Substrate Stiffness and Oxygen as Regulators of Stem Cell Differentiation during Skeletal Tissue Regeneration: A Mechanobiological Model

Extrinsic mechanical signals have been implicated as key regulators of mesenchymal stem cell (MSC) differentiation. It has been possible to test different hypotheses for mechano-regulated MSC differentiation by attempting to simulate regenerative events such as bone fracture repair, where repeatable spatial and temporal patterns of tissue differentiation occur. More recently, in vitro studies have identified other environmental cues such as substrate stiffness and oxygen tension as key regulators of MSC differentiation; however it remains unclear if and how such cues determine stem cell fate in vivo. As part of this study, a computational model was developed to test the hypothesis that substrate stiffness and oxygen tension regulate stem cell differentiation during fracture healing. Rather than assuming mechanical signals act directly on stem cells to determine their differentiation pathway, it is postulated that they act indirectly to regulate angiogenesis and hence partially determine the local oxygen environment within a regenerating tissue. Chondrogenesis of MSCs was hypothesized to occur in low oxygen regions, while in well vascularised regions of the regenerating tissue a soft local substrate was hypothesised to facilitate adipogenesis while a stiff substrate facilitated osteogenesis. Predictions from the model were compared to both experimental data and to predictions of a well established computational mechanobiological model where tissue differentiation is assumed to be regulated directly by the local mechanical environment. The model predicted all the major events of fracture repair, including cartilaginous bridging, endosteal and periosteal bony bridging and bone remodelling. It therefore provides support for the hypothesis that substrate stiffness and oxygen play a key role in regulating MSC fate during regenerative events such as fracture healing.     

Compressive mechanical properties of the intraluminal thrombus in abdominal aortic aneurysms and fibrin-based thrombus mimics

An intraluminal thrombus (ILT) forms in the majority of abdominal aortic aneurysms (AAAs). While the ILT has traditionally been perceived as a byproduct of aneurysmal disease, the mechanical environment within the ILT may contribute to the degeneration of the aortic wall by affecting biological events of cells embedded within the ILT. In this study, the drained secant modulus (E₅～modulus at 5% strain) of ILT specimens (luminal, medial, and abluminal) procured from elective open repair was measured and compared using unconfined compression. Five groups of fibrin-based thrombus mimics were also synthesized by mixing various combinations of fibrinogen, thrombin, and calcium. Drained secant moduli were compared to determine the effect of the components’ concentrations on mimic stiffness. The stiffness of mimics was also compared to the native ILT. Preliminary data on the water content of the ILT layers and mimics was measured. It was found that the abluminal layer (E₅=19.3 kPa) is stiffer than the medial (2.49 kPa) and luminal (1.54 kPa) layers, both of which are statistically similar. E₅ of the mimics (0.63, 0.22, 0.23, 0.87, and 2.54 kPa) is dependent on the concentration of all three components: E₅ decreases with a decrease in fibrinogen (60–20 and 20–15 mg/ml) and a decrease in thrombin (3–0.3 units/ml), and E₅ increases with a decrease in calcium (0.1–0.01 M). E₅ from two of the mimics were not statistically different than the medial and luminal layers of ILT. A thrombus mimic with similar biochemical components, structure, and mechanical properties as native ILT would provide an appropriate test medium for AAA mechanobiology studies.     

Community annotation in biology

   

Attempts to engage the scientific community to annotate biological data (such as protein/gene function) stored in databases have not been overly successful. There are several hypotheses on why this has not been successful but it is not clear which of these hypotheses are correct. In this study we have surveyed 50 biologists (who have recently published a paper characterizing a gene or protein) to better understand what would make them interested in providing input/contributions to biological databases. Based on our survey two things become clear: a) database managers need to proactively contact biologists to solicit contributions; and b) potential contributors need to be provided with an easy-to-use interface and clear instructions on what to annotate. Other factors such as 'reward' and 'employer/funding agency recognition' previously perceived as motivators was found to be less important. Based on this study we propose community annotation projects should devote resources to direct solicitation for input and streamlining of the processes or interfaces used to collect this input.     

Synthesis and evaluation of an anti-MLC1 × anti-CD90 bispecific antibody for targeting and retaining bone-marrow-derived multipotent stromal cells in infarcted myocardium

A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues. Here, we have generated and assessed a bispecific antibody heterodimer designed to improve the retention of bone-marrow-derived multipotent stromal cells (BMMSC) in cardiac tissue damaged by myocardial infarction. The heterodimer comprises an anti-human CD90 monoclonal antibody (mAb) (clone 5E10) and an anti-myosin light chain 1 (MLC1) mAb (clone MLM508) covalently cross-linked by a bis-arylhydrazone. We modified the anti-CD90 antibody with a pegylated-4-formylbenzamide moiety to a molar substitution ratio (MSR) of 2.6 and the anti-MLC1 antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9. The covalent modifications had no significant deleterious effect on mAb epitope binding. Furthermore, the binding of anti-CD90 antibody to BMMSCs did not prevent their differentiation into adipo-, chondro-, or osteogenic lineages. Modified antibodies were combined under mild conditions (room temperature, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-arylhydrazone, which was monitored at A(354). We evaluated epitope immunoreactivity for each mAb in the construct. Flow cytometry demonstrated binding of the bispecific construct to BMMSCs that was competed by free anti-CD90 mAb, verifying that modification and cross-linking were not detrimental to the anti-CD90 complementarity-determining region. Similarly, ELISA-based assays demonstrated bispecific antibody binding to plastic-immobilized recombinant MLC1. Excess anti-MLC1 mAb competed for bispecific antibody binding. Finally, the anti-CD90 × anti-MLC1 bispecific antibody construct induced BMMSC adhesion to plastic-immobilized MLC1 that was resistant to shear stress, as measured in parallel-plate flow chamber assays. We used mAbs that bind both human antigens and the respective pig homologues. Thus, the anti-CD90 × anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.