The mechanical and enzymatic digestion during pancreatic islet isolation leads to 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 the viability and functionality of human pancreatic islets of Langerhans. For this purpose, the islets in different groups the 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; the 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.
Evaluation of the 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 the 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 the 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, were 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 have shown 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 the 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 consisted 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 could help to increase the islets survival, and adding laminin and IV collagen cannot promote survival in this hydrogel (12). In a previous study, perfluorodecalin-enriched fibrin matrix led to the reduction of caspase-3 activation in the 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).
Given 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, we observed an increase in the BCL2 gene and a decrease in the BAX gene and BAX/BCL2 ratio. On the other hand, the evaluation of BAX, BCL2 and active caspase-3 proteins indicated significant differences between the 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, we measured the number of positive TUNEL cells. 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 evidence is 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 did not have an adverse effect on the secretory function of these islets.
With respect to the application of collagen microspheres, Wang et al. in their study suggested that collagen microspheres, compared with single-layer cultures, increased 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.
In the present project, our main problem was shortage of human pancreas for research. Obtaining appropriate pancreas for such studies is so difficult and costly. Because there is an inclusion and exclusion criteria for selecting the suitable pancreases. Moreover, we processed the pancreases which gained informed consent for research. Actually, the superiority of the present study was using human pancreas. The previous studies were mainly on animal sources and cell lines. On the other hand, we had to performed different analysis techniques on the cells in different groups, including viability, gene expression, immunohistochemistry, TUNEL assay, and insulin and C-peptide secretion. Therefore, we limited our target groups to what was mentioned in the manuscript. Moreover, we applied immunocytochemistry in order to examine the protein expression. It would be worthwhile to evaluate the protein levels quantitatively using western blotting if we get access to enough islets to isolate a pool of proteins.
In terms of the lack of significant improvement in the stimulation indexes, it seems that focusing on the scaffold structure is useful in further studies. Previously, researchers explained that high concentration of alginate can increase the scaffold stiffness and cause substances diffusion problems (32). Moreover, Hart et al. reported that densely encapsulated islets leaded to postponed and restricted insulin secretion (33). Importantly, although we did not observe negative effects of the mentioned scaffolds on the stimulation indexes, we need to design an effective encapsulation system that does not compromise the diffusion of secreted insulin from the islets. In this regard, it is important to examine alginate hydrogel enriched itself with the extracellular matrix components, without encapsulating in microspheres, to improve the results in future studies.