Abstract
The development of a bioartificial pancreas (BAP) with immunoisolating fashion has been gaining attention as a new method for treating diabetes. We have been proceeding with the development of a bag-type BAP that can be easily implanted and that allows for the optional injection or rejection of cells at any time. If fibrosis develops around a BAP device, then the permeability of substances transmitted through a semipermeable membrane will decrease, thereby reducing the reactivity with glucose, so it is necessary for the material of the device to have an excellent histocompatibility. Furthermore, in order to improve the efficacy of BAP treatment, it is important to maintain an environment of ample blood flow around the device. We have created a bag-type device for BAP that is 20 × 20 mm in size and comprises two layers of membranes. We have used an EVAL membrane for the outer membrane of the two layers. The EVAL membrane is a semipermeable membrane with good insulin permeability, which functions as an immunoisolation membrane. The inner membrane consists of PAU-coated HD-PE (nonwoven material processed with polyaminourethan) and it is designed to function as a scaffold for cells. We used Lewis rats to determine whether the effectiveness of fibroblast growth factor 2 (bFGF) can be improved by concomitantly using bFGF with a capacity for blood vessel regeneration as well as bFGF immersed in a sheet of gelatin. We placed the BAP in the abdominal cavity and covered it with the greater omentum. We were able to significantly increase the blood flow and the number of new blood vessels in the tissue surrounding the BAP device by using gelatinized bFGF. There were only a few instances of fibrosis as a biological reaction to the EVAL membrane, and the infiltration of inflammatory cells was mild. There were no adverse effects related to implantation of the device. We confirmed in this study that the use of an implantable BAP device and bFGF allowed for a better blood flow around the BAP device. There were only minor instances of fibrosis and inflammation reaction around the BAP, thus indicating the BAP that we are currently developing to have an excellent histocompatibility.
Introduction
In recent years, the implantation of pancreatic islets as treatment for diabetic patients has been attracting attention worldwide as an effective treatment and it has so far been used at many facilities (15,20,22). The rate of withdrawal of insulin by 1 year after implantation of pancreatic islets is reportedly 44%, and the performance of pancreatic islets implants has been improving over the past several years (12,13,21). Possible reasons include multiple implantations of pancreatic islets in a single patient and improvements in the use of immunosuppressive drugs. However, from a long-term perspective, the state of insulin withdrawal is not always maintained. Due to immune reactions, inflammation reactions, and side effects caused by immunosuppressive drugs in the chronic phase after an implantation, the functionality of an implanted pancreatic islets decrease (9,16,23). Furthermore, the use of immunosuppressive drugs not only has adverse effects on the implanted pancreatic islets but it also decreases the immune function of the patient and increases their susceptibility to infections and carcinogens (3,4). In order to solve these problems, the research and development of an artificial pancreas has been proactively pursued. The types of bioartificial pancreas (BAP) that have been reported thus far include an artificial blood vessel type, a microcapsule type, and a macrocapsule type (2,6–8,14,17,18,24). The BAP that we have been seeking to develop may be classified as a macrocapsule type.
One advantage of our BAP is that the use of semipermeable membranes maintains glucose, insulin, and oxygen permeability while also maintaining an immunoisolation state, because cells, antibodies, and complements related to immune reactions cannot permeate the semipermeable membranes. Therefore, it is not necessary to administer any immunosuppressive drugs. In addition, if fibrosis develops around a BAP device after the implantation of the BAP, then the permeability of substances passing through the semipermeable membrane decreases, thereby reducing the reactivity with glucose and further lowering insulin or oxygen permeability. Therefore, the use of a material with a good histocompatibility for the BAP device has made it possible to maintain functionality over long periods without the development of fibrosis around the BAP. In addition, another advantage is that it is equipped with a port for injecting cells to allow for the collecting and refilling of cells when the functionality of the insulin-secreting cells filling the BAP decreases(5,10).
The most important aspect for improving the performance of BAP treatment is the peripheral blood flow environment. We therefore focused on the greater omentum, which has an abundant blood flow. We devised a scheme for maintaining a favorable blood flow environment around the BAP by covering the BAP with the greater omentum. We also looked into increasing the blood flow by concomitantly using basic fibroblast growth factor 2 (bFGF) with blood vessel regeneration capacity (1,19). Because the bFGF has a short half-life, we also examined whether the effectiveness of bFGF can be improved by using bFGF immersed in a sheet of gelatin to add sustained release.
Materials and Methods
Method of Constructing a BAP Device
We created a bag-type device for BAP that is 20 × 20 mm in size and comprises two layers of membranes. We used an EVAL membrane for the outer membrane of the two layers. The EVAL membrane is a semipermeable membrane with good insulin permeability and it functions as an immunoisolation membrane. The inner membrane consists of PAU-coated HD-PE (nonwoven material processed with polyaminourethan) and is designed to function as a scaffold for cells. We also attached a port to this bag-type device for injecting cells in order to fill it with cells.
Treatment of the BAP Device with bFGF
After immersing 20 × 20-mm sheets of gelatin with bFGF (50 μg), the sheets were placed on both surface sides of the BAP device (Fig. 1).
Preparation of gelatinized bFGF for applying the BAP device. (A) Gelatin sheets of 20 × 20-mm in size were immersed with bFGF (50 μg). (B) The sheets were placed on both surface sides of the BAP device.
Animal Experiments
We used Lewis rats for this study. Under ether anesthesia, we made a small incision that was approximately 20 mm in size in the middle of the rats' abdomen and opened the abdominal cavity. We then placed the BAP device in the abdominal cavity and covered it with the greater omentum (Fig. 2A). After closing the abdominal rectus muscle using sutures, we guided and secured a port for injecting cells subcutaneously and then closed the skin by sewing it up (Fig. 2B, C). We performed the following two experimental groups: group 1: BAP independent (without bFGF) implant group and group 2: bFGF-covered BAP implant rat group. Body weight of the rats was 280.5 ± 1.9 g (mean ± SD) in group 1 and 269.0 ± 3.6 g in group 2.
Procedure of an implantation of the BAP device. (A) The BAP device was implanted in the abdominal cavity and covered with the greater omentum. (B) After closing the abdominal rectus muscle we guided and secured a cell injection port subcutaneously. (C) The skin was closed.
Measurement of Blood Flow in the Tissue Surrounding the BAP Device
For both groups, we opened the abdomen 2 weeks after the implantation of the BAP devices and measured the blood flow in the tissues using a laser doppler tissue blood flow imager (Advance, Tokyo, Japan) and quantification was determined using Laser FlowGraphy (LFG-1 version 1.0, Softcare).
Histological Examination
For both groups, 2 weeks after implanting the BAP devices, we removed them with the surrounding tissues. These samples were stained with hematoxylin and eosin (H&E) and Elastica van Gieson (EVG) stains to identify the fibrils of elastin in the blood vessels. The number of the induced blood vessels around the BAP device was counted in ten different fields of view.
Statistical Analyses
Mean values are presented with SDs. A Student's t-test was used to calculate the significance of difference in mean values. A value of p < 0.05 was considered statistically significant.
Results
Use of bFGF Increased Blood Flow in the Tissue Surrounding the BAP Device
Two weeks after implanting the BAP devices, we removed them with the surrounding tissues. We measured the skin blood flow and the blood flow in the BAP implant sites by using a laser blood-flow meter, and we calculated the ratio of BAP site/skin blood flow. Group 1 was 72.6 ± 1.7%, while group 2 was 89.4 ± 3.4% (Fig. 3A). The use of bFGF increased blood flow at the BAP implant site.
Evaluation of neovascularization around the BAP device. (A) Two weeks after BAP device implantation, we removed them with the surrounding tissues. At the time of removal, we carefully observed the status of vascularization around the devices and then measured blood flow by using a laser blood-flow meter. The ratio of BAP site/skin blood flow was 72.6 ± 1.7% for BAP only (group 1) and 89.4 ± 3.4% for BAP + bFGF (group 2). The use of bFGF significantly increased the blood flow at the BAP implant site. (B) The samples were stained with H&E and EVG stains to identify the blood vessels. The number of the induced blood vessels around the BAP device was counted in 10 different fields of view. bFGF treatment significantly increased the newly induced blood vessels.
bFGF Treatment Increased the Number of New Blood Vessels around the BAP Device
After formalin fixation of the extracted BAP and the surrounding tissue, we dyed them with EVG to identify the blood vessel fibrils of elastin, and we observed them at a magnification of ?40 to measure the number of blood vessels per field of view. Group 1 was 41.5 ± 3.0, while group 2 was 52 ± 4.28, thus indicating an increase in the number of blood vessels around the BAP (Fig. 3B).
The Biocompatibility of the EVAL Membrane Was Excellent
In both group 1 and group 2, there were no instances of fibrosis as a biological reaction to the EVAL membrane of the BAP surface, and the infiltration of inflammatory cells was not observed at all. The sheets of gelatin were completely absorbed by the body and disappeared (Fig. 4). There were also no adverse effects, such as the formation of abscesses or skin necrosis, in either group. These findings indicated that biocompatibility of the BAP device was excellent.
Biocompatibility of the BAP device. In both group 1 and group 2, there were no instances of fibrosis as a biological reaction to the EVAL membrane of the BAP surface, and the infiltration of inflammatory cells was not observed at all. The sheets of gelatin were completely absorbed by the body and disappeared. These findings indicated that biocompatibility of the BAP device was excellent.
Discussion
The measurements of the laser blood-flow meter indicated that the bFGF concomitant use group observed an increase in the blood flow of approximately 20% in comparison to the BAP independent implantation group. In addition, similar results were obtained in the measurements of the number of blood vessels by EVG staining study. We therefore clarified that the use of bFGF enables the blood flow around the BAP device to increase.
Generally, when a foreign object is implanted into a body, inflammation occurs and fibroblasts proliferate around the foreign object. If fibrosis occurs around the BAP device, then insulin-secreting cells in the BAP become damaged, because the ability to modulate blood glucose decreases due to glucose and insulin permeability, and oxygen permeation becomes difficult. There were no instances of fibrosis around the BAP devices, and the inflammation reaction was not observed, thus suggesting that the EVAL membrane that was used as an immunoisolation membrane of the BAP device had a good histocompatibility and that it may be possible to maintain its functionality over a long period after BAP implantation. In the future, we plan to examine the long-term biocompatibility of the BAP device. In addition, because favorable blood vessels were induced around the BAP device at 2 weeks after the implantation, it would be an appropriate time to inject insulin-secreting cells into the device. The advantage of our BAP device includes an aspect that it is easy to implant and easy to remove. In contrast, the macrocapsule type BAP devices, such as ours, are generally inferior to the microcapsule type ones in terms of oxygen permeability and blood glucose response. In this study, we succeeded in inducing the formation of excellent blood vessels by the use of gelatinized bFGF. Now we plan to first evaluate the performance of syngeneic rat islet transplantation as a source of insulin-secreting cells in our BAP device. Such experiments will provide important clues to create an optimum environment outside and inside the BAP device, while ignoring any immunological reactions. In order to maintain cell function over a long period, it is important to integrate cell matrix biology technologies into the BAP device development (11).
The ultimate aim of this study is to facilitate BAP therapy in diabetic patients using genetically modified artificial cells or xenogenic pancreatic islets or by utilizing its immunoisolation capacity, which is the greatest advantage of BAP. Once when we confirm that xenogenic pancreatic islets can be used as a cell source of BAP, the issue of a shortage of donors, which is one of the most serious issues that we are currently facing in transplantation, can be greatly addressed. Furthermore, when a method of inducing functional differentiated cells from embryonic stem cells or induced pluripotent cells can be developed in the near future, the use of such differentiated insulin-secreting cells will be an attractive source for immunoisolatory BAP, while ignoring the fear of malignant formation of the cells.