Printing with Collagen
Addition of Collagen to hydrogels in 3D printing improves stem cell differentiation in osteogenesis
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http://www.chemistryviews.org/details/news/9268521/Printing_with_Collagen.html
- Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering,
Daniela Filipa Duarte Campos, Andreas Blaeser, Kate Buellesbach, Kshama Shree Sen, Weiwei Xun, Walter Tillmann, Horst Fischer,
Adv. Healthcare Mater. 2016.
DOI: 10.1002/adhm.201501033
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Collagen
Tropocollagen molecule: three left-handed procollagens (red, green, blue) join to form a right handed triple helical tropocollagen.
Collagen is the most common protein found in mammals.
Collagen /ˈkɒlədʒᵻn/ is the main structural protein in the extracellular space in the various connective tissues in animal bodies. As the main component of connective tissue, it is the most abundant protein in mammals,[1] making up from 25% to 35% of the whole-body protein content. Depending upon the degree of mineralization, collagen tissues may be rigid (bone), compliant (tendon), or have a gradient from rigid to compliant (cartilage).[2] Collagen, in the form of elongated fibrils, is mostly found in fibrous tissues such as tendons, ligaments and skin. It is also abundant incorneas, cartilage, bones, blood vessels, the gut, intervertebral discs and the dentin in teeth.[3] In muscle tissue, it serves as a major component of the endomysium. Collagen constitutes one to two percent of muscle tissue, and accounts for 6% of the weight of strong, tendinous muscles.[4] Thefibroblast is the most common cell that creates collagen.
Gelatin, which is used in food and industry, is collagen that has been irreversibly hydrolyzed.[5] Collagen also has many medical uses in treating complications of the bones and skin.
The name collagen comes from the Greek κόλλα (kólla), meaning "glue", and suffix -γέν, -gen, denoting "producing".[6][7] This refers to the compound's early use in the process of boiling the skin and sinews of horses and other animals to obtain glue.
Collagen injections can be used in cosmetic procedures to improve the contours of aging skin.
Types of collagen
Collagen occurs in many places throughout the body. Over 90% of the collagen in the human body, however, is type I.[8]
So far, 28 types of collagen have been identified and described. They can be divided into several groups according to the structure they form:[2]
- Fibrillar (Type I, II, III, V, XI)
- Non-fibrillar
- FACIT (Fibril Associated Collagens with Interrupted Triple Helices) (Type IX, XII, XIV, XVI, XIX)
- Short chain (Type VIII, X)
- Basement membrane (Type IV)
- Multiplexin (Multiple Triple Helix domains with Interruptions) (Type XV, XVIII)
- MACIT (Membrane Associated Collagens with Interrupted Triple Helices) (Type XIII, XVII)
- Other (Type VI, VII)
The five most common types are:
- Type I: skin, tendon, vascular ligature, organs, bone (main component of the organic part of bone)
- Type II: cartilage (main collagenous component of cartilage)
- Type III: reticulate (main component of reticular fibers), commonly found alongside type I.
- Type IV: forms basal lamina, the epithelium-secreted layer of the basement membrane.
- Type V: cell surfaces, hair and placenta
Tobacco contains chemicals that damage collagen
Medical uses
Cardiac applications
The collagenous cardiac skeleton which includes the four heart valve rings, is histologically and uniquely bound to cardiac muscle. The cardiac skeleton also includes the separating septa of the heart chambers – the interventricular septum and the atrioventricular septum. Collagen contribution to the measure of cardiac performance summarily represents a continuous torsional force opposed to the fluid mechanics of blood pressure emitted from the heart. The collagenous structure that divides the upper chambers of the heart from the lower chambers is an impermeable membrane that excludes both blood and electrical impulses through typical physiological means. With support from collagen, atrial fibrillation should never deteriorate to ventricular fibrillation. Collagen is layered in variable densities with cardiac muscle mass. The mass, distribution, age and density of collagen all contribute to the compliance required to move blood back and forth. Individual cardiac valvular leaflets are folded into shape by specialized collagen under variable pressure. Gradual calcium deposition within collagen occurs as a natural function of aging. Calcified points within collagen matrices show contrast in a moving display of blood and muscle, enabling methods of cardiac imaging technology to arrive at ratios essentially stating blood in (cardiac input) and blood out (cardiac output). Pathology of the collagen underpinning of the heart is understood within the category of connective tissue disease.
Hydrolyzed type II collagen and osteoarthritis
A published study[9] reports that ingestion of a novel low molecular weight hydrolyzed chicken sternal cartilage extract, containing a matrix of hydrolyzed type II collagen,chondroitin sulfate, and hyaluronic acid, relieves joint discomfort associated with osteoarthritis. A randomized controlled trial (RCT) enrolling 80 subjects demonstrated that it was well tolerated with no serious adverse event and led to a significant improvement in joint mobility compared to the placebo group on days 35 (p = 0.007) and 70 (p < 0.001).
Fast facts on collagen
Here are some key points about collagen. More detail and supporting information is in the main article.25-27
- Protein makes up around 20% of the body's mass, and collagen makes up around 30% of the protein in the human body.
- There are at least 16 types of collagen, but 80-90% of the collagen in the body consists of types I, II, and III.
- Type I collagen fibrils are stronger than steel (gram for gram).
- Collagen is most commonly found within the body in the skin, bones and connective tissues.
- The word "collagen" is derived from the Greek "kolla," meaning glue.
- Collagen gives the skin its strength and structure, and also plays a role in the replacement of dead skin cells.
- Collagen production declines with age (as part of intrinsic aging), and is reduced by exposure to ultraviolet light and other environmental factors (extrinsic aging).
- Collagen in medical products can be derived from human, bovine, porcine and ovine sources.
- Collagen dressings attract new skin cells to wound sites.
- Cosmetic products such as revitalizing lotions that claim to increase collagen levels are unlikely to do so, as collagen molecules are too large to be absorbed through the skin.
- Collagen production can be stimulated through the use of laser therapy and the use of all-trans retinoic acid (a form ofvitamin A).
- Controllable factors that damage the production of collagen include sunlight, smoking and high sugar consumption.
Cosmetic surgery
Collagen has been widely used in cosmetic surgery, as a healing aid for burn patients for reconstruction of bone and a wide variety of dental, orthopedic, and surgical purposes. Both human and bovine collagen is widely used as dermal fillers for treatment of wrinkles and skin aging.[10] Some points of interest are:
- When used cosmetically, there is a chance of allergic reactions causing prolonged redness; however, this can be virtually eliminated by simple and inconspicuous patch testing prior to cosmetic use.
- Most medical collagen is derived from young beef cattle (bovine) from certified BSE-free animals. Most manufacturers use donor animals from either "closed herds", or from countries which have never had a reported case of BSE such as Australia, Brazil, and New Zealand.
Bone grafts
As the skeleton forms the structure of the body, it is vital that it maintains its strength, even after breaks and injuries. Collagen is used in bone grafting as it has a triple helical structure, making it a very strong molecule. It is ideal for use in bones, as it does not compromise the structural integrity of the skeleton. The triple helical structure of collagen prevents it from being broken down by enzymes, it enables adhesiveness of cells and it is important for the proper assembly of the extracellular matrix.[11]
Tissue regeneration
Collagen scaffolds are used in tissue regeneration, whether in sponges, thin sheets, or gels. Collagen has the correct properties for tissue regeneration such as pore structure, permeability, hydrophilicity and it is stable in vivo. Collagen scaffolds are also ideal for the deposition of cells, such as osteoblasts and fibroblasts and once inserted, growth is able to continue as normal in the tissue.[12]
Reconstructive surgical uses
Collagens are widely employed in the construction of the artificial skin substitutes used in the management of severe burns. These collagens may be derived from bovine, equine, porcine, or even human sources; and are sometimes used in combination with silicones, glycosaminoglycans, fibroblasts, growth factors and other substances.
Collagen is also sold commercially in pill form as a supplement to aid joint mobility. However, because proteins are broken down into amino acids before absorption, there is no reason for orally ingested collagen to affect connective tissue in the body, except through the effect of individual amino acid supplementation.
Collagen is also frequently used in scientific research applications for cell culture, studying cell behavior and cellular interactions with the extracellular environment.[13]
Wound care
Collagen is one of the body’s key natural resources and a component of skin tissue that can benefit all stages of the wound healing process.[14] When collagen is made available to the wound bed, closure can occur. Wound deterioration, followed sometimes by procedures such as amputation, can thus be avoided.
Collagen is a natural product, therefore it is used as a natural wound dressing and has properties that artificial wound dressings do not have. It is resistant against bacteria, which is of vital importance in a wound dressing. It helps to keep the wound sterile, because of its natural ability to fight infection. When collagen is used as a burn dressing, healthygranulation tissue is able to form very quickly over the burn, helping it to heal rapidly.[15]
Throughout the 4 phases of wound healing, collagen performs the following functions in wound healing:
- Guiding function: Collagen fibers serve to guide fibroblasts. Fibroblasts migrate along a connective tissue matrix.
- Chemotactic properties: The large surface area available on collagen fibers can attract fibrogenic cells which help in healing.
- Nucleation: Collagen, in the presence of certain neutral salt molecules can act as a nucleating agent causing formation of fibrillar structures. A collagen wound dressing might serve as a guide for orienting new collagen deposition and capillary growth.
- Hemostatic properties: Blood platelets interact with the collagen to make a hemostatic plug.
Chemistry
The collagen protein is composed of a triple helix, which generally consists of two identical chains (α1) and an additional chain that differs slightly in its chemical composition (α2).[16] The amino acid composition of collagen is atypical for proteins, particularly with respect to its high hydroxyproline content. The most common motifs in the amino acid sequence of collagen are glycine-proline-X and glycine-X-hydroxyproline, where X is any amino acid other than glycine, proline or hydroxyproline. The average amino acid composition for fish and mammal skin is given.[16]
| Amino acid | Abundance in mammal skin (residues/1000) | Abundance in fish skin (residues/1000) |
|---|---|---|
| Glycine | 329 | 339 |
| Proline | 126 | 108 |
| Alanine | 109 | 114 |
| Hydroxyproline | 95 | 67 |
| Glutamic acid | 74 | 76 |
| Arginine | 49 | 52 |
| Aspartic acid | 47 | 47 |
| Serine | 36 | 46 |
| Lysine | 29 | 26 |
| Leucine | 24 | 23 |
| Valine | 22 | 21 |
| Threonine | 19 | 26 |
| Phenylalanine | 13 | 14 |
| Isoleucine | 11 | 11 |
| Hydroxylysine | 6 | 8 |
| Methionine | 6 | 13 |
| Histidine | 5 | 7 |
| Tyrosine | 3 | 3 |
| Cysteine | 1 | 1 |
| Tryptophan | 0 | 0 |
Synthesis
First, a three-dimensional stranded structure is assembled, with the amino acids glycine and proline as its principal components. This is not yet collagen but its precursor, procollagen. Procollagen is then modified by the addition of hydroxyl groups to the amino acids proline and lysine. This step is important for later glycosylation and the formation of the triple helix structure of collagen. The hydroxylase enzymes that perform these reactions require Vitamin C as a cofactor, and a deficiency in this vitamin results in impaired collagen synthesis and the resulting disease scurvy[17] These hydroxylation reactions are catalyzed by two different enzymes: prolyl-4-hydroxylase[18] and lysyl-hydroxylase. Vitamin C also serves with them in inducing these reactions. In this service, one molecule of vitamin C is destroyed for each H replaced by OH. [19] The synthesis of collagen occurs inside and outside of the cell. The formation of collagen which results in fibrillary collagen (most common form) is discussed here. Meshwork collagen, which is often involved in the formation of filtration systems, is the other form of collagen. All types of collagens are triple helices, and the differences lie in the make-up of the alpha peptides created in step 2.
- Transcription of mRNA: About 34 genes are associated with collagen formation, each coding for a specific mRNA sequence, and typically have the "COL" prefix. The beginning of collagen synthesis begins with turning on genes which are associated with the formation of a particular alpha peptide (typically alpha 1, 2 or 3).
- Pre-pro-peptide formation: Once the final mRNA exits from the cell nucleus and enters into the cytoplasm, it links with the ribosomal subunits and the process of translation occurs. The early/first part of the new peptide is known as the signal sequence. The signal sequence on the N-terminal of the peptide is recognized by a signal recognition particle on the endoplasmic reticulum, which will be responsible for directing the pre-pro-peptide into the endoplasmic reticulum. Therefore, once the synthesis of new peptide is finished, it goes directly into the endoplasmic reticulum for post-translational processing. It is now known as pre-pro-collagen.
- Pre-pro-peptide to pro-collagen: Three modifications of the pre-pro-peptide occur leading to the formation of the alpha peptide:
- The signal peptide on the N-terminal is dissolved, and the molecule is now known as propeptide (not procollagen).
- Hydroxylation of lysines and prolines on propeptide by the enzymes 'prolyl hydroxylase' and 'lysyl hydroxylase' (to produce hydroxyproline and hydroxylysine) occurs to aid cross-linking of the alpha peptides. This enzymatic step requires vitamin C as a cofactor. In scurvy, the lack of hydroxylation of prolines and lysines causes a looser triple helix (which is formed by three alpha peptides).
- Glycosylation occurs by adding either glucose or galactose monomers onto the hydroxyl groups that were placed onto lysines, but not on prolines.
- Once these modifications have taken place, three of the hydroxylated and glycosylated propeptides twist into a triple helix forming procollagen. Procollagen still has unwound ends, which will be later trimmed. At this point, the procollagen is packaged into a transfer vesicle destined for the Golgi apparatus.
- Golgi apparatus modification: In the Golgi apparatus, the procollagen goes through one last post-translational modification before being secreted out of the cell. In this step, oligosaccharides (not monosaccharides as in step 3) are added, and then the procollagen is packaged into a secretory vesicle destined for the extracellular space.
- Formation of tropocollagen: Once outside the cell, membrane bound enzymes known as 'collagen peptidases', remove the "loose ends" of the procollagen molecule. What is left is known as tropocollagen. Defects in this step produce one of the many collagenopathies known as Ehlers-Danlos syndrome. This step is absent when synthesizing type III, a type of fibrilar collagen.
- Formation of the collagen fibril: 'Lysyl oxidase', an extracellular enzyme, produces the final step in the collagen synthesis pathway. This enzyme acts on lysines and hydroxylysines producing aldehyde groups, which will eventually undergo covalent bonding between tropocollagen molecules. This polymer of tropocollogen is known as a collagen fibril.
Amino acids
Collagen has an unusual amino acid composition and sequence:
- Glycine is found at almost every third residue.
- Proline makes up about 17% of collagen.
- Collagen contains two uncommon derivative amino acids not directly inserted during translation. These amino acids are found at specific locations relative to glycine and are modified post-translationally by different enzymes, both of which require vitamin C as acofactor.
- Hydroxyproline derived from proline
- Hydroxylysine derived from lysine - depending on the type of collagen, varying numbers of hydroxylysines are glycosylated (mostly having disaccharides attached).
Cortisol stimulates degradation of (skin) collagen into amino acids.[20]
Collagen I formation
Most collagen forms in a similar manner, but the following process is typical for type I:
- Inside the cell
- Two types of alpha chains are formed during translation on ribosomes along the rough endoplasmic reticulum (RER): alpha-1 and alpha-2 chains. These peptide chains (known as preprocollagen) have registration peptides on each end and a signal peptide.
- Polypeptide chains are released into the lumen of the RER.
- Signal pe
