Jung Yul Lim

Human foetal osteoblastic cell response to polymer-demixed nanotopographic interfaces

Nanoscale cell–substratum interactions are of significant interest in various biomedical applications. We investigated human foetal osteoblastic cell response to randomly distributed nanoisland topography with varying heights (11, 38 and 85 nm) produced by a polystyrene (PS)/polybromostyrene polymer-demixing technique. Cells displayed island-conforming lamellipodia spreading, and filopodia projections appeared to play a role in sensing the nanotopography. Cells cultured on 11 nm high islands displayed significantly enhanced cell spreading and larger cell dimensions than cells on larger nanoislands or flat PS control, on which cells often displayed a stellate shape. Development of signal transmitting structures such as focal adhesive vinculin protein and cytoskeletal actin stress fibres was more pronounced, as was their colocalization, in cells cultured on smaller nanoisland surfaces. Cell adhesion and proliferation were greater with decreasing island height. Alkaline phosphatase (AP) activity, an early stage marker of bone cell differentiation, also exhibited nanotopography dependence, i.e. higher AP activity on 11 nm islands compared with that on larger islands or flat PS. Therefore, randomly distributed island topography with varying nanoscale heights not only affect adhesion-related cell behaviour but also bone cell phenotype. Our results suggest that modulation of nanoscale topography may be exploited to control cell function at cell–biomaterial interfaces.

Surface energy effects on osteoblast spatial growth and mineralization

While short-term surface energy effects on cell adhesion are relatively well known, little is revealed as regards its later stage effects on cell behavior. We examined surface energy effects on osteoblastic cell growth and mineralization by using human fetal osteoblastic (hFOB) cells cultured on plasma-treated quartz (contact angle, theta=0 degrees) and octadecyltrichlorosilane (OTS)-treated quartz (theta=113 degrees). hFOB cells formed a homogeneous cell layer on plasma-treated quartz, while those cultured on OTS-treated quartz produced randomly distributed clump-like structures that were filled with cells (confirmed by confocal microscopy). Mineral deposition by hFOB cells was spatially homogeneous when cultured on hydrophilic surfaces. Furthermore, cells on hydrophilic surfaces exhibited increased mineralized area as well as enhanced mineral-to-matrix ratio (assessed by Fourier transform infrared spectroscopy), relative to cells on hydrophobic surfaces. Experiments using other types of osteoblast-like cells (MC3T3-E1, MG63, and SAOS-2) revealed more or less similar effects in spatial growth morphology. It was concluded that hydrophilic surfaces induce homogeneous spatial osteoblastic cell growth and mineral deposition and enhance the quantity (e.g., area) and quality (e.g., mineral-to-matrix ratio) of mineralization relative to hydrophobic surfaces. Our data suggest that surface energy effects on osteoblastic cell differentiation, especially mineralization, may be correlated with surface energy dependent changes in spatial cell growth.

Optimizing the osteogenic potential of adult stem cells for skeletal regeneration

Adult stem cells, including mesenchymal stem cells, display plasticity in that they can differentiate toward various lineages including bone cells, cartilage cells, fat cells, and other types of connective tissue cells. However, it is not clear what factors direct adult stem cell lineage commitment and terminal differentiation. Emerging evidence suggests that extracellular physical cues have the potential to control stem cell lineage specification. In this perspective article, we review recent findings on biomaterial surface and mechanical signal regulation of stem cell differentiation. Specifically, we focus on stem cell response to substrate nanoscale topography and fluid flow induced shear stress and how these physical factors may regulate stem cell osteoblastic differentiation in vitro.

Increased mechanosensitivity of cells cultured on nanotopographies.

Enhancing cellular mechanosensitivity is recognized as a novel tool for successful musculoskeletal tissue engineering. We examined the hypothesis that mechanosensitivity of human mesenchymal stem cells (hMSCs) is enhanced on nanotopographic substrates relative to flat surfaces. hMSCs were cultured on polymer-demixed, randomly distributed nanoisland surfaces with varying island heights and changes in intracellular calcium concentration, [Ca(2+)](i), in response to fluid flow induced shear stress were quantifide. Stem cells cultured on specific scale nanotopographies displayed greater intracellular calcium responses to fluid flow. hMSCs cultured on 10-20nm high nanoislands displayed a greater percentage of cells responding in calcium relative to cells cultured on flat control, and showed greater average [Ca(2+)](i) increase relative to cells cultured on other nanoislands (45-80nm high nanoislands). As [Ca(2+)](i) is an important regulator of downstream signaling, as well as proliferation and differentiation of hMSCs, this observation suggests that specific scale nanotopographies provide an optimal milieu for promoting stem cell mechanotransduction activity. That mechanical signals and substrate nanotopography may synergistically regulate cell behavior is of significant interest in the development of regenerative medicine protocols.

Mechanical stretch suppresses BMP4 induction of stem cell adipogenesis via upregulating ERK but not through downregulating Smad or p38

Bone morphogenetic proteins (BMPs) are also implicated in the commitment of mesenchymal stem cells (MSCs) toward adipocytes. We tested that stretching of cells may downregulate BMP4 induction of MSC adipogenesis. C3H10T1/2 MSCs were pretreated with BMP4 and induced to differentiate to adipocytes using adipogenic hormonal inducers. To test the stretch effect on BMP4 function, cells were exposed to cyclic tensile stretch (10% strain, 0.25Hz, 120min/day) during the BMP4 pretreatment period. BMP4 induced MSC adipocytic commitment. Stretching during the BMP4 exposure could suppress BMP4 induction of MSC adipogenesis, as assessed by downregulated adipogenic transcription factors (PPARγ, C/EBPα, aP2) and decreased lipid accumulation. BMP4 signaled through Smad1/5/8 and p38MAPK, whereas cell stretch did not affect BMP4-induced activation in Smad or p38. On the other hand, cell stretch triggered significant ERK1/2 phosphorylation relative to BMP4 treatment alone cells. Further, stretch suppression of BMP4-induced MSC adipogenesis was significantly deteriorated if cells were stretched with ERK blocked by PD98059. Combined, these suggest that cell stretch suppresses the BMP4 induction of MSC adipogenesis potentially via upregulating ERK but not through the downregulation of Smad or p38. Our data on inhibiting MSC adipogenesis will be of significant interest for obesity and developmental mechanobiology studies.

Retinoic acid inhibits BMP4-induced C3H10T1/2 stem cell commitment to adipocyte via downregulating Smad/p38MAPK signaling.

Increased adipocyte formation from mesenchymal stem cells (MSCs) is typical for obesity. It is recently observed that bone morphogenetic proteins (BMPs) provide instructive signals for the commitment of MSCs to adipocytes. We examined potential role of retinoic acid (RA) in inhibiting the BMP4 induction of MSC commitment toward adipocyte. BMP4-treated C3H10T1/2 MSCs, when further exposed to adipogenic differentiation media, displayed distinct adipocytic commitment and differentiation. This could be inhibited by RA exposure during the BMP4 treatment stage (commitment stage before adipogenic hormonal inducers were given), as was observed by reductions in key adipogenic genes/transcription factors (C/EBPα, PPARγ, aP2), lipogenic genes (LPL, FAS, GLUT4), and lipid accumulation. Among RA receptors (RARs) screened, RARβ was mainly upregulated under RA exposure. BMP4 signaled through both Smad1/5/8 and p38 mitogen-activated protein kinase (MAPK) and RA significantly suppressed the BMP4-triggered phosphorylation of both Smad1/5/8 and p38MAPK. These data suggest that RA has inhibitory effects on the BMP4 induction of C3H10T1/2 adipocytic commitment via downregulating Smad/p38MAPK signaling. How to inhibit MSC adipocytic commitment, as partly revealed in this study, will have a significant impact on treating obesity and related diseases.

The role of RhoA kinase (ROCK) in cell alignment on nanofibers

While the potential of nanofibers as tissue engineering scaffolds has been demonstrated, very little has been revealed as regards the molecular mechanism by which cells sense and respond to nanofibers. It was hypothesized that RhoA kinase (ROCK), one of the vital cell tension signaling cascades, plays a role in regulating cell alignment on nanofibers. To test this, unidirectionally aligned and randomly distributed nanofibers, both with an average diameter of ∼130nm, were fabricated with poly(l-lactic acid) (PLLA). A flat PLLA film was used as the control. Mesenchymal stem cells (MSCs, C3H10T1/2) displayed high fidelity in cell orientation along aligned nanofibers, and showed an increased cell spreading area on random nanofibers. Interestingly, cells cultured on aligned nanofibers displayed significantly greater ROCK expression relative to cells on a flat surface, as assessed by immunoblotting. To further test the role of ROCK, MSCs with ROCK small hairpin RNA (shRNA) were established. It is notable that, even when ROCK was stably knocked down via shRNA, cells could still display preferred orientation along aligned nanofibers. However, MSCs with shRNA-ROCK displayed a significantly decreased cell major axis length following aligned nanofibers compared with shRNA vector control, suggesting that ROCK may be involved in cell elongation on aligned nanofibers. Along with the reduction in cell length, cell area was decreased with ROCK silencing. These cell morphological changes induced by shRNA-ROCK were generally maintained on a flat surface and random nanofibers. A pharmacological ROCK inhibitor, Y-27632, produced results similar to those of shRNA-ROCK. The data on the role of ROCK in regulating cell alignment on nanofibers may provide a new mechanistic insight into nanofiber control of cells.

Inducing neurite outgrowth by mechanical cell stretch.

Abstract Establishing extracellular milieus to stimulate neuronal regeneration is a critical need in neuronal tissue engineering. Many studies have used a soluble factor (such as nerve growth factor or retinoic acid [RA]), micropatterned substrate, and electrical stimulation to induce enhanced neurogenesis in neuronal precursor cells. However, little attention has been paid to mechanical stimulation because neuronal cells are not generally recognized as being mechanically functional, a characteristic of mechanoresponsive cells such as osteoblasts, chondrocytes, and muscle cells. In this study, we performed proof-of-concept experiments to demonstrate the potential anabolic effects of mechanical stretch to enhance cellular neurogenesis. We cultured human neuroblastoma (SH-SY5Y) cells on collagen-coated membrane and applied 10% equibiaxial dynamic stretch (0.25 Hz, 120 min/d for 7 days) using a Flexcell device. Interestingly, cell stretch alone, even without a soluble neurogenic stimulatory factor (RA), produced significantly more and longer neurites than the non–RA-treated, static control. Specific neuronal differentiation and cytoskeletal markers (e.g., microtubule-associated protein 2 and neurofilament light chain) displayed compatible variations with respect to stretch stimulation.

Micropatterning-retinoic acid co-control of neuronal cell morphology and neurite outgrowth

Creating physical-biochemical superposed microenvironments optimal for stimulating neurite outgrowth would be beneficial for neuronal regenerative medicine. We investigated potential co-regulatory effects of cell micropatterning and retinoic acid (RA) soluble factor on neuronal cell morphology and neurite outgrowth. Human neuroblastoma (SH-SY5Y) cell patterning sensitivity could be enhanced by poly-L-lysine-g-polyethylene glycol cell-repellent back-filling, enabling cell confinement in lanes as narrow as 5 μm. Cells patterned on narrow (5 and 10 μm) lanes showed preferred nucleus orientation following the patterning direction. These cells also showed high nucleus aspect ratio but constrained nucleus spreading. On the other hand, cells on wide (20 μm and above) lanes showed random nucleus orientation and cell and nucleus sizes similar to those on unpatterned controls. All these changes were generally maintained with or without RA. Confining cells on narrow (5 and 10 μm) lanes, even without RA, significantly enhanced neurite extension relative to unpatterned control, which was further stimulated by RA. Interestingly, cell patterning on 5 and 10 μm lanes without RA produced longer neurites relative to the RA treatment alone case. Our data on the potential interplay between microscale physical cell confinement and RA-soluble stimulation may provide a new, integrative insight on how to trigger neurite/axon formation for neuronal regenerative medicine.

Graphene substrate for inducing neurite outgrowth.

A few recent studies demonstrated that graphene may have cytocompatibility with several cell types. However, when assessing cell behavior on graphene, there has been no precise control over the quality of graphene, number of graphene layers, and substrate surface coverage by graphene. In this study, using well-controlled monolayer graphene film substrates we tested the cytocompatibility of graphene for human neuroblastoma (SH-SY5Y) cell culture. A large-scale monolayer graphene film grown on Cu foils by chemical vapor deposition (CVD) could be successfully transferred onto glass substrates by wet transfer technique. We observed that graphene substrate could induce enhanced neurite outgrowth, both in neurite length and number, compared with control glass substrate. Interestingly, the positive stimulatory effect by graphene was achieved even in the absence of soluble neurogenic factor, retinoic acid (RA). Key genes relevant to cell neurogenesis, e.g., neurofilament light chain (NFL), were also upregulated on graphene. Inhibitor studies suggested that the graphene stimulation of cellular neurogenesis may be achieved through focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) cascades. Our data indicate that graphene may be exploited as a platform for neural regenerative medicine, and the suggested molecular mechanism may provide an insight into the graphene control of neural cells.