Molecular Retention Limitations for Prevascularized Subcutaneous Sites for Islet Transplantation.

Beta cell replacement therapies utilizing the subcutaneous space have inherent advantages to other sites: the potential for increased accessibility, noninvasive monitoring, and graft extraction. Site prevascularization has been developed to enhance islet survivability in the subcutaneous zone while minimizing potential foreign body immune responses. Molecular communication between the host and prevascularized implant site remains ill-defined. Poly(ethylene oxide)s (PEOs) of various hydrated radii (i.e., ∼11-62 Å) were injected into prevascularized subcutaneous sites in C57BL/6 mice, and the clearance and organ biodistribution were characterized. Prevascularization formed a barrier that confined the molecules compared with the unmodified site. Molecular clearance from the prevascularized site was inversely proportional to the molecular weight. The upper limit in molecular size for entering the vasculature to be cleared was determined to be 35 kDa MW PEO. These findings provide insight into the impact of vascularization on molecular retention at the injection site and the effect of molecular size on the mobility of hydrophilic molecules from the prevascularized site to the host. This information is necessary for optimizing the transplantation site for increasing the beta cell graft survival.

Long-Term Survival and Induction of Operational Tolerance to Murine Islet Allografts by Co-Transplanting Cyclosporine A Microparticles and CTLA4-Ig.

One strategy to prevent islet rejection is to create a favorable immune-protective local environment at the transplant site. Herein, we utilize localized cyclosporine A (CsA) delivery to islet grafts via poly(lactic-co-glycolic acid) (PLGA) microparticles to attenuate allograft rejection. CsA-eluting PLGA microparticles were prepared using a single emulsion (oil-in-water) solvent evaporation technique. CsA microparticles alone significantly delayed islet allograft rejection compared to islets alone (p < 0.05). Over 50% (6/11) of recipients receiving CsA microparticles and short-term cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig) therapy displayed prolonged allograft survival for 214 days, compared to 25% (2/8) receiving CTLA4-Ig alone. CsA microparticles alone and CsA microparticles + CTLA4-Ig islet allografts exhibited reduced T-cell (CD4+ and CD8+ cells, p < 0.001) and macrophage (CD68+ cells, p < 0.001) infiltration compared to islets alone. We observed the reduced mRNA expression of proinflammatory cytokines (IL-6, IL-10, INF-γ, and TNF-α; p < 0.05) and chemokines (CCL2, CCL5, CCL22, and CXCL10; p < 0.05) in CsA microparticles + CTLA4-Ig allografts compared to islets alone. Long-term islet allografts contained insulin+ and intra-graft FoxP3+ T regulatory cells. The rapid rejection of third-party skin grafts (C3H) in islet allograft recipients suggests that CsA microparticles + CTLA4-Ig therapy induced operational tolerance. This study demonstrates that localized CsA drug delivery plus short-course systemic immunosuppression promotes an immune protective transplant niche for allogeneic islets.

Nanothin Conformal Coating with Poly(N-vinylpyrrolidone) and Tannic Acid (PVPON/TA) Preserves Murine and Human Pancreatic Islets Function.

Beta cell replacement therapies can restore glycemic control to select individuals living with type 1 diabetes. However, the obligation of lifelong immunosuppression restricts cell therapies from replacing exogenous insulin administration. Encapsulation strategies can reduce the inherent adaptive immune response; however, few are successfully translated into clinical testing. Herein, we evaluated if the conformal coating of islets with poly(N-vinylpyrrolidone) (PVPON) and tannic acid (TA) (PVPON/TA) could preserve murine and human islet function while conferring islet allograft protection. In vitro function was evaluated using static glucose-stimulated insulin secretion, oxygen consumption rates, and islet membrane integrity. In vivo function was evaluated by transplanting human islets into diabetic immunodeficient B6.129S7-Rag1tm1Mom/J (Rag-/-) mice. The immunoprotective capacity of the PVPON/TA-coating was assessed by transplanting BALB/c islets into diabetic C57BL/6 mice. Graft function was evaluated by non-fasting blood glucose measurements and glucose tolerance testing. Both coated and non-coated murine and human islets exhibited indistinguishable in vitro potency. PVPON/TA-coated and control human islets were able to restore euglycemia post-transplant. The PVPON/TA-coating as monotherapy and adjuvant to systemic immunosuppression reduced intragraft inflammation and delayed murine allograft rejection. This study demonstrates that PVPON/TA-coated islets may be clinically relevant as they retain their in vitro and in vivo function while modulating post-transplant immune responses.

Bioabsorption of Subcutaneous Nanofibrous Scaffolds Influences the Engraftment and Function of Neonatal Porcine Islets.

The subcutaneous space is currently being pursued as an alternative transplant site for ß-cell replacement therapies due to its retrievability, minimally invasive procedure and potential for graft imaging. However, implantation of ß-cells into an unmodified subcutaneous niche fails to reverse diabetes due to a lack of adequate blood supply. Herein, poly (ε-caprolactone) (PCL) and poly (lactic-co-glycolic acid) (PLGA) polymers were used to make scaffolds and were functionalized with peptides (RGD (Arginine-glycine-aspartate), VEGF (Vascular endothelial growth factor), laminin) or gelatin to augment engraftment. PCL, PCL + RGD + VEGF (PCL + R + V), PCL + RGD + Laminin (PCL + R + L), PLGA and PLGA + Gelatin (PLGA + G) scaffolds were implanted into the subcutaneous space of immunodeficient Rag mice. After four weeks, neonatal porcine islets (NPIs) were transplanted within the lumen of the scaffolds or under the kidney capsule (KC). Graft function was evaluated by blood glucose, serum porcine insulin, glucose tolerance tests, graft cellular insulin content and histologically. PLGA and PLGA + G scaffold recipients achieved significantly superior euglycemia rates (86% and 100%, respectively) compared to PCL scaffold recipients (0% euglycemic) (* p < 0.05, ** p < 0.01, respectively). PLGA scaffolds exhibited superior glucose tolerance (* p < 0.05) and serum porcine insulin secretion (* p < 0.05) compared to PCL scaffolds. Functionalized PLGA + G scaffold recipients exhibited higher total cellular insulin contents compared to PLGA-only recipients (* p < 0.05). This study demonstrates that the bioabsorption of PLGA-based fibrous scaffolds is a key factor that facilitates the function of NPIs transplanted subcutaneously in diabetic mice.

Xenotransplantation of tannic acid-encapsulated neonatal porcine islets decreases proinflammatory innate immune responses.

BACKGROUND: Islet transplantation with neonatal porcine islets (NPIs) is a promising treatment for type 1 diabetes (T1D), but immune rejection poses a major hurdle for clinical use. Innate immune-derived reactive oxygen species (ROS) synthesis can facilitate islet xenograft destruction and enhance adaptive immune responses. METHODS: To suppress ROS-mediated xenograft destruction, we utilized nanothin encapsulation materials composed of multilayers of tannic acid (TA), an antioxidant, and a neutral polymer, poly(N-vinylpyrrolidone) (PVPON). We hypothesized that (PVPON/TA)-encapsulated NPIs will maintain euglycemia and dampen proinflammatory innate immune responses following xenotransplantation. RESULTS: (PVPON/TA)-encapsulated NPIs were viable and glucose-responsive similar to non-encapsulated NPIs. Transplantation of (PVPON/TA)-encapsulated NPIs into hyperglycemic C57BL/6.Rag or NOD.Rag mice restored euglycemia, exhibited glucose tolerance, and maintained islet-specific transcription factor levels similar to non-encapsulated NPIs. Gene expression analysis of (PVPON/TA)-encapsulated grafts post-transplantation displayed reduced proinflammatory Ccl5, Cxcl10, Tnf, and Stat1 while enhancing alternatively activated macrophage Retnla, Arg1, and Stat6 mRNA accumulation compared with controls. Flow cytometry analysis demonstrated significantly reduced innate immune infiltration, MHC-II, co-stimulatory molecule, and TNF expression with concomitant increases in arginase-1+ macrophages and dendritic cells. Similar alterations in immune responses were observed following xenotransplantation into immunocompetent NOD mice. CONCLUSION: Our data suggest that (PVPON/TA) encapsulation of NPIs is an effective strategy to decrease inflammatory innate immune signals involved in NPI xenograft responses through STAT1/6 modulation without compromising islet function.

Co-transplantation of human adipose-derived mesenchymal stem cells with neonatal porcine islets within a prevascularized subcutaneous space augments the xenograft function.

BACKGROUND: Cell transplantation has been widely recognized as a curative treatment strategy for variety of diseases including type I diabetes (T1D). Broader patient inclusion for this therapeutic option is restricted by a limited supply of healthy human islet donors and significant loss of islets immediately postintrahepatic transplant due to immune activation. Neonatal porcine islets (NPIs) are a potential ubiquitous ß-cell source for treating T1D. Mesenchymal stem cells (MSCs) have the inherent capacity to secrete immunoregulatory, anti-inflammatory, and proangiogenic factors and, thus, have the potential to improve islet engraftment, survival, and function. METHODS: Herein, we assessed the effect of human adipose-derived MSCs (AdMSCs) on NPI metabolic outcomes in diabetic mice when co-transplanted within the prevascularized subcutaneous deviceless (DL) space or kidney capsule (KC). Graft function has been evaluated by weekly blood glucose, stimulated porcine insulin, glucose tolerance, and total cellular graft insulin content. RESULTS: Compared with NPI alone, co-transplantation of NPIs and AdMSCs resulted in significantly earlier normoglycemia (*P < .05), improved glucose tolerance (*P < .05), superior stimulated serum porcine insulin (**P < .01), and increased graft insulin content (*P < .05) in the DL site and not the KC. CONCLUSIONS: Thus, our study demonstrates that co-transplantation of human AdMSCs with NPIs is an effective tactic to augment islet xenograft function in a clinically relevant extrahepatic site.

Co-localized immune protection using dexamethasone-eluting micelles in a murine islet allograft model.

The broad application of ß cell transplantation for type 1 diabetes is hindered by the requisite of lifelong systemic immunosuppression. This study examines the utility of localized islet graft drug delivery to subvert the inflammatory and adaptive immune responses. Herein, we have developed and characterized dexamethasone (Dex) eluting Food and Drug Administration-approved micro-Poly(lactic-co-glycolic acid) micelles and examined their efficacy in a fully major histocompatibility complex-mismatch murine islet allograft model. A clinically relevant dose of 46.6 ± 2.8 µg Dex per graft was confirmed when 2 mg of micelles was implemented. Dex-micelles + CTLA-4-Ig (n = 10) resulted in prolonged allograft function with 80% of the recipients demonstrating insulin independence for 60 days posttransplant compared to 40% in empty micelles + CTLA-4-Ig recipients (n = 10, P = .06). Recipients of this combination therapy (n = 8) demonstrated superior glucose tolerance profiles, compared to empty micelles + CTLA-4-Ig recipients (n = 4, P < .05), and significantly reduced localized intragraft proinflammatory cytokine expression. Histologically, increased insulin positive and FOXP3+ T cells were observed in Dex-micelles + CTLA-4-Ig grafts compared to empty micelles + CTLA-4-Ig grafts (P < .01 and P < .05, respectively). Localized drug delivery via micelles elution has the potential to alter the inflammatory environment, enhances allograft survival, and may be an important adjuvant approach to improve clinical islet transplantation outcomes.

In vitro co-culture of epithelial cells and smooth muscle cells on aligned nanofibrous scaffolds.

Esophagus is a complex, hollow organ consisting of epithelial cells in the inner mucosal layer and smooth muscle cells in the outer muscle layer. In the present study, we have evaluated the in vitro co-culture of epithelial cells and smooth muscle cells on the aligned nanofibrous scaffold made of PHBV, PHBV-gelatin, PCL and PCL-gelatin developed through electrospinning using rotating drum collector. Epithelial cells were labeled with cell tracker green while the smooth muscle cells were labeled with cell tracker red. Labeled cells were seeded on the aligned nanofibers matrices and tracked using laser scanning confocal microscopy. The results demonstrate that both epithelial and smooth muscle cells attach, extend, and proliferate over these nanofibrous matrices. Confocal z-sectioning shows that epithelial and smooth muscle cells tend to separate into two distinct layers on a single nanofiber system mimicking the in vivo anatomy. Cell viability assay showed that both types of cells are viable and also interact with each other. The functional gene expression of respective cell types demonstrates that both epithelial and smooth muscle cells are phenotypically as well as functionally active when they were co-cultured. Thus the study highlighted that aligned nanofibrous scaffolds could be potential alternative graft for esophageal tissue regeneration.

Cotransplantation of Mesenchymal Stem Cells With Neonatal Porcine Islets Improve Graft Function in Diabetic Mice.

Mesenchymal stem cells (MSCs) possess immunoregulatory, anti-inflammatory, and proangiogenic properties and, therefore, have the potential to improve islet engraftment and survival. We assessed the effect human bone marrow-derived MSCs have on neonatal porcine islets (NPIs) in vitro and determined islet engraftment and metabolic outcomes when cotransplanted in a mouse model. NPIs cocultured with MSCs had greater cellular insulin content and increased glucose-stimulated insulin secretion. NPIs were cotransplanted with or without MSCs in diabetic B6.129S7-Rag1tm1Mom/J mice. Blood glucose and weight were monitored until reversal of diabetes; mice were then given an oral glucose tolerance test. Islet grafts were assessed for the degree of vascularization and total cellular insulin content. Cotransplantation of NPIs and MSCs resulted in significantly earlier normoglycemia and vascularization, improved glucose tolerance, and increased insulin content. One experiment conducted with MSCs from a donor with an autoimmune disorder had no positive effects on transplant outcomes. Cotransplantation of human MSCs with NPIs demonstrated a beneficial metabolic effect likely as a result of earlier islet vascularization and improved islet engraftment. In addition, donor pathology of MSCs can influence the functional capacity of MSCs.

Developing Hybrid Polymer Scaffolds Using Peptide Modified Biopolymers for Cell Implantation.

Polymeric scaffolds containing biomimics offer exciting therapies with broad potential impact for cellular therapies and thereby potentially improve success rates. Here we report the designing and fabrication of a hybrid scaffold that can prevent a foreign body reaction and maintain cell viability. A biodegradable acrylic based cross-linkable polycaprolactone based polymer was developed and using a multihead electrospinning station to fabricate hybrid scaffolds. This consists of cell growth factor mimics and factors to prevent a foreign body reaction. Transplantation studies were performed subcutaneously and in epididymal fat pad of immuno-competent Balb/c mice and immuno-suppressed B6 Rag1 mice and we demonstrated extensive neo-vascularization and maintenance of islet cell viability in subcutaneously implanted neonatal porcine islet cells for up to 20 weeks of post-transplant. This novel approach for cell transplantation can improve the revascularization and allow the integration of bioactive molecules such as cell adhesion molecules, growth factors, etc.

Interaction of human smooth muscle cells with nanofibrous scaffolds: Effect of fiber orientation on cell adhesion, proliferation, and functional gene expression.

Poly(ɛ-caprolactone) (PCL) and PCL-gelatin random and aligned nanofibers with diameters in the range of 200-400 nm were developed through electrospinning. Mechanical properties of aligned PCL and PCL-gelatin nanofibers were compared, and it was found that aligned PCL nanofibers showed significantly higher tensile strength and Young's modulus than the PCL-gelatin nanofiber system (p < 0.05). The in vitro degradation of aligned nanofibers showed that PCL-gelatin nanofibers degrade faster than aligned PCL nanofibers. Further, human smooth muscle cells were cultured on the random and aligned PCL-gelatin nanofibers and evaluated for adhesion, orientation, morphology, viability, proliferation and gene expression. Our results demonstrate that PCL-gelatin promotes higher cell adhesion and proliferation than the PCL nanofibers after 3, 7, and 10 days of culture. Aligned topography favored orientation of the cells along their directions and cell stretching was better in aligned nanofibers than the random nanofibers. The upregulation of α-actin, myosin heavy chain, collagen type I, and elastin genes demonstrate good cell-matrix interactions in both random and aligned scaffolds. Therefore, the present study concludes that aligned PCL-gelatin nanofibers could serve as potential scaffolding for culture of smooth muscle cells and may promote functional regeneration of tubular organs.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-based nanofibrous scaffolds to support functional esophageal epithelial cells towards engineering the esophagus.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV-gelatin were electrospun to obtain defect-free nanofibers by optimizing various process and solution parameters. Tensile strength, Young's modulus, and wettability of PHBV-gelatin nanofibrous scaffold were determined and compared with PHBV nanofibrous scaffold. Our results demonstrate that PHBV-gelatin nanofibers exhibited higher tensile strength and Young's modulus than the PHBV nanofibers. Human esophageal epithelial cells (HEEpiC) were cultured on PHBV and PHBV-gelatin nanofiber showed better cell proliferation in PHBV nanofibrous scaffold than the PHBV-gelatin scaffold after 7 days of culture. HEEpiC cultured on PHBV and PHBV-gelatin nanofibrous scaffold exhibited characteristic epithelial cobblestone morphology after 3 days of culture. Further, the HEEpiC extracellular matrix (ECM) proteins (collagen type IV and laminin) and phenotypic marker proteins (cytokeratin-4 and 14) expressions were significantly higher in PHBV-gelatin nanofibrous scaffold than the PHBV nanofiber scaffold. However, the long-term stability and functional state of the cells on the PHBV scaffold give it an edge over the blend scaffolds. Thus, PHBV-based nanofibrous scaffolds could be explored further as ECM substitutes for the regeneration of esophageal tissue.

PCL and PCL-gelatin nanofibers as esophageal tissue scaffolds: optimization, characterization and cell-matrix interactions.

Nanofiber based scaffolds offer great promise in regeneration of various tissues including esophagus. Diseases of the esophagus such as malignancy and strictures require surgical intervention to repair the affected region using an appropriate substitute. Long gap esophageal atresia poses a clinical challenge to bridge the gap. In this study, nanofibrous scaffolds made of PCL and PCL-gelatin were fabricated through electrospinning. The average diameter of PCL and PCL-gelatin nanofibers were found to be 324 +/- 50 nm and 242 +/- 30 nm respectively. PCL-gelatin nanofibers was characterized using FTIR, DSC, UTM, Goniometer, suture retention strength and in vitro degradation and the results were compared with the PCL nanofibers. PCL nanofiber characterization results showed that it exhibited higher tensile strength, suture retention strength, contact angle and slower degradation when compared with the PCL-gelatin nanofibers. Further, the interaction of human esophageal epithelial cells with PCL and PCL-gelatin nanofibrous scaffold was determined by cell adhesion, proliferation and gene expression studies. Our results demonstrated that the epithelial cells adhered and proliferated well on both PCL and PCL-gelatin nanofibrous scaffolds and also exhibited the characteristic cobblestone morphology. Cell proliferation on PCL-gelatin nanofibrous scaffold was significantly higher than the PCL nanofibrous scaffold (*p <0.05). Therefore, these scaffolds could be explored as potential candidates for regeneration of functional esophagus.

Electrospun nanostructured chitosan-poly(vinyl alcohol) scaffolds: a biomimetic extracellular matrix as dermal substitute.

Electrospinning is a versatile technique to make biomimetic and nanostructured scaffolds for skin tissue engineering. In this study we have electrospun and characterized chitosan (C)-poly(vinyl alcohol) (PVA) blend nanofibers as dermal substitutes and compared with 2D C-PVA films. The in vitro characterization of the C-PVA nanofibers and 2D films were evaluated using mouse 3T3 fibroblast cells and our results demonstrated that the cells adhered and proliferated on the surface of C-PVA nanofibers. In our animal studies, the implantation of C-PVA nanofibers along with topical administration of growth factor R-Spondin 1 on full thickness wounds created on rats showed 98.6% wound closure after two weeks post-surgery. The catalase and superoxide dismutase activity of the healing tissue was significantly higher in the groups treated with topical administration of growth factor and C-PVA nanofibers (p < 0.05). Thus these C-PVA nanofibers along with novel growth factor are promising new biomaterials that could be used as dermal substitutes for accelerated wound healing.

Tissue engineering interventions for esophageal disorders--promises and challenges.

The diseases of the esophagus include congenital defects like atresia, tracheoesophageal fistula as well as others such as gastro-esophageal reflux disease (GERD), Barrett's esophagus, carcinoma and strictures. All esophageal disorders require surgical intervention and reconstruction with appropriate substitutes. Primary anastomosis is used to treat most cases but treatment of long gap atresia still remains a clinical challenge. Autologous graft therapies using tissues from colon, and small and large intestine or gastric transplantations have been attempted but have constraints like leakage, infection and stenosis at the implanted site, which leads to severe morbidity and mortality. An alternative for autologous grafts are allogenic and xenogenic grafts, which have better availability but disease transmission and immunogenicity limit their applications. Use of biodegradable and biocompatible scaffolds to engineer the esophagus promises to be an effective regenerative strategy for treatment of esophageal disorders. Nanotopography of the fibrous scaffolds mimics the natural extracellular matrix (ECM) of the tissue and incorporation of chemical cues and tailoring mechanical properties provide the right microenvironment for co-culture of different cell types. Scaffolds cultured with esophageal cells (epithelial cells, fibroblast and smooth muscle cells) might show enhancement of the biofunctionality in vivo. This review attempts to address the various strategies and challenges involved in successful tissue engineering of the esophagus.

Development of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers for skin tissue engineering: effects of topography, mechanical, and chemical stimuli.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, was electrospun to form defect-free fibers with high surface-area-to-volume ratio for skin regeneration. Several parameters such as solvent ratio, polymer concentration, applied voltage, flow rate, and tip-to-target distance were optimized to achieve defect-free morphology. The average diameter of the PHBV fibers was 724 ± 91 nm. PHBV was also solvent-cast to form 2-D films, and its mechanical properties, porosity, and degradation rates were compared with PHBV fibers. Our results demonstrate that PHBV fibers exhibited higher porosity, increased ductility, and faster degradation rate when compared with PHBV 2-D films (p < 0.05). In vitro studies with PHBV fibers and 2-D films were carried out to evaluate the adhesion, viability, proliferation, and gene expression of human skin fibroblasts. Cells adhered and proliferated on both PHBV fibers and 2-D films. However, the proliferation of cells on the surface of PHBV fibers was comparable to tissue culture polystyrene (TCPS, control) (p > 0.05). The gene expression of collagen I and elastin was significantly up-regulated when compared with TCPS control, whereas collagen III was down-regulated on PHBV fibers and 2-D film after 14 days in culture. The less ductile PHBV 2-D films showed higher levels of elastin expression. Furthermore, the PHBV fibers in the presence and absence of an angiogenesis factor (R-Spondin 1) were evaluated for their wound healing capacity in a rat model. The wound contracture in R-Spondin-1-loaded PHBV fibers was found to be significantly higher when compared with PHBV fibers alone after 7 days (p < 0.05). Furthermore, the presence of fibers promoted an increase in collagen and aided re-epithelialization. Thus our results demonstrate that the topography and mechanical and chemical stimuli have a pronounced influence on the cell proliferation, gene expression, and wound healing.