Mechanical and Vascular Cues Synergistically Enhance Osteogenesis in Human Mesenchymal Stem Cells

Development and maintenance of a vascular network are critical for bone growth and homeostasis; strategies that promote vascular function are critical for clinical success of tissue-engineered bone constructs. Co-culture of endothelial cells (ECs) with mesenchymal stem cells (MSCs) and exposure to 10% cyclic tensile strain have both been shown to regulate osteogenesis in isolation, but potential synergistic effects have yet to be explored. The objective of this study was to expose an MSC-EC co-culture to 10% cyclic tensile strain to examine the role of this mechanical stimulus on MSC-EC behavior. We hypothesized that paracrine signaling from ECs would stimulate osteogenesis of MSCs, and exposure to 10% cyclic tensile strain would enhance this anabolic signal. Human umbilical vein ECs and human bone marrow-derived MSCs were either monocultured or co-cultured at a 1:1 ratio in a mixed osteo/angiogenic medium, exposed to 10% cyclic tensile strain at 1 Hz for 4 h/day for 2 weeks, and biochemically and histologically analyzed for endothelial and osteogenic markers. While neither 10% cyclic tensile strain nor co-culture alone had a significant effect on osteogenesis, the concurrent application of strain to an MSC-EC co-culture resulted in a significant increase in calcium accretion and mineral deposition, suggesting that co-culture and strain synergistically enhance osteogenesis. Neither co-culture, 10% cyclic tensile strain, nor a combination of these stimuli affected endothelial markers, indicating that the endothelial phenotype remained stable, but unresponsive to the stimuli evaluated in this study. This study is the first to investigate the role of cyclic tensile strain on the complex interplay between ECs and MSCs in co-culture. The results of this study provide key insights into the synergistic effects of 10% cyclic tensile strain and co-culture on osteogenesis. Understanding mechanobiological factors affecting MSC-EC crosstalk will help enhance strategies for creating vascularized tissues in tissue engineering and regenerative medicine

Image-Guided Raman Spectroscopic Recovery of Canine Cortical Bone Contrast in Situ

Raman scattering provides valuable biochemical and molecular markers for studying bone tissue composition with use in predicting fracture risk in osteoporosis. Raman tomography can image through a few centimeters of tissue but is limited by low spatial resolution. X-ray computed tomography (CT) imaging can provide high-resolution image-guidance of the Raman spectroscopic characterization, which enhances the quantitative recovery of the Raman signals, and this technique provides additional information to standard imaging methods. This hypothesis was tested in data measured from Teflon tissue phantoms and from a canine limb. Image-guided Raman spectroscopy (IG-RS) of the canine limb using CT images of the tissue to guide the recovery recovered a contrast of 145:1 between the cortical bone and background. Considerably less contrast was found without the CT image to guide recovery. This study presents the first known IG-RS results from tissue and indicates that intrinsically high contrasts (on the order of a hundred fold) are available

Non-Invasive Raman Tomographic Imaging of Canine Bone Tissue

Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images. With recent advances, diffuse tomography shows promise for in vivo clinical imaging.1, 2 In principle, algorithms developed for fluorescence imaging in tissue can be applied to Raman signals. Although the Raman effect is weaker than fluorescence, the scattered signal is detectable, and thus tomography is achievable. Here we demonstrate the first diffuse tomography reconstructions based on Raman scatter. Raman mapping and imaging are well-established techniques for examining material surfaces.3 Subsurface mapping of simple planar objects was reported recently4, 5 using fiber optic probes with spatially separated injection and collection fibers.6 Noninvasive measurements of bone Raman spectra were demonstrated at depths of5mm role= presentation \u3e5mm below the skin.5 Bone is promising for Raman tomography because the spectra are rich in compositional information,7 which reflects bone maturity and health. Spectroscopically measured bone composition changes have been correlated with aging8 and susceptibility to osteoporotic fracture.9 The Raman spectrum of bone mineral is easily distinguished from the spectra of proteins and other organic tissue constituents, facilitating recovery of even weak signals by multivariate techniques. Assessments of bone quantity and quality are essential to detect and monitor fracture risk and fracture healing with disease or injury. Common sites for fracture with osteoporosis are the spine, proximal femur, and distal radius. Stress fractures are most frequently seen in the weight-bearing sites of the tibia and metatarsals. Fracture risk depends on bone geometry, architecture, and material properties, as well as the nature of applied load (magnitude, rate, and direction). As a result, noninvasive imaging and nondestructive analysis methods have been developed to assess many of these bone attributes that are increasingly important to clinical practice and basic research in orthopedics.10 Current clinical in vivo methods include dual-energy x-ray absorptiometry (DXA), quantitative computed tomography (QCT), magnetic resonance imaging (MRI), ultrasound, and most recently, high-resolution peripheral QCT. Ex vivo analyses of bone specimens from patients or animals have also utilized these and other techniques. In this study, we couple micro-computed tomography (micro-CT) and diffuse optical tomography with Raman spectroscopy to recover spatial and composition information from bone tissue ex vivo. We demonstrate the first reconstruction-based recovery of Raman signals through thick tissues to yield molecular information about subsurface bone tissue. Reconstructions from transcutaneous Raman measurements are challenging, because layers of skin, muscle, fat, and connective tissue lie over the bone sites of interest. These layers have different optical properties and thus variably scatter and polarize the injected light. We chose a canine model because of specimen availability and a bone size similar to human bone. We selected the tibia, a site that is clinically important and has relatively few overlying soft tissues. Measurements were made on the medial surface, where the only additional optical barrier is the crural extensor retinaculum ligament. The canine hind limb was harvested from an animal euthanized in an approved (UCUCA) University of Michigan study. The section of the limb distal to the knee was excised and scanned using in vivo micro-CT (eXplore Locus RS, GE Healthcare, Ontario, Canada). The tibia was scanned at80kV role= presentation \u3e80kV and 450μA role= presentation \u3e450μA with an exposure time of 100ms role= presentation \u3e100ms using a 360-deg scan technique. The image was reconstructed at a 93-μm role= presentation \u3e93-μm voxel resolution [Fig. 1a ]

Tissue-Engineered Constructs of Human Oral Mucosa Examined by Raman Spectroscopy

A noninvasive quality monitoring of tissue-engineered constructs is a required component of any successful tissue-engineering technique. During a 2-week production period, ex vivo produced oral mucosa-equivalent constructs (EVPOMEs) may encounter adverse culturing conditions that might compromise their quality and render them ineffective. We demonstrate the application of near-infrared Raman spectroscopy to in vitro monitoring of EVPOMEs during their manufacturing process, with the ultimate goal of applying this technology in situ to monitor the grafted EVPOMEs. We identify Raman spectroscopic failure indicators for less-than optimal EVPOMEs that are stressed by higher temperature and exposure to higher than normal concentration of calcium ions. Raman spectra of EVPOMEs exposed to thermal and calcium stress showed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs. We compared these results to histology and glucose consumption measurements, demonstrating that Raman spectroscopy is more sensitive and specific to changes in proteins' secondary structure not visible by HandE histology. We also exposed the EVPOMEs to rapamycin, a cell growth inhibitor and cell proliferation capacity preserver, and distinguished between EVPOMEs pretreated with 2?nM rapamycin and controls, using the ratio of the Amide III envelope to the phenylalanine band as an indicator.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140240/1/ten.tec.2012.0287.pd

Reduced Tissue-Level Stiffness and Mineralization in Osteoporotic Cancellous Bone

Osteoporosis alters bone mass and composition ultimately increasing the fragility of primarily cancellous skeletal sites; however, effects of osteoporosis on tissue-level mechanical properties of cancellous bone are unknown. Dual-energy x-ray absorptiometry (DXA) scans are the clinical standard for diagnosing osteoporosis though changes in cancellous bone mass and mineralization are difficult to separate using this method. The goal of this study was to investigate possible difference in tissue-level properties with osteoporosis as defined by donor T-scores. Spine segments from Caucasian female cadavers (58–92 yrs) were used. A T-score for each donor was calculated from DXA scans to determine osteoporotic status. Tissue level composition and mechanical properties of vertebrae adjacent to the scan region were measured using nanoindentation and Raman spectroscopy. Based on T-scores, six samples were in the Osteoporotic group (58–74 yrs) and four samples were in the Not Osteoporotic group (65–92 yrs). The indentation modulus and mineral to matrix ratio (mineral:matrix) were lower in the Osteoporotic group than the Not Osteoporotic group. Mineral:matrix ratio decreased with age (r2 = 0.35, p = 0.05), and the indentation modulus increased with a real bone mineral density (aBMD) (r2 = 0.41, p = 0.04)

The FENIKS Survey: Spectroscopic Confirmation of Massive Quiescent Galaxies at z ~ 3-5

The measured ages of massive, quiescent galaxies at $z\sim 3-4$ imply that massive galaxies quench as early as $z\sim 6$. While the number of spectroscopic confirmations of quiescent galaxies at $z < 3$ has increased over the years, there are only a handful at $z > 3.5$. We report spectroscopic redshifts of one secure ($z=3.757$) and two tentative ($z = 3.336$, $z=4.673$) massive ($\log(M_\ast/M_\odot) > 10.3$) quiescent galaxies with 11 hours of Keck/MOSFIRE $K$-band observations. Our candidates were selected from the FENIKS survey, which uses deep Gemini/Flamingos-2 $K_b$ $K_r$ imaging optimized for increased sensitivity to the characteristic red colors of galaxies at $z > 3$ with strong Balmer/4000 \AA\ breaks. The rest-frame $UVJ$ and $(ugi)_s$ colors of 3/4 quiescent candidates are consistent with $1-2$ Gyr old stellar populations. This places these galaxies as the oldest objects at these redshifts, and challenges the notion that quiescent galaxies at $z > 3$ are all recently-quenched, "post-starburst'' galaxies. Our spectroscopy shows that the other quiescent-galaxy candidate is a broad-line AGN ($z = 3.594$) with strong, redshifted $H\beta$+[O III] emission with a velocity offset $>1000$ km/s, indicative of a powerful outflow. The star-formation history of our highest redshift candidate suggests that its progenitor was already in place by $z \sim 7-11$, reaching $\sim$ 10$^{11} M_{\odot}$ by $z \simeq 10$. These observations reveal the limit of what is possible with deep near-infrared photometry and targeted spectroscopy from the ground and demonstrate that secure spectroscopic confirmation of quiescent galaxies at $z > 4$ is only feasible with JWST.Comment: 20 pages, 11 figures, submitted to Ap

Enhanced cellular infiltration of human adipose-derived stem cells in allograft menisci using a needle-punch method

Abstract Background The meniscus plays a crucial role in knee joint stability, load transmission, and stress distribution. Meniscal tears are the most common reported knee injuries, and the current standard treatment for meniscal deficiency is meniscal allograft transplantation. A major limitation of this approach is that meniscal allografts do not have the capacity to remodel and maintain tissue homeostasis due to a lack of cellular infiltration. The purpose of this study was to provide a new method for enhanced cellular infiltration in meniscal allografts. Methods Twenty medial menisci were collected from cadaveric human sources and split into five experimental groups: (1) control native menisci, (2) decellularized menisci, (3) decellularized menisci seeded with human adipose-derived stem cells (hASC), (4) decellularized needle-punched menisci, and (5) decellularized needle-punched menisci seeded with hASC. All experimental allografts were decellularized using a combined method with trypsin EDTA and peracetic acid. Needle punching (1-mm spacing, 28 G microneedle) was utilized to improve porosity of the allograft. Samples were recellularized with hASC at a density of 250 k/g of tissue. After 28 days of in vitro culture, menisci were analyzed for mechanical, biochemical, and histological characteristics. Results Menisci maintained structural integrity and material properties (compressive equilibrium and dynamic moduli) throughout preparations. Increased DNA content was observed in the needle-punched menisci but not in the samples without needle punching. Histology confirmed these results, showing enhanced cellular infiltration in needle-punched samples. Conclusions The enhanced infiltration achieved in this study could help meniscal allografts better remodel post-surgery. The integration of autologous adipose-derived stem cells could improve long-term efficacy of meniscal transplantation procedures by helping to maintain the meniscus in vivo

Thrombin-Responsive Transcutaneous Patch for Auto-Anticoagulant Regulation

A thrombin-responsive closed-loop patch is developed for prolonged heparin delivery in a feedback-controlled manner. This microneedle-based patch can sense activated thrombin and subsequently releases heparin to prevent coagulation in the blood flow. This "smart" heparin patch can be transcutaneously inserted into skin without drug leakage and can sustainably regulate blood coagulation in response to thrombin

Diminished 11β-Hydroxysteroid Dehydrogenase Type 2 Activity Is Associated With Decreased Weight and Weight Gain Across the First Year of Life

Context: Low birth weight is associated with adverse metabolic outcome in adulthood. Exposure to glucocorticoid (GC) excess in utero is associated with decreased birth weight, but the prospective longitudinal relationship between GC metabolism and growth has not been examined. Objective: We have hypothesized that changes in GC metabolism leading to increased availability may impair growth. Design: This was a prospective, longitudinal study with clinical measurements and 24-hour urinary steroid metabolite analysis at 1, 4, 12, 26, and 52 weeks after delivery in mothers and their babies. Setting: The study was conducted with observations and samples collected in the volunteers' own homes. Participants: Healthy mothers and newborn babies/infants participated in the study. Interventions: There were no interventions. Main outcome measures: Urinary steroid metabolite excretion quantified by gas chromatography/mass spectroscopy across the first year of life in relation to change in weight was measured. Results: The total production of the GC metabolites quantified increased across the first year of life. Markers of 11β-hydroxysteroid dehydrogenase type 1 activity increased from the age of 3 months as did those of 5α-reductase activity. After correcting for confounding variables, low markers of 11β-hydroxysteroid dehydrogenase type 2 activity was associated with reduced absolute weight and decreased weight gain over the first year of life. In the mothers, 5α-reductase activity was low at birth and progressively increased to normal over the first 6 months postpartum. Conclusions: Increased GC exposure as a consequence of reduced 11β-hydroxysteroid dehydrogenase type 2 activity is likely to be a critical determinant of growth in early life. This not only highlights the central role of GCs and their metabolism, but also emphasizes the need for detailed longitudinal analyses