1C Mechanobiology Part 1

Tissue Engineering

• Mechanobiology at Johns Hopkins (Gilman Hall, Whiting School of Engineering).

Flat Biology: Cell Polarization

• Petri Dish

  • Invented around 1879 by Julius Richard Petri.

  • Exploration of how cells adhere to tissue culture plastic and what cell adhesion molecules bind.

Mechanotransduction

• Cells respond to mechanical stimulation with adaptive changes in function.

  • Short-term responses include:

    • Changes in intracellular tension.

    • Variations in adhesion, spreading, or migration.

  • Long-term effects:

    • Altered protein synthesis and secretion.

    • Changes in structural organization, proliferation, and viability.

• Responses are mediated through overlapping signaling pathways (Jaalouk & Lammerding, Nat. Rev., 2009).

Do Mechanics Drive Cell Differentiation?

• Matrix Elasticity directs stem cell lineage specification.

• Authors: Adam J. Engler, Shamik Sen, H. Lee Sweeney, Dennis E. Discher.

  • Published in Cell, 2006.

• Contact: discher@seas.upenn.edu

Mesenchymal Stem Cells (MSCs)

Proliferation and Lineage Specification

• MSC Self-Renewal and Proliferation

  • Differentiation into various lineages:

    • Osteogenesis (bone).

    • Chondrogenesis (cartilage).

    • Myogenesis (muscle).

    • Other lineages include adipogenesis, ligamentogenesis.

• Commitment through various transitionary states (e.g., pre-adipocyte, fibroblast).

• Schematic illustrates progression through unique micro-niches in development.

Bone Marrow-Derived MSCs

• Capable of differentiating into classic mesenchymal lineages (bone, cartilage, fat) and other germ layers (neurons, endothelial cells).

  • Different biochemical microenvironments of bone, fat, muscle, and brain influence lineage commitment.

• Lineage-specific commitment influenced by distinct biological and biochemical factors.

Influence of Substrate Rigidity on Differentiation

Rigidities in Tissues

• Different tissues feature varying elastic moduli.

• Hypothesis: Substrate rigidity can change lineage specification of multipotent MSCs.

• Elastic Materials

  • Normal stress as a function of normal strain.

Substrate Rigidity and Cell Culture

• Polyacrylamide (PA) gels provide compliant surfaces for cell culture.

  • Cross-linking agents create gels with adjustable stiffness.

  • Adhesive molecules facilitate cell attachment.

Questions and Answers on Substrate Properties

• Questions regarding modulus of polystyrene dishes/glass coverslips and physiological stiffness ranges for soft tissues, cartilage, and bone.

Mimicking Tissue-Specific Stiffnesses

• Maintain biochemical medium consistency.

• Prior to bone mineralization, collagen-rich matrix ('osteoid') is deposited.

Effects on Cell Shape and Adaptation

• Substrate stiffness causes changes in cell shape.

  • More organized cytoskeletal structures on stiffer substrates.

Lineage Commitment Influenced by Stiffness

• Staining for specific proteins to assess lineage commitment:

  • β3-tubulin (neuronal), MyoD (myogenic), CBFα-1 (osteogenic).

Stochasticity in Marker Expression

• Not all cells express differentiation markers (P-NFH, MyoD, CBFα-1).

Matrix Elasticity in Lineage Specification

• Absence of soluble cues can direct MSC differentiation with various modulus ranges (0.1 to 40 kPa) towards neural, myogenic, and osteogenic lineages.

Mechanotransduction and Signal Transduction

General Mechanisms

• Cell fate decisions are transcription-based; biophysical cues transmitted to the nucleus.

• Family of Rho-GTPases regulates the actin cytoskeleton.

• RhoA signaling promotes stress fiber development.

Role of Mechanosensing in Gene Responses

• Mechanotransduction pathways can be blocked to assess significance in responses, involving various inhibitors.

Focal Adhesions (FAs)

• Nascent FAs transmit external forces, growing in size due to mechanical resistance.

• Mature FAs act as traction points for cell spreading and migrate; they recruit signaling molecules to relay cues.

Cytoskeletal Forces and Nucleus Interaction

• External forces propagate to the nuclear surface, affecting gene expression through mechanical deformation.

Structure of the Nuclear Envelope

• Comprised of an outer layer continuous with rough ER and inner membranes providing structural support via intermediate filaments.

LINC Complexes

• The only known structure for transmitting cytoskeletal stresses to the nuclear surface, enabling linkage between the nucleus and cytoskeleton.

Summary of Cytoskeletal Forces

• Integrins link ECM to the cytoskeleton allowing force transmission which can affect nuclear conformations and gene expression.

Effects of Substrate Stiffness on Cell Shape

• Observing differences in cell shape due to changes in substrate stiffness over time (96 hrs to 4 hrs).

Changes in Actin Assembly

• Substrate stiffness alters actin assembly, shifting from diffuse to more organized structures on stiffer surfaces.

Changes in Focal Adhesion Size

• Compliance of substrate influences focal adhesion size and integrin aggregation in response to mechanical forces.