Journal of Diabetes Research

Journal of Diabetes Research / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 7262680 |

J. Matthew Rhett, Hongjun Wang, Heather Bainbridge, Lili Song, Michael J. Yost, "Connexin-Based Therapeutics and Tissue Engineering Approaches to the Amelioration of Chronic Pancreatitis and Type I Diabetes: Construction and Characterization of a Novel Prevascularized Bioartificial Pancreas", Journal of Diabetes Research, vol. 2016, Article ID 7262680, 12 pages, 2016.

Connexin-Based Therapeutics and Tissue Engineering Approaches to the Amelioration of Chronic Pancreatitis and Type I Diabetes: Construction and Characterization of a Novel Prevascularized Bioartificial Pancreas

Academic Editor: Urs Christen
Received08 May 2015
Revised31 Jul 2015
Accepted18 Aug 2015
Published14 Dec 2015


Total pancreatectomy and islet autotransplantation is a cutting-edge technique to treat chronic pancreatitis and postoperative diabetes. A major obstacle has been low islet cell survival due largely to the innate inflammatory response. Connexin43 (Cx43) channels play a key role in early inflammation and have proven to be viable therapeutic targets. Even if cell death due to early inflammation is avoided, insufficient vascularization is a primary obstacle to maintaining the viability of implanted cells. We have invented technologies targeting the inflammatory response and poor vascularization: a Cx43 mimetic peptide that inhibits inflammation and a novel prevascularized tissue engineered construct. We combined these technologies with isolated islets to create a prevascularized bioartificial pancreas that is resistant to the innate inflammatory response. Immunoconfocal microscopy showed that constructs containing islets express insulin and possess a vascular network similar to constructs without islets. Glucose stimulated islet-containing constructs displayed reduced insulin secretion compared to islets alone. However, labeling for insulin post-glucose stimulation revealed that the constructs expressed abundant levels of insulin. This discrepancy was found to be due to the expression of insulin degrading enzyme. These results suggest that the prevascularized bioartificial pancreas is potentially a tool for improving long-term islet cell survival in vivo.

1. Introduction

Chronic pancreatitis is a serious inflammatory condition resulting in fibrotic replacement and loss of function of the pancreas. In addition to causing severe pain and discomfort for the patient, left untreated, chronic pancreatitis will result in the inability to produce insulin and subsequent diabetes [1]. Total pancreatectomy and islet autotransplantation (TP-IAT) is a state-of-the-art procedure to treat the intractable pain in patients with chronic pancreatitis and to ameliorate postoperative diabetes [2]. It involves removal of the pancreas, enzymatic digestion of the extracellular matrix, purification of the β-cell-containing islets, and reintroduction of the islets to the patient by infusion into the hepatic portal vein, after which the islets take up residence in the liver [2]. However, more than half of islet cells die immediately after intraportal infusion [3, 4]. Although the quality of life is significantly improved in many patients, only about 1/3 of them become insulin independent, 1/3 require minimal insulin replacement (<10 u/day), and the rest develop pancreatogenic diabetes after surgery due to low islet cell survival [2]. Furthermore, even in patients that become insulin independent, many patients become dependent within a period of years [5, 6].

Type 1 diabetes is an autoimmune disease defined by pancreatic β-cell death, resulting in insulin deficiency and hyperglycemia [7]. Improved islet cell survival would also beneficially impact allogenic islet transplantation therapy for type 1 diabetes. Unfortunately, pancreatic islet allotransplantation is currently labeled an experimental procedure in the US, and therefore it is generally not covered by health insurance or Medicare [8]. Improving islet cell survival would help to transition the procedure from an experimental to a therapeutic status by increasing the successfulness of the technique.

This issue underlies a common basic challenge facing tissue engineers, namely, the survival of implanted cells and living tissue engineered constructs (TECs) in vivo. Implanted devices, cells, and exogenous tissues are often rejected by the patient’s body [9]. This can result in serious medical consequences such as physical disfigurement and discomfort, device malfunction, the need for implant removal and tissue reconstruction, and, in the case of TP-IAT, “brittle” diabetes [1013]. A major factor contributing to poor implanted cell survival is the common host response to implanted devices, TECs, or cells which initially involves coagulation and inflammation, including the infiltration of neutrophils within minutes of the initial injury [14]. Neutrophils are well documented to cause collateral tissue damage during the wound healing response [1518] and to correlate with increased capsule size and contractility in response to a silicone implant [19]. Importantly, neutrophil infiltration of intraportally introduced islets during the immediate blood mediated inflammatory reaction (IBMIR) has long been documented [2022]. Additionally, islets can embolize and cause liver necrosis, generating further inflammation [4].

Extracellular ATP (purinergic signaling) is increasingly being recognized as a key proximate signal for inflammation following tissue injury [23, 24] and a major conduit for these signals is connexin proteins [25]. The gap junction (GJ) protein connexin43 (Cx43) is the most ubiquitously expressed isoform of connexin [26]. Cx43 monomers oligomerize to form half-channels (called hemichannels) that are delivered to the plasma membrane [27, 28]. Cx43 hemichannels have been demonstrated to mediate ATP release in a number of scenarios and a number of cell types including vascular endothelial cells [2935], making hemichannels a prime suspect in the origin of injury-related extracellular ATP signals. Importantly, it has also been shown that purinergic signaling directly activates the clotting response during IBMIR [36].

We and others have shown that manipulation of Cx43 is an effective means to reduce inflammation, accelerate healing, and reduce scarring and capsule formation [15, 1719]. These studies have culminated in recent efficacious clinical trials for the Cx43 mimetic peptide ACT1 in healing chronic venous leg ulcers and diabetic leg ulcers [37, 38]. We have developed another Cx43 peptidomimetic, named JM2, based on the JuxtaMembrane microtubule binding motif on the Cx43 carboxy-terminus. We have recently demonstrated that JM2 blocks ATP release and strongly inhibits the inflammatory response to a silicone implant in an ATP-dependent manner [39]. Importantly, the Cx43 microtubule binding motif is not conserved between connexin isoforms, making JM2 a specific inhibitor of the Cx43 isoform [40]. Pancreatic β-cells solely express Cx36 and are therefore hypothesized to be unaffected by JM2, as opposed to pan-connexin channel blockers that could potentially affect insulin production [41].

Once the initial events of the foreign body response have been negotiated, long-term survival and engraftment of TECs requires host support of the implanted tissue. As stated by Fisher and colleagues in a recent Tissue Engineering review article [42], “Complete vascularization of engineered tissues is currently a major hurdle in the field of tissue engineering, inhibiting successful postimplantation viability.” Recently, we have applied a fundamentally different methodology to developing prevascularized tissues. Specifically, 3D endothelial cell-fibroblast constructs were generated in a scaffold-free environment by seeding cells in a nonadherent agarose mold [43, 44]. Importantly, these constructs displayed biophysical properties and histological characteristics of vascular networks, and we are here adopting the nomenclature of self-organizing prevascularized endothelial-fibroblast constructs (SPECs). Crucially, we have recently found that SPECs rapidly anastomose with host vasculature within 3 days and promote tissue repair [45]. In this study, we have combined the two separate, but proven, SPEC and JM2 technologies in a novel configuration to create a prevascularized bioartificial pancreas in order to address a critical need in the treatment of chronic pancreatitis and type 1 diabetes.

2. Materials and Methods

2.1. Cell Culture and Treatments

Cells were cultured as previously described [44]. Adult normal human dermal fibroblasts (NHDFs) were purchased from Lonza (Walkersville, MD), and human adipose microvascular endothelial cells (HAMECs) were purchased from ScienCell (Carlsbad, CA). The endothelial cells were grown in Medium 200 (M200; Gibco) supplemented with low serum growth supplement (LSGS; Gibco) and 1% penicillin streptomycin solution (PS; Cellgro). Fibroblasts were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (FBS; Corning), and 1% PS.

JM2 peptide was generated by Petron Inc. (Daejeon, Korea) and was composed, from N-terminus to C-terminus, of a biotin tag, an 8-mer poly-D-arginine internalization vector [46], and the Cx43 C-terminal amino acids 231–245 (VFFKGVKDRVKGRSD) that encompass the microtubule binding sequence (amino acids 234–243 [40]), biotin-rrrrrrrr-VFFKGVKDRVKGRSD. JM2 was solubilized in sterile distilled H2O and used at a final concentration of 50 μM. Flufenamic acid (FFA) was purchased from Abcam (Cambridge, MA), diluted in ethanol, and used at a concentration of 50 μM.

2.2. Islet Isolation

Islets were isolated from C57BL/6 mice as previously described [47, 48]. Briefly, mice were anesthetized, and the pancreas was perfused with collagenase (type V, 0.6 mg/mL, Sigma Aldrich, St. Louis, MO) through the pancreatic ducts. The dissected enzyme-containing pancreas was then incubated at 37°C with constant shaking to release the islets. Density gradient separation was used to isolate the islets, which were cultured in vitro in Dulbecco’s Modified Eagles Medium (DMEM; Gibco, Grand Island, NY) plus 10% of fetal bovine serum (Corning, Tewksbury, MA) at 37°C, 5% CO2 on low attachment cell culture plates (Corning).

2.3. SPEC Fabrication

SPECs were created as previously described [4345]. Specifically, 7.2 × 105 NHDFs and 1.8 × 105 HAMECs were combined and seeded into nonadherent agarose troughs 9 mm long × 1 mm wide × 1 mm deep. In the case of islet-containing SPECs, 50 islets were combined with the fibroblasts and endothelial cells prior to seeding. The suspensions were cultured for 3 days in a 1 : 2 mixture of M200 (Gibco) media plus LSGS (Gibco) : DMEM (Gibco) plus 10% fetal bovine serum (Corning).

2.4. Microscopic Analysis

After 3 days in culture SPECs were either fixed in 4% paraformaldehyde for 1 hr, or cyroembedded in Tissue-Tek O.C.T. Compound (VWR, Radnor, PA). Cryoembedded SPECs were sectioned at 10 μM and fixed in 2% paraformaldehyde. Fixed sections and whole-mount SPECs were labeled with antibodies against insulin (Sigma Aldrich, Cat. number I2018) and CD31 (Abcam, Cat. number ab28364) and labeled for actin using phalloidin (Life Technologies, Grand Island, NY, Cat. number A22284), and the nucleus was labeled with Hoechst stain (Life Technologies, Cat. number H3570). Whole-mount SPECs were imaged on a TCS SP5 laser scanning confocal microscope (LSCM) equipped with 20x/0.75 numerical aperture (NA) and 63x/1.2 NA water objectives (Leica, Buffalo Grove, IL). Sectioned SPECs were also imaged on a TCS SP5 LSCM equipped with a 20x/0.70 NA dry objective and a 63x/1.4 NA oil objective. All figures are maximum projections of z-stacks.

2.5. Glucose-Stimulated Insulin Secretion (GSIS) Assay

GSIS assay was performed as previously described [47]. Islets alone, SPECs, and SPECs containing islets were first equilibrated in Krebs buffer (25 mM HEPES, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 0.1% BSA, and 2.5 mM CaCl2) with low glucose (2.8 mM) and connexin inhibitors (JM2 or FFA, both used at 50 μM) or vehicle for 2 hrs. The islets/constructs were then treated with Krebs buffer with low glucose (2.8 mM) for 1 hr. The buffer was then collected and replaced with Krebs buffer with high glucose (28 mM) for a second hour, and the buffer was again collected. SPECs with and without islets were then fixed in 4% paraformaldehyde, labeled, and imaged as above. The concentration of insulin released into the buffer was measured using a STELLUX Chemi Rodent Insulin ELISA kit (ALPCO, Salem, NH).

2.6. Western Blot

SPECs were homogenized in 250 μL of tricine sample loading buffer (BioRad, Hercules, CA) by shearing with progressively smaller gauge needles beginning with 20 g and ending with 26 g. Confluent cultures of NHDFs and HAMECs were lysed in an equivalent volume of loading buffer using a cell scraper. Samples were resolved by 4–15% SDS-PAGE (BioRad), followed by immunoblotting for insulin degrading enzyme (Abcam, Cat. number 25733) and α-tubulin (Sigma Aldrich, Cat. number T8203).

2.7. Statistics

Statistics were performed in Prism 5, Version 5.0f (GraphPad Software, Inc., La Jolla, CA). Two-way ANOVA with Bonferroni post hoc test was used to compare the interaction effect between glucose concentration and treatment on insulin secretion, and direct comparisons of the concentration of secreted insulin within treatment groups from low to high glucose were made by paired -tests. One-way ANOVA with Bonferroni post hoc test was used to compare the fold change in insulin secretion from low to high glucose.

3. Results

3.1. Insulin Expression and Vascular Network Formation Are Maintained in Islet-Containing SPECs

Our first goal was to determine the effects of seeding islets together with endothelial cells (HAMECs) and fibroblasts (NHDFs) as previously described to fabricate SPECs [43]. Specifically, we wanted to determine if (a) the islets would be included or excluded from the SPECs and (b) if inclusion of islets would affect vascular network formation, as it has been shown that altering the ratio of HAMECs : NHDFs affects the vascular network [44]. To that end, we constructed SPECs with the previously determined optimal ratio of 1 : 4 HAMECs : NHDFs, and in half of the SPECs we added 50 islets per construct. These constructs were then fixed and labeled for insulin as a marker for islets [47] and CD31 as a marker for endothelial cells [49]. In whole-mount SPECs, insulin-expressing islets were observed to be embedded in the exterior of the SPECs (Figure 1). Furthermore, we also found islets in the interior of SPECs in fresh-frozen tissue sections (Figure 2). Importantly, a vascular network was discernable on the surface of SPECs both with and without islets (Figure 1). In tissue sections, this network could be seen extending to the interior of SPECs irrespective of the presence of islets (Figure 2). Taken together, these results demonstrate that SPECs incorporate islets without affecting vascular network formation, and the β-cells in the incorporated islets continue to express insulin.

3.2. Prevascularized Bioartificial Pancreases Display Reduced Insulin Secretion

We next tested whether islet-containing SPECs, or prevascularized bioartificial pancreases, secreted insulin and responded to high glucose challenge by GSIS assay. Islets alone, SPECs without islets, and islet-containing SPECs pretreated for 2 hrs with ether vehicle, 50 μM JM2, or 50 μM FFA were first subjected to low glucose (2.8 mM) for 1 hr and then high glucose (28 mM) for a second hour. Following high glucose challenge, the constructs were fixed and the low and high glucose buffers were analyzed for insulin concentration by ELISA. Islets alone displayed normal function, producing nanogram levels of insulin per islet (Figure 3(a)) and responding to high glucose by significantly doubling insulin secretion (Figures 3(a) and 3(c)). In contrast, the prevascularized bioartificial pancreases secreted very low levels of insulin that were significantly less than islets alone (Figure 3(a)). Surprisingly, islet-containing SPECs were not only unresponsive to high glucose but even displayed a trend toward reduced insulin secretion (Figure 3(b)), although this effect was not statistically significant. However, when compared to islets alone, the response to high glucose by islet-containing SPECs was significantly reduced (Figure 3(c)).

In order to gain further insight, we limited the statistical comparisons to prevascularized bioartificial pancreases. Two-way ANOVA revealed that there was a significant effect by treatment group. Consistent with this, we noted that FFA appeared to further suppress insulin secretion relative to vehicle and JM2-treated islet-containing SPECs. However, Bonferroni post hoc analysis failed to confirm that this was a statistically significant effect (Figure 3(b)). These data suggest that insulin expression or secretion by islets incorporated in prevascularized bioartificial pancreases was inhibited. Alternatively, the fibroblast and endothelial cells of the SPEC may have sequestered or degraded the secreted insulin.

3.3. Insulin Expression Is Maintained in JM2-Treated, Prevascularized Bioartificial Pancreases

We explored the observed lack of secreted insulin by immunolabeling the SPECs containing islets and SPECs without islets subjected to GSIS assay. As above, the constructs were labeled with antibodies against insulin and CD31 to identify islets and endothelial cells, respectively. All constructs displayed an intact vascular network (Figure 4), indicating that neither the GSIS assay nor the connexin inhibitors affected maintenance of the vascular network. Unexpectedly and in contrast to the level of insulin secretion, insulin expression by islets in vehicle and JM2-treated prevascularized bioartificial pancreases was comparable to constructs imaged without being subjected to GSIS assay (Figure 4). To confirm that this level of insulin expression was comparable to free islets, sectioned islet-containing SPECs and free islets were labeled for insulin, and no differences were observed (Supplemental Figure  1) (see Supplementary Material available online at In contrast, consistent with the trend toward reduced insulin secretion observed for FFA-treated prevascularized bioartificial pancreases, FFA-treated constructs displayed reduced insulin expression post-GSIS assay (Figure 4).

Due to the reduced insulin secretion observed in islet-containing SPECs (Figure 3) and findings that show that while Cx36 is the predominant isoform Cx43 is also expressed in islets [50], we sought to directly test whether or not JM2 and FFA affect insulin secretion. As such, free islets pretreated with JM2 and FFA were subjected to GSIS assay. We found that vehicle and JM2-treated islets responded normally to high glucose, approximately doubling their insulin secretion (Supplemental Figure  2). Consistent with the results of the GSIS assay performed on islet-containing SPECs and the associated post-GSIS assay labeling, FFA failed to increase insulin secretion in response to high glucose (Supplemental Figure  2). Also consistent with the results of the GSIS assay performed on islet-containing SPECs, two-way ANOVA revealed that there was a significant effect by treatment group. Together, these results indicated that neither the presence of islets, GSIS assay, nor connexin inhibitors affected vascular network formation. However, pan-connexin blockers, like FFA, inhibit insulin expression and/or secretion, while the targeted inhibitor JM2 did not. Additionally, incorporation of islets into SPECs inhibited insulin secretion.

3.4. SPECs Express Insulin Degrading Enzyme

Notably, we did not detect insulin sequestered or “trapped” within the body of islet-containing SPECs. In combination with the data indicating that insulin expression was not affected by vehicle or JM2 treatment in islet-containing SPECs (Figure 4 and Supplemental Figure  1) and insulin secretion was not affected by vehicle or JM2 treatment in free islets (Supplemental Figure  2), this suggested that insulin may be degraded in insulin-containing SPECs. Therefore, we tested for the presence of insulin degrading enzyme in SPECs. To this end, SPECs were homogenized in sample loading buffer and 2D cultures of HAMECs and NHDFs were lysed in sample loading buffer. Western analysis of the samples showed that insulin degrading enzyme was robustly expressed in both cultures of primary cells as well as the SPEC constructs themselves (Figure 5). These results indicate that insulin secreted by islets embedded in SPECs may be degraded by insulin degrading enzyme expressed by the other component cells of the construct.

4. Discussion

To our knowledge, this is the first demonstration of a prevascularized artificial organ constructed with both parenchymal cells and stromal cells using scaffold-free technology (Figures 1 and 2). The novelty of this finding stems directly from the delicate and highly orchestrated nature of vascular network formation. A number of studies have shown that the formation of a vascular network is dependent on the combination of the presence of a balanced cocktail of growth factors with an appropriate environmental context of specific extracellular matrix molecules and stromal cells [44, 5154]. Therefore it would have been unsurprising if the addition of parenchymal cells negatively impacted the ability of the construct to form vascular networks.

This is a particularly important finding in the context of transplanted islet cells. In vivo, pancreatic islets are normally highly vascularized structures, which is crucial for proper function [55]. Using current protocols, transplanted islets may not become vascularized for a period of days to weeks [56]. Importantly, the robust SPEC vascular network displayed a resemblance to the vasculature of pancreatic islets in vivo (e.g., compare Figure 1 with the live, in vivo imaging of Powers and coworkers [57]). Given the ability of SPECs to rapidly anastomose with host vasculature [45], this work represents a major step forward in developing the ability to more effectively transplant isolated islets.

Equally important was the finding that connexin inhibitors did not affect the integrity of the already formed microvasculature (Figure 4). This was somewhat unexpected in that connexins are important in cell migration and vascular network formation [5860]. However, the SPECs were only exposed to JM2 and FFA for a relatively short period of time (2 hrs), and it is unknown if longer exposures would have a different effect on the vascular network. We and others have previously shown that targeted inhibition of Cx43 channels improves wound healing and the response to implanted materials [15, 18, 19, 38, 39, 61]. These effects appear to be mediated through dampening of purinergic signals that attract neutrophils to injury sites [25, 39]. The significance of this observation is that only a limited window of exposure to connexin-based therapeutics is necessary to reduce tissue damage caused by the innate inflammatory response. Therefore, our results suggest that the acute treatment of an implant site with targeted Cx43 inhibitors could improve integration of artificial tissues (such as the islet-containing SPECs described herein) without disrupting a preformed vascular network.

In addition to a lack of effect on vascular network formation, the targeted Cx43 inhibitor JM2 did not affect insulin expression. In contrast, reduced insulin levels were observed in FFA-treated SPECs (Figures 3 and 4 and Supplemental Figure  2). This highlights a benefit of targeted connexin inhibitors such as JM2 and ACT-1. Namely, while pan-connexin inhibitors like FFA and mefloquine do inhibit hemichannel function and dampen the inflammatory response [39, 62], they can also have unintended side effects such as effects on insulin expression [41, 63, 64]. Connexin-mimetic peptides such as JM2 and ACT-1 are short-lived and have limited side effects even when administered systemically [65, 66].

Unexpectedly, we found that islets incorporated in SPECs displayed reduced insulin secretion (Figure 3). Labeling of the constructs post-GSIS assay suggest that the constructs themselves were not sequestering insulin secreted by the islets because insulin labeling was restricted to the islets (Figure 4). This left the possibilities that the other cells in the construct degraded the insulin, or that insulin secretion was inhibited by interactions between the β-cells of the islets and the cells making up the rest of the SPEC. Insulin degrading enzyme has been shown to be expressed by a number of cell types including endothelial cells [67]. Importantly, it has been localized to the cell surface of cerebrovascular endothelial cells [68] and is secreted by a number of other cell types [69]. Consistently, our results show that both the endothelial cells and fibroblasts used in constructing the SPECs robustly express insulin degrading enzyme, and this expression is maintained in assembled SPECs (Figure 5). Therefore, it is likely that the apparent level of insulin secreted by our prevascularized bioartificial pancreases was low due to degradation by insulin degrading enzyme expressed by the endothelial cells and/or fibroblasts in the construct.

Alternative strategies to improving insulin secretion may therefore need to be implemented before our prevascularized bioartificial pancreas technology can be implemented. One such approach would involve gene targeting in order to knock down the expression of insulin degrading enzyme. For example, the CRISPR/Cas9 system is a new technology that has the power to rapidly and efficiently edit the genome of cultured cells in a targeted manner [70], and this technique could be employed to block the expression of insulin degrading enzyme in our constructs.

Another strategy may be to employ different cell types and/or a higher ratio of islets to construct cells. A similar approach to ours has been previously attempted by Johansson et al. in which isolated islets were “coated” with endothelial cells and mesenchymal stem cells, and the constructs were found to secrete insulin at the same level as control free islets [71]. In Johansson and coworker’s study mesenchymal stem cells and dermal endothelial cells were used as opposed to dermal fibroblasts and adipose-derived endothelial cells in our study. It is possible that mesenchymal stem cells and dermal endothelial cells express lower levels of insulin degrading enzyme than the NHDFs and HAMECs used in our study. In addition, the islets used in the Johansson et al. study were merely coated with the construct cells [71], while our islets were embedded in a larger construct. Therefore, even if the mesenchymal stem cells and endothelial cells used by Johansson et al. express levels of insulin degrading enzyme comparable to the cells in our constructs, there would be less insulin degrading enzyme present relative to the level of insulin produced in their constructs. Thus a possible solution to improving insulin secretion by our constructs would be to increase the number of islets incorporated per construct; however, there is likely a critical threshold of islets that can be embedded in each SPEC that will prevent vascular network formation and/or reduce the constructs’ anastomotic potential. This is a critical point because while Johansson and coworker’s constructs did appear to form “vascular sprouts,” our approach differs significantly in that the islets in our constructs are embedded in a much larger vascularized structure that has been demonstrated to anastomose with host vasculature when implanted [45]. Additionally, immunoisolation is enhanced in our prevascularized bioartificial pancreases by incorporating the anti-inflammatory peptide, JM2, which could be “loaded” into the constructs by pretreating the islet-containing SPECs prior to implantation [39].

Another aspect of the Johansson et al. study that differed from ours was the use of a dynamic perfusion GSIS assay [71]. By perfusing the constructs, the media is continuously sampled, and therefore insulin secreted by the their constructs may have been collected before it could be degraded by the construct cells. In the GSIS assay we employed, free islets or constructs were bathed in static high and low glucose buffers, which were collected at the end of an hour. Thus, our static conditions would cause any insulin secreted by our constructs to be continuously exposed to the insulin degrading enzyme-expressing construct cells during the hour-long incubations, resulting in degradation of the majority of the secreted insulin. If this is the case, then the constructs we developed may not need any modification because in vivo the job of perfusion would be carried out by the host vasculature. In other words, rapid anastomosis of the host vasculature with that of our prevascularized bioartificial pancreases would be sufficient to transport the insulin secreted by our constructs to the proper destination tissues before the insulin could be degraded.

The concept of the bioartificial pancreas has existed since the 1980s [72]. The principal behind the bioartificial pancreas is that islets encapsulated in a microsphere of hydrogel material (typically alginate) will be “immunoisolated” (i.e., protected from the immune system) but maintain the ability to take in oxygen and nutrients and secrete insulin [73]. However, clinical implementation of this technology has been hampered by lack of revascularization of these constructs [55]. The prevascularized bioartificial pancreases described herein provide a potential solution to this critical hurdle common to the tissue engineering field in general [42].

5. Conclusions

Connexin-based peptides are gaining headway as potential therapeutics with novel applications from the prevention of arrhythmia to cancer treatments [18, 37, 38, 61, 65, 66, 74, 75]. We have here combined it with a novel technology to promote vascularization and perfusion of implanted tissue engineered constructs and isolated islets to create a prevascularized bioartificial pancreas. These constructs may eventually provide a more viable alternative to current islet transplant procedures to meet an urgent need for long-term solutions to type 1 diabetes and postoperative “brittle” diabetes subsequent to pancreatectomy.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


This work was supported by Grants from CTSA NIH UL1 TR000062 to J. Matthew Rhett and NIH NIDCR R01-DE019355 to Michael J. Yost. This study used the services of the Morphology, Imaging and Instrumentation Core, which is supported by NIH-NIGMS P30 GM103342 to the South Carolina COBRE for Developmentally Based Cardiovascular Diseases. The authors would also like to thank Caitlin Czajka for technical assistance.

Supplementary Materials

Figure 1: Demonstrates that insulin levels are similar in free islets and islets embedded in SPECs.

Figure 2: Shows that while the broad-spectrum connexin inhibitor flufenamic acid inhibits insulin secretion, JM2 does not affect insulin secretion in free islets.

  1. Supplementary Material


  1. C. E. Forsmark, “Management of chronic pancreatitis,” Gastroenterology, vol. 144, no. 6, pp. 1282–1291.e3, 2013. View at: Publisher Site | Google Scholar
  2. S. J. Kesseli, K. A. Smith, and T. B. Gardner, “Total pancreatectomy with islet autologous transplantation: the cure for chronic pancreatitis?” Clinical and Translational Gastroenterology, vol. 6, no. 1, article e73, 2015. View at: Publisher Site | Google Scholar
  3. M. Biarnés, M. Montolio, V. Nacher, M. Raurell, J. Soler, and E. Montanya, “β-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia,” Diabetes, vol. 51, no. 1, pp. 66–72, 2002. View at: Publisher Site | Google Scholar
  4. D. Yin, J. W. Ding, J. Shen, L. Ma, M. Hara, and A. S. Chong, “Liver ischemia contributes to early islet failure following intraportal transplantation: benefits of liver ischemic-preconditioning,” American Journal of Transplantation, vol. 6, no. 1, pp. 60–68, 2006. View at: Publisher Site | Google Scholar
  5. D. E. R. Sutherland, D. M. Radosevich, M. D. Bellin et al., “Total pancreatectomy and islet autotransplantation for chronic pancreatitis,” Journal of the American College of Surgeons, vol. 214, no. 4, pp. 409–424, 2012. View at: Publisher Site | Google Scholar
  6. T. Berney, T. Rudisuhli, J. Oberholzer, A. Caulfield, and P. Morel, “Long-term metabolic results after pancreatic resection for severe chronic pancreatitis,” Archives of Surgery, vol. 135, no. 9, pp. 1106–1111, 2000. View at: Publisher Site | Google Scholar
  7. J. Stone, D. Fitchett, S. Grover et al., “Canadian Diabetes Association 2013 clinical practice guidelines for the prevention and management of diabetes in Canada: vascular protection in people with diabetes,” Canadian Journal of Diabetes, vol. 37, supplement 1, pp. S100–S104, 2013. View at: Google Scholar
  8. NDIC, Pancreatic Islet Transplantation, 2013,
  9. J. M. Morais, F. Papadimitrakopoulos, and D. J. Burgess, “Biomaterials/tissue interactions: possible solutions to overcome foreign body response,” AAPS Journal, vol. 12, no. 2, pp. 188–196, 2010. View at: Publisher Site | Google Scholar
  10. S. M. Levine, M. E. Lester, B. Fontenot, and R. J. Allen Sr., “Perforator flap breast reconstruction after unsatisfactory implant reconstruction,” Annals of Plastic Surgery, vol. 66, no. 5, pp. 513–517, 2011. View at: Publisher Site | Google Scholar
  11. M. Gerritsen, A. Kros, J. A. Lutterman, R. J. M. Nolte, and J. A. Jansen, “A percutaneous device as model to study the in vivo performance of implantable amperometric glucose sensors,” Journal of Materials Science: Materials in Medicine, vol. 12, no. 2, pp. 129–134, 2001. View at: Publisher Site | Google Scholar
  12. T. F. Henriksen, J. P. Fryzek, L. R. Hölmich et al., “Surgical intervention and capsular contracture after breast augmentation: a prospective study of risk factors,” Annals of Plastic Surgery, vol. 54, no. 4, pp. 343–351, 2005. View at: Publisher Site | Google Scholar
  13. M. Dorlon, S. Owczarski, H. Wang, D. Adams, and K. Morgan, “Increase in postoperative insulin requirements does not lead to decreased quality of life after total pancreatectomy with islet cell autotransplantation for chronic pancreatitis,” American Surgeon, vol. 79, no. 7, pp. 676–680, 2013. View at: Google Scholar
  14. S. Franz, S. Rammelt, D. Scharnweber, and J. C. Simon, “Immune responses to implants: a review of the implications for the design of immunomodulatory biomaterials,” Biomaterials, vol. 32, no. 28, pp. 6692–6709, 2011. View at: Publisher Site | Google Scholar
  15. C. Qiu, P. Coutinho, S. Frank et al., “Targeting connexin43 expression accelerates the rate of wound repair,” Current Biology, vol. 13, no. 19, pp. 1697–1703, 2003. View at: Publisher Site | Google Scholar
  16. J. V. Dovi, L.-K. He, and L. A. DiPietro, “Accelerated wound closure in neutrophil-depleted mice,” Journal of Leukocyte Biology, vol. 73, no. 4, pp. 448–455, 2003. View at: Publisher Site | Google Scholar
  17. K. E. Degen and R. G. Gourdie, “Embryonic wound healing: a primer for engineering novel therapies for tissue repair,” Birth Defects Research Part C—Embryo Today: Reviews, vol. 96, no. 3, pp. 258–270, 2012. View at: Publisher Site | Google Scholar
  18. G. S. Ghatnekar, M. P. O'Quinn, L. J. Jourdan, A. A. Gurjarpadhye, R. L. Draugh, and R. G. Gourdie, “Connexin43 carboxyl-terminal peptides reduce scar progenitor and promote regenerative healing following skin wounding,” Regenerative Medicine, vol. 4, no. 2, pp. 205–223, 2009. View at: Publisher Site | Google Scholar
  19. B. L. Soder, J. T. Propst, T. M. Brooks et al., “The connexin43 carboxyl-terminal peptide ACT1 modulates the biological response to silicone implants,” Plastic & Reconstructive Surgery, vol. 123, no. 5, pp. 1440–1451, 2009. View at: Publisher Site | Google Scholar
  20. W. Bennet, B. Sundberg, C.-G. Groth et al., “Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation?” Diabetes, vol. 48, no. 10, pp. 1907–1914, 1999. View at: Publisher Site | Google Scholar
  21. D. J. van der Windt, R. Bottino, A. Casu, N. Campanile, and D. K. C. Cooper, “Rapid loss of intraportally transplanted islets: an overview of pathophysiology and preventive strategies,” Xenotransplantation, vol. 14, no. 4, pp. 288–297, 2007. View at: Publisher Site | Google Scholar
  22. B. Naziruddin, S. Iwahashi, M. A. Kanak, M. Takita, T. Itoh, and M. F. Levy, “Evidence for instant blood-mediated inflammatory reaction in clinical autologous islet transplantation,” American Journal of Transplantation, vol. 14, no. 2, pp. 428–437, 2014. View at: Publisher Site | Google Scholar
  23. B. McDonald, K. Pittman, G. B. Menezes et al., “Intravascular danger signals guide neutrophils to sites of sterile inflammation,” Science, vol. 330, no. 6002, pp. 362–366, 2010. View at: Publisher Site | Google Scholar
  24. Y. Chen, R. Corriden, Y. Inoue et al., “ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors,” Science, vol. 314, no. 5806, pp. 1792–1795, 2006. View at: Publisher Site | Google Scholar
  25. J. M. Rhett, S. A. Fann, and M. J. Yost, “Purinergic signaling in early inflammatory events of the foreign body response: modulating extracellular ATP as an enabling technology for engineered implants and tissues,” Tissue Engineering Part B: Reviews, vol. 20, no. 5, pp. 392–402, 2014. View at: Publisher Site | Google Scholar
  26. J. L. Solan and P. D. Lampe, “Connexin43 phosphorylation: structural changes and biological effects,” Biochemical Journal, vol. 419, no. 2, pp. 261–272, 2009. View at: Publisher Site | Google Scholar
  27. J. M. Rhett and R. G. Gourdie, “The perinexus: a new feature of Cx43 gap junction organization,” Heart Rhythm, vol. 9, no. 4, pp. 619–623, 2012. View at: Publisher Site | Google Scholar
  28. J. M. Rhett, E. L. Ongstad, J. Jourdan, and R. G. Gourdie, “Cx43 associates with Nav1.5 in the cardiomyocyte perinexus,” Journal of Membrane Biology, vol. 245, no. 7, pp. 411–422, 2012. View at: Publisher Site | Google Scholar
  29. M. V. L. Bennett, J. M. Garré, J. A. Orellana, F. F. Bukauskas, M. Nedergaard, and J. C. Sáez, “Connexin and pannexin hemichannels in inflammatory responses of glia and neurons,” Brain Research, vol. 1487, pp. 3–15, 2012. View at: Publisher Site | Google Scholar
  30. T. C. Clarke, O. J. S. Williams, P. E. M. Martin, and W. H. Evans, “ATP release by cardiac myocytes in a simulated ischaemia model: inhibition by a connexin mimetic and enhancement by an antiarrhythmic peptide,” European Journal of Pharmacology, vol. 605, no. 1–3, pp. 9–14, 2009. View at: Publisher Site | Google Scholar
  31. H. K. Eltzschig, T. Eckle, A. Mager et al., “ATP release from activated neutrophils occurs via connexin 43 and modulates adenosine-dependent endothelial cell function,” Circulation Research, vol. 99, no. 10, pp. 1100–1108, 2006. View at: Publisher Site | Google Scholar
  32. M. Faigle, J. Seessle, S. Zug, K. C. El Kasmi, and H. K. Eltzschig, “ATP release from vascular endothelia occurs across Cx43 hemichannels and is attenuated during hypoxia,” PLoS ONE, vol. 3, no. 7, Article ID e2801, 2008. View at: Publisher Site | Google Scholar
  33. P. Gomes, S. P. Srinivas, W. Van Driessche, J. Vereecke, and B. Himpens, “ATP release through connexin hemichannels in corneal endothelial cells,” Investigative Ophthalmology & Visual Science, vol. 46, no. 4, pp. 1208–1218, 2005. View at: Publisher Site | Google Scholar
  34. J. A. Orellana, N. Froger, P. Ezan et al., “ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels,” Journal of Neurochemistry, vol. 118, no. 5, pp. 826–840, 2011. View at: Publisher Site | Google Scholar
  35. E. Scemes, “Nature of plasmalemmal functional “hemichannels”,” Biochimica et Biophysica Acta—Biomembranes, vol. 1818, no. 8, pp. 1880–1883, 2012. View at: Publisher Site | Google Scholar
  36. K. M. Dwyer, T. B. Mysore, S. Crikis et al., “The transgenic expression of human CD39 on murine islets inhibits clotting of human blood,” Transplantation, vol. 82, no. 3, pp. 428–432, 2006. View at: Publisher Site | Google Scholar
  37. G. S. Ghatnekar, C. L. Grek, D. G. Armstrong, S. C. Desai, and R. G. Gourdie, “The effect of a connexin43-based peptide on the healing of chronic venous leg ulcers: a multicenter, randomized trial,” Journal of Investigative Dermatology, vol. 135, pp. 289–298, 2015. View at: Publisher Site | Google Scholar
  38. C. L. Grek, G. M. Prasad, V. Viswanathan, D. G. Armstrong, R. G. Gourdie, and G. S. Ghatnekar, “Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: a multicenter, randomized trial,” Wound Repair and Regeneration, vol. 23, no. 2, pp. 203–212, 2015. View at: Publisher Site | Google Scholar
  39. B. W. Calder, J. M. Rhett, H. Bainbridge, S. A. Fann, R. G. Gourdie, and M. J. Yost, “Inhibition of connexin 43 hemichannel-mediated ATP release attenuates early inflammation during the foreign body response,” Tissue Engineering Part A, vol. 21, no. 11-12, pp. 1752–1762, 2015. View at: Publisher Site | Google Scholar
  40. B. N. G. Giepmans, I. Verlaan, T. Hengeveld et al., “Gap junction protein connexin-43 interacts directly with microtubules,” Current Biology, vol. 11, no. 17, pp. 1364–1368, 2001. View at: Publisher Site | Google Scholar
  41. P. Meda, “Cx36 involvement in insulin secretion: characteristics and mechanism,” Cell Communication & Adhesion, vol. 10, no. 4–6, pp. 431–435, 2003. View at: Publisher Site | Google Scholar
  42. A. J. Melchiorri, B.-N. B. Nguyen, and J. P. Fisher, “Mesenchymal stem cells: roles and relationships in vascularization,” Tissue Engineering—Part B: Reviews, vol. 20, no. 3, pp. 218–228, 2014. View at: Publisher Site | Google Scholar
  43. C. A. Czajka, A. N. Mehesz, T. C. Trusk, M. J. Yost, and C. J. Drake, “Scaffold-free tissue engineering: organization of the tissue cytoskeleton and its effects on tissue shape,” Annals of Biomedical Engineering, vol. 42, no. 5, pp. 1049–1061, 2014. View at: Publisher Site | Google Scholar
  44. C. A. Czajka and C. J. Drake, “Self-Assembly of prevascular tissues from endothelial and fibroblast cells under scaffold-free, nonadherent conditions,” Tissue Engineering—Part A, vol. 21, no. 1-2, pp. 277–287, 2015. View at: Publisher Site | Google Scholar
  45. C. A. Czajka, B. W. Calder, M. J. Yost, and C. J. Drake, “Implanted scaffold-free prevascularized constructs promote tissue repair,” Annals of Plastic Surgery, vol. 74, no. 3, pp. 371–375, 2015. View at: Publisher Site | Google Scholar
  46. P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L. Steinman, and J. B. Rothbard, “The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 24, pp. 13003–13008, 2000. View at: Publisher Site | Google Scholar
  47. H. Dong, T. M. Fahmy, S. M. Metcalfe et al., “Immuno-isolation of pancreatic islet allografts using pegylated nanotherapy leads to long-term normoglycemia in full MHC mismatch recipient mice,” PLoS ONE, vol. 7, no. 12, Article ID e50265, 2012. View at: Publisher Site | Google Scholar
  48. H. Wang, S. S. Lee, W. Gao et al., “Donor treatment with carbon monoxide can yield islet allograft survival and tolerance,” Diabetes, vol. 54, no. 5, pp. 1400–1406, 2005. View at: Publisher Site | Google Scholar
  49. J. A. Ismail, V. Poppa, L. E. Kemper et al., “Immunohistologic labeling of murine endothelium,” Cardiovascular Pathology, vol. 12, no. 2, pp. 82–90, 2003. View at: Publisher Site | Google Scholar
  50. N. L. Farnsworth and R. K. P. Benninger, “New insights into the role of connexins in pancreatic islet function and diabetes,” The FEBS Letters, vol. 588, no. 8, pp. 1278–1287, 2014. View at: Publisher Site | Google Scholar
  51. A. C. LaRue, V. A. Mironov, W. S. Argraves, A. Czirók, P. A. Fleming, and C. J. Drake, “Patterning of embryonic blood vessels,” Developmental Dynamics, vol. 228, no. 1, pp. 21–29, 2003. View at: Publisher Site | Google Scholar
  52. A. C. LaRue, W. S. Argraves, M. H. Zile, and C. J. Drake, “Critical role for retinol in the generation/differentiation of angioblasts required for embryonic blood vessel formation,” Developmental Dynamics, vol. 230, no. 4, pp. 666–674, 2004. View at: Publisher Site | Google Scholar
  53. C. J. Drake and C. D. Little, “Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 17, pp. 7657–7661, 1995. View at: Publisher Site | Google Scholar
  54. M. L. Iruela-Arispe and G. E. Davis, “Cellular and molecular mechanisms of vascular lumen formation,” Developmental Cell, vol. 16, no. 2, pp. 222–231, 2009. View at: Publisher Site | Google Scholar
  55. M. Brissova and A. C. Powers, “Revascularization of transplanted islets: can it be improved?” Diabetes, vol. 57, no. 9, pp. 2269–2271, 2008. View at: Publisher Site | Google Scholar
  56. A. R. Pepper, B. Gala-Lopez, O. Ziff, and A. M. J. Shapiro, “Revascularization of transplanted pancreatic islets and role of the transplantation site,” Clinical and Developmental Immunology, vol. 2013, Article ID 352315, 13 pages, 2013. View at: Publisher Site | Google Scholar
  57. L. R. Nyman, K. S. Wells, W. S. Head et al., “Real-time, multidimensional in vivo imaging used to investigate blood flow in mouse pancreatic islets,” The Journal of Clinical Investigation, vol. 118, no. 11, pp. 3790–3797, 2008. View at: Publisher Site | Google Scholar
  58. L. A. B. Elias, D. D. Wang, and A. R. Kriegstein, “Gap junction adhesion is necessary for radial migration in the neocortex,” Nature, vol. 448, no. 7156, pp. 901–907, 2007. View at: Publisher Site | Google Scholar
  59. R. Francis, X. Xu, H. Park et al., “Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics,” PLoS ONE, vol. 6, no. 10, Article ID e26379, 2011. View at: Publisher Site | Google Scholar
  60. W. S. Argraves and C. J. Drake, “Genes critical to vasculogenesis as defined by systematic analysis of vascular defects in knockout mice,” Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, vol. 286, no. 2, pp. 875–884, 2005. View at: Publisher Site | Google Scholar
  61. C. L. Grek, J. M. Rhett, J. S. Bruce, M. A. Abt, G. S. Ghatnekar, and E. S. Yeh, “Targeting connexin 43 with α–connexin carboxyl-terminal (ACT1) peptide enhances the activity of the targeted inhibitors, tamoxifen and lapatinib, in breast cancer: clinical implication for ACT1,” BMC Cancer, vol. 15, no. 1, 2015. View at: Publisher Site | Google Scholar
  62. J. M. Rhett, J. Jourdan, and R. G. Gourdie, “Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1,” Molecular Biology of the Cell, vol. 22, no. 9, pp. 1516–1528, 2011. View at: Publisher Site | Google Scholar
  63. R. Guinamard, C. Simard, and C. Del Negro, “Flufenamic acid as an ion channel modulator,” Pharmacology and Therapeutics, vol. 138, no. 2, pp. 272–284, 2013. View at: Publisher Site | Google Scholar
  64. E. C. Ritchie, J. Block, and R. L. Nevin, “Psychiatric side effects of mefloquine: applications to forensic psychiatry,” Journal of the American Academy of Psychiatry and the Law, vol. 41, no. 2, pp. 224–235, 2013. View at: Google Scholar
  65. M. P. O'Quinn, J. A. Palatinus, B. S. Harris, K. W. Hewett, and R. G. Gourdie, “A peptide mimetic of the connexin43 carboxyl terminus reduces gap junction remodeling and induced arrhythmia following ventricular injury,” Circulation Research, vol. 108, no. 6, pp. 704–715, 2011. View at: Publisher Site | Google Scholar
  66. C. L. Grek, J. M. Rhett, and G. S. Ghatnekar, “Cardiac to cancer: connecting connexins to clinical opportunity,” FEBS Letters, vol. 588, no. 8, pp. 1349–1364, 2014. View at: Publisher Site | Google Scholar
  67. W. Gao, P. B. Eisenhauer, K. Conn et al., “Insulin degrading enzyme is expressed in the human cerebrovascular endothelium and in cultured human cerebrovascular endothelial cells,” Neuroscience Letters, vol. 371, no. 1, pp. 6–11, 2004. View at: Publisher Site | Google Scholar
  68. J. A. Lynch, A. M. George, P. B. Eisenhauer et al., “Insulin degrading enzyme is localized predominantly at the cell surface of polarized and unpolarized human cerebrovascular endothelial cell cultures,” Journal of Neuroscience Research, vol. 83, no. 7, pp. 1262–1270, 2006. View at: Publisher Site | Google Scholar
  69. J. Zhao, L. Li, and M. A. Leissring, “Insulin-degrading enzyme is exported via an unconventional protein secretion pathway,” Molecular Neurodegeneration, vol. 4, article 4, 2009. View at: Publisher Site | Google Scholar
  70. J. D. Sander and J. K. Joung, “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnology, vol. 32, no. 4, pp. 347–355, 2014. View at: Publisher Site | Google Scholar
  71. U. Johansson, I. Rasmusson, S. P. Niclou et al., “Formation of composite endothelial cell-mesenchymal stem cell islets: a novel approach to promote islet revascularization,” Diabetes, vol. 57, no. 9, pp. 2393–2401, 2008. View at: Publisher Site | Google Scholar
  72. F. Lim and A. M. Sun, “Microencapsulated islets as bioartificial endocrine pancreas,” Science, vol. 210, no. 4472, pp. 908–910, 1980. View at: Publisher Site | Google Scholar
  73. E. C. Opara, S. H. Mirmalek-Sani, O. Khanna, M. L. Moya, and E. M. Brey, “Design of a bioartificial pancreas(+),” Journal of Investigative Medicine, vol. 58, no. 7, pp. 831–837, 2010. View at: Google Scholar
  74. V. Abudara, J. Bechberger, M. Freitas-Andrade et al., “The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes,” Frontiers in Cellular Neuroscience, vol. 8, article 306, 2014. View at: Publisher Site | Google Scholar
  75. W. H. Evans, G. Bultynck, and L. Leybaert, “Manipulating connexin communication channels: use of peptidomimetics and the translational outputs,” Journal of Membrane Biology, vol. 245, no. 8, pp. 437–449, 2012. View at: Publisher Site | Google Scholar

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