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Original Articles: Experimental Transplantation

Reversal of Diabetes by Pancreatic Islet Transplantation into a Subcutaneous, Neovascularized Device

Pileggi, Antonello1,2; Molano, R Damaris1; Ricordi, Camillo1,2,3; Zahr, Elsie1; Collins, Jill1; Valdes, Rafael4; Inverardi, Luca1,3,5

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doi: 10.1097/01.tp.0000203858.41105.88
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Abstract

In type 1 diabetes mellitus (T1DM) the insulin-producing β-cells in the pancreatic islets are destroyed by an autoimmune process. Tight glycemic metabolic control by intense exogenous insulin treatment can reduce or prevent the progression of diabetes complications (1). Restoration of β-cell function by transplantation of allogeneic pancreatic islets represents a promising therapeutic option for patients with T1DM, providing a more physiologic metabolic control than exogenous insulin (2–6).

Numerous studies have attempted to identify the most suitable site of implant for islet grafts (7, 8), and clinical islet transplantation is currently performed by intrahepatic embolization through the portal system (2–6). Even if the safety of this technique has been established, specific infrastructure and expertise are needed. Risks inherent to the procedure are bleeding, thrombosis and elevation of portal pressure (5, 9, 10). Repeated infusions of islets isolated from more than one donor pancreas might increase the risks associated with the transplantation procedure. The inherent hyperglycemic environment and the relatively higher concentrations of drug metabolites in the liver (11, 12) may also contribute to the delayed loss of graft function observed in recent clinical trials (4, 5), possibly related to β-cell toxicity (13). Development of alternative implantation sites for pancreatic islets may be of assistance to reduce risks and improve the success rate of islet transplantation.

Tissue engineering approaches for the development of bioartificial organs, including the pancreas, have included invasive surgical procedures that are difficult to implement in the clinical setting, such as subcapsular kidney space, spleen (8), intra-abdominal vascularized bio-hybrid devices (14, 15), omental pouch (16), excluded intestinal segments (17) and intramuscular (18), amongst others. The subcutaneous site seems advantageous because of the potentially less invasive procedure that could be performed under local anesthesia. However, subcutaneous islet implantation has been hampered so far by inherent limitations of this site (lack of early vascularization, induction of local inflammation, and mechanical stress on the graft) resulting in primary non-function (7, 19–22). Subcutaneous implantation of biocompatible materials (i.e. diffusion chambers, hollow fibers) for tissue and cellular transplantation has been performed, but disadvantages included degradation of biomaterials and generation of intense avascular fibrotic reactions limiting oxygen availability to the grafted tissue and reducing diffusion of substances from the device (19–24).

In the present study, we have assessed the engraftment and long-term function of syngeneic pancreatic islets transplanted into diabetic rats using a newly designed biocompatible device. Subcutaneous implantation of the device prior to islet transplantation allows for the development of a rich vascular bed embedding the device which favors islet engraftment and results in sustained graft function for over five months. Our data suggests that this approach may offer a viable alternative site for the implantation of pancreatic islets.

METHODS

Pancreatic Islet Isolation

All animal procedures were performed at the Translational Research Laboratory of the Cell Transplant Center under protocols approved by the Institutional Animal Care Committee. Islets were obtained from male Lewis rat (Harlan, Indianapolis, IN) donor pancreata by a mechanically-enhanced enzymatic digestion using Liberase (Roche; Indianapolis, IN) followed by separation on discontinuous density gradients (Mediatech; Herndon, VA) (25). After overnight culture (37°C, 5% CO2) in supplemented CMRL-1066 medium (Gibco-Invitrogen; Carlsbad, CA), islet grafts of 3,000 islet equivalents (IEQ) were aliquoted in nontissue-culture Petri dishes.

Biocompatible Implantable Device

The implantable device developed at the National Autonomous University of Mexico and manufactured at Mecánica de Precisión (México D.F., Mexico) (26), consisted of a 2 cm-long cylindrical stainless-steel mesh (450 μm pore size) with 0.6 cm diameter and two stoppers (s) in polytetrafluoroethylene (PTFE) at the extremities (Fig. 1A). Forty days prior to islet transplantation, the devices (sterilized by autoclaving) were implanted subcutaneously in the dorsal region of the recipients under general anesthesia (isoflurane USP; Baxter, Deerfield, IL). Connective tissue rich in vascular structures embedded the device through the pores of the mesh (Fig. 1B). In order to prevent complete occlusion of the device lumen by the recipient's tissue, a PTFE plunger (p) was inserted filling the lumen at the time of implant (Fig. 1A).

FIGURE 1.
FIGURE 1.:
Biocompatible implantable device. (A) The device consists of a stainless-steel biocompatible mesh with removable PTFE stoppers (s) on each end. At the time of implantation, one of the stoppers is replaced by a PTFE plunger (p) filling the lumen, in order to prevent its occlusion by the host connective tissue. The plunger is then removed at the time of islet transplantation, and the device sealed with a stopper. The ruler indicates size in cm. (B) Macroscopic appearance of the implantable device 80 days after islet transplantation showing abundant vascular structures and connective tissue embedding it. Vascular structures are evident also in the internal walls of the device (insert). The same pattern was observed in all devices removed at >160 days after islet transplantation.

Islet Transplantation

Male Lewis rats were rendered diabetic by intravenous administration of the β-cell toxin streptozotocin (two intravenous injections of 60 mg/Kg two-three days apart; Sigma-Aldrich, St. Louis, MO) and were used as recipients of syngeneic islets only if overtly diabetic (non-fasting blood glucose levels ≥350 mg/dl). At the time of islet transplantation, islets were collected by the means of a precision syringe (Hamilton; Reno, NV) and transferred to a polyethylene catheter that was used to implant them.

A small cutaneous incision allowed access to one extremity of the device, to remove the PTFE plunger, and islets (3,000 IEQ per recipient) were implanted into the lumen of the device in a small volume of saline solution. The device was then sealed by placing a PTFE stopper, and the skin sutured. Control animals received 3,000 IEQ syngeneic islet grafts implanted into the liver through the portal vein, as previously described (27). Antibiotic prophylaxis consisted of amoxicillin 250 mg/L in drinking water for up to 2 weeks from surgical procedures.

Assessment of Graft Function

Graft function was defined as non-fasting glycemic levels <200 mg/dl and assessed using a portable blood glucose meter (Bayer; Tarrytown, NY) on whole blood samples obtained from the tail of the animals. A glucose tolerance test was performed after 164 days from transplantation in selected animals, in order to assess the metabolic performance of the islets grafted in the device. After overnight fasting, animals received an intravenous glucose bolus (2 g/kg in saline) and blood glucose was monitored for up to 120 min. Glucose clearance was assessed by calculating the rate of fall of blood sugar per minute (k-glucose value, kg), as previously described (28, 29). In order to exclude residual function of the native pancreas after long-term follow-up, the graft-bearing devices were explanted and non-fasting glycemic values monitored in the subsequent days to observe a prompt return to hyperglycemia confirming that euglycemia after transplantation was indeed maintained by the grafted islets.

Histopathological Analysis

The explanted devices were collected in 10% formalin buffer. After fixation, the tissue contained in the device was dissected-free from the metal mesh and embedded in paraffin blocks. Histopathological assessment was performed at the Imaging Core at the Diabetes Research Institute. Tissue sections (4-μm thick) were stained with hematoxylin and eosin and the grafts assessed morphologically. Masson trichrome staining (Chromaview; Richard-Allan Scientific, Kalamazoo, MI) was also performed on selected grafts. This procedure results in a blue stain of collagen, while muscle fibers and cytoplasm stain red.

In order to confirm the function of implanted islets, expression of insulin was assessed by immunohistochemistry using a guinea pig anti-insulin antibody (Dako, Carpinteria, CA) and a biotinilated donkey anti-guinea pig immunoglobulin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), followed by streptavidin-horseradish peroxidase and revealed by aminoethylcarbazole (Zymed Laboratories, Inc., South San Francisco, CA). Alternatively, secondary Alexa-568-conjugated goat-anti-guinea pig antibody (Molecular Probes, Eugene, OR) was used for immunofluorescence microscopy. Endothelial cells were identified using a rabbit anti-vonWillerbrand Factor (vWF) antibody (Chemicon International; Temecula, CA) followed by Alexa-488 goat anti-rabbit antibody (Molecular Probes). Nuclear staining was obtained with 4′,6 diamino-2-phenilindole (DAPI; Molecular Probes). Immunofluorescence analysis was performed using confocal microscopy (Zeiss LSM-510).

RESULTS

The biocompatible device consisted of a cylindrical stainless-steel mesh with a PTFE stopper at each extremity (Fig. 1A). The device was implanted subcutaneously in the intrascapular region 40 days prior to islet transplantation. This step allowed for the growth of recipient's tissue and vascular structures embedding the device. In order to prevent occlusion, a PTFE plunger was inserted into the device's lumen (Fig. 1A) at the time of implantation. Subcutaneous implantation of the device was well tolerated, and consisted of a quick, minimally-invasive procedure requiring only brief anesthesia. No adverse reactions to the implanted materials were observed.

At the time of islet transplantation, a small cutaneous incision was sufficient to access the device that was surrounded by highly vascularized connective tissue, and presented a visible capillary network crossing the pores of the metal mesh (Fig. 1B). Upon gentle removal of the plunger, facilitated by the lack of adherence of connective tissue to PTFE, a richly vascularized connective layer was also observed inside the device surrounding a well-defined central lumen. The islet implantation procedure was simple to perform and uneventful. The device was then sealed with a PTFE stopper and the skin sutured.

Transplantation of syngeneic islets in the metal devices resulted in reversal of diabetes in seven of the eight recipients with a median time of 6 days (range 1–13; n=7; Fig. 2). Three animals achieving normoglycemia soon after islet implantation (day 1 ×2, and day 4) were followed-up short-term (10 days). The remaining four animals were followed-up long-term (>160 days) and showed correction of diabetes (on days 6, 7, and 13 ×2, respectively), and sustained euglycemia until removal of the graft-bearing devices that invariably resulted in hyperglycemia (Fig. 2).

FIGURE 2.
FIGURE 2.:
Glycemic control after islet transplantation. Reversal of diabetes and maintenance of normoglycemia was measured in chemically diabetic recipients of syngeneic islets into either the subcutaneous implanted device (excluding one animal that did not achieve euglycemia; n=7, n=4 after day 10; ○) or the liver (n=4; •). After islet transplantation, non-fasting blood glucose levels returned to physiological ranges (<200 mg/dl) and remained stable long-term in both groups. Hyperglycemia promptly resumed upon removal of the graft-bearing devices (arrows) in all animals, demonstrating that long-term glycemic control was due to the islets transplanted into the devices.

Additional animals received the same mass of syngeneic islets into the liver (n=4) and achieved normoglycemia on the day after implant. After reversal of diabetes, metabolic function of implanted islets was comparable in both groups, as measured by the ability to maintain glycemic values within physiological ranges over time (Fig. 2).

Animals in both groups showed improved clinical condition and steady body weight gain after transplantation (Fig. 3). This clinical pattern was observed also in the only recipient that did not reach euglycemia in the device group, indicating partial function of the graft possibly due to a suboptimal islet mass implanted.

FIGURE 3.
FIGURE 3.:
Body weight monitoring after islet transplantation. Body weight of recipients of syngeneic islet grafts intrahepatically (n=4; •) and into the subcutaneous device (n=5; ○), including the animal not achieving euglycemia after islet transplantation, comparable weight gain in both groups over time.

Transplanted islets showed good response during metabolic challenge (performed on day >160), with rapid glucose clearance and return to normoglycemia following intravenous glucose bolus. Glucose disposal rate was 2.53±0.09, 1.24±0.10, and 1.77±0.19 for non-diabetic controls, device and intrahepatic grafts, respectively. Glucose levels were slightly higher in the device group demonstrating delayed disposal when compared to non-diabetic controls and to recipients of intra-hepatic islets (Fig. 4).

FIGURE 4.
FIGURE 4.:
Intravenous glucose tolerance test was performed in animals bearing a functional graft long-term. After >160 days from islet transplantation, islets grafted into the devices (n=2, •) and intrahepatically (n=4; ○) were able to promptly restore normoglycemia upon glucose challenge. Nondiabetic controls are shown (n=2; □). Mean (± SD) glycemic values for each experimental group are plotted.

Removal of the device was performed at day 80 (n=1) and on day 170 (n=4) after islet transplantation. Invariably, all animals showed return to hyperglycemia within 24 hr from device removal, confirming that euglycemia was indeed sustained by the islets implanted into the devices (Fig. 2).

Histopathological assessment of explanted grafts showed well-preserved islet structures embedded in connective tissue (Figs. 5 and 6). A prerequisite for the survival and proper function of islets is that early revascularization of the graft occurs. Abundant vascular structures (both arteries and veins) were observed throughout the sections as early as 10 days after transplantation (Fig. 5) and after long-term follow-up (Fig. 6), suggesting that neovascularization of the graft had indeed occurred through vessels arising from the wall of the device.

FIGURE 5.
FIGURE 5.:
Composite photomicrograph of a device removed 10 days after islet transplantation. Hematoxylin & eosin (A) and insulin staining (B) of a cross section (5×) of tissue retrieved from the subcutaneous device 10 days after islet implant. Collagen constitutes most of the section. A clot is present in the central area of the explanted tissue. The inserts show magnification of an area containing islet tissue (insulin positive, brown color) and evident vascular structures including arteries (triangles) and veins (arrows).
FIGURE 6.
FIGURE 6.:
Histopathological assessment of explanted islet grafts >160 days after transplantation. (A) Masson's trichrome staining shows collagen in blue, while cytoplasm, erythrocytes and smooth muscle in red. The central area contains agglomerated islets with well-preserved morphology surrounded by collagen (5×). Numerous vascular structures are visible throughout the section in both connective and endocrine tissue, including arteries (triangles) and veins (arrows). Higher magnification (40×) of the indicated areas is shown in the inserts. (B) Immunohistochemistry demonstrates positive signal for insulin (brown) in the β-cells of the transplanted islets (5×). Higher magnification of the indicated areas is shown in the insert (40×). (C) Confocal microscopy analysis showing immunofluorescence for the endothelial marker von Willerbrand factor (green fluorescence) reveals the lumen of the vascular structures within the tissue contained into the device (insulin=red fluorescence; nuclei=DAPI blue fluorescence; 40×). Similar patterns were observed in all devices explanted after 80 (n=1) and 170 days (n=4).

Immunohistochemical analysis for insulin was performed on the harvested tissue sections in order to assess the ability of implanted islet β-cells to synthesize and store insulin. Clusters of well-preserved islets with strong insulin immunostaining were observed in all grafts, a hallmark of healthy and functional insulin-producing cells (Figs. 5 and 6). Additionally, immunofluorescence for the endothelial marker vWF showed endothelial lining within islet structures (insulin positive) in the specimens obtained from the devices after long-term follow-up, therefore confirming the presence of vascular structures with evident endothelial lining inside the grafted tissue (Fig. 6C).

DISCUSSION

Implementation of efficient tissue harvesting, transplantation techniques and availability of novel biocompatible materials for tissue engineering are opening new avenues for the treatment of diseases characterized by loss of function of organs or tissues. Development of a bioartificial endocrine pancreas is a highly desirable goal that may have an impact for the treatment of patients with diabetes. In order to be clinically applicable, it is required that a bioartificial pancreas: i) is implantable using minimally-invasive procedures, ii) provides a well defined space for the grafted tissue, iii) favors engraftment and function of the transplanted tissue, and iv) can be easily accessible to biopsy or removed, if necessary.

The subcutaneous space may represent a valuable site for implantation of bioartificial organs because of the easy access for both implant and removal. However, engraftment of pancreatic islets in the subcutaneous site has been difficult to achieve. Possible limitations of this implantation site include the generation of local inflammation at the time of islet transplantation and the mechanical stress resulting in primary non-function (7, 19–22). Indeed subcutaneous transplantation of islets invariably fails to restore normoglycemia (7, 19–22) unless islet encapsulation or induction of prevascularization of the site of implant is used (19–22, 30, 31). Subcutaneous devices and polymers tested in the past presented multiple limitations including poor oxygen availability to the grafted tissue and reduced diffusion of substances from the device (19–24). Flat devices with few millimeters (<2 mm) thickness have been tested for islet implant aiming at minimizing the distance between graft and recipient's vascular bed (19). The use of polymers able to prevent islet aggregation (and possibly necrosis) after implantation, and/or of factors promoting neo-vascularization was generally required to obtain measurable function although for limited periods of time in these devices (19, 20, 30, 31).

Delivery of pro-angiogenic factors in the subcutaneous implantation site using devices that were then removed at the time of syngeneic islet implantation resulted in reversal of diabetes and sustained function, while islets transplanted into a non-prevascularized subcutaneous space failed to correct diabetes (21, 22). Pitfall of such ingenious approach is the damage of the integrity of the vascular structures and generation of inflammation at the time of device removal, which may compromise islet engraftment (21, 22).

The design of the device utilized in the present study allows for the preservation of the vascular structures at the time of accessing the lumen of device for islet implant. The recipient's connective tissue coating the walls of the device creates a well-defined, highly vascularized environment that allows for islet engraftment and function. The implantable device utilized in the present study has a unique geometry consisting of an internal diameter of 0.6 cm which is clinically relevant as it would allow loading a sufficient number of islets to reverse diabetes in a patient while keeping the device length within an acceptable range. Our study shows for the first time that a device with such characteristics allows for islet cell survival and early graft vascularization (10 days after implant), and supports long-term (>5 months) survival and function of pancreatic islets (which are particularly susceptible to hypoxia) in the unfavorable subcutaneous implantation site. A rich network of vascular structures was also observed within the grafted tissue in the devices removed after 5 months, which was associated with the presence of viable insulin rich-endocrine tissue.

Nonfasting glycemic values over time remained within physiological ranges in animals receiving islets into the device, and were comparable to that of intrahepatic islet graft recipients. Glucose clearance during intravenous challenge showed good graft performance with prompt return to normoglycemia in the device group, although slightly delayed when compared to controls, which may be a consequence of the different anatomical location of implanted islets. When assessing the function of implanted islets in the experimental setting, it is of utmost importance demonstrating that euglycemia is maintained by implanted islets and to exclude residual function of the native pancreas. In our study, removal of the long-term functioning graft-bearing devices resulted in hyperglycemia in all cases, therefore confirming unequivocally that euglycemia was sustained by the islet grafts.

Although not formally addressed in the present study, absence of islet-blood contact may represent a substantial advantage of the implantable device over the intrahepatic site, as it would prevent loss of islet cells due to blood-mediated inflammation reaction early after transplantation (2, 32). Additionally, the relatively hyperglycemic hepatic environment and exposure to high drug metabolite concentrations in the liver (11, 12) may lead to exhaustion and islet cell toxicity resulting in altered graft potency, making it preferable to use alternative sites of implantation in the clinical setting.

The random distribution of islets in the liver precludes the use of graft biopsies to guide timely therapeutic interventions and prolong graft survival. The well-confined site of implant in this device allows performing biopsies or to retrieve the device if necessary.

Notably, the device used in the present study is not designed to confer immunoisolation to the transplanted tissue, but rather as an alternative implantation site for islet transplantation. Therefore, it could be utilized in the allogeneic transplantation setting in combination with conventional immunosuppression. Alternatively, our approach could be combined with immunoprotective strategies including encapsulation (19, 20, 30) and/or co-transplantation with other cells (e.g. Sertoli cells) (33). Furthermore, the use of proangiogenic compounds might be of assistance in reducing the time between device implantation and islet transplantation utilizing our device (21, 30).

Taken together, our study describes a potentially clinically applicable alternative for islet transplantation and potentially for other cellular therapies in reparative medicine. Implanted by a minimally invasive approach, the device provides an excellent environment for the engraftment, revascularization and long-term function of implanted tissues, while complying with biocompatibility, safety, and confining of the transplanted tissue that are required for clinical use.

ACKNOWLEDGMENTS

This study was included in the thesis work of A.P. for the Ph.D. program in Experimental Surgery and Microsurgery (Ciclo XVIII) at the Department of Surgery of the University of Pavia Medical School, Pavia, Italy. The authors are grateful to Yelena Gadea for technical aid and to Kevin Johnson for the excellent assistance with the challenging histological sample processing.

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