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The forces generated by gravity have shaped life on Earth and impact gene expression and morphogenesis during early development. Conversely, disuse on Earth or during spaceflight, reduces normal mechanical loading of organisms, resulting in altered cell and tissue function. Although gravity mechanical loading in adult mammals is known to promote increased cell proliferation and differentiation, little is known about how distinct cell types respond to gravity mechanostimulation during early development. In this study we sought to understand, with single cell RNA-sequencing resolution, how a 60-min pulse of 50 g hypergravity (HG)/5 kPa hydrostatic pressure, influences transcriptomic regulation of developmental processes in the embryoid body (EB) model. Our study included both day-9 EBs and progenitor mouse embryonic stem cells (ESCs) with or without the HG pulse. Single cell t-distributed stochastic neighbor mapping shows limited transcriptome shifts in response to the HG pulse in either ESCs or EBs; this pulse however, induces greater positional shifts in EB mapping compared to ESCs, indicating the influence of mechanotransduction is more pronounced in later states of cell commitment within the developmental program. More specifically, HG resulted in upregulation of self-renewal and angiogenesis genes in ESCs, while in EBs, HG loading was associated with upregulation of Gene Ontology-pathways for multicellular development, mechanical signal transduction, and DNA damage repair. Cluster transcriptome analysis of the EBs show HG promotes maintenance of transitory cell phenotypes in early development; including EB cluster co-expression of markers for progenitor, post-implant epiblast, and primitive endoderm phenotypes with HG pulse but expression exclusivity in the non-pulsed clusters. Pseudotime analysis identified three branching cell types susceptible to HG induction of cell fate decisions. In totality, this study provides novel evidence that ESC maintenance and EB development can be regulated by gravity mechanostimulation and that stem cells committed to a differentiation program are more sensitive to gravity-induced changes to their transcriptome.

CDKN1A/P21 is a potent inhibitor of cell cycle progression and its overexpression is thought to be associated with inhibition of normal bone regenerative osteogenesis during spaceflight. To test whether CDKN1A/P21 regulates osteogenesis in response to mechanical loading we studied cyclic stretch versus static culture of Cdkn1a-/- (null) or wildtype primary mouse bone marrow osteoprogenitors during 21-day ex-vivo mineralization assays. Cyclically stretched Cdkn1a-/- cells are 3.95-fold more proliferative than wildtype, while static Cdkn1a-/- cells show a 2.50-fold increase. Furthermore, stage-specific single cell RNAseq analyses show expression of Cdkn1a is strongly suppressed by cyclic stretch in early and late osteoblasts, and minimally in the progenitor population. Lastly, both stretch and/or Cdkn1a deletion cause population shift from osteoprogenitors to osteoblasts, also indicating increased differentiation. Collectively, our results support the hypothesis that Cdkn1a constitutively plays a mechano-reversible anti-proliferative role during osteogenesis and suggests a new molecular target to counter regenerative deficits caused by disuse.

The temporomandibular joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment options and debilitating nature of severe temporomandibular disorders has been the primary driving force for the introduction and development of TMJ disc tissue engineering as an approach to alleviate this important clinical issue. This study aimed to evaluate the efficacy of laser micropatterning (LMP) ex vivo-derived TMJ disc scaffolds to enhance cellular integration, a major limitation to the development of whole tissue implant technology. LMP was incorporated into the decellularized extracellular matrix scaffold structure using a 40 W CO2 laser ablation system to drill an 8×16 pattern with a bore diameter of 120 μm through the scaffold thickness. Disc scaffolds were seeded with human neonatal-derived umbilical cord mesenchymal stem cells differentiated into chondrocytes at a density of 900 cells per mm(2) and then assessed on days 1, 7, 14, and 21 of culture. Results derived from histology, PicoGreen DNA quantification, and cellular metabolism assays indicate that the LMP scaffolds improve cellular remodeling compared to the unworked scaffold over the 21-day culture period. Mechanical analysis further supports the use of the LMP showing the compressive properties of the LMP constructs closely represent native disc mechanics. The addition of an artificial path of infiltration by LMP culminated in improved chondrocyte adhesion, dispersion, and migration after extended culture aiding in recapitulating the native TMJ disc characteristics.

Human perinatal tissues have been used for over a century as allogeneic biomaterials. Due to their advantageous properties including angiogenecity, anti-inflammation, anti-microbial, and immune privilege, these tissues are being utilized for novel applications across wide-ranging medical disciplines. Given continued clinical success, increased adoption of perinatal tissues as a disruptive technology platform has allowed for significant penetration into the multi-billion dollar biologics market. Here, we review current progress and future applications of perinatal biomaterials, as well as associated regulatory issues.

The temporomandibular joint (TMJ) is a complex hinge and gliding joint that induces significant shear loads onto the fibrocartilage TMJ disc during jaw motion. The purpose of this study was to assess regional variation in the disc’s shear loading characteristics under physiologically relevant loads and to associate those mechanical findings with common clinical observations of disc fatigue and damage. Porcine TMJ discs were compressed between an axially translating bottom platen and a 2.5-cm-diameter indenter within a hydrated testing chamber. Discs were cyclically sheared at 0.5, 1, or 5 Hz to 1, 3, or 5% shear strain. Within the anterior and intermediate regions of the disc when sheared in the anteroposterior direction, both shear and compressive moduli experienced a significant decrease from instantaneous to steady state, while the posterior region’s compressive modulus decreased approximately 5%, and no significant loss of shear modulus was noted. All regions retained their shear modulus within 0.5% of instantaneous values when shear was applied in the mediolateral direction. The results of the disc’s regional shear mechanics suggest an observable and predictable link with the common clinical observation that the posterior region of the disc is most often the zone in which fatigue occurs, which may lead to disc damage and perforation.

Material compliance has been shown to be a predictor of vascular graft patency and as such is a critical parameter when designing new materials. Although ex vivo derived materials have been clinically successful in a number of applications their mechanical properties are a direct function of the original vessel and are not easily controllable. These investigations describe an approach to modulate the mechanical properties of an ex vivo derived scaffold by machining variable (discrete) wall thicknesses to control compliance. Human umbilical arteries (HUAs) were machine lathed directly from the umbilical cord at wall thicknesses of 250, 500, 750, and 1000 μm then decellularized using 1% sodium dodecyl sulfate. Compliance over physiological pressures, increased from 3.08 ± 1.84% to 11.47 ± 4.11% as direct function of each discrete vessel diameter. Radial stress strain analysis revealed primary and secondary failure points attributed to the discrete layers within the anisotropic scaffold. Maximum strength and suture retention were shown to increase with increasing wall thickness, by contrast stress failure decreased with increasing thickness due to increasing proportions of the mechanically weaker amorphous Wharton's jelly. Reseeded smooth muscle cells were shown to adhere, proliferate, and migrate from the scaffold surface showing the potential of the HUA as a mechanically "tunable" material with applications as an acellular implant or as a tissue engineered construct. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3189-3196, 2012.