The eye is a vital organ, and a part of it which is very important is the cornea. The cornea is composed of three layers, the epithelium, stroma, and endothelium. All three are composed of cells and extracellular matrix that contains lots of protein. The cornea has a protective function, preventing dust, foreign matter, and UV rays from entering the eye. Corneal damage can cause vision problems until blindness.
Until now, blindness cases always increase every year. One cause of blindness is corneal ulcer due to exposure to bacteria, fungi, and herpes viruses. Corneal damage is treated by transplantation or known as keratoplasty. However, this method still has weaknesses such as rejection from the patient’s body, limited donors, and a long duration of healing. For this reason, new corneal replacement technology with excellent compatibility like the human cornea and does not require a donor is needed.
A natural material that can be used for the development of artificial corneas is collagen because of its biocompatible nature, thereby reducing the potential of rejection from the recipient’s body. Chitosan which is a derivative of chitin and found in many crustaceans can trigger regeneration of the cornea. Glycerol provides anti-bacterial, anti-fungal, and anti-viral properties to reduce the causes of corneal ulcers. Meanwhile, hydroxypropyl methylcellulose (HPMC) is used to increase the transparency of the cornea.
The study was conducted to determine whether the collagen-chitosan-HMPC material is toxic or not through cytotoxicity testing, whether all materials show a suitable functional group and whether new compounds are formed through functional group tests, biomaterial pore sizes through morphological tests, and whether the material is antibacterial through antibacterial testing. Artificial corneas are synthesized by mixing collagen and chitosan into acetic acid. Then glycerol is added until the solution is homogeneous, molded, and dried. There are two samples, samples without HPMC and samples with HPMC.
With Fourier Transform InfraRed (FTIR), the functional group of the material for artificial cornea can be known with certainty. Each composing material has a unique group that can be detected so it becomes a reference whether the synthesis has been successful. FTIR results showed typical chitosan functional groups, hydroxyl (OH) at 3427.2 cm -1 wave number , -CH 2 and -CH 3 groups at 1458.23 cm -1 , C = O group at 1647.14 cm -1 , CO group at 1406.22 cm -1 , and -COC group at 1044.22 cm -1 .
While the typical collagen functional groups were shown by absorption of -OH at wave number 3427.2 cm -1 , amide group I at 1647 cm -1 , and amide group II at 1458.23 cm -1 . The addition of HPMC raises the absorption at wave numbers 2942.13 cm -1 , 1647.14 cm -1 , and 1458.23 cm -1 , respectively showing the groups CH, C = O and CC, and CH 2 and C-CH 3 .
All foreign substances to be implanted in humans must pass a cytotoxicity test to ensure the implant is not toxic to the body. Cytotoxicity tests were carried out using the MTT method and Huh7it liver cells. The artificial cornea in this study had a percentage of living cells above 85%. It showed that the artificial cornea is not toxic because it has exceeded the 50% limit of living cells. The addition of HPMC also did not affect the percentage of living cells.
The artificial cornea’s surface state (morphology) was also identified using the Scanning Electron Microscopy (SEM) instrument. This morphological test aims to see the surface quality and pore size of the artificial cornea. Pores in the cornea play a role to support cell proliferation including blood vessel pathways and nutrition. In this study, samples with HPMC had more varied pore sizes compared to samples without HPMC, respectively 68.37 µm and 33.27 µm.
The standard pore diameters in the cornea range from 33-38 nm for the Descemet’s membrane and 88- 92 nm for the anterior basement membrane. Corneal biomaterials without HPMC are in accordance with the standard pore diameter of Descemet’s membrane. Descemet’s membrane is the thinnest and strongest tissue in the cornea. This membrane is made of collagen and serves as a resting place for endothelial cells while protecting these cells from infection and injury. Whereas biomaterials with HPMC still require optimization to increase the pore size of the artificial cornea to be able to comply with the pore standards of the Descemet’s membrane or anterior basement epithelium.
One of the causes of corneal ulcers is bacteria so artificial corneas must have anti-bacterial properties. This property can be confirmed using an anti-bacterial test using the Mueller Hinton Agar (MHA) diffusion method. This test will produce a clear zone (zone of inhibition) between the sample and the bacteria Staphylococcus aureus and Escherichia colli. If the diameter of the clear zone is 20 mm, the inhibitory capacity of the sample bacteria is very strong, 10-20 mm means strong, 5-10 mm means medium, while below 5 mm means weak.
Based on anti-bacterial testing, all artificial corneal samples showed clear zone diameters above 10 mm so the sample has strong bacterial inhibition. It is supported by the use of chitosan which can form amine groups that can damage bacterial cell walls (lysis). In conclusion, collagen – chitosan – glycerol – HPMC can be a material for artificial corneas because it is compatible with the corneal biocompatibility and anti-bacterial properties. (*)
Author: Prihartini Widiyanti
Details of research available at: https://www.researchgate.net/publication/334630620_Collagen-Chitosan-_Glycerol-HPMC_Composite_as_Cornea_Artificial_Candidate