Poster N3 - HABP conjugated PCL Nanofibrous Membranes for Synovial Membrane Regeneration

Introduction: The intimal layer of the synovium, also known as the synovial membrane, lines articular joints, and the cells that compose it play a crucial role in regulating the composition of the synovial fluid within the joint. Notably, the intimal layer of the synovial membrane is enriched with substantial amounts of hyaluronic acid (HA), which is absent in the deeper subintimal layer of the synovium [1, 2]. While attempts have been made to tissue engineer an artificial synovial membrane[3], not many alternative biomimetic approaches exist in literature. In the present study, we report our initial steps toward generating a biomimetic artificial synovial membrane by recapitulating key components of the structure and composition of synovial membranes using electrospun poly-ε-caprolactone (PCL) fibers functionalized with a hyaluronic acid binding peptide (HABP) sequence. A key advantage of using HABP over exogenous HA is that even the highest molecular weight commercially available HA products have significantly lower molecular weights than endogenous HA, which can potentially induce inflammatory responses in cells [4].

Materials and

Methods: Using electrospinning (Figure 1A), Polycaprolactone nanofibrous membranes were fabricated. Membranes were then cut in 10 mm circular discs and treated with hexamethylenediamine (HMD) and further Sulfo-SMCC. The Hyaluronan binding peptides (HABP) and scrambled counterparts were conjugated onto the PCL nanofibers to attract endogenous Hyaluronan onto the scaffold. The following experiments were designed to understand whether HABP coated PCL nanofibrous membranes are effective in retaining HA onto the membrane.In each scaffold, 50,000 hDF cells were seeded on the surface. Several tests were conducted in the day 1, day 7 and day 14 of the cell culture. Presto blue assays were performed to exam the metabolic activity of the cells on the scaffolds’ surface. By using the PicoGreen assay, the total DNA of the cells were quantified and further indicating the proliferation of the cells. Also, BCA assays were conducted in order to find out the total protein mass on the scaffolds’ surface, and thus calculating the cell numbers. To quantify the total lubricin produced by the cells, human lubricin ELISA tests were conducted and normalized with total mass of the protein to get the production rate of lubricin. HA deposition was investigated by using Alcian blue stain on the cells on the scaffolds’ surface. Furthermore, α-SMA and Collagen 1 immunostaining were operated to determine the inflammatory condition and the ECM composition.

Results, Conclusions, and Discussions: In the Alcian Blue assay at day 1, HABP group showed 33.39% higher than control group, as well as 41.44% higher than the scr-HABP group in histology. Moreover, at day 7, the histological result presented that the HABP group is 64.44% higher than the control group, and 3.2% higher than the scr-HABP group group (Figure 1B). The results of this study will be used towards successful design strategies for a tissue engineered model of synovial membranes for patients suffering Osteoarthritis.

Acknowledgements (Optional): [1] Lee, Yunsup, Jaehoon Choi, and Nathaniel S. Hwang. "Regulation of lubricin for functional cartilage tissue regeneration: a review." Biomaterials research 22.1 (2018): 9. [2] Smith, M. D. "The normal synovium, Open Rheumatol." J 5.1 (2011): 100. [3] Stefani, Robert M., et al. "A functional tissue-engineered synovium model to study osteoarthritis progression and treatment." Tissue Engineering Part A 25.7-8 (2019): 538-553.. [4] Faust, Heather J., et al. "A hyaluronic acid binding peptide-polymer system for treating osteoarthritis." Biomaterials 183 (2018): 93-101