(B-65) Biocompatibility of Gelatin Macroporous Scaffolds for Localized Immunomodulation in Type 1 Diabetes

Introduction:: Current treatments for autoimmune diseases mute inflammation systemically and nonspecifically often leading to off-target effects and higher susceptibility to infection. In Type 1 Diabetes (T1D), autoimmune attacks on pancreatic islet cells cause severe insulin deficiency in patients. Teplizumab is the only approved systemic and nonspecific treatment for T1D which targets CD3 to dampen T cell autoreactivity and delay T1D onset [1]. Localized treatments that specifically target autoreactive T cells would be more effective in preventing treatment systemic effects. Through freeze-drying, we previously fabricated gelatin scaffolds of varying macropore sizes to model the different stages of lymph node inflammation during immune responses as a biomaterial platform for localized immunomodulation in T1D. These macroporous scaffolds were designed with the goal of promoting the immunomodulation of lymph node stromal cells in a physiologically relevant environment. However, before a biomaterial can be used for an implantable tissue engineering construct, its biocompatibility needs to be evaluated. Here, we aim to characterize the biocompatibility of our macroporous gelatin scaffolds in vitro and in vivo to validate their application as a tissue-engineered construct that could provide localized, specific immunomodulatory treatment in T1D. Conditions of biocompatibility for our scaffolds include the ability to promote vascularization, deliver cells viably, effectively recruit and modulate immune cells, and integrate with host tissue.

Materials and Methods:: Splenocytes were isolated for in vitro culture from a 13-week-old female NOD mouse, and they were seeded in 4mm diameter macroporous gelatin scaffolds with small (< 50 µm), medium (< 150 µm), or large pores ( >300 µm) fabricated through freeze-drying in liquid nitrogen at -80C or -20C, respectively. Seeded scaffolds were cultured in 300 µL of 5% FBS media supplemented with IL-2  cytokine. As positive controls, LPS or αCD3 was added to splenocytes in medium pore scaffolds to stimulate macrophage and T cell activation and proliferation, respectively. Medium pore scaffolds were chosen for positive controls because pilot testing showed that the pore size of scaffolds alone did not significantly affect splenocyte proliferation. As a negative control, splenocytes were cultured in 5% FBS media supplemented with IL-2 in 2D conditions. Following 72 hours of culture, 200 µL of conditioned media from each experimental group and control was collected for IL-6, IL-10, and TNF-α ELISA assays (RnD) per manufacturer protocol. The remaining 100 µL of media was retained for MTS assay to quantify splenocyte proliferation by a proliferative index [2]. ELISA assay data were analyzed via Student’s t test whereas MTS assay data were analyzed via Mann-Whitney testing.
To assess in vivo biocompatibility, acellular scaffolds (small, medium, large pores) were implanted in the dorsal subcutaneous site of 8-week-old female NOD mice for 21 days. Then, scaffolds and surrounding tissue were harvested, processed for histological examination (H&E) and immunofluorescence with endothelial, lymphocyte, and macrophage markers, and imaged through confocal microscopy.

Results, Conclusions, and Discussions:: When compared to the positive control (medium scaffold supplemented with αCD3), there was significantly less splenocyte proliferation in all three scaffold types analyzed. Thus, these gelatin macroporous scaffolds do not stimulate immune cell proliferation in vitro. Moreover, scaffold pore size did not significantly affect splenocyte proliferation.
Secretion of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines IL-6 and TNF-α measured via ELISA was significantly lower in splenocyte cultured in scaffolds without LPS than any of the LPS conditions, as expected. However, IL-6 and TNF-α secretions were not significantly different across scaffolds of different pore sizes. Thus, the gelatin macroporous scaffolds do not stimulate immune cell activation and pro-inflammatory cytokine secretion in vitro. Additionally, the small scaffolds did elicit a significant anti-inflammatory as increased IL-10 secretion compared to the negative control; so, it is possible that the scaffolds themselves could have immunomodulatory effects in vitro. To further support this hypothesis, IL-6 and TNF-α levels were significantly higher in 2D splenocyte culture with LPS than in 3D (medium scaffolds) culture with LPS. TNF-α levels were significantly elevated in small and large pore scaffolds compared to the 2D negative control indicating that independently from scaffold composition (gelatin), pore architecture may affect immune cell behavior which remains to be investigated.
Qualitative in vivo assessment of scaffold integration 21 days after implantation in NOD mice shows that host cells were able to migrate into all three types of scaffolds, though small regions of fibrosis are visible around all scaffolds. Further testing with immunofluorescence staining is currently being conducted to quantify immune cell response, fibrosis, and vascularization of scaffolds as more comprehensive assessment of in vivo scaffold biocompatibility.
Through assessment of biocompatibility of the freeze-dried gelatin biomaterial composing our scaffolds, this study contributes to developing a novel immunomodulatory platform for treating Type 1 Diabetes.

Acknowledgements (Optional): :

References (Optional): : [1]       U. B. Masharani and J. Becker, “Teplizumab therapy for type 1 diabetes,” Expert Opin. Biol. Ther., vol. 10, no. 3, pp. 459–465, Mar. 2010, doi: 10.1517/14712591003598843.

[2]       P. J. VandeVord, H. W. T. Matthew, S. P. DeSilva, L. Mayton, B. Wu, and P. H. Wooley, “Evaluation of the biocompatibility of a chitosan scaffold in mice,” J. Biomed. Mater. Res., vol. 59, no. 3, pp. 585–590, 2002, doi: 10.1002/jbm.1270