imaging and Spectroscopy Lab, Department of Bioengineering, Temple University, Philadelphia, Pennsylvania.

Near infrared (NIR) spectroscopy using a fiber optic probe shows great promise for the nondestructive monitoring of tissue engineered construct development; however, the NIR evaluation of matrix components in samples with high water content is challenging, as water absorbances overwhelm the spectra. In this study, we established approaches by which NIR spectroscopy can be used to select optimal individual engineered hydrogel constructs based on matrix content and mechanical properties. NIR spectroscopy of dry standard compounds allowed identification of several absorbances related to collagen and/or proteoglycan (PG), of which only two could be identified in spectra obtained from hydrated constructs, at ∼5940 and 5800 cm. In dry sample mixtures, the ratio of these peaks correlated positively to collagen and negatively to PG. In NIR spectra from engineered cartilage hydrogels, these peaks reflected higher collagen and PG content and dynamic modulus values, permitting the differentiation of constructs with poor and good matrix development. Similarly, the increasing baseline offset in raw NIR spectra also reflected matrix development in hydrated constructs. However, weekly monitoring of NIR spectra and the peaks at ∼5940 and 5800 cm was not adequate to differentiate individual constructs based on matrix composition. Interestingly, changes in the baseline offset of raw spectra could be used to evaluate the growth trajectory of individual constructs. These results demonstrate an optimal approach for the use of fiber optic NIR spectroscopy for monitoring of the development of engineered cartilage, which will aid in identifying individual constructs for implantation. Impact statement A current demand in tissue engineering is the establishment of nondestructive approaches to evaluate construct development during growth . In this article, we demonstrate original nondestructive approaches by which fiber optic NIR spectroscopy can be used to assess matrix (PG and collagen) formation and mechanical properties in hydrogel-based constructs. Our data provide a cohesive molecular-based approach for longitudinal evaluation of construct development during growth The establishment of these approaches is a valuable step toward the real-time identification and selection of constructs with optimal properties, which may lead to successful tissue integration upon implantation.
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http://dx.doi.org/10.1089/ten.TEC.2020.0014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187967PMC
April 2020

Clinical application of near infrared fiber optic spectroscopy for noninvasive bone assessment.

J Biophotonics 2020 04 5;13(4):e201960172. Epub 2020 Feb 5.

Department for Bioengineering, Temple University, Philadelphia, Pennsylvania.

Approaches for noninvasive bone quality assessment are of great clinical need, particularly in individuals that require close monitoring of disease progression. X-ray measurements are standard approaches to assess bone quality; however, they have several disadvantages. Here, a nonionizing approach for noninvasive assessment of the second metacarpal bone based on near infrared (NIR) spectroscopy was investigated. Transcutaneous bone signal detection was experimentally confirmed with cadaveric hand data, and Monte Carlo modeling further indicated that 50% of the measured signals arise from bone. Spectral data were collected via a NIR fiber optic from the bone of individuals with osteogenesis imperfecta, a disease marked by frequent bone fractures and fragility. Multiple significant correlations were found between spectral parameters related to water, protein and fat, and standard bone quality parameters obtained by X-ray measurements. The results from this preliminary study highlight the potential application of NIR spectroscopy for the noninvasive assessment of bone quality.
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http://dx.doi.org/10.1002/jbio.201960172DOI Listing
April 2020

Fourier transform infrared spectroscopy of developing bone mineral: from amorphous precursor to mature crystal.

Analyst 2020 Feb;145(3):764-776

Department of Bioengineering, Temple University, Philadelphia, Pennsylvania 19122, USA.

Bone mineral development has been described to proceed through an amorphous precursor prior to apatite crystallization. However, further analytical approaches are necessary to identify specific markers of amorphous mineral components in bone. Here, we establish an original Fourier transform infrared (FTIR) spectroscopy approach to allow the specific identification of the amorphous and/or crystalline nature of bone mineral. Using a series of standards, our results demonstrate that obtaining the second derivative of the FTIR spectra could reveal a peak specifically corresponding to amorphous calcium phosphate (ACP) at ∼992 cm-1. The intensity of this peak was strongly correlated to ACP content in standard mixtures. The analysis of a variety of bones showed that a clear ACP peak could be identified as a specific marker of the existence of an amorphous mineral component in developing bones. In contrast, the ACP peak was not detected in the mature bones. Moreover, subjecting developing bones to ex vivo crystallization conditions led to a clear reduction of the ACP peak, further substantiating the conversion of amorphous mineral precursor into mature apatite crystals. Analysis of mineralization in osteogenic cell cultures corroborated our observations, showing the presence of ACP as a major transient component in early mineralization, but not in the mature matrix. Additionally, FTIR imaging revealed that ACP was present in areas of matrix development, distributed around the edges of mineralizing nodules. Using an original analytical approach, this work provides strong evidence to support that bone mineral development is initiated by an amorphous precursor prior to apatite crystallization.
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http://dx.doi.org/10.1039/c9an01588dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046087PMC
February 2020

MRI-derived bone porosity index correlates to bone composition and mechanical stiffness.

Bone Rep 2019 Dec 26;11:100213. Epub 2019 Jun 26.

Department of Radiology, University of Pennsylvania, United States of America.

The MRI-derived porosity index (PI) is a non-invasively obtained biomarker based on an ultrashort echo time sequence that images both bound and pore water protons in bone, corresponding to water bound to organic collagenous matrix and freely moving water, respectively. This measure is known to strongly correlate with the actual volumetric cortical bone porosity. However, it is unknown whether PI may also be able to directly quantify bone organic composition and/or mechanical properties. We investigated this in human cadaveric tibiae by comparing PI values to near infrared spectral imaging (NIRSI) compositional data and mechanical compression data. Data were obtained from a cohort of eighteen tibiae from male and female donors with a mean ± SD age of 70 ± 21 years. Biomechanical stiffness in compression and NIRSI-derived collagen and bound water content all had significant inverse correlations with PI ( = -0.79, -0.73, and -0.95 and  = 0.002, 0.007, and <0.001, respectively). The MRI-derived bone PI alone was a moderate predictor of bone stiffness (  = 0.63,  = 0.002), and multivariate analyses showed that neither cortical bone cross-sectional area nor NIRSI values improved bone stiffness prediction compared to PI alone. However, NIRSI-obtained collagen and water data together were a moderate predictor of bone stiffness (R = 0.52,  = 0.04). Our data validates the MRI-derived porosity index as a strong predictor of organic composition of bone and a moderate predictor of bone stiffness, and also provides preliminary evidence that NIRSI measures may be useful in future pre-clinical studies on bone pathology.
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http://dx.doi.org/10.1016/j.bonr.2019.100213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660551PMC
December 2019

Environmentally-Controlled Near Infrared Spectroscopic Imaging of Bone Water.

Sci Rep 2019 07 15;9(1):10199. Epub 2019 Jul 15.

Department of Bioengineering, Temple University, Philadelphia, USA.

We have designed an environmentally-controlled chamber for near infrared spectroscopic imaging (NIRSI) to monitor changes in cortical bone water content, an emerging biomarker related to bone quality assessment. The chamber is required to ensure repeatable spectroscopic measurements of tissues without the influence of atmospheric moisture. A calibration curve to predict gravimetric water content from human cadaveric cortical bone was created using NIRSI data obtained at six different lyophilization time points. Partial least squares (PLS) models successfully predicted bone water content that ranged from 0-10% (R = 0.96, p < 0.05, root mean square error of prediction (RMSEP) = 7.39%), as well as in the physiologic range of 4-10% of wet tissue weight (R = 0.87, p < 0.05, RMSEP = 14.5%). Similar results were obtained with univariate and bivariate regression models for prediction of water in the 0-10% range. Further, we identified two new NIR bone absorbances, at 6560 cm and 6688 cm, associated with water and collagen respectively. Such data will be useful in pre-clinical studies that investigate changes in bone quality with disease, aging and with therapeutic use.
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http://dx.doi.org/10.1038/s41598-019-45897-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629628PMC
July 2019

Spectroscopic Analysis of Human Tracheal Tissue during Decellularization.

Otolaryngol Head Neck Surg 2019 02 16;160(2):302-309. Epub 2018 Oct 16.

6 Department of Bioengineering, Temple University, Philadelphia, Pennsylvania, USA.

Objective: To use mid-infrared (IR) spectroscopy to assess changes in the cartilaginous framework of human trachea during decellularization.

Study Design: Laboratory-based study.

Setting: Research laboratory.

Methods: Six cadaveric human tracheas were decellularized using a detergent enzymatic method (DEM). Tissue samples were obtained from each specimen after 0, 1, 10, and 25 DEM cycles for histologic and spectroscopic analysis. Decellularization was confirmed using hematoxylin and eosin (H&E) and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) staining. Changes in cartilaginous framework were examined using Fourier transform infrared imaging spectroscopy (FT-IRIS) and an attenuated total reflectance (ATR) probe in the mid-IR frequencies. Results were statistically analyzed using 1-way analysis of variance (ANOVA) and principal component analysis (PCA).

Results: Six decellularized tracheal scaffolds were successfully created using a DEM protocol. Histologic examination showed near-complete nuclear loss following 25 DEM cycles. As observed with FT-IRIS analysis, the collagen absorbance signal (1336 cm) was predominantly in the perichondria and remained stable after 25 DEM cycles ( P = .132), while the absorbance from sugar rings in proteoglycans and nucleic acids in hyaline cartilage (1080 cm) showed a significant decrease after 1 DEM cycle ( P = .0007). Examination of the luminal surface of the trachea with an ATR probe showed raw mid-IR spectra consistent with cartilage. PCA showed significant separation of spectra corresponding to treatment cycle along the principal components 1 and 2.

Conclusion: Mid-IR spectroscopy is a viable method of monitoring changes in extracellular matrix components during the decellularization of human trachea.
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http://dx.doi.org/10.1177/0194599818806271DOI Listing
February 2019

Matrix mineralization controls gene expression in osteoblastic cells.

Exp Cell Res 2018 11 5;372(1):25-34. Epub 2018 Sep 5.

Department of Orthopedics, Klinikum rechts der Isar, Technical University Munich, 81675 Munich, Germany. Electronic address:

Osteoblasts are adherent cells, and under physiological conditions they attach to both mineralized and non-mineralized osseous surfaces. However, how exactly osteoblasts respond to these different osseous surfaces is largely unknown. Our hypothesis was that the state of matrix mineralization provides a functional signal to osteoblasts. To assess the osteoblast response to mineralized compared to demineralized osseous surfaces, we developed and validated a novel tissue surface model. We demonstrated that with the exception of the absence of mineral, the mineralized and demineralized surfaces were similar in molecular composition as determined, for example, by collagen content and maturity. Subsequently, we used the human osteoblastic cell line MG63 in combination with genome-wide gene set enrichment analysis (GSEA) to record and compare the gene expression signatures on mineralized and demineralized surfaces. Assessment of the 5 most significant gene sets showed on mineralized surfaces an enrichment exclusively of genes sets linked to protein synthesis, while on the demineralized surfaces 3 of the 5 enriched gene sets were associated with the matrix. Focusing on these three gene sets, we observed not only the expected structural components of the bone matrix, but also gene products, such as HMCN1 or NID2, that are likely to act as temporal migration guides. Together, these findings suggest that in osteoblasts mineralized and demineralized osseous surfaces favor intracellular protein production and matrix formation, respectively. Further, they demonstrate that the mineralization state of bone independently controls gene expression in osteoblastic cells.
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http://dx.doi.org/10.1016/j.yexcr.2018.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185740PMC
November 2018

Infrared Spectroscopic Quantification of Methacrylation of Hyaluronic Acid: A Scaffold for Tissue Engineering Applications.

Appl Spectrosc 2018 Oct 10;72(10):1455-1466. Epub 2018 Aug 10.

Tissue Imaging and Spectroscopy Lab, Department of Bioengineering, Temple University, Philadelphia, PA, USA.

Methacrylated hyaluronic acid (MeHA) has been used extensively in tissue engineering and drug delivery applications. The degree of methacrylation (DM) of HA impacts hydrogel crosslinking, which is of pivotal importance for cell interactions. The methacrylation reaction occurs over several hours, and DM is currently assessed post reaction and after dialysis of the solution, using nuclear magnetic resonance (H NMR) data. Thus, there is little control over exact DM in a specific reaction. Here, infrared (IR) spectroscopy in attenuated total reflection (ATR) mode was investigated as an alternate modality for assessment of the DM of HA hydrogels, including during the reaction progression. Attenuated total reflection is a low-cost technique that is widely available in research and industry labs that can be used online during the reaction process. Strong correlations were achieved with IR-derived peak heights from dialyzed and lyophilized samples at 1708 cm (from the methacrylic ester carbonyl vibration), and H NMR values ( R = 0.92, P = 6.56E-11). Additional IR peaks of importance were identified using principal component analysis and resulted in significant correlations with the H NMR DM parameter: 1454 cm ( R = 0.85, P = 2.81E-8), 1300 cm ( R = 0.95, P = 4.50E-14), 950 ( R = 0.85, P = 3.55E-8), 856 cm ( R = 0.94, P = 1.20E-12), and 809 cm ( R = 0.93, P = 3.54E-12). A multiple linear regression model to predict H NMR-derived DM using the 1708, 1300, and 1200 cm peak heights as independent variables resulted in prediction with an error of 3.2% using dialyzed and lyophilized samples ( P < 0.001). Additionally, a multilinear regression model to predict the DM in undialyzed liquid MeHA samples obtained during the reaction process using similar peak height positions as independent variables resulted in a prediction error of 0.81% ( P < 0.05). Thus, IR spectroscopy can be utilized as an alternate modality to H NMR for quantification of the DM of MeHA while sampling either on-line during the methacrylation reaction as well as in post-lyophilized products. This could greatly simplify workflow for tissue engineering and other applications.
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http://dx.doi.org/10.1177/0003702818785353DOI Listing
October 2018

Spatial correlation of native and engineered cartilage components at micron resolution.

Ann N Y Acad Sci 2019 04 29;1442(1):104-117. Epub 2018 Jul 29.

Department of Bioengineering, Temple University, Philadelphia, Pennsylvania.

Tissue engineering (TE) approaches are being widely investigated for repair of focal defects in articular cartilage. However, the amount and/or type of extracellular matrix (ECM) produced in engineered constructs does not always correlate with the resultant mechanical properties. This could be related to the specifics of ECM distribution throughout the construct. Here, we present data on the amount and distribution of the primary components of native and engineered cartilage (i.e., collagen, proteoglycan (PG), and water) using Fourier transform infrared imaging spectroscopy (FT-IRIS). These data permit visualization of matrix and water at 25 μm resolution throughout the tissues, and subsequent colocalization of these components using image processing methods. Native and engineered cartilage were cryosectioned at 80 μm for evaluation by FT-IRIS in the mid-infrared (MIR) and near-infrared (NIR) regions. PG distribution correlated strongly with water in native and engineered cartilage, supporting the binding of water to PG in both tissues. In addition, NIR-derived matrix peaks correlated significantly with MIR-derived collagen peaks, confirming the interpretation that these absorbances arise primarily from collagen and not PG. The combined use of MIR and NIR permits assessment of ECM and water spatial distribution at the micron level, which may aid in improved development of TE techniques.
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http://dx.doi.org/10.1111/nyas.13934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351229PMC
April 2019

Validated Approaches for Quantification of Bone Mineral Crystallinity Using Transmission Fourier Transform Infrared (FT-IR), Attenuated Total Reflection (ATR) FT-IR, and Raman Spectroscopy.

Appl Spectrosc 2018 Nov 20;72(11):1581-1593. Epub 2018 Jul 20.

Department of Bioengineering, Temple University, Philadelphia, PA, USA.

Bone mineral crystallinity is an important factor determining bone quality and strength. The gold standard method to quantify crystallinity is X-ray diffraction (XRD), but vibrational spectroscopic methods present powerful alternatives to evaluate a greater variety of sample types. We describe original approaches by which transmission Fourier transform infrared (FT-IR), attenuated total reflection (ATR) FT-IR, and Raman spectroscopy can be confidently used to quantify bone mineral crystallinity. We analyzed a range of biological and synthetic apatite nanocrystals (10-25 nm) and found strong correlations between different spectral factors and the XRD determination of crystallinity. We highlight striking differences between FT-IR spectra obtained by transmission and ATR. In particular, we show for the first time the absence of the 1030 cm crystalline apatite peak in ATR FT-IR spectra, which excludes its use for analyzing crystallinity using the traditional 1030/1020 cm ratio. The νPO splitting ratio was also not adequate to evaluate crystallinity using ATR FT-IR. However, we established original approaches by which ATR FT-IR can be used to determine apatite crystallinity, such as the 1095/1115 and 960/1115 cm peak ratios in the second derivative spectra. Moreover, we found a simple unified approach that can be applied for all three vibrational spectroscopy modalities: evaluation of the νPO peak position. Our results allow the recommendation of the most reliable analytical methods to estimate bone mineral crystallinity by vibrational spectroscopy, which can be readily implemented in many biomineralization, archeological and orthopedic studies. In particular, we present a step forward in advancing the use of the increasingly utilized ATR FT-IR modality for mineral research.
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http://dx.doi.org/10.1177/0003702818789165DOI Listing
November 2018

Identification of the effector domain of biglycan that facilitates BMP-2 osteogenic function.

Sci Rep 2018 05 4;8(1):7022. Epub 2018 May 4.

Oral and Craniofacial Health Sciences, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC-27599, USA.

We have reported that recombinant biglycan (BGN) core protein accelerates bone formation in vivo by enhancing bone morphogenetic protein (BMP)-2 function. The purpose of the present study was to identify the specific domain ("effector") within the BGN core protein that facilitates BMP-2 osteogenic function. Thus, we generated various recombinant and synthetic peptides corresponding to several domains of BGN, and tested their effects on BMP-2 functions in vitro. The results demonstrated that the leucine-rich repeats 2-3 domain (LRR2-3) of BGN significantly enhanced the BMP-2 induced Smad1/5/9 phosphorylation, osteogenic gene expression, and alkaline phosphatase activity in myogenic C2C12 cells. Furthermore, addition of LRR2-3 to osteoblastic MC3T3-E1 cells accelerated in vitro mineralization without compromising the quality of the mineral and matrix. These data indicate that LRR2-3 is, at least in part, responsible for BGN's ability to enhance BMP-2 osteogenic function, and it could be useful for bone tissue regeneration.
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http://dx.doi.org/10.1038/s41598-018-25279-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935668PMC
May 2018

Non-Destructive Spectroscopic Assessment of High and Low Weight Bearing Articular Cartilage Correlates with Mechanical Properties.

Cartilage 2019 10 24;10(4):480-490. Epub 2018 Apr 24.

Department of Bioengineering, Temple University, Philadelphia, PA, USA.

Objective: Autologous articular cartilage (AC) harvested for repair procedures of high weight bearing (HWB) regions of the femoral condyles is typically obtained from low weight bearing (LWB) regions, in part due to the lack of non-destructive techniques for cartilage composition assessment. Here, we demonstrate that infrared fiber optic spectroscopy can be used to non-destructively evaluate variations in compositional and mechanical properties of AC across LWB and HWB regions.

Design: AC plugs ( = 72) were harvested from the patellofemoral groove of juvenile bovine stifle joints, a LWB region, and femoral condyles, a HWB region. Near-infrared (NIR) and mid-infrared (MIR) fiber optic spectra were collected from plugs, and indentation tests were performed to determine the short-term and equilibrium moduli, followed by gravimetric water and biochemical analysis.

Results: LWB tissues had a significantly greater amount of water determined by NIR and gravimetric assay. The moduli generally increased in tissues from the patellofemoral groove to the condyles, with HWB condyle cartilage having significantly higher moduli. A greater amount of proteoglycan content was also found in HWB tissues, but no differences in collagen content. In addition, NIR-determined water correlated with short-term modulus and proteoglycan content ( = -0.40 and -0.31, respectively), and a multivariate model with NIR data was able to predict short-term modulus within 15% error.

Conclusions: The properties of tissues from LWB regions differ from HWB tissues and can be determined non-destructively by infrared fiber optic spectroscopy. Clinicians may be able to use this modality to assess AC prior to harvesting osteochondral grafts for focal defect repair.
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http://dx.doi.org/10.1177/1947603518764269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755878PMC
October 2019

A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis.

Matrix Biol 2018 09 29;70:102-122. Epub 2018 Mar 29.

Department of Orthopaedic Surgery, Graduate Program in Cell Biology and Regenerative Medicine, Thomas Jefferson University, Philadelphia, PA, USA. Electronic address:

Intervertebral disc degeneration and associated low back and neck pain is a ubiquitous health condition that affects millions of people world-wide, and causes high incidence of disability and enormous medical/societal costs. However, lack of appropriate small animal models with spontaneous disease onset has impeded our ability to understand the pathogenetic mechanisms that characterize and drive the degenerative process. We report, for the first time, early onset spontaneous disc degeneration in SM/J mice known for their poor regenerative capacities compared to "super-healer" LG/J mice. In SM/J mice, degenerative process was marked by decreased nucleus pulposus (NP) cellularity and changes in matrix composition at P7, 4, and 8 weeks with increased severity by 17 weeks. Distinctions between NP and annulus fibrosus (AF) or endplate cartilage were lost, and NP and AF of SM/J mice showed higher histological grades. There was increased NP cell death in SM/J mice with decreased phenotypic marker expression. Polarized microscopy and FTIR spectroscopy demonstrated replacement of glycosaminoglycan-rich NP matrix with collagenous fibrous tissue. The levels of ARGxx were increased in, indicating higher aggrecan turnover. Furthermore, an aberrant expression of collagen X and MMP13 was observed in the NP of SM/J mice, along with elevated expression of Col10a1, Ctgf, and Runx2, markers of chondrocyte hypertrophy. Likewise, expression of Enpp1 as well as Alpl was higher, suggesting NP cells of SM/J mice promote dystrophic mineralization. There was also a decrease in several pathways necessary for NP cell survival and function including Wnt and VEGF signaling. Importantly, SM/J discs were stiffer, had decreased height, and poor vertebral bone quality, suggesting compromised motion segment mechanical functionality. Taken together, our results clearly demonstrate that SM/J mouse strain recapitulates many salient features of human disc degeneration, and serves as a novel small animal model.
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http://dx.doi.org/10.1016/j.matbio.2018.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081256PMC
September 2018

Compositional Assessment of Human Tracheal Cartilage by Infrared Spectroscopy.

Otolaryngol Head Neck Surg 2018 04 16;158(4):688-694. Epub 2018 Jan 16.

2 Tissue Imaging and Spectroscopy Laboratory, Department of Bioengineer-ing, Temple University, Philadelphia, Pennsylvania, USA.

Objectives To assess the potential of infrared fiber-optic spectroscopy to evaluate the compositional properties of human tracheal cartilage. Study Design Laboratory-based study. Methods Twenty human cadaveric distal tracheas were harvested (age range 20-78 years; 6 females, 14 males) for compositional analysis. Histologic staining, Fourier transform infrared imaging spectroscopy data on collagen and proteoglycan (PG) content, and near-infrared (NIR) fiber-optic probe spectroscopic data that reflect protein and water content were evaluated. NIR fiber-optic probe data were also obtained from the proximal trachea in 4 human cadavers (age range 51-65 years; 2 females, 2 males) in situ for comparison to distal trachea spectral data. Results In the distal trachea cohort, the spectroscopic-determined ratio of PG/amide I, indicative of the relative amount of PG, was significantly higher in the tissues from the younger group compared to the older group (0.37 ± 0.08 vs 0.32 ± 0.05, P = .05). A principal component analysis of the NIR spectral data enabled separation of spectra based on tracheal location, likely due to differences in both protein and water content. The NIR-determined water content based on the 5200-cm peak was significantly higher in the distal trachea compared to the proximal trachea ( P < .001). Conclusions Establishment of normative compositional values and further elucidating differences between the segments of trachea will enable more directed research toward appropriate compositional end points in regenerative medicine for tracheal repair.
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http://dx.doi.org/10.1177/0194599817752310DOI Listing
April 2018

Vibrational spectroscopy and imaging: applications for tissue engineering.

Analyst 2017 Oct;142(21):4005-4017

Department of Bioengineering, Temple University, Philadelphia, PA, USA.

Tissue engineering (TE) approaches strive to regenerate or replace an organ or tissue. The successful development and subsequent integration of a TE construct is contingent on a series of in vitro and in vivo events that result in an optimal construct for implantation. Current widely used methods for evaluation of constructs are incapable of providing an accurate compositional assessment without destruction of the construct. In this review, we discuss the contributions of vibrational spectroscopic assessment for evaluation of tissue engineered construct composition, both during development and post-implantation. Fourier transform infrared (FTIR) spectroscopy in the mid and near-infrared range, as well as Raman spectroscopy, are intrinsically label free, can be non-destructive, and provide specific information on the chemical composition of tissues. Overall, we examine the contribution that vibrational spectroscopy via fiber optics and imaging have to tissue engineering approaches.
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http://dx.doi.org/10.1039/c7an01055aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653442PMC
October 2017

Non-destructive NIR spectral imaging assessment of bone water: Comparison to MRI measurements.

Bone 2017 Oct 28;103:116-124. Epub 2017 Jun 28.

Department of Bioengineering, Temple University, 1947 N. 12th St, Philadelphia, PA, USA. Electronic address:

Bone fracture risk increases with age, disease states, and with use of certain therapeutics, such as acid-suppressive drugs, steroids and high-dose bisphosphonates. Historically, investigations into factors that underlie bone fracture risk have focused on evaluation of bone mineral density (BMD). However, numerous studies have pointed to factors other than BMD that contribute to fragility, including changes in bone collagen and water. The goal of this study is to investigate the feasibility of using near infrared spectral imaging (NIRSI) to determine the spatial distribution and relative amount of water and organic components in whole cross-sections of bone, and to compare those results to those obtained using magnetic resonance imaging (MRI) methods. Cadaver human whole-section tibiae samples harvested from 18 donors of ages 27-97years underwent NIRSI and ultrashort echo time (UTE) MRI. As NIRSI data is comprised of broad absorbances, second derivative processing was evaluated as a means to narrow peaks and obtain compositional information. The (inverted) second derivative peak heights of the NIRSI absorbances correlated significantly with the mean peak integration of the water, collagen and fat NIR absorbances, respectively, indicating that either processing method could be used for compositional assessment. The 5797cm absorbance was validated as arising from the fat present in bone marrow, as it completely disappeared after ultrasonication. The MRI UTE-determined bound water content in tibial cortical bone samples ranged from 62 to 91%. The NIRSI water peaks at 5152cm and at 7008cm correlated significantly with the UTE data, with r=0.735, p=0.016, and r=0.71, p=0.0096, respectively. There was also a strong correlation between the intensity of the NIRSI water peak at 7008cm and the intensity of the collagen peak at 4608cm (r=0.69, p=0.004). Since NIRSI requires minimal to no sample preparation, this approach has great potential to become a gold standard modality for the investigation of changes in water content, distribution, and environment in pre-clinical studies of bone pathology and therapeutics.
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http://dx.doi.org/10.1016/j.bone.2017.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572678PMC
October 2017

Near-Infrared Spectroscopy Predicts Compositional and Mechanical Properties of Hyaluronic Acid-Based Engineered Cartilage Constructs.

Tissue Eng Part A 2018 01 15;24(1-2):106-116. Epub 2017 May 15.

1 Tissue Imaging and Spectroscopy Lab, Department of Bioengineering, Temple University , Philadelphia, Pennsylvania.

Hyaluronic acid (HA) has been widely used for cartilage tissue engineering applications. However, the optimal time point to harvest HA-based engineered constructs for cartilage repair is still under investigation. In this study, we investigated the ability of a nondestructive modality, near-infrared spectroscopic (NIR) analysis, to predict compositional and mechanical properties of HA-based engineered cartilage constructs. NIR spectral data were collected from control, unseeded constructs, and twice per week by fiber optic from constructs seeded with chondrocytes during their development over an 8-week period. Constructs were harvested at 2, 4, 6, and 8 weeks, collagen and sulfated glycosaminoglycan content measured using biochemical assays, and the mechanical properties of the constructs evaluated using unconfined compression tests. NIR absorbances associated with the scaffold material, water, and engineered cartilage matrix, were identified. The NIR-determined matrix absorbance plateaued after 4 weeks of culture, which was in agreement with the biochemical assay results. Similarly, the mechanical properties of the constructs also plateaued at 4 weeks. A multivariate partial least square model based on NIR spectral input was developed to predict the moduli of the constructs, which resulted in a prediction error of 10% and R value of 0.88 for predicted versus actual values of dynamic modulus. Furthermore, the maximum increase in moduli was calculated from the first derivative of the curve fit of NIR-predicted and actual moduli values over time, and both occurred at ∼2 weeks. Collectively, these data suggest that NIR spectral data analysis could be an alternative to destructive biochemical and mechanical methods for evaluation of HA-based engineered cartilage construct properties.
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http://dx.doi.org/10.1089/ten.TEA.2017.0035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770116PMC
January 2018

Compositional mapping of the mature anterior cruciate ligament-to-bone insertion.

J Orthop Res 2017 11 2;35(11):2513-2523. Epub 2017 Mar 2.

Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, New York 10027.

The anterior cruciate ligament (ACL)-to-bone interface constitutes a complex, multi-tissue structure comprised of contiguous ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone regions. This composite structure enables load transfer between structurally and functionally dissimilar tissues and is critical for ligament homeostasis and joint stability. Presently, there is a lack of quantitative understanding of the matrix composition and organization across this junction, especially after the onset of skeletal maturity. The objective of this study is to characterize the adult bovine ACL-to-bone interface using Fourier transform infrared spectroscopic imaging (FTIRI), testing the hypothesis that regional changes in collagen, proteoglycan, and mineral distribution, as well as matrix organization, persist at the mature insertion. It was observed that while collagen content increases continuously across the adult interface, collagen alignment decreases between ligament and bone. Proteoglycans were primarily localized to the fibrocartilage region and an exponential increase in mineral content was observed between the non-mineralized and mineralized regions. These observations reveal significant changes in collagen distribution and alignment with maturity, and these trends underscore the role of physiologic loading in postnatal matrix remodeling. Findings from this study provide new insights into interface organization and serve as benchmark design criteria for interface regeneration and integrative soft tissue repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2513-2523, 2017.
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http://dx.doi.org/10.1002/jor.23539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548644PMC
November 2017

Fourier Transform Infrared Spectroscopic Imaging-Derived Collagen Content and Maturity Correlates with Stress in the Aortic Wall of Abdominal Aortic Aneurysm Patients.

Cardiovasc Eng Technol 2017 03 19;8(1):70-80. Epub 2016 Dec 19.

Department of Bioengineering, Temple University, Philadelphia, PA, USA.

Abdominal aortic aneurysm (AAA) is a degenerative disease of the aorta characterized by severe disruption of the structural integrity of the aortic wall and its major molecular constituents. From the early stages of disease, elastin in the aorta becomes highly degraded and is replaced by collagen. Questions persist as to the contribution of collagen content, quality and maturity to the potential for rupture. Here, using our recently developed Fourier transform infrared imaging spectroscopy (FT-IRIS) method, we quantified collagen content and maturity in the wall of AAA tissues in pairs of specimens with different wall stresses. CT scans of AAAs from 12 patients were used to create finite element models to estimate stress in different regions of tissue. Each patient underwent elective repair of the AAA, and two segments of the AAA tissues from anatomic regions more proximal or distal with different wall stresses were evaluated by histology and FT-IRIS after excision. For each patient, collagen content was generally greater in the tissue location with lower wall stress, which corresponded to the more distal anatomic regions. The wall stress/collagen ratio was greater in the higher stress region compared to the lower stress region (1.01 ± 1.09 vs. 0.55 ± 0.084, p = 0.02). The higher stress region also corresponded to the location with reduced intraluminal thrombus thickness. Further, collagen maturity tended to decrease with increased collagen content (p = 0.068, R = 0.38). Together, these results suggest that an increase in less mature collagen content in AAA patients does not effectively compensate for the loss of elastin in the aortic wall, and results in a reduced capability to endure wall stresses.
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http://dx.doi.org/10.1007/s13239-016-0289-3DOI Listing
March 2017

Near infrared spectroscopic assessment of developing engineered tissues: correlations with compositional and mechanical properties.

Analyst 2017 Apr;142(8):1320-1332

Department of Bioengineering, Temple University, Philadelphia, PA, USA.

Articular cartilage degeneration causes pain and reduces the mobility of millions of people annually. Regeneration of cartilage is challenging, due in part to its avascular nature, and thus tissue engineering approaches for cartilage repair have been studied extensively. Current techniques to assess the composition and integrity of engineered tissues, including histology, biochemical evaluation, and mechanical testing, are destructive, which limits real-time monitoring of engineered cartilage tissue development in vitro and in vivo. Near infrared spectroscopy (NIRS) has been proposed as a non-destructive technique to characterize cartilage. In the current study, we describe a non-destructive NIRS approach for assessment of engineered cartilage during development, and demonstrate correlation of these data to gold standard mid infrared spectroscopic measurements, and to mechanical properties of constructs. Cartilage constructs were generated using bovine chondrocyte culture on polyglycolic acid (PGA) scaffolds for six weeks. BMP-4 growth factor and ultrasound mechanical stimulation were used to provide a greater dynamic range of tissue properties and outcome variables. NIR spectra were collected daily using an infrared fiber optic probe in diffuse reflectance mode. Constructs were harvested after three and six weeks of culture and evaluated by the correlative modalities of mid infrared (MIR) spectroscopy, histology, and mechanical testing (equilibrium and dynamic stiffness). We found that specific NIR spectral absorbances correlated with MIR measurements of chemical composition, including relative amount of PGA (R = 0.86, p = 0.02), collagen (R = 0.88, p = 0.03), and proteoglycan (R = 0.83, p = 0.01). In addition, NIR-derived water content correlated with MIR-derived proteoglycan content (R = 0.76, p = 0.04). Both equilibrium and dynamic mechanical properties generally improved with cartilage growth from three to six weeks. In addition, significant correlations between NIRS-derived parameters and mechanical properties were found for constructs that were not treated with ultrasound (PGA (R = 0.71, p = 0.01), water (R = 0.74, p = 0.02), collagen (R = 0.69, p = 0.04), and proteoglycan (R = 0.62, p = 0.05)). These results lay the groundwork for extension to arthroscopic engineered cartilage assessment in clinical studies.
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http://dx.doi.org/10.1039/c6an02167kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386819PMC
April 2017

Early Alterations in Bone Characteristics of Type I Diabetic Rat Femur: A Fourier Transform Infrared (FT-IR) Imaging Study.

Appl Spectrosc 2016 Dec 28;70(12):2005-2015. Epub 2016 Sep 28.

Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

Alterations in microstructure and mineral features can affect the mechanical and chemical properties of bones and their capacity to resist mechanical forces. Controversial results on diabetic bone mineral content have been reported and little is known about the structural alterations in collagen, maturation of apatite crystals, and carbonate content in diabetic bone. This current study is the first to report the mineral and organic properties of cortical, trabecular, and growth plate regions of diabetic rat femurs using Fourier transform infrared (FT-IR) microspectroscopy and the Vickers microhardness test. Femurs of type I diabetic rats were embedded into polymethylmethacrylate blocks, which were used for FT-IR imaging and microhardness studies. A lower mineral content and microhardness, a higher carbonate content especially labile type carbonate content, and an increase in size and maturation of hydroxyapatite crystals were observed in diabetic femurs, which indicate that diabetes has detrimental effects on bone just like osteoporosis. There was a decrease in the level of collagen maturity in diabetic femurs, implying a decrease in bone collagen quality that may contribute to the decrease in tensile strength and bone fragility. Taken together, the findings revealed alterations in structure and composition of mineral and matrix components, and an altered quality and mechanical strength of rat femurs in an early stage of type I diabetes. The results contribute to the knowledge of structure-function relationship of mineral and matrix components in diabetic bone disorder and can further be used for diagnostic or therapeutic purposes.
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http://dx.doi.org/10.1177/0003702816671059DOI Listing
December 2016

The Effect of Stontium Ranelate on Fracture Reduction in Osteogenesis Imperfecta is Comparable to Recent Bisphosphonate Data.

J Bone Miner Res 2016 11 7;31(11):2065. Epub 2016 Sep 7.

Musculoskeletal Integrity Program, Hospital for Special Surgery, New York, NY, USA.

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http://dx.doi.org/10.1002/jbmr.2976DOI Listing
November 2016

Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

J Tissue Eng Regen Med 2016 09 11;10(9):772-82. Epub 2016 Aug 11.

Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA.

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on μsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683170PMC
September 2016
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