Lecture 9 Polypeptides- Collagen, Keratin, ECM and Silk BME 515 Natural Polymers

What are polypeptides and how are they formed?

Polypeptides - Chains of amino acids linked by peptide bonds.

Forms when a carboxyl group of one amino acid reacts with the amino group of another, releasing H2O.

What is alginate and how does it form a hydrogel?

Alginate is a linear copolymer of β-D-mannuronate (M) and α-L-guluronate (G) in GGGG, MMMM, or GMGM blocks.

It forms a gel only when the G-blocks crosslink with divalent cations (e.g., Ca2+)

What determines the properties of an alginate hydrogel?

1. M/G ratio,

2. block sequence,

3. G-block length, (More and longer G-blocks means more crosslinking with Ca2+, so the gel is affected directly by G-block content.)

4. molecular weight.

What is hyaluronic acid (HA) and where is it found?

HA is - a long, unbranched glycosaminoglycan of N-acetylglucosamine and glucuronic acid. It plays a role in cell movement and proliferation.

It is found naturally in the ECM of skin, cartilage, synovial fluid, vitreous humor, and umbilical cord.

Why is HA useful for joints, eyes, and skin?

Its high amount of negative charges makes it hydrophilic with high viscosity, lubrication, and water retention. That is why it works as a as a moisturizer.

:PPPP How is HA degraded and why does that matter clinically?

By hyaluronidase. It lowers HA viscosity and increases tissue permeability, so it is given with other drugs to speed their dispersion. Some bacteria (Staph aureus) use it to spread through tissue. HA breakdown products are proangiogenic.

(dont think she ask) How does HA molecular weight determine its use?

10-20 kDa: acts like toll-like receptors. 50 kDa: optimal moisturizing. 300 kDa: anti-atrophic and anti-aging. 500 kDa: immunomodulatory. 1500-1700 kDa: adjuvant therapy in rheumatoid arthritis. Bioavailability also depends on MW.

What other applications does HA have?

• Ophthalmic (eye) surgery (with local anesthetics),

• osteoarthritis joint injections,

• reducing nerve sensitivity and pain.

What is the extracellular matrix (ECM)?

A non-cellular, three-dimensional network of proteins and carbohydrates found in most tissues and directly connected to the cells it surrounds. Its major component is collagen.

What are the three main functions of the ECM?

1) Support scaffold for cells;

2) controls communication between cells; (Blood clotting is an example)

3) regulates cell growth, migration, and differentiation.

What are the main components of the ECM?

Glycosaminoglycans, collagen and other glycoproteins, proteoglycans.

What are the general properties of the ECM?

• Gives structural and biochemical support (scaffolding);

  • rigid, dense, porous, insoluble;

• a reservoir of growth factors;

  • biodegradable and biocompatible;

• controls cell proliferation, adhesion, migration,

What is decellularization (dECM) and why is it used? (*unsure like actually abt)

Isolating the ECM from its native cells and genetic material (membranes, nucleic acids, mitochondria) while keeping its functional components.

It gives a natural scaffold that mimics the structure and composition of native ECM and can come from many species.

What are the steps of making a dECM scaffold and what does each do?

Decellularization (detergents, enzymes, osmotic solutions, or perfusion) removes cells; crosslinking improves mechanical properties; sterilization (ethylene oxide, gamma ray, e-beam) makes it safe; preservation (deep-freezing/cryopreservation) allows storage.

Why is crosslinking used on dECM?

It is the key step to improve mechanical properties (strength, hardness) after decellularization and it increases resistance to degradation.

Why is glutaraldehyde a problem as a crosslinker?

It reacts with amino groups and increases strength and anti-degradation, but it is toxic and easily causes tissue calcification. Epoxy compounds and HMDC also have toxicity.

What are the advantages and drawbacks of the natural crosslinkers?

Genipin: stable, robust anti-enzymatic ability, low toxicity, but expensive and leaves a dark blue color. Proanthocyanidins: non-toxic, anti-calcification, anti-inflammatory, easily acquired, but leave a red-brown color. Tannic acid: low cost, antioxidant, but carcinogenic effects.

What are the clinical uses of dECM materials?

Soft tissue and tendon repair, chronic wounds and burns, breast reconstruction, dura mater repair,

What are other applications of dECM?

Therapeutic drug delivery, tissue engineering (e.g., cartilage: decellularize, recellularize, implant), 3D cell culture, and modeling in vivo systems to study signaling pathways.

How is the ECM degraded and how can its properties be altered?

Degraded by proteases, mainly matrix metalloproteinases (MMPs). Properties are altered by synthesizing or degrading ECM components, varying the degree of crosslinking, solubilizing from decellularized samples, and using detergents (e.g., deoxycholic acid) to extract ECM.

What are the limitations of dECM?

Tumor-derived dECM showed cancer progression while healthy dECM inhibited cancer growth; current decellularization methods damage the ECM; hard to retain active components through processing, sterilization, and preservation; cell-ECM interactions are not fully understood.

What is collagen and why is it important?

The most abundant animal protein (about 30% of body protein) and the main structural protein of connective tissue: ligaments, tendons, cartilage, skin, bone, and blood vessels. In skin it provides support and strength, and declining production with age causes aging effects.

What is the structure of collagen?

A triple helix of three α chains (tropocollagen) rich in glycine, proline, and hydroxyproline (Gly-Pro-X or Gly-Hyp-X), stabilized by hydrogen bonding and cross-linked to neighbors. Tropocollagen assembles into fibrils (about 67 nm banding) and then fibers.

What are the main collagen types?

Type I: skin, tendon, bone, ligaments. Type II: cartilage and vitreous humor. Type III: skin, muscle, blood vessels. Type IV: sheet-forming 2D network in basal laminae.

What are the general properties of collagen?

Soluble in acidic aqueous solutions; type I fibrils have enormous tensile strength and can be stretched without breaking; forms strong fibers through self-aggregation and cross-linking.

What are the biomedical applications of collagen?

Drug delivery, sponges for burns and wounds, hemostatic sealant , wound dressings, and tissue engineering scaffolds.

How does collagen degrade and how can the rate be controlled?

Biodegradable and bioresorbable.

Cross-linking regulates the rate

What are the limitations of collagen?

Supply is hard to ensure;

sutures can increase infection and inflammation;

ineffective at infected sites;

hydrophilicity causes swelling and faster release;

very expensive.

What is keratin and what is its structure?

A fibrous structural protein in hair, feathers, wool, and horns, with α-helix (coiled) and β-pleated sheet secondary structures. It contains C, O, N, H, and S (sulfur, from disulfide bonds).

What are the general properties of keratin?

Biodegradable, biocompatible, insoluble in water, high stability, high toughness and modulus; absorbs heavy metal ions and formaldehyde. It protects epithelial cells and strengthens organs, and plays a role in wound healing.

What are the applications of keratin and what property explains each?

Water purification and air cleansing (absorbs heavy metals and formaldehyde); wound healing, bone regeneration, hemostasis, and nerve repair (biocompatible, biodegradable, role in wound healing).

How is keratin degraded and altered?

Degrades enzymatically and by oxidation (breaks disulfide bonds, proteolysis). It can be extracted by oxidation/reduction, purified into dressings and creams, rehydrated easily, and cross-linked.

What are the limitations of keratin?

Low molecular weight, poor mechanical properties for some applications, and regenerated keratin is very fragile.

What is silk and what are its two components?

Silkworm silk has silk fibroin (SF), the core that gives toughness and load-bearing, and silk sericin (SS), a water-soluble glycoprotein gumming agent (25-30% of the cocoon). SF comes from Bombyx mori and spiders such as Nephila clavipes.

What is the structure of silk fibroin?

A peptide copolymer: hydrophobic blocks of short amino acids that form H-bonded crystals, and hydrophilic blocks with charged amino acids. B. mori SF has a heavy chain (390 kDa), light chain (26 kDa), and P25 (about 25 kDa) in a 6:6:1 ratio; P25 maintains complex integrity.

Why is silk so strong?

The hydrophobic heavy chain domains (Gly-Ala-Gly-Ala-Gly-Ser repeats) form antiparallel β-sheet crystallites. High glycine and alanine content lets the sheets pack tightly, giving rigidity and tensile strength.

What are the general properties of silk?

Biocompatible, durable, lightweight (1.3 g/cm3), very high strength (up to 4.8 GPa), high toughness and elasticity (4-7x Kevlar, up to 35%), and thermally stable up to 250 °C.

How can silk be processed?

Gas forming, salt leaching, electrospinning, electrospraying, 3D printing, nano/microspheres, and casting films, giving scaffolds, sponges, films, fibers, hydrogels, and particles.

What are the applications of silk?

Tissue scaffolds (electrospun mats resembling ECM topography, nerve regeneration), endothelialization and skin/bone repair (fibronectin-coated mats), wound healing and skin regeneration, bone and cartilage engineering, drug/gene delivery, and use in food, cosmetics, and sutures.