Material Processing -
Collagen, Elastin, & Their Monomers
Read on to hear more about the synthesis of collagen & elastin in the body, as well as the formation of their monomers.
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Note: we will not be discussing processing of collagen & elastin to form tissues in tissue engineering; refer back to our page on Synthetic Tissue Engineering to read more about how tissue is made. We will, however, address scaffolds and addition of additives, in brief.
Collagen
Unlike many polymers, collagen is best gotten from nature - either directly from animals or in the form of genetically engineered “recombinant” human sources. Simply put, the process of making the proteins is very complicated and requires proteins & enzymes to be done successfully. Therefore, an explanation of its synthesis in the body is required, as well as an explanation of how that collagen is then extracted and purified for use in tissue engineering.
A little bit of information on the three most common monomers: glycine, proline, and hydroxyproline.
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Glycine: “is the simplest (and only achiral) proteinogenic amino acid,” an important amino acid and precursor for many macromolecules (PubChem, n.d.). Glycine is an inhibitory neurotransmitter in the central nervous system, a sweetener, and an important molecule in the synthesis of proteins, peptides, and purines… in fact, it has been hypothesized to be “the amino acid that may have kick-started life on Earth” (Cotton, 2010). Glycine typically appears as a white crystalline powder, soluble in water and slightly more dense (PubChem, “Proline,” n.d.). Glycine is typically synthesized one of four ways: 1) in the body, the enzyme serine hydroxymethyltransferase catalyzes the combination of serine and tetrahydrofolate to form glycine and N5,N10-methylenetetrahydrofolate; 2) in the laboratory, chloroethanoic acid can be combined with ammonia to form glycine and ammonium chloride; 3) do the classic Strecker synthesis; 4) the Miller-Urey method, an experiment that supposedly explains how life was made from water, methane, ammonia, and hydrogen (Cotton, 2010).
Proline: is one of the twenty amino acids in proteins - but actually, technically an imino acid. It is a cyclic aliphatic acid that is synthesized from glutamic acid. First, glutamic acid is converted to a semialdehyde through loss of one of its oxygens. Then, the imine on the alpha carbon of the glutamate semialdehyde forms a Schiff base, and the molecule self-reduces into a five-member ring ("Amino acids - proline," n.d.). That five-member ring then shifts one of its double bonds to a single bond, protonating the nitrogen and forming proline (see below):
Above: The formation of proline from glutamic acid ("Amino acids - proline," n.d.)
Hydroxyproline: is formed during the hydroxylation of proline by the enzyme prolyl hydroxylase. Hydroxyproline is formed in one of the last steps of collagen formation, and is key to ensuring the stability of the triple helix. Connective tissue typically has somewhat high levels of hydroxyproline: should it be found in the urine, or a vitamin C deficiency be found, it should be noted that the body’s collagen and therefore structural components are likely becoming unstable and breaking (PubChem, “Hydroxyproline,” n.d.)
On to collagen itself:
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In the body, the process of making collagen starts within the endoreticulum with the formation of polypeptide chains (done via messenger RNA translation). These polypeptide chains have identifying amino acids that determine which type of collagen will be made. After the polypeptide chains are assembled, the strands (“proα chains”) find their correct partners (different collagen types have helices with three of the same or three different strands). They are then folded and assembled into groups of three. These three strands then twist into a triple helix at a defined starting place, with some translational modifications taking place (amino acids move around in order to stabilize the helical shape, as a slightly irregular helix is more stable than a completely perfect one) (Koide & Nagata, 2005). The result of this twisting is called procollagen - essentially the twisted helical domains of collagen with additional propeptides on the ends of the helix.
Once they have been formed, procollagen molecules are transported to the golgi where they are packaged into stacks and secreted from the endoplasmic reticulum. In the final step of collagen formation, enzymes in the space beyond the eukaryotic cell “cleave” the propeptides off of the procollagen, leaving just the helix behind - collagen (Koide & Nagata, 2005).
In order to make collagen for tissue engineering, researchers typically start with material from nature - it is much easier to extract, and purify the collagen than to have to build the molecules from scratch and assemble them into the proper macroscopic structures.
Collagen is pretty much ubiquitous in nature: purified collagen (fibrous collagen, especially) is easily extracted from rats, cows, pigs, sheep, human placentas, human peripheral nerve tissues, and more recently - fish. Most of this collagen is insoluble, however strong enzymes and alkali can be used to cleave crosslinks and make collagen soluble (it is far easier to work with water-soluble collagen) (Dong & Lv, 2016).
One problem with extracting collagen from animal tissues is the risk of disease and allergy. However, Dong & Lv note that researchers are attempting to overcome limitations by making recombinant (genetically engineer-
Above: “Procollagen biosynthesis” (Koide & Nagata, 2005, pp. 88, Figure 2)
-ed) human collagens; researchers culture human skin-fibroblasts and transport the genes to host cells such as yeast, bacteria, insects, animals, and plants. So far, recombinant human collagens’ triple helices have poor stability, though researchers are introducing P4H to do the translational modification/proline hydroxylation (see above) to stabilize the helices (Dong & Lv, 2016).
Another important consideration for collagen sources and preparation methodology is collagen’s inherent antigenicity and immunogenicity; collagen both provokes an immune response within the body and is the recognized target of antibodies goi-
-ng after it (Ilinskaya & Dobrovolskaia, 2016). Antigenicity and immunogenicity are very important within the context of organ transplantation and tissue engineering; new tissue is of no use if the body decides to attack or reject it. Collagen’s antigenicity/immunogenicity can be (somewhat) managed through cross-linking and removing non-helical regions (Don & Lv, 2016). Also, recall the nature video, “The Heart Makers,” from our page on synthetic tissue engineering… for complex organs, the decellularization process is a very important step in ensuring immune system acceptance (Maher 2013).
Due to the lack of hydrogen bond on its amino group, proline forms a slight bend when found in a helix, and is often thus found in turns or loops. Its ability to isomerize between the cis and trans (typically - unstable and stable, same side and opposite side) conformations contributes to the folding of proteins (“Amino acids - proline,” n.d.).
Elastin
Elastin is typically found and derived from nature, much like collagen. In order to produce elastin, it is more difficult than collagen due to its unstable nature and less abundance within mammal bodies. In some cases, the processing of soluble elastin has come from the deposition of elastin by neonatal rats which have been beneficial in comparison to other methods. The rat method has been able to show comparable results compared to other derivation with the necessities of cleaning the protein of any possible bacteria and to be shown as a precursor to tropoelastin moiety (Chipman et al., 1985).
In addition to this, while typically not 100% medical-grade, studies have shown that poultry skin could be used to produce elastin which would create a pathway for the material to be used in commercial aspects which can save a lot of time if the material is gained cost-effectively and properly cleansed of any probable disease (Nadalian et al., 2013). While this is a novel idea to derive elastin rather than other ways, it might not be the safest due to the chicken fat that can sometimes combine with the elastin monomer chains
Above: Necessities inorder to produce a 3D construct for implantation of cell and organ material (Reid et al., 2020, Figure 3)
within the cross-linked cell.
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In the case of processing, Elastin is unlikely to be used in its natural form and must be changed in order to change the structural value of the elastin product. This is usually done through making an elastin scaffolding which is combined with another material or by using elastin as an additive to another scaffold. Either way the processing of elastin to go from natural material to a material that is able to be used in surgery is a necessity in order for elastin to be able to be used.
Additives - How fibers are added to scaffolding
Typically, fibers are either added to scaffolding through an adhesive material which could be from a protein or natural material; just as long as the material is able to break down in the body safely (MIT 2015). Other options for adding fibers to scaffolding also are placing the scaffold and additive in the same area and supplying nutrients so that the additive is able to grow while taking shape of the scaffolding created for a purpose. Throughout this whole process, things like temperature, humidity, and dryness must also be regulated for the product to come out correctly.
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Above: “MIT 3.054 Cellular Solids: Structure, Properties, and Applications, Spring 2015" taught by Laura Gibson (MIT 2015). Watch minutes 40:00-50:00 for scaffolding explanations.