Quiz 8 Cell & Tissue Eng.

Lectures 17 & 18

Microtechnologies in TE

• Used to engineer cellular function & control the behavior of cells in order to assemble functional tissue.

Cons of Microtechnologies

• Perfect control of cell microenvironments

• Emerging tools for tissue engineering enabling control of cell spatial arrangements.

Types of Microtechnologies (Direct)

• Controlled scaffolds

• Improved bioreactors

Types of Microtechnologies (Indirect)

• Controlled in vitro microenvironments to understand biological mechanism

• Perform high-throughput assays

• Examples:

  • Microfluidics

  • Patterned co-culture

  • Surface Patterning

  • Mechanical / shear forces

Micropatterning cells

• Control of cell spatial organization is enabled by the following parameters OR by using gradient of adhesion molecules (RGD).

  • Mechanical:

    • Shape, roughness of the surface

  • Soluble/linked:

    • GFs

  • Adhesive ligands:

    • Biomolecules for attachment, ECM or polymeric ECM mimics

Micropatterning techniques

• Soft lithography

• Self-assembled monolayers

• Microcontact printing

• Microfluidic patterning

Soft lithography

• Molding a polymer using photoresist master mold

• Fast, cheap methods

• Device features only limited by the complexity of the mold

  • Down to 10 nm feature resolution

Self-assemebled mono layers (SAM)

• SAMs are organized organic monolayer films formed on a gold surface

• Example:

  • Alkanethiol (Thiolate-gold bond)

  • Can be used to graft other biomolecules using various chemistry

Microcontact Printing

• A technique that uses topographic patterns on the surface of an elastomeric PDMS stamp.

  • Stamp is inked with a solution containing the patterning component.

  • Transfer of the patterned component to the substrate.

  • Used for patterning proteins such as fibronectin, streptavidin.

Microfluidic patterning

• Allows patterning surfaces by restricting the flow of fluids to desired regions of a substrate.

• Components such as ligands, proteins, and cells are deoposited from the solution to create a pattern on the subtrate

Host Immune System

• Plays an important role for scaffold implant integration and tissue regeneration.

• Controlling the Immune System to induce Tissue Regeneration

Innate Immunity

• Early, limited specificity. Considered as the first line of defense.

• Examples:

  • Skin, epitheliums, tears

  • Cell types such as; Phagocytes, neutrophils and macrophages.

Adaptive Immunity

• Later response, variable response depending on the trigger

• Highly specific and can target 1 particular antigens on 1 given pathogen.

• Memory of infections

• Cell types such as; B cell, plasma cells, T cells

Sequence of host reaction after injury

• Injury

• Acute inflammation

• Chronic inflammation

• Scaring or Fibrosis

Injury

• Caused by tissue damage, surgery, implantation

Acute Inflammation

• Neutrophils ( polymorphonuclear leukocyte)

Chronic inflammation

• Monocytes and Lymphocytes

Scaring or Fibrosis

• Fibrous capsule (recruitment of fibroblast)

Inflammation Response

• Complex biological response of vascular tissues to harmful stimuli.

• To contain, neutralize injuries or processes.

• Events that lead to healing and reconstruction of injured tissues around implantation site.

Acute Inflammation

• Immediate response to tissue injury

• Occur in the first minutes to days after injury

4 Signs of Acute Inflammation

1. Redness

2. Swelling

3. Local heat

4. Pain

Acute Inflammation Steps

1. Phagocytic neutrophils from blood stream infiltrate the wound.

2. Neutrophils engulf debris and foreign organisms

3. Signals released by neutrophils and the recruitment of monocytes at the injury, 5-6 hours after process had started.

4. Neutrophils and monocytes start to enlarge to form tissue macrophages.

5. Macrophages have similar functions as neutrophil but have more sustainable killing capacities.

6. Macrophages and trapped platelets secrete cytokines promoting angiogenesis, chemotaxis and mitosis for surrounding fibroblast, and recruiting Lymphocytes.

7. Lymphocytes infiltrate the wound to reduce the risk of infections.

Resolution ~ Acute Inflammation

• Requires:

  • Cells which can repair (skin vs brain)

  • Minimal exudate

  • Mild tissue damage

Chronic Inflammation

• Pathological condition

• Characterized by active inflammation and tissue destruction

• Not characterized by the classic signs observed in acute inflammation. Instead:

  • Infiltration of mononuclear immune cells

  • Tissue destruction

  • Proliferation of blood vessels and connective tissue (fibrosis)

Tissue implant leading to immune system activation

• Synthetic biomaterials are not bearing immunologically recognizable biological motifs and are not attacked by the immune system as opposite to transplanted donor tissue.

Biological responses by the body that reduce efficiency of implanted biomaterials

• Inflammation

• Wound Healing

• Foreign body reaction

Controlling biomaterials integration

• Can be degraded by M1 macrophages

• Can be encapsulated in fibrosis capsule

• Biomaterials trigger inflammation at the site of implantation.

• Needs balance between pro- and anti- inflammatory response for integration

M1/M2 balance in bone repair mechanism (Young)

• M1 differentiate in response to INF-y from Th cell 1.

• M2 differentiate in response to IL-4 from Th cell 2

• Chronic inflammation stimulates production of pro-inflammatory cytokines in M1, followed by production of anti-inflammatory cytokines in M2.

  • Net tissue adaptation

M1/M2 balance in bone repair mechanism (Old)

• M1 pro-inflammatory cytokines are inherently increased due to reversal of Th1/Th2 ratio.

• Tissue injury progressively increases M1 and decreases M2 responses, with, a net loss of the adaptive response, resulting in bone resorption.

Include Research Example takeaways

Yes