Dynamically Modulated Core-Shell Microfibers to Study the Effect of Depth Sensing of Matrix Stiffness on Stem Cell Fate.

It is well known that extracellular matrix stiffness can affect cell fate and change dynamically during many biological processes. Existing experimental means for in situ matrix stiffness modulation often alters its structure, which could induce additional undesirable effects on cells. Inspired by the phenomenon of depth sensing by cells, we introduce here core-shell microfibers with a thin collagen core for cell growth and an alginate shell that can be dynamically stiffened to deliver mechanical stimuli. This allows for the maintenance of biochemical properties and structure of the surrounding microenvironment, while dynamically modulating the effective modulus "felt" by cells. We show that simple addition of Sr2+ in media can easily increase the stiffness of initially Ca2+ cross-linked alginate shells. Thus, despite the low stiffness of collagen cores (<5 kPa), the effective modulus of the matrix "felt" by cells are substantially higher, which promotes osteogenesis differentiation of human mesenchymal stem cells. We show this effect is more prominent in the stiffening microfiber compared to a static microfiber control. This approach provides a versatile platform to independently and dynamically modulate cellular microenvironments with desirable biochemical, physical, and mechanical stimuli without an unintended interplay of effects, facilitating investigations of a wide range of dynamic cellular processes.

Development of a Virtual Cell Model to Predict Cell Response to Substrate Topography.

Cells can sense and respond to changes in the topographical, chemical, and mechanical information in their environment. Engineered substrates are increasingly being developed that exploit these physical attributes to direct cell responses (most notably mesenchymal stem cells) and therefore control cell behavior toward desired applications. However, there are very few methods available for robust and accurate modeling that can predict cell behavior prior to experimental evaluations, and this typically means that many cell test iterations are needed to identify best material features. Here, we developed a unifying computational framework to create a multicomponent cell model, called the "virtual cell model" that has the capability to predict changes in whole cell and cell nucleus characteristics (in terms of shape, direction, and even chromatin conformation) on a range of cell substrates. Modeling data were correlated with cell culture experimental outcomes in order to confirm the applicability of the virtual cell model and demonstrating the ability to reflect the qualitative behavior of mesenchymal stem cells. This may provide a reliable, efficient, and fast high-throughput approach for the development of optimized substrates for a broad range of cellular applications including stem cell differentiation.

Nacre Topography Produces Higher Crystallinity in Bone than Chemically Induced Osteogenesis.

It is counterintuitive that invertebrate shells can induce bone formation, yet nacre, or mother of pearl, from marine shells is both osteoinductive and osteointegrative. Nacre is composed of aragonite (calcium carbonate) and induces production of vertebrate bone (calcium phosphate). Exploited by the Mayans for dental implants, this remarkable phenomenon has been confirmed in vitro and in vivo, yet the characteristic of nacre that induces bone formation remains unknown. By isolating nacre topography from its inherent chemistry in the production of polycaprolactone (PCL) nacre replica, we show that, for mesenchymal stem cells, nacre topography is osteoinductive. Gene expression of specific bone marker proteins, osteopontin, osteocalcin, osteonectin, and osterix, is increased 10-, 2-, 1.7-, and 1.8-fold, respectively, when compared to planar PCL. Furthermore, we demonstrate that bone tissue that forms in response to the physical topographical features of nacre has a higher crystallinity than bone formed in response to chemical cues with a full width half-maximum for PO43- Raman shift of 7.6 ± 0.7 for mineral produced in response to nacre replica compared to a much broader 34.6 ± 10.1 in response to standard osteoinductive medium. These differences in mineral product are underpinned by differences in cellular metabolism. This observation can be exploited in the design of bone therapies; a matter that is most pressing in light of a rapidly aging human population.

Continuous Fabrication and Assembly of Spatial Cell-Laden Fibers for a Tissue-Like Construct via a Photolithographic-Based Microfluidic Chip.

Engineering three-dimensional (3D) scaffolds with in vivo like architecture and function has shown great potential for tissue regeneration. Here we developed a facile microfluidic-based strategy for the continuous fabrication of cell-laden microfibers with hierarchically organized architecture. We show that photolithographically fabricated microfluidic devices offer a simple and reliable way to create anatomically inspired complex structures. Furthermore, the use of photo-cross-linkable methacrylated alginate allows modulation of both the mechanical properties and biological activity of the hydrogels for targeted applications. Via this approach, multilayered hollow microfibers were continuously fabricated, which can be easily assembled in situ, using 3D printing, into a larger, tissue-like construct. Importantly, this biomimetic approach promoted the development of phenotypical functions of the target tissue. As a model to engineer a complex tissue construct, osteon-like fiber was biomimetically engineered, and enhanced vasculogenic and osteogenic expression were observed in the encapsulated human umbilical cord vein endothelial cells and osteoblast-like MG63 cells respectively within the osteon fibers. The capability of this approach to create functional building blocks will be advantageous for bottom-up regeneration of complex, large tissue defects and, more broadly, will benefit a variety of applications in tissue engineering and biomedical research.

A Quiescent, Regeneration-Responsive Tissue Engineered Mesenchymal Stem Cell Bone Marrow Niche Model via Magnetic Levitation.

The bone marrow niche represents a specialized environment that regulates mesenchymal stem cell quiescence and self-renewal, yet fosters stem cell migration and differentiation upon demand. An in vitro model that embodies these features would open up the ability to perform detailed study of stem cell behavior. In this paper we present a simple bone marrow-like niche model, which comprises of nanomagnetically levitated stem cells cultured as multicellular spheroids within a type I collagen gel. The stem cells maintained are nestin positive and remain quiescent until regenerative demand is placed upon them. In response to coculture wounding, they migrate and appropriately differentiate upon engraftment. This tissue engineered regeneration-responsive bone marrow-like niche model will allow for greater understanding of stem cell response to injury and also facilitate as a testing platform for drug candidates in a multiwell plate format.

Osteoprogenitor response to semi-ordered and random nanotopographies.

In bone tissue engineering, it is desirable to use materials to control the differentiation of mesenchymal stem cell populations in order to gain direct bone apposition to implant materials. It has been known for a number of years that microtopography can alter cell adhesion, proliferation and gene expression. More recently, the literature reveals that nanotopography is also of importance. Here, the reaction of primary human osteoprogenitor cell populations to nanotopographies down to 10 nm in size is considered. The topographies were originally produced by colloidal lithography and polymer demixing on silicon and then embossed (through an intermediate nickel shim) into polymethylmethacrylate. The biological testing considered cell morphology (image analysis of cell spreading and scanning electron microscopy), cell cytoskleton and adhesion formation (fluorescent staining of actin, tubulin, vimentin and vinculin) and then subsequent cell growth and differentiation (fluorescent staining of osteocalcin and osteopontin). The results demonstrated that the nanotopographies stimulated the osteoprogenitor cell differentiation towards an osteoblastic phenotype.

Osteoprogenitor response to defined topographies with nanoscale depths.

In the development of the next generation of orthopaedic implants for load-bearing applications, an ability to influence osteoprogenitor population activity and function will be highly desirable. This will allow the formation of hard-tissue directly onto the implant, i.e. osseointegration, rather than the formation of fibrous capsules which form around many of the current, non-bioactive, prosthesis. The formation of capsules leads to micromotion due to modulus mismatch and ultimately to fracture and the need for revision surgery. Microtopography and latterly nanotopography have been shown to elicit influence over adhesion, proliferation and gene expression of a wide number of cell types. This study has examined the possibility of modulating cell adhesion using a range of nanometric scale shallow pits and grooves. The topographies were manufactured using photolithography followed by the production of nickel shims and finally embossing into polymethylmethacrylate. Cell testing with human osteoprogenitor populations showed that the nanotopographies allowed control of cell adhesion, cytoskeleton, growth and production of the osteoblastic markers osteocalcin and osteopontin. It is concluded that the human bone marrow stromal cells are highly responsive to nanoscale features.

Changes in fibroblast morphology in response to nano-columns produced by colloidal lithography.

In designing new biomaterials, specific chemical and topographical cues will be important in guiding cell response. Filopodia are actin-driven structures produced by cells and speculated to be involved in cell sensing of the three-dimensional environment. This report quantifies filopodia response to cylindrical nano-columns (100 nm diameter, 160 nm high) produced by colloidal lithography. Also observed were actin cytoskeleton morphology by fluorescence microscopy and filopodia morphology by electron microscopy (scanning and transmission). The results showed that the fibroblasts used produced more filopodia per microm of cell perimeter and that filopodia could often be seen to interact with the cells' nano-environment. By understanding as to which features evoke spatial reactions in cells, it may be possible to design better biomaterials.