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International Journal of Bioprinting Printing collagen type IV membrane

matrix protein in basement membranes, the use of Col- There is a challenge in developing printable collagen
IV in tissue bioengineering is imperative. In ocular ink due to its variable fibrillogenesis/self-assembly process
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tissue bioengineering, Col-IV could significantly generate in vitro at neutral pH. This process leads to collagen
clinically viable ocular tissue, such as a bioengineered precipitation and has inconsistent speed that can take
corneal endothelium, for use in corneal endothelial up to 30 min. This property prevents the development
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transplants. of high-concentration collagen-only bioinks because the
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Corneal endothelial diseases and dystrophies are ability to have consistent gel-sol time is critical in the
associated with dysfunctional endothelium, resulting in printing process. Previously, a printable Col-I of high
visual loss and pain. In severe cases, they may even lead concentrations (up to 18 mg/mL) at neutral pH without
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to blindness. They affect all ethnic groups, with a higher fibrillogenesis was developed. In this study, we employed
prevalence rate in Caucasians at 7–21.6%. Corneal a similar methodology to create printable Col-IV ink that
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endothelial transplantation using donor corneal tissue is can be kept at room temperature (RT) without gelation
the gold standard for restoring vision in patients suffering for bioengineering a corneal endothelium and evaluated
from corneal endothelial diseases. There is an increasing its mechanical suitability and cellular biocompatibility.
number of corneal endothelial transplantations being Additionally, we compared the wound healing ability
reported, a trend likely associated with the significantly of corneal endothelial cells cultured on Col-I and Col-
higher risk of corneal endothelial diseases in our aging IV membranes and conducted mock surgical tests to
population. As a result, there is subsequently an increased examine the compatibility of the bioengineered Col-IV
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need for donor tissue. Globally, a donor cornea is available endothelium in donor corneas.
for only 1 in 70 patients. Bioengineering a corneal 2. Materials and methods
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endothelium can help tissue supply catch up with demand.
One of the key challenges is to develop a suitable material 2.1. Materials
carrier that is similar to Descemet’s membrane, where Unless noted otherwise, all chemical reagents and collagen
endothelial cells are located. were purchased from Sigma-Aldrich (United States of
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America [USA]). Cell culture-related reagents and tissue
Descemet’s membrane is an acellular and lamellar
structure consisting of four key extracellular matrix culture vessels were sourced from Gibco (USA).
(ECM) proteins: Col-IV, laminins, nidogens, and 2.2. Developing a photo-crosslinkable collagen
perlecan. To date, the primary materials used to replicate type IV ink
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Descemet’s membrane include decellularized corneal Based on the previously published protocol on Col-I
stroma lamellae where Col-I was the main protein, fish ink, Col-IV solution was made by mixing acidic
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scale, decellularized Descemet’s membrane, crystalline human Col-IV solution (12 mg/mL) with 5 M NaOH,
lens, silk fibroin, Col-I, gelatin, and polycaprolactone. CaCl , and riboflavin (Table 1) to generate a stable and
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2
Despite Col-IV being the key collagen type in Descemet’s neutral solution. The final pH was between 6.7 and
membrane, its use as a biomaterial has been limited, 7.4. The solution can be kept in the dark at RT without
and few fabrication methods for generating Col- precipitation or fibrillogenesis for at least one month
IV membranes have been reported. For instance, and can be crosslinked when brought under ultraviolet
Palchesko et al. engineered a basement membrane (UV) light.
for corneal endothelial cells by coating Col-IV onto a
polydimethylsiloxane (PDMS) stamp, then transferring
the Col-IV layer onto a compressed Col-I gel using the Table 1. Composition of collagen type IV (Col-IV) solutions
for photo-crosslinking
surface-initiated assembly (SIA) technique. Similarly,
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Sorkio et al. used the Langmuir-Schefer technique to Composition Quantity Concentration
construct a Col-I/IV double layer to mimic the native
Bruch’s membrane for retinal pigmented epithelial cells. Acidic Col-IV solution (Cat# 90 μL 12 mg/mL
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C7521; Sigma-Aldrich, United
Both methods used Col-I in their membrane construction. States of America [USA])
However, it is important to note that Col-I, typically CaCl (Cat# C5670; Sigma- 7.3 μL 27.4 mg/mL
found in the interstitial matrix, is not present in basement Aldrich, USA)
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membranes, including Descemet’s membrane.
4,13
Therefore, developing a printable Col-IV biomaterial NaOH (Cat# 567530; Sigma- 1.9 μL 5 M
Aldrich, USA)
without the support of Col-I could greatly advance the
biofabrication of structures that more closely resemble Riboflavin (Cat# PHR1054; 0.1 mg 0.1% (w/v)
the native matrix composition of basement membranes. Sigma-Aldrich, USA)

Volume 10 Issue 4 (2024) 158 doi: 10.36922/ijb.3258