The mechanical and enzymatic digestion during pancreatic islet isolation leads to the destruction of ECM, resulting in loss of biological and mechanical protection. In this regard, tissue engineering is a promising way for restoring ECM by maintaining the structure and function of the cells. Recent studies have shown that the culture of human islets in 3D scaffolds improves the morphology, viability, and performance of the islets during pre-transplant stage.
In the present study, the effect of 3D hydrogel microenvironment with different compounds was used to evaluate viability and functionality of human pancreatic islets of Langerhans. For this purpose, the islets in different groups (Con.), (Alg.), (Coll. I & Alg.), (Coll. I, IV & Alg.), (Coll. I, Lam & Alg.), and (Coll. I, IV, Lam & Alg.) were incubated for one week and then examined for morphology, viability and functionality. In order to culture the islets in alginate hydrogel scaffolds, in four groups of; (Coll. I & Alg.), (Coll. I, IV & Alg.), (Coll. I, Lam & Alg.), and (Coll. I, IV, Lam & Alg.), first, the islets were encapsulated in the collagen I microspheres in the presence or absence of collagen IV and laminin, and finally placed in the alginate hydrogel platform.
The evaluation of islets structure with inverted optical microscopy showed the maintenance of the cells native morphology in the hydrogel scaffolds. The obtained results were confirmed by SEM. Our findings showed that spherical-3D morphology and integrated pancreatic islet structures were preserved in all hydrogel scaffolds groups. On the other hand, it seemed that structural integrity of the islets in the control group was relatively disturbed.
According to the Nomenclature Committee on Cell Death (NCCD), using fluorescent dyes as one of the criteria to confirm lack of membrane integrity and cell death, in this study, cell viability was examined with PI. It was clearly observed that the viability of the pancreatic islets in the course of one week in the control group was significantly reduced, while in hydrogel groups, it was completely preserved.
Previous studies showed that hydrogels, by simulating hydrophilic elements of the ECM, are desired tools for the encapsulation of the pancreatic islets. Recently, researchers have used various biomaterials and methods to achieve an optimal and efficient system for the reconstruction of the human pancreatic islets ECM. Cellular scaffolds design should be done in a way that they can mimic the inherent ECM, by maintaining cellular viability.
In this study, the main substrate of the hydrogels was alginate. Studies showed that the use of alginate polysaccharide has many advantages. Alginate is the most common biomaterial for islets encapsulation. Alginate capsule processing is possible in physiological conditions, including body temperature, physiological osmolarity and pH (21, 22). Moreover, alginate is generally biocompatible and biodegradable. It has been shown that alginate is suitable for encapsulating cells and controlled delivery of bioactive molecules. In addition, this biomaterial can affect cell growth, metabolism and insulin production (8, 23, 24). Investigation of the intrinsic microenvironment of human pancreatic islets showed that their ECM consists of collagen I, III, IV, V and VI, laminin and fibronectin (25, 26).
Zhang et al. studied the survival of pancreatic islets in a collagen matrix of type I for 7 days. They found that cellular viability was significantly higher than that of the control group. In addition, the islets enclosed in the collagen matrix had a much lower number of active caspase-expressing cells than the control group (27). Lee et al. also made a comparison between the rat pancreatic islets cultured in the alginate and the alginate-collagen type I composite, and observed that the used composite remarkably improved the cells viability (11). On the application of ECM factors, researchers examined the survival rate of mice Islets in silk hydrogel containing laminin and collagen IV. This study showed that Silk alone can help to increase islets survival, and adding laminin and IV collagen cannot promote this hydrogel (12). In a previous study, perfluorodecalin-enriched fibrin matrix led to the reduction of caspase-3 activation in cultured human pancreatic islets. This structure provided a favorable chemical and physical environment for these cells, but the study was only performed within 24 hours (28). In general, studies on the evaluation of apoptosis following the use of 3D scaffolds in human pancreatic islets are very limited.
In the present study, we observed that in all groups with hydrogel scaffolds the viability of human pancreatic islets was significantly higher than the control group, and the presence of collagen and laminin did not have any superiority over pure alginate hydrogel. In total, all scaffolds designed in this study were able to fully maintain the viability of the islets (Fig. 11).
Considering the importance of cellular life in the prognosis of pancreatic islet transplantation, it seems essential to investigate the cause of islets’ death in order to prevent this destructive process. In this regard, the present study focused on the internal pathway of apoptosis - as the main cause of pancreatic islets death. Therefore, the expression of genes and proteins involved in the process of apoptosis and the number of positive TUNEL cells was evaluated.
Although the analysis of BAX and BCL2 genes expression and the BAX/BCL2 ratio reveled that there was no significant difference between different groups of scaffolds, the increased of BCL2 gene and the decreased of BAX gene and BAX/BCL2 ratio was visible. On the other hand, the evaluation of BAX, BCL2 and active caspase-3 proteins indicated significant differences between hydrogel scaffold groups and non-scaffold group. BAX expression decreased in all scaffold groups, but contrary to the expectation, the expression of BCL2 showed a non-significant reduction. Furthermore, we found out that the expression of active caspase-3 was reduced in all hydrogel scaffold groups. This reduction was significant in the surrounded microspheres by alginate hydrogel. The contradiction between significant differences at the level of genes and proteins expression might have been due to the effect of scaffolds on post-translational proteins modifications.
In order to further clarify the cell death pathway, after the evaluation of active caspase-3 protein expression, the number of positive TUNEL cells were measured. In the control group, a large number of cells were TUNEL positive, as the high expression of active caspase-3 and enormous cell death was evident. However, the findings showed that although caspase-3 expression was slightly expressed in 3D-scaffold groups, very limited number of cells were TUNEL positive. This discrepancy may suggests that caspase-3 activation is not necessarily indicative of apoptosis (29). On the other hand, it seems that there is a threshold for the destructive activity of active caspase-3. Geske et al. showed that apoptotic cells induced by p53 could be released from apoptosis during the removal of apoptotic stimulus. Their study suggested that DNA repair could be activated in this process and in some cases results in a return from the cell death pathway (30). All in all, studies suggest that the mechanisms involved in apoptosis are complex and a cascade of molecular events guides intrinsic and extrinsic apoptosis. Previous evidences are based on the fact that these two pathways are interconnected and the molecules involved in one path can affect the other (31).
The performance of pancreatic islets is an indicator of the islets transplantation prognosis. In this study, the evaluation of insulin and C-peptide secretion indexes suggested that hydrogel scaffolds do not have an adverse effect on the secretory function of these islets. Although the differences were not statistically significant, the improvement in secretion indices was visible in some groups. The islets of the (Alg.) group showed a higher level of both indices. Thus, the alginate hydrogel microenvironment might provide a favorable 3D-microenvironment for these cells.
With respect to the application of collagen microspheres, Wang et al. study suggested that collagen microspheres, compared with single-layer cultures, increases the production and secretion of GDNF from HEK293 cells (13). Subsequently, Lee et al. embedded these cells in collagen microspheres, incorporated in alginate hydrogel and compared GDNF secretion in these conditions with cells embedded in collagen I and alginate composites (13). They found that in the composite, stable release of GDNF occurred throughout the entire culture period, and its release level was controlled by various concentrations of alginate. On the other hand, due to proliferation of HEK293 cells in collagen microspheres enclosed in alginate scaffolds, GDNF release was increased steadily.
In the present study, there was no significant differences in the index of insulin and C-peptide secretion between the groups containing microspheres and the control group. Overall, the 3D scaffolds designed in this study had no adverse effect on the activity of pancreatic islands.