Critical Frequency and Critical Stretching Rate for Reorientation of Cells on a Cyclically Stretched Polymer in a Microfluidic Chip.

The ability of cells to sense and respond to mechanical signals from their surrounding microenvironments is one of the key issues in tissue engineering and regeneration, yet a fundamental study of cells with both cell observation and mechanical stimulus is challenging and should be based upon an appropriate microdevice. Herein we designed and fabricated a two-layer microfluidic chip to enable simultaneous observation of live cells and cyclic stretching of an elastic polymer, polydimethylsiloxane (PDMS), with a modified surface for enhanced cell adhesion. Human mesenchymal stem cells (hMSCs) were examined with a series of frequencies from 0.00003 to 2 Hz and varied amplitudes of 2%, 5%, or 10%. The cells with an initial random orientation were confirmed to be reoriented perpendicular to the stretching direction at frequencies greater than a threshold value, which we term critical frequency (fc); additionally, the critical frequency fc was amplitude-dependent. We further introduced the concept of critical stretching rate (Rc) and found that this quantity can unify both frequency and amplitude dependences. The reciprocal value of Rc in this study reads 8.3 min, which is consistent with the turnover time of actin filaments reported in the literature, suggesting that the supramolecular relaxation in the cytoskeleton within a cell might be responsible for the underlying cell mechanotransduction. The theoretical calculation of cell reorientation based on a two-dimensional tensegrity model under uniaxial cyclic stretching is well consistent with our experiments. The above findings provide new insight into the crucial role of critical frequency and critical stretching rate in regulating cells on biomaterials under biomechanical stimuli.

A simplified yet enhanced and versatile microfluidic platform for cyclic cell stretching on an elastic polymer.

While the microfluidic chips for cell stretching and real-time cell observations have so far been composed of three layers, the present work reports a two-layer one, which is, on the surface, not available due to the 'inherent' difficulty of unstable focusing on cells in the microscopic observation under the stretching operation, etc. Herein, this difficulty was overcome to a large extent, in the case of appropriate device parameters, which were determined based upon finite element analysis and orthogonal experimental design. The novel chip was fabricated and confirmed to work in frequency up to 2 Hz and stretching ratio up to 20%. We further performed uniaxial stretching experiments of human mesenchymal stem cells on an elastic polymer, polydimethylsiloxane, and the cells were found to be highly oriented perpendicular to the stretching direction. The short working distance on this simplified two-layer chip enabled clear observation of microtubules and stress fibers of cells under an optical microscope. We also tested radial stretching and gradient stretching as proofs of concept of the extendibility of this type of chip. Therefore, in spite of being simpler, the two-layer chip suggested in this study exhibited enhanced and versatile functions, and the present work has thus afforded a new methodology of fabrication of microfluidic chips for the study of cells on biomaterials under a mechanical stimulus.

Cell migration regulated by RGD nanospacing and enhanced under moderate cell adhesion on biomaterials.

While nanoscale modification of a biomaterial surface is known to influence various cell behaviors, it is unclear whether there is an optimal nanospacing of a bioactive ligand with respect to cell migration. Herein, we investigated the effects of nanospacing of arginine-glycine-aspartate (RGD) peptide on cell migration and its relation to cell adhesion. To this end, we prepared RGD nanopatterns with varied nanospacings (31-125 nm) against the nonfouling background of poly(ethylene glycol), and employed human umbilical vein endothelial cells (HUVECs) to examine cell behaviors on the nanopatterned surfaces. While HUVECs adhered well on surfaces of RGD nanospacing less than 70 nm and exhibited a monotonic decrease of adhesion with the increase of RGD nanospacing, cell migration exhibited a nonmonotonic change with the ligand nanospacing: the maximum migration velocity was observed around 90 nm of nanospacing, and slow or very slow migration occurred in the cases of small or large RGD nanospacings. Therefore, moderate cell adhesion is beneficial for fast cell migration. Further molecular biology studies revealed that attenuated cell adhesion and activated dynamic actin rearrangement accounted for the promotion of cell migration, and the genes of small G proteins such as Cdc42 were upregulated correspondingly. The present study sheds new light on cell migration and its relation to cell adhesion, and paves a way for designing biomaterials for applications in regenerative medicine.

Nonmonotonic Self-Deformation of Cell Nuclei on Topological Surfaces with Micropillar Array.

Cells respond to the mechanical signals from their surroundings and integrate physiochemical signals to initiate intricate mechanochemical processes. While many studies indicate that topological features of biomaterials impact cellular behaviors profoundly, little research has focused on the nuclear response to a mechanical force generated by a topological surface. Here, we fabricated a polymeric micropillar array with an appropriate dimension to induce a severe self-deformation of cell nuclei and investigated how the nuclear shape changed over time. Intriguingly, the nuclei of mesenchymal stem cells (MSCs) on the poly(lactide-co-glycolide) (PLGA) micropillars exhibited a significant initial deformation followed by a partial recovery, which led to an "overshoot" phenomenon. The treatment of cytochalasin D suppressed the recovery of nuclei, which indicated the involvement of actin cytoskeleton in regulating the recovery at the second stage of nuclear deformation. Additionally, we found that MSCs exhibited different overshoot extents from their differentiated lineage, osteoblasts. These findings enrich the understanding of the role of the cell nucleus in mechanotransduction. As the first quantitative report on nonmonotonic kinetic process of self-deformation of a cell organelle on biomaterials with unique topological surfaces, this study sheds new insight into cell-biomaterial interactions.

Subcellular cell geometry on micropillars regulates stem cell differentiation.

While various material factors have been shown to influence cell behaviors, recent studies started to pay attention to the effects of some material cues on "subcellular" geometry of cells, such as self-deformation of cell nuclei. It is particularly interesting to examine whether a self deformation happens discontinuously like a first-order transition and whether subcellular geometry influences significantly the extent of stem cell differentiation. Herein we prepared a series of micropillar arrays of poly(lactide-co-glycolide) and discovered a first-order transition of nuclear shape as a function of micropillar height under the examined section area and interspacing of the pillars. The deformed state of the nuclei of mesenchymal stem cells (MSCs) was well maintained even after osteogenic or adipogenic induction for several days. The nuclear deformation on the micropillar arrays was accompanied with smaller projected areas of cells, but led to an enhanced osteogenesis and attenuated adipogenesis of the MSCs, which is different from the previously known relationship between morphology and differentiation of stem cells on flat substrates. Hence, the present study reveals that the geometry of cell nuclei may afford a new cue to regulate the lineage commitment of stem cells on the subcellular level.

Microfluidic 3D bone tissue model for high-throughput evaluation of wound-healing and infection-preventing biomaterials.

We report the use of a microfluidic 3D bone tissue model, as a high-throughput means of evaluating the efficacy of biomaterials aimed at accelerating orthopaedic implant-related wound-healing while preventing bacterial infection. As an example of such biomaterials, inkjet-printed micropatterns were prepared to contain antibiotic and biphasic calcium phosphate (BCP) nanoparticles dispersed in a poly(D,L-lactic-co-glycolic) acid matrix. The micropatterns were integrated with a microfluidic device consisting of eight culture chambers. The micropatterns immediately and completely killed Staphylococcus epidermidis upon inoculation, and enhanced the calcified extracellular matrix production of osteoblasts. Without antibiotic elution, bacteria rapidly proliferated to result in an acidic microenvironment which was detrimental to osteoblasts. These results were used to demonstrate the tissue model's potential in: (i) significantly reducing the number of biomaterial samples and culture experiments required to assess in vitro efficacy for wound-healing and infection prevention and (ii) in situ monitoring of dynamic interactions of biomaterials with bacteria as wells as with tissue cells simultaneously.

Inkjet printed antibiotic- and calcium-eluting bioresorbable nanocomposite micropatterns for orthopedic implants.

Inkjet printing of antibiotic- and calcium-eluting micropatterns was explored as a novel means of preventing the formation of biofilm colonies and facilitating osteogenic cell development on orthopedic implant surfaces. The micropatterns consisted of a periodic array of ∼50 µm circular dots separated by ∼150 µm. The composition of the micropatterns was controlled by formulating inks with rifampicin (RFP) and poly(D,L-lactic-co-glycolic) acid (PLGA) dissolved in an organic solvent with ∼100 nm biphasic calcium phosphate (BCP) nanoparticles suspended in the solution. During printing RFP and PLGA co-precipitated to form a nanocomposite structure with ∼10-100 nm RFP and the BCP particles dispersed in the PLGA matrix. The rate of RFP release was strongly influenced by the RFP loading in the micropattern, particularly on the first day. The RFP-containing micropatterns effectively prevented the formation of Staphylococcus epidermidis biofilm colonies due to their ability to kill bacteria prior to forming colonies on the patterned surfaces. The BCP-containing micropatterns printed on the surface of the alloy TiAl6V4 significantly accelerated osteoblast cell differentiation, as measured by alkaline phosphatase expression and calcium deposition, without compromising cell proliferation.