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

Loss of BMP signaling through BMPR1A in osteoblasts leads to greater collagen cross-link maturation and material-level mechanical properties in mouse femoral trabecular compartments

Bone morphogenetic protein (BMP) signaling pathways play critical roles in skeletal development and new bone formation. Our previous study, however, showed a negative impact of BMP signaling on bone mass because of the osteoblast-specific loss of a BMP receptor (i.e. BMPR1A) showing increased trabecular bone volume and mineral density in mice. Here, we investigated the bone quality and biomechanical properties of the higher bone mass associated with BMPR1A deficiency using the osteoblast-specific Bmpr1a conditional knockout (cKO) mouse model. Collagen biochemical analysis revealed greater levels of the mature cross-link pyridinoline in the cKO bones, in parallel with upregulation of collagen modifying enzymes. Raman spectroscopy distinguished increases in the mature to immature cross-link ratio and mineral to matrix ratio in the trabecular compartments of cKO femora, but not in the cortical compartments. The mineral crystallinity was unchanged in the cKO in either the trabecular or cortical compartments. Further, we tested the intrinsic material properties by nanoindentation and found significantly higher hardness and elastic modulus in the cKO trabecular compartments, but not in the cortical compartments. Four point bending tests of cortical compartments showed lower structural biomechanical properties (i.e. strength and stiffness) in the cKO bones due to the smaller cortical areas. However, there were no significant differences in biomechanical performance at the material level, which was consistent with the nanoindentation test results on the cortical compartment. These studies emphasize the pivotal role of BMPR1A in the determination of bone quality and mechanical integrity under physiological conditions, with different impact on femoral cortical and trabecular compartments

Investigation of Mineral and Collagen Organization in Bone using Raman Spectroscopy.

The extraordinary toughness and stiffness of bone are associated with its three main constituents - apatite mineral, collagen protein and water. Variations in composition and organization of these constituents are known to exist as a function of disease and aging. These variations greatly influence bone quality and need to be understood in greater detail. This thesis advances the understanding of molecular organization in bone along three directions: quantification of molecular orientation, analysis of mineral deformation in response to hydration changes and loading and investigation of age-dependent bone quality. First, polarized Raman spectroscopy was adapted for bone tissue applications to quantify molecular organization in non-deproteinated, turbid tissue. This enabled the simultaneous quantitative measurements of altered mineral and collagen orientations in Osteogenesis Imperfecta, a bone disease associated with collagen mutations. Second, the effect of distorting the water environment in bone was investigated by replacing matrix water with deuterium oxide. Changes in hydrogen bonding affected collagen secondary structure, resulting in compression of the mineral lattice as evidenced by changes in peak positions and widths of mineral Raman bands. Further, polarized Raman spectroscopy was used to probe nano-scale deformations due to tensile loading and orientation-dependent strains within the mineral lattice were observed. These results demonstrate the potential of Raman spectroscopy to provide insights on molecular orientation and interaction at the nano-scale. Third, exploratory data mining tools were employed to identify tissue-level compositional (Raman) and mechanical (nanoindentation) metrics that predict bone quality, instead of the traditionally used linear regressions. The results showed that compositional properties offer only a partial understanding of mechanical properties at the tissue-level and vice versa. Hence, a specific combination of compositional and mechanical metrics was required to reliably classify femoral specimens according to age. These findings suggest that combined metrics will better predict transformations in bone quality than individual metrics and call for novel techniques to explore the complex multi-scale interactions in bone. The multiple lines of evidence presented in this thesis provide an insight into the complex roles that mineral, collagen and water play in governing tissue quality and mechanical properties of bone.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/84443/1/mekhala_1.pd

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 CH(2) 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 H&E 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

Loss of BMP signaling through BMPR1A in osteoblasts leads to greater collagen cross-link maturation and material-level mechanical properties in mouse femoral trabecular compartments

Bone morphogenetic protein (BMP) signaling pathways play critical roles in skeletal development and new bone formation. Our previous study, however, showed a negative impact of BMP signaling on bone mass because of the osteoblast-specific loss of a BMP receptor (i.e. BMPR1A) showing increased trabecular bone volume and mineral density in mice. Here, we investigated the bone quality and biomechanical properties of the higher bone mass associated with BMPR1A deficiency using the osteoblast-specific Bmpr1a conditional knockout (cKO) mouse model. Collagen biochemical analysis revealed greater levels of the mature cross-link pyridinoline in the cKO bones, in parallel with upregulation of collagen modifying enzymes. Raman spectroscopy distinguished increases in the mature to immature cross-link ratio and mineral to matrix ratio in the trabecular compartments of cKO femora, but not in the cortical compartments. The mineral crystallinity was unchanged in the cKO in either the trabecular or cortical compartments. Further, we tested the intrinsic material properties by nanoindentation and found significantly higher hardness and elastic modulus in the cKO trabecular compartments, but not in the cortical compartments. Four point bending tests of cortical compartments showed lower structural biomechanical properties (i.e. strength and stiffness) in the cKO bones due to the smaller cortical areas. However, there were no significant differences in biomechanical performance at the material level, which was consistent with the nanoindentation test results on the cortical compartment. These studies emphasize the pivotal role of BMPR1A in the determination of bone quality and mechanical integrity under physiological conditions, with different impact on femoral cortical and trabecular compartments