A Tissue-Engineered Construct Based on a Decellularized Scaffold and the Islets of Langerhans: A Streptozotocin-Induced Diabetic Model

Abstract

Producing a tissue-engineered pancreas based on a tissue-specific scaffold from a decellularized pancreas, imitating the natural pancreatic tissue microenvironment and the islets of Langerhans, is one of the approaches to treating patients with type 1 diabetes mellitus (T1DM). The aim of this work was to investigate the ability of a fine-dispersed tissue-specific scaffold (DP scaffold) from decellularized human pancreas fragments to support the islets' survival and insulin-producing function when injected in a streptozotocin-induced diabetic rat model. The developed decellularization protocol allows us to obtain a scaffold with a low DNA content (33 [26; 38] ng/mg of tissue, p < 0.05) and with the preservation of GAGs (0.92 [0.84; 1.16] µg/mg, p < 0.05) and fibrillar collagen (273.7 [241.2; 303.0] µg/mg, p < 0.05). Rat islets of Langerhans were seeded in the obtained scaffolds. The rats with stable T1DM were treated by intraperitoneal injections of rat islets alone and islets seeded on the DP scaffold. The blood glucose level was determined for 10 weeks with a histological examination of experimental animals' pancreas. A more pronounced decrease in the recipient rats' glycemia was detected after comparing the islets seeded on the DP scaffold with the control injection (by 71.4% and 51.2%, respectively). It has been shown that the DP scaffold facilitates a longer survival and the efficient function of pancreatic islets in vivo and can be used to engineer a pancreas.

Keywords: decellularized scaffold; islet transplantation; islets of Langerhans; streptozotocin-induced diabetes mellitus.

Figure 1

Research design.

Figure 2

The DP-scaffold characterization. (a–f) A histological picture of a tissue-specific scaffold from a decellularized pancreas (DP scaffold); (a) H&E staining; (b) Masson’s staining; (c) alcian blue staining; (d) immunohistochemical staining with antibodies to type I collagen; (e) staining with orcein for elastic fibers; (f) DAPI staining (scale bar = 100 µm); (g) GAG content; (h) collagen content; (i) DNA content; (j–o) a DP-scaffold cytotoxicity study in vitro; (j,m) negative control; (k,n) positive control; (l,o) a DP scaffold on the surface of the NIH/3T3 monolayer; (j–l) phase contrast microscopy; (m–o) fluorescence LIVE/DEAD; scale bar = 100 µm.

Figure 3

(a–c) Isolated healthy rat islets of Langerhans: (a) phase contrast microscopy; (b) dithizone staining; (c) islets cultured for 24 h, acridine orange and propidium iodide (AO/PI) fluorescent staining; (d) functional activity of isolated healthy rat islets of Langerhans cultured for 24 h; (e,f) islets seeded on DP scaffold: (e) phase contrast microscopy; (f) AO/PI fluorescent staining; scale bar = 100 µm.

Figure 4

(a–c) A pancreas of a healthy rat; (d–f) a pancreas of a rat from the control group with streptozotocin-induced T1DM, 0 week; (g–i) a pancreas of a rat from the control group with streptozotocin-induced T1DM, 10 weeks; (j–l) a pancreas of a rat from experimental group 1 with streptozotocin-induced T1DM after an intraperitoneal injection of the islets of Langerhans; (m–o) a pancreas of a rat from experimental group 2 with streptozotocin-induced T1DM after an intraperitoneal injection of the islets of Langerhans with a scaffold; (a,d,g,j,m) H&E staining; (b,e,h,k,n) insulin immunohistochemical staining; (c,f,i,l,o) glucagon immunohistochemical staining; scale bar = 100 µm.

Figure 5

The dynamics of blood glucose levels in rats with streptozotocin-induced T1DM from the control (without treatment) and experimental groups after an intraperitoneal injection of the islets of Langerhans or the islets of Langerhans seeded on a DP scaffold. ^∆ p < 0.05: control group vs. experimental group 1; ^# p < 0.05: control group vs. experimental group 2; * p < 0.05: experimental group 1 vs. experimental group 2.