Pressureless Mechanical Induction of Stem Cell Differentiation Is Dose and Frequency Dependent

<div><p>Movement is a key characteristic of higher organisms. During mammalian embryogenesis fetal movements have been found critical to normal tissue development. On the single cell level, however, our current understanding of stem cell differentiation concentrates on inducing factors through cytokine mediated biochemical signaling. In this study, human mesenchymal stem cells and chondrogenesis were investigated as representative examples. We show that pressureless, soft mechanical stimulation precipitated by the cyclic deformation of soft, magnetic hydrogel scaffolds with an external magnetic field, can induce chondrogenesis in mesenchymal stem cells without any additional chondrogenesis transcription factors (TGF-β1 and dexamethasone). A systematic study on the role of movement frequency revealed a classical dose-response relationship for human mesenchymal stem cells differentiation towards cartilage using mere mechanical stimulation. This effect could even be synergistically amplified when exogenous chondrogenic factors and movement were combined.</p> </div

Chondrogenesis on magnetic hydrogels with and without mechanical stimulation.

<p>Aggrecan (antibody labeling, red), SOX9 (antibody labeling, green) and Collagen II expression (green) immunohistochemistry of hMSC over a period of 5 weeks. Cell cultures were either not stimulated or underwent repeated mechanical stimulation (left). The role of movement was investigated both in control medium (top rows) and in standard chondrogenic medium (bottom rows). Samples were counterstained with DAPI to make cell nuclei visible. Mechanical stimulation resulted in clear up-regulation of all chondrogenic markers if compared to the non-stimulated control cultures.</p

Frequency dependent hMSC differentiation.

<p>Mechanical stimulation frequency influences the differentiation and formation of tissue-typical extracellular matrix (amount of GAG formed) in both control and chondrogenic medium. Cells were pressure-free stretched on soft scaffolds for 2 seconds (stimulation period). Non-moved scaffolds (left) served as additional controls. The amount of GAG deposition indicated differentiation on all mechanical stimulated scaffolds particularly at high frequency. This behavior shows that mechanical soft movement follows a dose-effect type response similar to a classical response of specific cells to a given biochemical factor.</p

Mechanical stimulation induced chondrogenesis.

<p>a) Cell numbers (DNA amount per scaffold) confirmed good cell expansion and growth. Below is the glycosaminoglycan (GAG) deposition per scaffold over a period of 5 weeks. Control medium (white bars) and chondrogenic medium (grey bars) were applied on cells seeded into either tissue culture plate (no scaffold), hydrogel scaffold (no nanomagnets, i.e. no movement is possible) or magnetic hydrogel. Mechanical stimulation (arrow) triggered higher GAG deposition. b) Comparable DNA amount indicated good cell growth for cells seeded into magnetic hydrogels with both medium types and no negative effects from mechanical stimulation. GAG deposition using diluted chondrogenic (grey) versus control medium (white bars). Mechanically stimulated hMSC in control medium showed comparable GAG deposition as in standard chondrogenic medium under magnetic actuation (indicated by ↕). * p < 0.01 cells cultured with control medium under mechanical stimulation versus non stimulated and mechanically stimulated hydrogel using both cell culture media.</p

Efficient Magnetic Recycling of Covalently Attached Enzymes on Carbon-Coated Metallic Nanomagnets

In the pursuit of robust and reusable biocatalysts for industrial synthetic chemistry, nanobiotechnology is currently taking a significant part. Recently, enzymes have been immobilized on different nanoscaffold supports. Carbon coated metallic nanoparticles were found to be a practically useful support for enzyme immobilization due to their large surface area, high magnetic saturation, and manipulatable surface chemistry. In this study carbon coated cobalt nanoparticles were chemically functionalized (diazonium chemistry), activated for bioconjugation (<i>N,N</i>-disuccinimidyl carbonate), and subsequently used in enzyme immobilization. Three enzymes, β-glucosidase, α-chymotrypsin, and lipase B were successfully covalently immobilized on the magnetic nonsupport. The enzyme–particle conjugates formed retained their activity and stability after immobilization and were efficiently recycled from milliliter to liter scales in short recycle times

Efficient Magnetic Recycling of Covalently Attached Enzymes on Carbon-Coated Metallic Nanomagnets

In the pursuit of robust and reusable biocatalysts for industrial synthetic chemistry, nanobiotechnology is currently taking a significant part. Recently, enzymes have been immobilized on different nanoscaffold supports. Carbon coated metallic nanoparticles were found to be a practically useful support for enzyme immobilization due to their large surface area, high magnetic saturation, and manipulatable surface chemistry. In this study carbon coated cobalt nanoparticles were chemically functionalized (diazonium chemistry), activated for bioconjugation (<i>N,N</i>-disuccinimidyl carbonate), and subsequently used in enzyme immobilization. Three enzymes, β-glucosidase, α-chymotrypsin, and lipase B were successfully covalently immobilized on the magnetic nonsupport. The enzyme–particle conjugates formed retained their activity and stability after immobilization and were efficiently recycled from milliliter to liter scales in short recycle times