Mechanoregulation of Proliferation, Differentiation, Senescence and Survival of Bone Marrow Primary Osteoprecursor Cells

Cell and animal studies conducted onboard the International Space Station and during the Shuttle program have provided extensive data illustrating bone degenerative responses to mechanical unloading in microgravity. Specifically CDKN1a/p21, an inhibitory modulator of cell cycle progression, is upregulated in osteoprecursor cells of the femur during 15-day spaceflight, suggesting that microgravity can block stem cell-based tissue regenerative process at the level of progenitor proliferation and differentiation. To study a potential role for CDKN1a/p21 in regulating osteogenic mechanosensitivity, we cultured primary bone marrow osteoprogenitor cells from CDKN1a/p21-null (p21-null) and wildtype mice with and without mechanical stimulation, and compared their morphological, proliferative, and in-vitro mineralization responses. Structural cell alterations due to mechanical stimulation were assessed by florescence labeling of f-actin cytoskeleton and focal adhesions. Mechanical stimulation of p21-null cells resulted in more pronounced cytoskeletal alignment with the axis of stretch than for wildtype cells. In addition, p21-null cells subjected to stretch loading also formed significantly more focal adhesions than wildtype cells. Combined these findings suggest that p21-null cells are structurally more responsive to stretch stimulation than the wildtype cells. Because osteoprogenitor cells are well known to respond to mechanical stimulation with increased proliferation, we also tested this response in p21-null cells. Results from those experiments show the proliferative capacity of mechanically stimulated p21-null cells far exceeded that of wildtype controls. Specifically, cell counts from 14, and 21 days post mechanical stimulation, show that p21- null cells to have a 4-fold increase in proliferation compared to wildtype. When the p21-null cell differentiation response to mechanical stimulation was evaluated, the p21-null cultuers elicited more extensive mineralization at earlier assessed timepoints than control cultures. Specifically, Von Kossa staining for mineralized matrix showed that the p21-null cells produced more than twice the mineralized surface area of wildtype cells, and at an earlier 7-day time point in culture. Taken together these results suggest that CDKN1a/p21 normally plays a role in negatively regulating osteoprogenitor proliferation and differentiation responses to mechanostimulation in bone. Findings of CDKN1a/p21's increased expression during spaceflight in microgravity also suggest not only a potential molecular mechanism for arresting regenerative bone growth in space, but potentially also a reduced impact for bone-formation-promoting exercise mechanostimulation. The findings described here constitute a novel role for p21 as a regulator of tissue regeneration in response to mechanical load stimulation, and also suggest a new promising molecular target to promote regenerative health in disuse conditions

Osteogenic Transcription Regulated by Exaggerated Stretch Loading via Convergent Wnt Signaling

Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided data illuminating the deleterious biological response of bone to mechanical unloading. Down regulation of proliferative mechanisms within stem cell populations of the osteogenic niche is a suggested mechanism for loss of bone mass. However the intercellular communicative cues from osteoblasts and osteocytes in managing stem cell proliferation and osteogenic differentiation are largely unknown. In this investigation, MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells, are co-culture under dynamic tensile conditions and evaluated for phenotypic expression of biochemical signaling proteins influential in driving stem cell differentiation. MLO-Y4 and MC3T3-E1 were co-cultured on polyethersulfone membrane with a 0.45m porosity to permit soluble factor transfer and direct cell-cell gap junction signaling. Cyclic tensile stimulation was applied for 48 h at a frequency of 0.1Hz and strain of 0.1. Total Live cell counts indicate mechanical activation of MC3T3-E1s inhibits proliferation while MLO-Y4s increase in number. However, the percent of live MLO-Y4s within the population is low (46.3 total count, *p0.05, n4) suggesting a potential apoptotic signaling cascade. Immunofluorescence demonstrated that stimulation of co-cultures elicits increased gap junction communication. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggest that an osteocyte-osteoblast signaling feedback mechanism may regulate mechanotransduction of an apoptotic cascade within osteocytes and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed

Novel Approach to Quantification of Telomere Length with Direct Nanopore Sequencing and PCR Amplification

The ends of human chromosomes contain telomeres, or tandem arrays of repeating DNA sequences capped by multiple associated proteins that protect chromosomal ends from degradation. Telomeres function to preserve genomic stability by preventing natural chromosomal ends from being recognized as broken DNA double-strand breaks and triggering inappropriate DNA damage responses. Mounting evidence shows telomere length is an inherited trait that decreases with cellular division and normal aging. In addition, telomere length also appears to be influenced by other factors such as cellular oxidative stress, radiation and mechanical unloading of tissues as in microgravity. To measure these potential effects of the space environment on telomere lengths and cellular aging and regenerative potential we developed a novel telomere measurement approach based on nanopore sequencing of PCR amplified bar-coded chromosome termini. Specifically, telomeres can be directly enriched using barcode sequences ligated to the end of a free end- repaired telomere using the WetLab-2 facility SmartCycler on ISS. Prior to the ligation and amplification protocol a proteinase K digestion of capping proteins followed by a single 95-degree C heat denaturation of the protease is included. After digestion and bar-code ligation, PCR amplification will initiate with the ligated barcoded sequence, suppressing amplification of intra-genomic fragments and resulting in long read barcoded telomere amplicons including the nanopore motor protein sequences. Purified PCR amplicons are then used for nanopore sequencing library generation by simple addition of motor proteins and sequencing library is loaded into the MinION nanopore DNA-sequencer. Amplicon sequence reads from the nanopore device can be base-called quickly on ISS due to barcoding ligation and subsequent PCR amplification enhancing the telomere sequence resolution. If successfully implemented on ISS this technique will provide a novel means of measuring regenerative ability of somatic stem cells in astronauts, and of determining whether spaceflight in microgravity alters their telomere lengths and causes premature cellular aging

Seismic Retrofitting Using Micropile Systems Centrifugal Model Studies

A series of centrifuge tests were conducted on micropile group and network systems in order to investigate the response to earthquake loading and soil-micropile interaction behavior. Model tests on group and network systems embedded in loose to medium dry sand are described. Micropile bending moment, deflection, and acceleration were measured during testing. Dynamic p-y curves were derived from the measurements for low and high levels of shaking and were compared with the backbone p-y curves for sand recommended by API and other published data. Group and network effects were investigated for different configurations and at different levels of loading. For the selected frequency of excitation, the results indicate a positive group effect increasing with the number of piles and the batter angle. This paper describes the experimental procedures used to carry out the centrifugal model tests and summarizes the main preliminary results

The Role of Gravity Mechanotransduction in Regulating Stem Cell Tissue Regenerative Potential at the Single Cell Expressome Level

Gravity is an omnipresent force on Earth, and all living organisms have evolved under the influence of constant gravity. Mechanical forces generated by gravity are potent modulators of stem cell based tissue regenerative mechanisms, inducing cell fate decisions and tissue specific commitment. A novel mechanical unloading investigation assessed the formation, morphology, and gene expression of embryoid bodies (EB), a transitory cell model of early differentiation. After 15 days of spaceflight, the mechanotransduction-null EB cells showed upregulated proliferative mechanisms while differentiation cues were silenced

The strategic function of quality in the management of innovation

Quality management can be used to support strategically the management of innovation. Tools in strategic quality management can be useful in: creating the organizational conditions in which innovations can be developed; supervising and initiating innovation processes; producing innovation content; and implementing innovations in the primary processes of the organization. This conclusion is based on the results of a research project in the Dutch construction industry. In a large-scale house-building project the supportive use of quality tools in the management of innovation was studied. The study indicates that quality tools are used implicitly and sometimes explicitly to manage innovation processes

Interpretation and reporting of process capability results: focus on improvement

A global financial services company followed a software-mediated process assessment (SMPA) approach based on ISO/IEC 15504, ISO/IEC 20000 and the IT Infrastructure Library (ITIL®). Using an action research approach, the Incident Management, Problem Management, and Change Management processes were assessed at two points in time during an ITSM process improvement project. This paper analyzes the results of the process assessments, highlights issues with the interpretation of the results, and offers an alternative method to report process capability results to motivate process improvement. The study found that by using the proportion of SMPA recommendations as a proxy measure for process improvement, the processes did improve yielding fewer recommendations in cycle 2 when compared to cycle 1 of the action research

Drilled and Grouted Micropiles: State-of-Practice Review, Volume I: Background, Classifications, Cost

DTFH61-93-C-00128Micropiles are small-diameter, drilled and grouted reinforced piles used for both structural support and in situ earth reinforcement. They were conceived in Italy in 1952, but have become popular in the United States only since the mid-1980s. This report provides a comprehensive state-of-practice review, drawing on data from an international basis. This volume, Volume I, provides a general and historical framework and new classifications of type and application. Cost and feasibility are also discussed

Drilled and Grouted Micropiles: State-of-Practice Review, Volume III: Construction, QA/QC, and Testing

DTFH61-93-C-00128Micropiles are small-diameter, drilled and grouted reinforced piles used for both structural support and in-situ earth reinforcement. They were conceived in Italy in 1952 but have become popular in the United States only since the mid 1980s. This report provides a comprehensive state-of-practice review, drawing on data from an international basis. This volume, Volume IV, provides summaries of 20 major case histories illustrating the principles and procedures of volumes I, II, and III

Drilled and Grouted Micropiles: State-of-Practice Review, Volume II: Design

DTFH61-93-C-00128Micropiles are small-diameter, drilled and grouted reinforced piles used for both structural support and in situ earth reinforcement. They were conceived in Italy in 1952, but have become popular in the United States only since the mid-1980s. This report provides a comprehensive state-of-practice review, drawing on data from an international basis. This volume, Volume II, deals with the design of single piles, and groups and networks of piles