Cell metabolism in response to biomaterial mechanics

This project assessed the use of short chain peptide (F2/S) hydrogel biomaterial substrates as an instructional tool for driving stem cell differentiation through fine-tuning of the substrate mechanical properties (altered elasticity or stiffness) to mimic that of naturally occurring tissue types. By doing this, differentiation of mesenchymal stem cells (MSCs) into neuronal cells on a 2 kPa (soft) substrate, chondrocytes on 6 kPa (medium) substrate and osteoblasts on 38 kPa (rigid) substrates was achieved. This non-invasive procedure of influencing stem cell behaviour allows a means of exploring innate cell behaviour as they adopt different cell lineages on differentiation. As such, an LC-MS based metabolomics study was used to profile differences in cell behaviour. Stem cells were observed as having increased metabolic activity when undergoing differentiation compared to their ‘resting’ state when they are observed as metabolically quiescent or relatively inactive. As such, the metabolome, as a reflection of the current state of cell metabolism, was used to illustrate the observed divergence of phenotypes as differentiation occurs on each substrate F2/S type. The project further investigated the potential of endogenous small molecules (metabolites) identified using metabolomics, as effective compounds in driving or supporting cell differentiation in vitro. From this, the compounds cholesterol sulphate and sphinganine were found to induce MSC differentiation along the osteogenic and neurogenic routes respectively. A third compound, GP18:0, was observed to have influence on promoting both osteo- and chondrogenic development. These results highlight the potential role a broad based metabolomics study plays in the identification of endogenous metabolites and ascertaining the role(s) they play in cellular differentiation and subsequent tissue development. Lastly, the use of F2/S substrates as a potential clinical scaffold for the regeneration of cartilage tissue was explored. Long term differentiation of pericytes into chondrocytes cultured in 20 kPa F2/S substrates was assessed and the cellular phenotype of the resultant chondrocytes compared to the more conventionally used induction media method. Pericytes cultured within the biomaterial alone showed a balanced expressed of type II collagen and aggrecan with lessened type X collagen expression compared to the coupled use of induction media which showed a bias towards collagen (both type II and type X) gene expression. This observation suggests that in order to mimic native hyaline cartilage tissue in vitro, the use of biomaterial mechanics is potentially a better approach in guiding stem cell differentiation than the use of chemical cues

Improving cartilage phenotype from differentiated pericytes in tunable peptide hydrogels

Differentiation of stem cells to chondrocytes in vitro usually results in a heterogeneous phenotype. This is evident in the often detected over expression of type X collagen which, in hyaline cartilage structure is not characteristic of the mid-zone but of the deep-zone ossifying tissue. Methods to better match cartilage developed in vitro to characteristic in vivo features are therefore highly desirable in regenerative medicine. This study compares phenotype characteristics between pericytes, obtained from human adipose tissue, differentiated using diphenylalanine/serine (F2/S) peptide hydrogels with the more widely used chemical induced method for chondrogenesis. Significantly higher levels of type II collagen were noted when pericytes undergo chondrogenesis in the hydrogel in the absence of induction media. There is also a balanced expression of collagen relative to aggrecan production, a feature which was biased toward collagen production when cells were cultured with induction media. Lastly, metabolic profiles of each system show considerable overlap between both differentiation methods but subtle differences which potentially give rise to their resultant phenotype can be ascertained. The study highlights how material and chemical alterations in the cellular microenvironment have wide ranging effects on resultant tissue type

Nacre Topography Produces Higher Crystallinity in Bone than Chemically Induced Osteogenesis

It is counter-intuitive 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 are 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 higher crystallinity than bone formed in response to chemical cues with full width half maximum for PO4 3- 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 ageing human population. Aragonite and calcite are the two calcium carbonate polymorphs that constitute the shell of molluscan bivalves conferring strength and resilience due to the nano- and microstructural assembly of the overall architecture. A small percentage of the invertebrate shell constitute the organic matrix which is responsible for the intricate processes of nucleation, growth and inhibition of calcium carbonate crystals resulting in the well-defined shell structure. The discovery of fully integrated shell dental implants in Mayan skulls initiated a number of studies showing that nacre, or mother of pearl, the aragonite calcium carbonate polymorph derived from the pearl oyster Pinctada maxima has good osteointegrative properties in vivo. Further exploration of this phenomenon in human jaw reconstructions and sheep femur implants confirm the osteointegrative properties of invertebrate shells. In addition, nacre initiates osteogenic differentiation in mesenchymal stem cells (MSCs) in vitro. This observation has led to a number of studies in which nacre and its chemistry have been incorporated into the design of existing biomaterials to induce bone formation. MSCs can be induced into undergoing osteogenesis in vitro by the use of pre-formulated soluble factors in the culture media, chemically defined surfaces, substrate matrix elasticity and the surface topography of the substrate. These approaches induce osteogenesis when presented in isolation or in combination. When these cues are presented in combination, surface patterning plays an important role and topography can have a stronger influence on cell behaviour when presented with effective surface chemistries. In vertebrate and invertebrate systems, the main requisites for forming hard tissue or biomineral structures are calcium phosphate and calcium carbonate respectively, both of which are assembled in a variety of ways generating an incredible amount of structural diversity. This juxtaposition of phosphate and carbonate is described as the “Bone-Shell Divide”. It is intriguing that mammalian cells respond to mineral on the shell side of the Bone-Shell Divide and this begs questions: which feature of nacre elicits this response and, in transcending the Bone-Shell Divide, do MSCs produce bone of similar or superior characteristics to that induced by other means? Addressing these questions has important implications in tissue engineering and biomaterial applications, especially with regards to orthopaedic applications where critical sized defects in trauma and reconstructive surgery demand large areas of intact bone usually acquired by creating a secondary injury site. By isolating the topographical features of nacre from its inherent chemistry, we show that the osteoinductive properties of nacre arise from the patterning of the surface presented to MSCs. Importantly, separating nacre topography from its inherent chemistry enhances the osteogenic response. In this report we dissect out the contribution of topography to nacre bioactivit

Biomimetic oyster shell–replicated topography alters the behaviour of human skeletal stem cells

The regenerative potential of skeletal stem cells provides an attractive prospect to generate bone tissue needed for musculoskeletal reparation. A central issue remains efficacious, controlled cell differentiation strategies to aid progression of cell therapies to the clinic. The nacre surface from Pinctada maxima shells is known to enhance bone formation. However, to date, there is a paucity of information on the role of the topography of P. maxima surfaces, nacre and prism. To investigate this, nacre and prism topographical features were replicated onto polycaprolactone and skeletal stem cell behaviour on the surfaces studied. Skeletal stem cells on nacre surfaces exhibited an increase in cell area, increase in expression of osteogenic markers ALP (p

Confined Sandwichlike Microenvironments Tune Myogenic Differentiation.

Sandwichlike (SW) cultures are engineered as a multilayer technology to simultaneously stimulate dorsal and ventral cell receptors, seeking to mimic cell adhesion in three-dimensional (3D) environments in a reductionist manner. The effect of this environment on cell differentiation was investigated for several cell types cultured in standard growth media, which promotes proliferation on two-dimensional (2D) surfaces and avoids any preferential differentiation. First, murine C2C12 myoblasts showed specific myogenic differentiation. Human mesenchymal stem cells (hMSCs) of adipose and bone marrow origin, which can differentiate toward a wider variety of lineages, showed again myodifferentiation. Overall, this study shows myogenic differentiation in normal growth media for several cell types under SW conditions, avoiding the use of growth factors and cytokines, i.e., solely by culturing cells within the SW environment. Mechanistically, it provides further insights into the balance between integrin adhesion to the dorsal substrate and the confinement imposed by the SW system

Nanotopographical induction of osteogenesis through adhesion, bone morphogenic protein cosignaling, and regulation of microRNAs

It is emerging that nanotopographical information can be used to induce osteogenesis from mesenchymal stromal cells from the bone marrow and it is hoped that this nanoscale bioactivity can be utilized to engineer next generation implants. However, the osteogenic mechanism of surfaces is currently poorly understood. In this report, we investigate mechanism and implicate bone morphogenic protein (BMP) in up-regulation of RUNX2 and show that RUNX2 and its regulatory miRNAs are BMP sensitive. Our data demonstrates that osteogenic nanotopography promotes co-localization of intergrins and BMP2 receptors in order to enhance osteogenic activity and that vitronectin is important in this interface. This provides insight that topographical regulation of adhesion can have effects on signaling cascades outside of cytoskeletal signaling and that adhesions can have roles in augmenting BMP signaling

Mehrstichproben-Rangtests bei einseitigen Alternativen zur statistischen Auswertung von Mutagenitaetsexperimenten

TIB: RN 6363 (1985,3) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Bioprinted schwann and mesenchymal stem cell co-cultures for enhanced spatial control of neurite outgrowth

Bioprinting nerve conduits supplemented with glial or stem cells is a promising approach to promote axonal regeneration in the injured nervous system. In this study, we examined the effects of different compositions of bioprinted fibrin hydrogels supplemented with Schwann cells and mesenchymal stem cells (MSCs) on cell viability, production of neurotrophic factors, and neurite outgrowth from adult sensory neurons. To reduce cell damage during bioprinting, we analyzed and optimized the shear stress magnitude and exposure time. The results demonstrated that fibrin hydrogel made from 9 mg/mL of fibrinogen and 50IE/mL of thrombin maintained the gel’s highest stability and cell viability. Gene transcription levels for neurotrophic factors were significantly higher in cultures containing Schwann cells. However, the amount of the secreted neurotrophic factors was similar in all co-cultures with the different ratios of Schwann cells and MSCs. By testing various co-culture combinations, we found that the number of Schwann cells can feasibly be reduced by half and still stimulate guided neurite outgrowth in a 3D-printed fibrin matrix. This study demonstrates that bioprinting can be used to develop nerve conduits with optimized cell compositions to guide axonal regeneration

Cell material interactions

Cells can interpret information from their extracellular environment by turning physical cues into biochemical signals (mechanotransduction). Such physical cues may be in the form of surface chemistry (inherent or grafted), topography (macro, micro, or nanoscale in size) and stiffness (tissues have varying hardness from soft fat or nerve tissue to hard bone tissue). Using cell adhesion sites, cells alter their adhesion and cytoskeleton in response to these extracellular cues. Adhesions and the cytoskeleton are intimately linked to biochemical effectors that can elicit control over cell migration, growth and even stem cell self-renewal and differentiation. This chapter will explore how we can use synthetic materials to explore the cell–material interface and how this has implications for tissue engineering next-generation scaffolds