2022 Annual Meeting

Evaluating Sampling Methods for Degradation of Lyophilized Silk Sponges in Vitro

Silk fibroin proteins, isolated from the cocoons of the Bombyx mori silkworm, have a useful set of properties when used to form a biomaterial, including tunable mechanical properties, limited ability to activate inflammatory immune cascades, non-thrombogenicity, and controllable biodegradability. Silk fibroins can be used to create sponge-like materials with a wide range of adjustable properties, including pore size, elastic modulus, and crystalline content. Silk fibroin sponges have the potential to serve as a tissue regeneration platform,1-2 and their customizability offers the capability to deliver more personalized treatments to patients. The use of silk scaffolds in tissue engineering and wound healing has been successful in previous investigations.1, 3 The sponges can enzymatically degrade in the body, and it is possible to produce scaffolds with extracellular matrix (ECM) components that encourage cell proliferation, migration, and infiltration.1, 3 However, we do not fully understand how to predict the rate of scaffold degradation in vivo.1 The objective of this study is to evaluate the validity of discrete vs continuous sampling when measuring sponge degradation, building on previous work,4to understand and determine if diffusion limitations play a role in our in vitro studies as a function of sample size. Following previously established protocols, the degradation of the sponges was tracked by measuring mass loss over time. In the discrete sampling method, each mass measurement was obtained from a unique sponge. Alternatively, for continuous sampling, the mass loss of the sponge was measured for duration of the experiment allowing one to collect many time points for a single sponge. The results exhibited a divergence in the degradation timeline between the discrete and continuous sampling methods. The discrete method degradation curve was fit with Michaelis-Menten kinetics and parameters KM = 25 mg/ml and kcat = 66.1 mg/(U*day) were found. We hypothesize that these differences are due to the negative influence of the drying and weighing steps during the degradation process using the continuous sampling method. We hypothesize that these steps disturb the scaffold integrity and alter the crystalline content. In conclusion, the discrete sampling method will be used moving forward as we continue to evaluate the influence of fabrication parameters on degradation kinetics, despite the substantially larger number of samples needed for these experiments.
  1. Jameson, J. F.; Pacheco, M. O.; Bender, E. C.; Kotta, N. M.; Black, L. D.; Kaplan, D. L.; Grasman, J. M.; Stoppel, W. L., Impact of bioactive molecule inclusion in lyophilized silk scaffolds varies between in vivo and in vitro assessments. bioRxiv 2022, 2022.05.24.493207. DOI: 10.1101/2022.05.24.493207.
  2. Stoppel, W. L.; Ghezzi, C. E.; McNamara, S. L.; Black, L. D., 3rd; Kaplan, D. L., Clinical applications of naturally derived biopolymer-based scaffolds for regenerative medicine. Ann Biomed Eng 2015, 43 (3), 657-80. DOI: 10.1007/s10439-014-1206-2.
  3. Stoppel, W. L.; Hu, D.; Domian, I. J.; Kaplan, D. L.; Black, L. D., 3rd, Anisotropic silk biomaterials containing cardiac extracellular matrix for cardiac tissue engineering. Biomed Mater 2015, 10 (3), 034105. DOI: 10.1088/1748-6041/10/3/034105.
  4. Jameson, J. F.; Pacheco, M. O.; Butler, J. E.; Stoppel, W. L., Estimating Kinetic Rate Parameters for Enzymatic Degradation of Lyophilized Silk Fibroin Sponges. Front Bioeng Biotechnol 2021, 9 (537), 664306. DOI: 10.3389/fbioe.2021.664306.