Machine Learned Composite Methods for Electronic Structure Theory.

Because of the prohibitive scaling of ab initio techniques for modeling chemical species with high accuracy, they are not generally tractable for large systems. It is therefore of considerable interest to develop high-accuracy computational models with low computational cost that can afford predictions of electronic structure and properties of macromolecular species. Composite methods, as first introduced by Pople [Pople, J. A.; Head-Gordon, M.; Fox, D. J.; Raghavachari, K.; Curtiss, L. A. J. Chem. Phys.1989, 90, 5622.], are an intuitive solution to this problem as they seek to systematically increase accuracy in model chemistries by taking advantage of favorable error cancellation among reasonably low-cost models. By linearly combining a series of carefully chosen model chemistries, the result of a prohibitive-scaling correlated model chemistry with a large basis set may be approximated with relatively good fidelity. However, the full extent to which the choice of low-cost models dictates the predictive accuracy of composite methods is not known, and a full exploration of all model chemistries would be advantageous for the design and validation of a generalizable composite method for widespread application. Here, we show that remarkable accuracy can be generally achieved with composite methods that are more judiciously constructed, leading to increased accuracy with significantly reduced computational cost. By designing a systematic procedure for the automated generation and assessment of over 10 billion unique composite methods, we have extensively explored the space of modern model chemistries to elucidate important design principles in the construction of reliable composite procedures. We anticipate our work to be the starting point in the pursuit of creative approaches to modeling large chemical systems with high accuracy by using novel combinatorial modeling.

Hierarchical biofabrication of biomimetic collagen-elastin vascular grafts with controllable properties via lyophilisation.

This article describes the development of a hierarchical biofabrication technique suitable to create large but complex structures, such as vascular mimicking grafts, using facile lyophilisation technology amenable to multiple other biomaterial classes. The combination of three fabrication techniques together, namely solvent evaporation, lyophilisation, and crosslinking together allows highly tailorable structures from the microstructure up to the macrostructure, and with the ability to independently crosslink each layer it allows great flexibility to match desired native mechanical properties independently of the micro/macrostructure. We have demonstrated the flexibility of this biofabrication technique by independently optimising each of the layers to create a multi-layered arterial structure with tailored architectural and biophysical/biochemical properties using a collagen-elastin composite. Taken together, the facile biofabrication methodology developed has led to the development of a biomimetic bilayered scaffold suitable for use as a tissue engineered vascular graft (for haemodialysis access or peripheral/coronary bypass), or as an in vitro test platform to examine disease progression, pharmacological toxicity, or cardiovascular medical device testing. STATEMENT OF SIGNIFICANCE: The ability to grow large complex tissues such as blood vessels for transplantation is often hampered by the limitations of the selected biofabrication technique. Here, we sought to overcome some of the fabrication limitations for naturally occurring cardiovascular polymers (collagen/elastin) via a hierarchical approach to fabrication where each layer is built upon the previous. This approach enabled the flexibility to modify and tailor each layer's properties independently via control over polymer concentration, microstructure, and crosslinking. This simple approach facilitated us to fabricate multi-layered vascular grafts which were remodelled into high-density vascular tissue after 21-days. The fabrication approach could be translated to a myriad of other tissues while the engineered vascular graft could also be used as a test platform for drugs/medical devices or as a tissue engineering scaffold for vascular grafting for different indications.

Material stiffness influences the polarization state, function and migration mode of macrophages.

Biomaterial implantation is followed by an inflammatory cascade dominated by macrophages, which determine implant acceptance or rejection through pro- and anti-inflammatory polarization states (Anderson et al., 2008; Brown and Badylak, 2013). It is known that chemical signals such as bacterial endotoxins and cytokines (IL4) can direct macrophage polarization (Mantovani et al., 2004); however, recent evidence implicates biophysical cues in this process (McWhorter et al., 2015; Patel et al., 2012). Here we report that THP-1 derived macrophages cultured on collagen-coated polyacrylamide gels of varying stiffness adapt their polarization state, functional roles and migration mode according to the stiffness of the underlying substrate. Through gene expression and protein secretion analysis, we show that stiff polyacrylamide gels (323 kPa) prime macrophages towards a pro-inflammatory phenotype with impaired phagocytosis in macrophages, while soft (11 kPa) and medium (88 kPa) stiffness gels prime cells towards an anti-inflammatory, highly phagocytic phenotype. Furthermore, we show that stiffness dictates the migration mode of macrophages; on soft and medium stiffness gels, cells display Rho-A kinase (ROCK)-dependent, podosome-independent fast amoeboid migration and on stiff gels they adopt a ROCK-independent, podosome-dependent slow mesenchymal migration mode. We also provide a mechanistic insight into this process by showing that the anti-inflammatory property of macrophages on soft and medium gels is ROCK-dependent and independent of the ligand presented to them. Together, our results demonstrate that macrophages adapt their polarization, function and migration mode in response to the stiffness of the underlying substrate and suggest that biomaterial stiffness is capable of directing macrophage behaviour independent of the biochemical cues being presented to them. The results from this study establish an important role for substrate stiffness in directing macrophage behaviour, and will lead to the design of immuno-informed biomaterials that are capable of modulating the macrophage response after implantation. STATEMENT OF SIGNIFICANCE: Biomaterial implantation is followed by an inflammatory cascade dominated by macrophages, which determine implant acceptance or rejection through pro- and anti-inflammatory polarization states. It is known that chemical signals can direct macrophage polarization; however, recent evidence implicates biophysical cues in this process. Here we report that macrophages cultured on gels of varying stiffness adapt their polarization state, functional roles and migration mode according to the stiffness of the underlying substrate. The results from this study establish an important role for substrate stiffness in directing macrophage behaviour, and will lead to the design of immuno-informed biomaterials that are capable of modulating the macrophage response after implantation.

Identification of the mechanisms by which age alters the mechanosensitivity of mesenchymal stromal cells on substrates of differing stiffness: Implications for osteogenesis and angiogenesis.

In order to identify the mechanisms by which skeletal maturity alters the mechanosensitivity of mesenchymal stromal cells (MSCs) and, the implications for osteogenesis and angiogenesis during bone formation, we compared the response of MSCs derived from children and skeletally-mature healthy adults cultured on soft and stiff collagen-coated polyacrylamide substrates. MSCs from children were more mechanosensitive, showing enhanced angiogenesis and osteogenesis on stiff substrates as indicated by increased endothelial tubule formation, PGF production, nuclear-translocation of YAP, ALP activity and mineralisation. To examine these mechanisms in more detail, a customised PCR array identified an age-dependent, stiffness-induced upregulation of NOX1, VEGFR1, VEGFR2, WIF1 and, of particular interest, JNK3 in cells from children compared to adults. When JNK3 activity was inhibited, a reduction in stiffness-induced driven osteogenesis was observed - suggesting that JNK3 might serve as a novel target for recapitulating the enhanced regenerative potential of children in adults suffering from bone degeneration. STATEMENT OF SIGNIFICANCE: We investigated the age-associated changes in the capacity of MSCs for bone regeneration involving the mechanosensitive signalling pathways, which reduce the ability of adult cells to respond to biophysical cues in comparison to cells from children, who are still undergoing bone development. Our results offer new insights into the mechanobiology of MSCs and sheds new light on age-altered mechanosensitivity and, on why children have such an immense capacity to regenerate their skeletal system. We have identified the mechanisms by which skeletal maturity alters the mechanosensitivity of mesenchymal stromal cells and an age-dependent, stiffness-induced upregulation of a number of prominent genes including, most notably, JNK3 in children cells, thus suggesting its potential to promote enhanced bone repair.

The effect of time-dependent deformation of viscoelastic hydrogels on myogenic induction and Rac1 activity in mesenchymal stem cells.

Cell behaviours within tissues are influenced by a broad array of physical and biochemical microenvironmental factors. Whilst 'stiffness' is a recognised physical property of substrates and tissue microenvironments that influences many cellular behaviours, tissues and their extracellular matrices are not purely rigid but 'viscoelastic' materials, composed of both rigid-like (elastic) and dissipative (viscous) elements. This viscoelasticity results in materials displaying increased deformation with time under the imposition of a defined force or stress, a phenomenon referred to as time-dependent deformation or 'creep'. Previously, we compared the behaviour of human mesenchymal stem cells (hMSCs) on hydrogels tailored to have a constant stiffness, but to display varying levels of creep in response to an applied force. Using polyacrylamide as a model material, we showed that on high-creep hydrogels (HCHs), hMSCs displayed increased proliferation, spread area and differentiation towards multiple lineages, compared to their purely stiff analogue, with a particular propensity for differentiation towards a smooth muscle cell (SMC) lineage. In this present study, we investigate the mechanisms behind this phenomenon and show that hMSCs adhered to HCHs have increased expression of SMC induction factors, including soluble factors, ECM proteins and the cell-cell adhesion molecule, N-Cadherin. Further, we identify a key role for Rac1 signalling in mediating this increased N-Cadherin expression. Using a real-time Rac1-FRET biosensor, we confirm increased Rac1 activation on HCHs, an observation that is further supported functionally by observed increases in motility and lamellipodial protrusion rates of hMSCs. Increased Rac1 activity in hMSCs on HCHs provides underlying mechanisms for enhanced commitment towards a SMC lineage and the compensatory increase in spread area (isotonic tension) after a creep-induced loss of cytoskeletal tension on viscoelastic substrates, in contrast to previous studies that have consistently demonstrated up-regulation of RhoA activity with increasing substrate stiffness. Tuning substrate viscoelasticity to introduce varying levels of creep thus equips the biomaterial scientist or engineer with a new tool with which to tune and direct stem cell outcomes.

Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration.

Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFß). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in increased matrix formation when compared to the addition of soluble PPS to the hydrogel, or the hydrogel alone. We believe that this new generation injectable, degradable hydrogel, incorporating now a covalently bound-PPS, when combined with MPCs, has the potential to assist cartilage regeneration in a multitude of therapeutic targets, including for intervertebral disc (IVD) degeneration.

An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration.

Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-ß. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.

The influence of substrate creep on mesenchymal stem cell behaviour and phenotype.

Human mesenchymal stem cells (hMSCs) are capable of probing and responding to the mechanical properties of their substrate. Although most biological and synthetic matrices are viscoelastic materials, previous studies have primarily focused upon substrate compressive modulus (rigidity), neglecting the relative contributions that the storage (elastic) and loss (viscous) moduli make to the summed compressive modulus. In this study we aimed to isolate and identify the effects of the viscous component of a substrate on hMSC behaviour. Using a polyacrlyamide gel system with constant compressive modulus and varying loss modulus we determined that changes to substrate loss modulus substantially affected hMSC morphology, proliferation and differentiation potential. In addition, we showed that the effect of substrate loss modulus on hMSC behaviour is due to a reduction in both passive and actively generated isometric cytoskeletal tension caused by the inherent creep of substrates with a high loss modulus. These findings highlight substrate creep, or more explicitly substrate loss modulus, as an important mechanical property of a biomaterial system that can be tailored to encourage the growth and differentiation of specific cell types.

Complex embryos displaying bilaterian characters from Precambrian Doushantuo phosphate deposits, Weng'an, Guizhou, China.

Three-dimensionally preserved embryos from the Precambrian Ediacaran Doushantuo Formation, Weng'an, Guizhou, southern China, have attracted great attention as the oldest fossil evidence yet found for multicellular animal life on Earth. Many embryos are early cleavage embryos and most of them yield a limited phylogenetic signal. Here we report the discovery of two Doushantuo embryos that are three-dimensionally preserved and complex. Imaging techniques using propagation phase-contrast based synchrotron radiation microtomography (PPC-SR-microCT) reveal that the organization of cells demonstrates several bilaterian features, including the formation of anterior-posterior, dorso-ventral, and right-left polarities, and cell differentiation. Unexpectedly, our observations show a noticeable difference in organization patterns between the embryos, suggesting that they represent two distinct taxa. These embryos provide further evidence for the presence of bilaterian animals in the Doushantuo biota. Furthermore, these bilaterians had already diverged into distantly related groups at least 40 million years before the Cambrian radiation, indicating that the last common ancestor of the bilaterians lived much earlier than is usually thought.

Germ line determinants are not localized early in sea urchin development, but do accumulate in the small micromere lineage.

Two distinct modes of germ line determination are used throughout the animal kingdom: conditional-an inductive mechanism, and autonomous-an inheritance of maternal factors in early development. This study identifies homologs of germ line determinants in the sea urchin Strongylocentrotus purpuratus to examine its mechanism of germ line determination. A list of conserved germ-line associated genes from diverse organisms was assembled to search the S. purpuratus genome for homologs, and the expression patterns of these genes were examined during embryogenesis by whole mount in situ RNA hybridization and QPCR. Of the 14 genes tested, all transcripts accumulate uniformly during oogenesis and Sp-pumilio, Sp-tudor, Sp-MSY, and Sp-CPEB1 transcripts are also uniformly distributed during embryonic development. Sp-nanos2, Sp-seawi, and Sp-ovo transcripts, however, are enriched in the vegetal plate of the mesenchyme blastula stage and Sp-vasa, Sp-nanos2, Sp-seawi, and Sp-SoxE transcripts are localized in small micromere descendents at the tip of the archenteron during gastrulation and are then enriched in the left coelomic pouch of larvae. The results of this screen suggest that sea urchins conditionally specify their germ line, and support the hypothesis that this mechanism is the basal mode of germ line determination amongst deuterostomes. Furthermore, accumulation of germ line determinants selectively in small micromere descendents supports the hypothesis that these cells contribute to the germ line.