(165ab) Crosslinking to Enhance the Mechanical Properties of 3D-Plotted Collagen-Based Scaffolds for Bone Tissue Engineering

Nonunion bone fractures represent a clinically important subject due to their morbidity and economic burden. Consequently, scaffold-based tissue engineering has become an increasingly prevalent topic in biomedical research. The design of 3D scaffolds serves a vital role in achieving desirable efficacy for bone reconstruction. We have investigated the effects of biomaterials and 3D-printing techniques on the degradation rate, porosity, and modulus of 3D scaffolds. Specifically, this project explores the effects of crosslinking on modulating the degradation of collagen-based scaffolds for bone tissue engineering. The first technique for crosslinking was plasma treatment with argon gas. The second technique being employed is treatment with a solution of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 2-(N-Morpholino)ethanesulfonic acid (MES). The scaffolds consisted of poly(lactic-co-glycolic acid) (PLGA), nano-hydroxyapatite (nHA), type I collagen, and polyethylene glycol (PEG), and they were processed in the solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFP). It has been shown that crosslinked collagen bolsters structural stability, resulting in improved rates of osteogenesis.

Using specific biomaterials, the scaffolds were formulated to imitate the extracellular matrix of bone. Solution homogeneity was ensured through mixing coupled with sonication at an amplitude of 40 μm. Room-temperature dispensing through a 3D-Bioplotter (EnvisionTEC) prevented collagen degradation from extreme temperatures. After scaffold production, the scaffolds (n=4) underwent argon plasma treatment (Harrick Plasma) for four minutes, which we hypothesized would promote collagen crosslinking within the scaffold matrix. Untreated scaffolds were used as control (n=4). Following plasma treatment, all scaffolds were submerged in vials of phosphate buffered saline containing calcium and magnesium (PBS++) for ten days at room temperature. The ten-day incubation period resulted in partial dissolution of collagen in the PBS++ solution, which was then analyzed through a bicinchoninic acid (BCA) assay to test for protein concentrations. If collagen was crosslinked as hypothesized, the protein concentration was expected to be lower for the plasma-treated scaffolds due to less collagen washing off of the scaffold.

It was found that the plasma-treated scaffolds had a lower collagen release into PBS++ (0.397 ± 0.238 μg/μL), whereas the untreated scaffolds had a higher release of collagen (0.655 ± 0.120 μg/μL); however, the difference was not statistically significant (p<0.05). Additionally, the weight loss of plasma-treated scaffolds was slightly less than the weight loss of untreated scaffolds. Nevertheless, the difference was not statistically significant, nor could it be confirmed that the weight loss was due entirely to collagen dissolution. In future trials, incubation periods will be increased to two, four, and six weeks, and additional scaffolds will be used to create a larger sample size. Crosslinking with EDC/NHS/MES solution is currently underway. Moreover, analytical methods, such as differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy, have been employed in order to gauge how effectively these techniques promote collagen crosslinking. DSC was used to analyze melting transitions and thermal stability of the scaffolds. FTIR is capable of analyzing collagen’s secondary structure, allowing for the identification of peptide bonds in characteristic vibrational bands.