Collagen, well-known as an anti-aging supplement, is an extracellular structural protein found in the connective tissues of bones, tendons, ligaments, cornea, cartilage, skin, intervertebral disc etc. Collagen fibrils are made up of triple helical units that are assembled together. There are different types of collagen classified on the basis of their structural differences. Among them, type I collagen accounts for almost 90% of the total collagen in the body and is known to have enormous tensile strength that is compared with the strength of steel. But collagen has poor regenerative capacity thus rendering the prognosis of collagen-related disease and degeneration complicated.
The US patent no. 9518106 titled, “Collagen fibrillar construction” discloses methods and compositions for producing an organized array of collagen fibrillar networks. The invention is based on the ability of collagen monomers to get organized into short or long range fibrillar networks when a solution of collagen monomers is confined within templates of predetermined shapes. In different embodiments, the shape of the templates corresponds to that of cornea, ligament, tendon, knee meniscus, intervertebral disc and cartilage. The collagen monomers in the solution are in liquid crystalline phase (nematic, smectic or cholesteric) and are precipitated to form load bearing fibrillar collagen which has applications in tissue engineering for e.g. “to produce both the short and long-range organization and morphology of collagen in highly-anisotropic native tissues such as tendon, ligament, bone, annulus fibrosus and cornea”. It is claimed that the collagen produced by this method has higher mechanical strength compared to the regular collagen gels that are currently available.
The in-vitro method further involves applying controlled tension to the collagen fibrils in the solution in the presence of enzymes such as collagen lytic proteases and optionally, supplemental collagen monomers. This leads to strain dependent polymerization and catabolism of the collagen fibrils thus “sculpting” them to form an organized load-bearing structure. The collagen degrading enzymes include Collagenase (Bacterial Collagenase), Cathepsin and Matrix Metallo Proteases (MMP). As can be seen from the figure given below, collagen fibrils in solution are strained between two micromanipulators in a special chamber. A fresh supply of collagen monomers and the enzyme MMP are added by micropipettes to the reaction buffer to allow for collagenous tissue growth which is directly observed under live DIC microscopy.
In some embodiments, the method further involves neutralising the pH of the reaction solution at about 100C – 390C. The collagen monomers that are used for fibrillogenesis are tropocollagen monomers, procollogen monomers or atelo-collagen monomers. In some embodiments, the reaction solution further comprises of co-nonsolvency agents, collagen binding agents and/or cross-linking agents.