Hydrogels have widely used applications in biomedical fields because of their biocompatibility and close resemblance to natural tissue. Drawing inspiration from the designer collagens [1] and gradient collagens [2], we focused on an orthopedic approach by constructing a hydrogel that would mimic tissue interfaces, such as bone-cartilage, tendon-bone, and ligament-bone interfaces.
In the lab we produced two different types of Scl2 proteins, one of which we combined with nHAP nanoparticles, particles used to replicate the physical gradient of tissue interfaces, to create our first gradient. We then used two different types of collagen (differentiated by their own binding sites) hydrogels to create a second gradient produced through our gradient maker (further explained in the gradient maker section).
After meeting with many experts in the field, including Dr. Lawrence Bonassar, whose research focuses on biomaterials and tissue engineering, and Jos Olijve, a member of the Scientific Development Team at Rousselot, we decided our best options for crosslinking would either be chemical crosslinking, which is a widely used method that has a high degree of crosslinking but has potentially cytotoxic effects, and UV photo-crosslinking, which enhances the mechanical properties of hydrogels but has a lower crosslinking density. Through trial and error, we decided to use UV photo-crosslinking for reasons described in later sections.
From our successful production of our gradient-mimetic-collagen hydrogels we hope that our hydrogels can potentially be used for injuries requiring bone-tissue healing.
We now describe the process of making Scl2 hydrogels. Although we were unable to obtain Scl2 proteins, we followed this process with rat-tail collagen and used the same process. First the Scl2 proteins are functionalized for photoreactive crosslinking by conjugation with acrylate-PEG-N-hydroxysuccinimide inside a buffer solution, then placed on a shaker for 48 hours at 4°C. Next, the proteins were diluted with acetic acid, and PEGDA power was added to each solution to 5 wt.%. For our bone-mimicking gel, we add nHAP power to 5wt.% for imaging purposes. Next, we create a UV photoinitiator solution of 2,2-dimethoxy-2-phenyl-acetophenone in N-vinylpyrrolidone, which is added to the Scl2 solution.
To create the final gels, we used our gradient machine to mix the viscous solutions into silicone molds, with a gradient generated over the distance of 1cm to 1mm depending on the machines settings. To crosslink the hydrogels, we exposed them for 5 minutes to longwave UV light (6 mW cm-2 , Spectroline). This UV irradiation time was based off of our rheological testing, with a longer exposure time due to the use of a lower power source. The two sources were balanced for total exposure energy. The hydrogels were then immersed in PBS for 24 hours.
Previous hydrogels designed as cell scaffolds lack specificity in catering to joint tissues, where cell types do not grow in even layers but in a gradient, such as with the bone-cartilage or bone-tendon interfaces [2]. Papers such as Bioactive hydrogels based on Designer Collagens [1] focus on the creation of hydrogels able to grow a single type of cell. Our gradient hydrogels encourage growth of the multiple cell types required for injuries in the knee and elbow joints. In addition to the previous papers mentioned, we used the results from Nanocomposite hydrogels for biomedical applications [3] and Biomimetic gradient hydrogels for tissue engineering [4] to create our procedure for the hydrogels.
We first created hydrogels out of gelatin to gain familiarity with the process and test our hardware devices. The process for gelatin hydrogels started with modeling the gels based off of a scientific paper describing designer collagen hydrogels [1]. This involved a two-step creation process in which the Scl2 proteins were functionalized with photoreactive crosslinking sites followed by collagen conjugation to polyethylene glycol (PEG) using photoreactive crosslinking as well. This procedure was then modified in a specialized manner according to the details of our project. We started off using chemical crosslinking and then made modifications from this. The gelatin hydrogels underwent multiple rounds of iteration and trials, but we finally made hydrogels using water, gelatin, nHAP particles, and a 2% glutaraldehyde solution.
We observed from our first trials that the nHAP nanoparticles were prone to clumping in the gelatin-glutaraldehyde solution, and after 10 hours of crosslinking, the hydrogels had a low viscosity with the nHAP particles settling at the bottom of the liquid. In contrast, the gelatin solution without the nHAP particles had a thicker consistency resembling that of a hydrogel. We used part of both of the hydrogels to attempt making a gradient hydrogel.
Figure 1 3% wv gelatin with nHAP particles made at a 1:10 ratio of glutaraldehyde to gelatin solution (left without flash, right with flash)
Figure 2 3% wv gelatin made at a 1:10 ratio of glutaraldehyde to gelatin solution (left without flash, right with flash)
Figure 3. Gradient hydrogels
With the remaining gels we froze them for 12 hours at 4°C and then lyophilized them for 48 hours. These gelatin hydrogels served as the basis for the experimenting done for the collagen hydrogels as these were a more general solution while we waited for the wet-lab subteam to engineer the collagen-secreting E. Coli.
In anticipation of our Scl2 proteins, we first created rat tail collagen hydrogels. Rat tail collagen, compared to gelatin, has the advantage of requiring the same procedure as Scl2 to create hydrogels. First, we separately created our two scaffold types - with and without nHAP, to encourage growth of bone and cartilage tissue, respectively. For the hydrogel with the nHAP, we first dissolved the rat tail collagen in 20 mM acetic acid, and then combined this with the PEGDA powder and nHAP powder. For the crosslinking, we added glutaraldehyde at a 1:1 ratio to the PEGDA. The hydrogel without nHAP followed the same procedure, excluding the nHAP.
Figure 4 First trial of making rat tail collagen hydrogels with nHAP particles (right without flash, left with flash)
Figure 5 Second trial of making collagen hydrogels (left side has nHAP, right side does not)
Figure 6 Slow motion video showing consistency of the collagen hydrogels pictured above.
Early on, our major issues were that the consistency of our hydrogels were not firm enough. Through testing, we determined that this resulted from issues within our procedure, specifically our crosslinking methods. We decided to test two different crosslinking methods: chemical and photocrosslinking.
During the design process, we tested multiple different amounts of each of our crosslinking chemicals and UV light time exposure, and ultimately chose the procedure yielding the firmest hydrogel, detailed above (refer to “Creation Process”). When choosing between different crosslinking methods, our team selected our photocrosslinking procedure for our final product, as it yielded firmer hydrogels. In addition, after doing more research and receiving feedback from Jos Olijve, a member of the Scientific Development Team at Rousselot, we learned that consideration of the cytotoxic effects of glutaraldehyde present in the chemically crosslinked hydrogels should be taken into consideration.
Once we decided on photocrosslinking, we further refined our procedure for this method. After imaging, we discovered that our nHAP particles still tended to clump to each other, as opposed to the even distribution we had intended. To solve this, we added the additional step of sonicating our nHAP solution before adding the collagen, which yielded improved distribution and imaging results. We then evaluated the quality of our hydrogels through imaging, mechanical testing, and cell cultures, described in our Proof of Concept section.
- 1. E. Cosgriff-Hernandez, M.S. Hahn, B. Russell, T. Wilems, D. Munoz-Pinto, M.B. Browning, J. Rivera, M. Höök, Bioactive hydrogels based on Designer Collagens, Acta Biomaterialia, Volume 6, Issue 10, 2010, Pages 3969-3977, ISSN 1742-7061, https://doi.org/10.1016/j.actbio.2010.05.002.
- 2. Lauren M. Cross, Ashish Thakur, Nima A. Jalili, Michael Detamore, Akhilesh K. Gaharwar, Nanoengineered biomaterials for repair and regeneration of orthopedic tissue interfaces, Acta Biomaterialia, Volume 42, 2016, Pages 2-17, ISSN 1742-7061, https://doi.org/10.1016/j.actbio.2016.06.023.
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