Neonatal Porcine Research Articles

Xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells improves murine hind limb ischemia through lymphangiogenesis and angiogenesis.

The clinical utility of stem cell therapy for peripheral artery disease has not been fully discussed, and one obstacle is limited donor supplies. In this study, we attempted to rescue mouse ischemic hind limb by xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs). Neonatal porcine bone marrow-derived mesenchymal stem cells were transplanted to ischemic hind limbs of male C57BL/6J mice (npBM-MSCs group). Mice with syngeneic transplantation of mouse BM-MSCs (mBM-MSCs group) were also prepared for comparison. The angiogenic effects were evaluated by recovery of blood flow on laser Doppler imaging, histologic findings, and genetic and protein levels of angiogenic factors. Regarding laser Doppler assessments, blood flow in the hind limb was rapidly recovered in the npBM-MSCs group, compared with that in the mBM-MSCs group (P=.016). Compared with the mBM-MSCs group, the npBM-MSCs group had early and prominent lymphangiogenesis [P<.05 on both post-operative days (PODs) 3 and 7] but had similar angiogenesis. Regarding genomic assessments, xenotransplantation of npBM-MSCs enhanced the expressions of both porcine and murine Vegfc in the hind limbs by POD 3. Interestingly, the level of murine Vegfc expression was significantly higher in the npBM-MSCs group than in the mBM-MSCs group on PODs 3 and 7 (P<.001 for both). Furthermore, the secreted VEGFC protein level was higher from npBM-MSCs than from mBM-MSCs (P<.001). Xenotransplantation of npBM-MSCs contributed to the improvement of hind limb ischemia by both angiogenesis and lymphangiogenesis, especially promotion of the latter. npBM-MSCs may provide an alternative to autologous and allogeneic MSCs for stem cell therapy of critical limb ischemia.

Xenogeneic Sertoli cells express complement inhibitory proteins necessary for their survival of hyperacute rejection

Abstract Transplantation is used to treat organ failure associated with various diseases, however there is a severe shortage of organs acceptable for transplant. Utilization of pig organs offers an endless supply of transplantable tissue, but xenografts are rapidly destroyed by hyperacute rejection (HAR). The primary humoral mediator of HAR is the activation of complement (C″) by preformed-antibody binding to xenoantigens, causing a proteolytic cascade that results in cell lysis by membrane attack complex (MAC) insertion. Sertoli cells (SCs) are immunoregulatory testicular cells that survive as xenografts for an extended period without the use of immunosuppressive drugs. The goal of this study was to examine the mechanisms for SC survival of C″. Neonatal porcine SCs (NPSCs) transplanted into rats survived as xenografts while porcine islets (NPIs) did not. Immunohistochemistry of the surviving grafts show deposited C3, but not MAC, suggesting that NPSCs inhibit C″ after activation. An in vitro human serum (HS)-C″ cytotoxicity assay confirmed that NPSCs survive activated human C″. Analysis of NPSC expression of complement inhibitory proteins (CIP) revealed higher levels of membrane cofactor protein (MCP) and decay accelerating factor (DAF) as compared to NPIs, which are killed by C″. Knocking down expression of MCP and DAF using shRNA confirmed their importance in NPSC survival of C″. Less than 10% of these cells survived exposure to the HS-C″ cytotoxicity assay, indicating MCP and DAF are important in NPSC xenograft survival in vitro.

Coagulation, inflammation, and CD46 transgene expression in neonatal porcine islet xenotransplantation.

Thrombosis is a known consequence of intraportal islet transplantation, particularly for xenogeneic islets. To define the origins of thrombosis after islet xenotransplantation and relate it to early inflammation, we examined porcine islets transplanted into non-human primates using a dual-transplant model to directly compare islet characteristics. α1,3-Galactosyltransferase gene-knockout (GTKO) islets with and without expression of the human complement regulatory transgene CD46 (hCD46) were studied. Biologically inert polyethylene microspheres were used to examine the generic pro-thrombotic effects of particle embolization. Immunohistochemistry was performed 1 and 24hours after transplantation. Xeno-islet transplantation activated both extrinsic and intrinsic coagulation pathways. The intrinsic pathway was also initiated by microsphere embolization, while extrinsic pathway tissue factor (TF) and platelet aggregation were more specific to engrafted islets. hCD46 expression significantly reduced TF, platelet, fibrin, and factor XIIIa accumulation in and around islets but did not alter intrinsic factor activation. Layers of TF+ cells emerged around islets within 24hours, particularly co-localized with vimentin, and identified as CD3+ and CD68+ cells inflammatory cells. These findings detail the origins of thrombosis following islet xenotransplantation, relate it to early immune activation, and suggest a role for transgenic hCD46 expression in its mitigation. Layers of TF-positive inflammatory cells and fibroblasts around islets at 24hours may have important roles in the progressive events of thrombosis, inflammatory cell recruitment, rejection, and the ultimate outcome of transplanted grafts. These suggest that the strategies targeting these elements could yield more progress toward successful xenogeneic islet engraftment and survival.

Multipotent mesenchymal stromal cells derived from porcine exocrine pancreas improve insulin secretion from juvenile porcine islet cell clusters.

Neonatal and juvenile porcine islet cell clusters (ICC) present an unlimited source for islet xenotransplantation to treat type 1 diabetes patients. We isolated ICC from pancreata of 14days old juvenile piglets and characterized their maturation by immunofluorescence and insulin secretion assays. Multipotent mesenchymal stromal cells derived from exocrine tissue of same pancreata (pMSC) were characterized for their differentiation potential and ability to sustain ICC insulin secretion in vitro and in vivo. Isolation of ICC resulted in 142±50×103 IEQ per pancreas. Immunofluorescence staining revealed increasing presence of insulin-positive beta cells between day 9 and 21 in culture and insulin content per 500IEC of ICC increased progressively over time from 1178.4±450µg/L to 4479.7±1954.2µg/L from day 7 to 14, P<.001. Highest glucose-induced insulin secretion by ICC was obtained at day 7 of culture and reached a fold increase of 2.9±0.4 compared to basal. Expansion of adherent cells from the pig exocrine tissue resulted in a homogenous CD90+ , CD34- , and CD45- fibroblast-like cell population and differentiation into adipocytes and chondrocytes demonstrated their multipotency. Insulin release from ICC was increased in the presence of pMSC and dependent on cell-cell contact (glucose-induced fold increase: ICC alone: 1.6±0.2; ICC+pMSC+contact: 3.2±0.5, P=.0057; ICC+pMSC no-contact: 1.9±0.3; theophylline stimulation: alone: 5.4±0.7; pMSC+contact: 8.4±0.9, P=.013; pMSC no-contact: 5.2±0.7). After transplantation of encapsulated ICC using Ca2+ -alginate (alg) microcapsules into streptozotocin-induced diabetic and immunocompetent mice, transient normalization of glycemia was obtained up to day 7 post-transplant, whereas ICC co-encapsulated with pMSC did not improve glycemia and showed increased pericapsular fibrosis. We conclude that pMSC derived from juvenile porcine exocrine pancreas improves insulin secretion of ICC by direct cell-cell contact. For transplantation purposes, the use of pMSC to support beta-cell function will depend on the development of new anti-fibrotic polymers and/or on genetically modified pigs with lower immunogenicity.

Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation.

Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non-human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof-of-concept experiments the potential of the cytoplasmic ubiquitin-editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF-stimulated NF-κB activation and NPI graft function. As adeno-associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. Forcing the expression of A20 in NPI with Ad5 vector blocked NF-κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro-inflammatory genes Cxcl10 and Icam1. A20-expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14-day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF-κB-dependent gene expression without adverse impact upon NPI maturation. We report a new protocol that allows for high-efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.

Versatility of hydrogel-forming microneedles in in vitro transdermal delivery of tuberculosis drugs

Current therapy of tuberculosis (TB) has several limitations, such as risk of liver injury and intestinal dysbiosis due to frequent oral administration of antibiotics. Transdermal administration could be used to improve antibiotic delivery for treatment of Mycobacterium tuberculosis infection. Therefore, we developed a novel approach, using hydrogel-forming microneedle (MN) arrays to transdermally deliver TB drugs, namely rifampicin, isoniazid, pyrazinamide and ethambutol, which have different physicochemical properties. These drugs were individually prepared into three types of drug reservoirs, including lyophilised tablets, directly compressed tablets and poly(ethylene glycol) tablets. Formulations of each drug reservoir type were optimised to achieve a rapidly dissolving tablet, and further integrated with hydrogel-forming MN arrays for in vitro permeation studies. Three types of hydrogel formulation were manufactured using different type of polymers and crosslinking processes. These MN arrays were then evaluated in terms of swelling ability, morphology and physical properties. Results of solute diffusion studies showed that drug permeation across the swollen hydrogel membrane was affected mostly by physiochemical properties and functional groups of each drug. In the in vitro studies, the amount of permeated drug through the hydrogel-forming MN arrays across the dermatomed neonatal porcine skin was affected by the drug solubility and reservoir design. The highest permeation of rifampicin (3.64 mg) and ethambutol (46.99 mg) were achieved using MN arrays combined with the poly(ethylene glycol) tablets and directly compressed tablet, respectively. For isoniazid and pyrazinamide, the highest drug permeation was attained using lyophilised reservoir with the amount of drug delivered approximately 58.45 mg and 20.08 mg, respectively. These equate to transdermal delivery of approximately 75% (rifampicin), 79% (isoniazid), 20% (pyrazinamide) and 47% (ethambutol) of the drugs loaded into the reservoirs on average. Importantly, the results of this work have demonstrated the versatility of hydrogel formulations to deliver a TB drug regime using MN arrays. Accordingly, this is a promising approach to deliver high dose of TB drugs.

Enhancing intradermal delivery of tofacitinib citrate: Comparison between powder-loaded hollow microneedle arrays and dissolving microneedle arrays.

Autoimmune-mediated inflammatory skin diseases, such as psoriasis, alopecia areata, and vitiligo, have been reported as the 4th leading cause of nonfatal disease burden worldwide. This is mainly related to the poor quality of life experienced by these patients. Although topical and systemic steroids represent the most common treatment, the variability in success rates and side effects often lead to treatment discontinuation. Recent off-label clinical studies using oral Janus Kinase (JAK) inhibitors (e.g., ruxolitinib, tofacitinib, baraticinib) have shown promising results. However, frequent side effects, such as infections and blood clots have been reported. Therefore, the aim of this research was to enhance the intradermal delivery of tofacitinib citrate with MN arrays. Using crosslinked hydrogels containing modifying agents (urea, sorbitol and sodium chloride), hollow MN arrays were fabricated and then loaded with tofacitinib citrate. Their efficiency in intradermal delivery of tofacitinib was compared with dissolving MN arrays and a control (Aqueous cream BP), using neonatal porcine skin. Despite the fact that the hydrogel was only present on the outer surface, hollow MN arrays showed comparable resistance to compression values and insertion capabilities to dissolving MN arrays. Although hollow MN arrays containing NaCl in the formulation led to slightly higher depositions of tofacitinib in epidermis and dermis of neonatal porcine skin when compared to a control cream, dissolving MN arrays showed superiority in terms of tofacitinib deposition in the dermis. Indeed, at 24h of the study, control cream and dissolving MN arrays delivered 143.98 ug/cm2 and 835 ug/cm2 of drug in the dermis, respectively, confirming the enhanced intradermal drug delivery capacity of MN arrays and their potential for treatment of autoimmune skin diseases.

INNATE IMMUNE SENSING AND TISSUE REMODELING OF A BIODEGRADABLE TEMPERING MATRIX SUPPORTED ISLET GRAFT

Introduction: The subcutaneous site represents an accessible alternative islet transplant site, but suffers from poor vascularisation. Here we test a clinically proven Biodegradable-Temporising-Matrix; NovoSorb™ (IDT) repurposed from use in burn and wound treatment that may provide a platform for subcutaneous islet-transplants. Materials and Methods: Human, neonatal porcine and murine islets were isolated using standard techniques. Islet viability & function was assessed using Annexin/Propodium iodide staining, Glucose Stimulated Insulin Secretion, ELISA and histology. Diabetes was induced using alloxan tetrahydrate i.v. in injectable grade water (20 mg/ml) at a dose of 110 mg/kg body weight. All transplants were surgically placed under the left renal capsule. Microscopy data was obtained using a Zeiss 7MP two-photon microscope. Results and Discussion: When tested in in-vitro co-culture experiments with human islets, IDT showed no impact on islet viability and function. Mouse islets were then transplanted under the renal capsule of diabetic recipients pre-implanted with IDT and resulted in complete recovery and long-term maintenance of euglycemia (>100 days). Also, neonatal porcine islets transplanted with IDT showed increasing porcine insulin production through time (>300 days). These data demonstrate that IDT is non-toxic for human, mouse and porcine islets and does not interfere with normal physiological function of islet grafts. Mice were then transplanted with IDT alone and the in-vivo foreign body reaction and tissue remodelling was assessed using transgenic (LysM-GFP+/tdTomTg) mice: blood vessels express tdTomato red and Lysozyme+ myeloid cells express GFP. Post-operative day (POD) 7 showed LysM-GFP+ myeloid cells infiltrating the graft. By POD 14, LysM-GFP+ myeloid cell-fusion events had occurred evidenced by the presence of giant cells and scattered immature vascular networks with dispersed loose collagen-fibrils. At POD 30, dendritic-like cells were observed, the vascular network was denser and the collagen fibres had increased in thickness. Further; to assess whether innate sensing of the islet graft would occur in the absence of a supportive scaffold, we transplanted islets from whole body tdTomTg mice (Tomato positive islets) into LysM-GFP/+ mice (syngeneic). 24hrs later LysM-GFP+ myeloid cells were migrating over the surface of the islets and were exhibiting neutrophil like ‘swarming’ behaviour with evident disruption of islet tissue. Conclusion: This analysis reveals that IDT can provide a neo-vascularization platform advantageous for subcutaneous islet transplantation and that there is a dynamic interaction between the innate-immune system and the syngeneic islet graft with or without IDT.

Reduced treatment frequencies with bumped kinase inhibitor 1369 are effective against porcine cystoisosporosis

Bumped kinase inhibitors (BKIs) are a new class of antiprotozoal drugs that target calcium-dependent protein kinase 1 (CDPK1) in various apicomplexan parasites. A multiple dose regimen of BKI 1369 has been shown to be highly effective against Cystoisospora suis (syn. Isospora suis), the causative agent of neonatal porcine coccidiosis. However, multiple dosing may not be widely applicable in the field. The present study aimed to determine the efficacy of reduced treatment frequencies with BKI 1369 against porcine cystoisosporosis in vitro and in vivo. Pre-incubation of sporozoites with BKI 1369 completely failed to inhibit the infection in vitro unless treatment was prolonged post-infection. Notably, a single treatment of infected cell cultures 2 days post-infection (dpi) resulted in a significant reduction of merozoite replication. In an experimental infection model, treatment of suckling piglets experimentally infected with C. suis 2 and 4 dpi with 20 mg BKI 1369/kg body weight completely suppressed oocyst excretion. A single treatment on the day of infection or 2 dpi suppressed oocyst excretion in 50% and 82% of the piglets and reduced the quantitative excretion in those that shed oocysts by 95.2% and 98.4%, respectively. Moreover, a significant increase in body weight gain and reduced number of diarrhea days were observed in BKI 1369 treated piglets compared to the control piglets, irrespective of time points and frequencies of treatment. Given that reduced treatment frequencies with BKI 1369 are comparable in efficacy to repeated applications without any adverse effects, this could be considered as a practical therapeutic alternative against porcine cystoisosporosis.

Long-Term Monitoring of Donor Xenogeneic Testis Tissue Grafts and Cell Implants in Recipient Mice Using Ultrasound Biomicroscopy

Testis tissue xenografting and testis cell aggregate implantation from various donor species into recipient mice are novel models for the study and manipulation of testis formation and function in target species. Thus far, the analysis of such studies has been limited to surgical or post-mortem retrieval of samples. Here we used ultrasound biomicroscopy (UBM) to monitor the development of neonatal porcine testis grafts and implants in host mice for 24 wk, and to correlate UBM and (immuno)histologic changes. This led to long-term visualization of gradual changes in volume, dimension and structure of grafts and implants; detection of a 4 wk developmental gap between grafts and implants; and revelation of differences in implant development depending on the craniocaudal site of implantation on the back of host mice. Our data support the reliability and precision of UBM for longitudinal study of transplants, which eliminates the need for frequent surgical sampling.

Development and Characterization of Gantrez&lt;sup&gt;®&lt;/sup&gt; S-97 and Hyaluronic Acid Microneedles for Transdermal Fluorescein Sodium Delivery

Microneedles (MNs) are attractive micron scale technology, which has been used as a physical force to create transport pathways and enhance the permeability of drugs into the skin. Fluorescein sodium (FS), a hydrophilic drug was loaded in MNs for transportation through skin. The purposes of this study were to develop and evaluate the optimal formulation of FS-loaded polymeric microneedles (MNs) as a device for transdermal drug delivery system. The FS-MNs were fabricated by micro-molding technique and prepared by using Gantrez® S-97 (G) and hyaluronic acid (HA). The physical appearances were observed under digital microscope. The mechanical properties were determined by a texture analyzer. The insertion study was tested on neonatal porcine skin. The MNs height changing after insertion into the skin at predetermined times was measured to show dissolution ability of MNs. Finally, the drug permeation profile of FS-MNs was investigated by Franz diffusion cell. For the results, all formulations were complete fabrication of conical microneedle array (11 rows x 11 columns in 10 mm2 patch area) with average 600 + 20 μm in height, 300 + 5 μm in width, and 600 + 10 μm in interspace. The percent decrease of MNs height in mechanical strength of 30%G+5%HA was significantly less than others at 1.8 to 8.8 N/121 array. The formulation mixing with 30% Gantrez® S-97 had 100% of penetration into porcine skin. The dissolution ability showed that MNs were completely dissolved within 60 minutes. At 24 h of skin permeation, the FS permeated through the skin from 1%FS solution, 30%G+1%FS MNs, and 30%G+5%HA+1%FS MNs was 1.00%, 4.27% and 7.53%, respectively. The flux values of 1%FS solution, 30%G+1%FS MNs, and 30%G+5%HA+1%FS MNs were 0.006 μg/cm2/min, 0.032 μg/cm2/min, and 0.037 μg/cm2/min, respectively, indicating the highest skin permeability of FS from 30%G+5%HA+1%FS MNs. In conclusion, the 30%G+5%HA+1%FS formulation presented appropriate MNs properties as a device for transdermal drug delivery system.

Protective effect of necrostatin-1 on the damage of pancreas islet cells induced by TNF-α.

To investigate whether necrostatin-1 (Nec-1) can protect islet cells from the damage induced by TNF-α. After isolation and purification, the neonatal porcine islet cell clusters (NICCs) were divided into 3 groups (islets 10 000 IEQ/group): a Nec-1 group (Nec-1+TNF-α was added to the culture medium), a TNF-α group (TNF-α was added to the culture medium), and a control group (pure medium). The number of cells was observed after 48 h of co-culture. The cell death was evaluated by AO/EB staining. Insulin secretion and DNA of islets were detected by chemiluminescence and nucleic acid quantitative analysis. RT-PCR assay was used to examine the mRNA expressions of insulin gene, glueogan gene and somatostatin gene. Flow cytometry analysis was used to detect the viability of B cells. The number of islets in Nec-1 group, TNF-α group and the control group were (8 425±2 187), (4 325±778), and (7 122±1 558) IEQ, respectively. Compared to the other two groups, the number of dead cells in TNF-α group was greatly increased. The insulin/DNA values in the Nec-1 group, TNF-α group and blank control group were (13.21±3.15), (2.47±0.45), and (7.44±0.97) mIU/mg, respectively. Compared to the TNF-α group and the control group, the mRNA relative expression levels of insulin gene (6.73±1.07), glucagon gene (10.13±1.98), somatostatin gene (8.57±1.11) were significantly increased in the Nec-1 group (all P<0.05), the rate of live cells (97.32±1.87)% and live B cells (90.86±3.68)% were increased significantly in the Nec-1 group (all P<0.05). TNF-α can induce neonatal porcine islet cells damage, which is attenuated in the presence of Nec-1. Nec-1 can increase the content of endocrine cells in NICCs.

Development, Evaluation, and Pharmacokinetic Assessment of Polymeric Microarray Patches for Transdermal Delivery of Vancomycin Hydrochloride.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause harmful and potentially deadly infections. Vancomycin remains the first-line antibiotic treatment for MRSA-derived infections. Nevertheless, as a peptide drug, it is poorly absorbed when administered orally because of its high molecular weight and low permeability in the gastrointestinal tract and is therefore administered intravenously for the treatment of systemic diseases. In order to circumvent some of the many drawbacks associated with intravenous injection, other routes of drug delivery should be investigated. One of the strategies which has been employed to enhance transdermal drug delivery is based on microarray patches (MAPs). This work, for the first time, describes successful transdermal delivery of vancomycin hydrochloride (VCL) using dissolving MAPs (DMAPs) and hydrogel-forming MAPs (HFMAPs). VCL was formulated into DMAPs and reservoirs [film dosage forms, lyophilized wafers, and compressed tablets (CSTs)] using excipients such as poly(vinyl pyrrolidone), poly(vinyl alcohol), sodium hyaluronate, d-sorbitol, and glycerol. In this study, HFMAPs were manufactured using aqueous blends containing poly(methylvinyl ether-co-maleic acid) cross-linked by esterification with poly(ethylene glycol). The VCL-loaded CSTs (60% w/w VCL) were the most promising reservoirs to be integrated with HFMAPs based on the physicochemical evaluations performed. Both HFMAPs and DMAPs successfully delivered VCL in ex vivo studies with the percentage of drug that permeated across the neonatal porcine skin recorded at 46.39 ± 8.04 and 7.99 ± 0.98%, respectively. In in vivo studies, the area under the plasma concentration time curve from time zero to infinity (AUC0-∞) values of 162.04 ± 61.84 and 61.01 ± 28.50 μg h/mL were achieved following the application of HFMAPs and DMAPs, respectively. In comparison, the AUC0-∞ of HFMAPs was significantly greater than that of the oral administration control group, which showed an AUC0-∞ of 30.50 ± 9.18 μg h/mL (p < 0.05). This work demonstrates that transdermal delivery of VCL is feasible using DMAPs and HFMAPs and could prove effective in the treatment of infectious diseases caused by MRSA, such as skin and soft tissue infections, lymphatic-related infections, and neonatal sepsis.

Localised and sustained intradermal delivery of methotrexate using nanocrystal-loaded microneedle arrays: Potential for enhanced treatment of psoriasis

Methotrexate (MTX), typically used as its sodium salt (MTX Na), is a first-line treatments for moderate to severe psoriasis, showing good efficacy. However, its systemic administration is associated with many side effects. Intradermal delivery into psoriatic tissue could offer an alternative approach. However, successful intradermal administration of MTX Na is currently precluded by its physicochemical properties. Moreover, due to its hydrophilic nature, MTX Na is swiftly cleared from the target tissue, necessitating frequent dosing which may affect patient compliance. To address these limitations, we investigated the combination of nanocrystal (NC) and dissolving microneedle (MN) technologies as an alternative approach for localised and sustained intradermal delivery of MTX Na. Poorly water-soluble MTX nanocrystals (MTX NC) were produced by a bottom-up technique with a mean particle size of 678 ± 15 nm. Sustained in vitro drug release was observed over 72 h. The MTX NC were then incorporated into the shafts of dissolving MN arrays with a drug loading of 2.48 mg/array. The MTX NC-loaded MN arrays exhibited satisfactory mechanical strength and insertion capabilities in the skin-simulant Parafilm M® and their shafts dissolved entirely in less than 20 min after insertion into excised neonatal porcine skin. Importantly, in vivo studies in Sprague Dawley rats revealed that the MN arrays were able to deposit approximately 25.1% of the loaded MTX NC in the skin, which acted, in turn, as a drug depot and released the MTX in a sustained manner over 72 h, while minimising MTX systemic exposure. Indeed, 24 h from MN application, 312.70 ± 161.95 µg/g of MTX was retained in the skin at the application site. This was approximately 322-fold higher than the amount of MTX (0.942 ± 0.59 µg/g) retained in the skin after oral administration of MTX Na. Interestingly, even after 72 h after MN application, around 12.5% of the MTX NC deposited in the skin by the MN was retained. In contrast, the maximal blood concentration of MTX achieved following MN application, was only 40% of that measured after oral administration of MTX Na. Accordingly, MTX NC-loaded dissolving MN arrays could be a promising approach for effective localised and sustained intradermal delivery of MTX as a potential enhanced treatment for psoriasis.

Cover page

XenotransplantationVolume 27, Issue 4 e12647 ISSUE INFORMATION Free Access Cover page First published: 07 September 2020 https://doi.org/10.1111/xen.12647 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The cover image is based on the Original Article Co-transplantation of human adipose-derived mesenchymal stem cells with neonatal porcine islets within a prevascularized subcutaneous space augments the xenograft function by Purushothaman Kuppan et al., https://doi.org/10.1111/xen.12581. Volume27, Issue4July/August 2020e12647 RelatedInformation

Development and characterisation of novel poly (vinyl alcohol)/poly (vinyl pyrrolidone)-based hydrogel-forming microneedle arrays for enhanced and sustained transdermal delivery of methotrexate.

Methotrexate (MTX) is one of the mainstays of treatment for rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) and it is mainly administered either orally or by subcutaneous (SC) injection, which are not so satisfactory. While orally administered MTX is associated with variable bioavailability and causes gastrointestinal side effects, including nausea and vomiting, SC injection is painful and produces high peak blood levels of MTX. Transdermal delivery presents an attractive alternative administration route. However, MTX passive permeation through the skin is hindered by the skin barrier and MTX physicochemical properties. To address these issues, hydrogel-forming microneedle arrays (HFMN) and a patch-like reservoir loaded with MTX (MTX-RV) were developed and combined to form a minimally invasive patch to deliver MTX transdermally in a sustained manner. HFMN were prepared from an aqueous blend of poly (vinyl alcohol) (PVA) and poly (vinyl pyrrolidone) (PVP) which was crosslinked chemically with citric acid (CA) at 130˚C. MTX-RV was prepared from hydroxypropyl methylcellulose (HPMC) and glycerol. Both the HFMN and MTX-RV were fully characterised and then combined to form an integrated patch, which was evaluated ex vivo and in preclinical studies. The HFMN demonstrated a satisfactory mechanical strength and insertion capability into excised neonatal porcine skin, as well as moderate swelling properties. The MTX-RV incorporated a high dose of MTX (150.3±5.3µg/mg) without precipitation. The integrated patch delivered MTX at a steady-state flux of 506.8±136.9µg.cm2/h in an ex vivo setup. Furthermore, in preclinical studies performed in Sprague Dawley rats, MTX appeared in blood after 1h from patch application at a concentration of 7.6±2.0nM. MTX blood level increased gradually to reach its peak, Cmax=35.1±5.1nM, at 24h. Importantly, the HFMN were removed intact from the skin with only mild erythema, despite the cytotoxic nature of MTX. Accordingly, the integrated patch produced in this work represents a promising minimally invasive transdermal drug delivery system that can overcome the skin barrier and deliver MTX in a sustained manner. This may help in minimising or even avoiding the nausea and vomiting, associated with the conventional administration routes.

A piglet model of iatrogenic rectosigmoid hypoganglionosis reveals the impact of the enteric nervous system on gut barrier function and microbiota postnatal development

BackgroundHirschsprung-associated enterocolitis physiopathology likely involves disturbed interactions between gut microbes and the host during the early neonatal period. Our objective was to create a neonatal porcine model of iatrogenic aganglionosis to evaluate the impact of the enteric nervous system (ENS) on microbiota and intestinal barrier postnatal development. MethodsUnder general anesthesia, the rectosigmoid serosa of 5-day-old suckling piglets was exposed to 0.5% benzalkonium chloride solution (BAC, n = 7) or saline (SHAM, n = 5) for 1 h. After surgery, animals returned to their home-cage with the sow and littermates and were studied 21 days later. ResultsBAC treatment induced partial aganglionosis with absence of myenteric plexus and reduced surface area of submucosal plexus ganglia (−58%, P < 0.05) in one third of the rectosigmoid circumference. Epithelial permeability of this zone was increased (conductance +63%, FITC-dextran flux +386%, horseradish-peroxidase flux +563%, P < 0.05). Tight junction protein remodeling was observed with decreased ZO-1 (−95%, P < 0.05) and increased claudin-3 and e-cadherin expressions (+197% and 61%, P < 0.05 and P = 0.06, respectively). BAC piglets harbored greater abundance of proinflammatory bacteria (Bilophila, Fusobacterium) compared to SHAM in the rectosigmoid lumen. ConclusionsThis large animal model demonstrates that hypoganglionosis is associated with dramatic defects of gut barrier function and establishment of proinflammatory bacteria.Level of evidence

Novel Design Approaches in the Fabrication of Polymeric Microarray Patches via Micromoulding.

The focus on novel systems for transdermal delivery of therapeutic agents has increased considerably over recent years, as this administration route comes with many advantages. Polymeric microarray patches (MAPs) are minimally invasive devices that enable systemic delivery of a wide range of drugs by overcoming the outer skin barrier. Conventionally, MAPs fabricated by micromoulding have a low needle density. In this study, the performance of hydrogel-forming MAPs cast using novel industrially manufactured micromoulds with a high needle density (600 needles/0.75 cm2) was compared to that of MAPs obtained using conventional moulds with a lower density (196 needles/0.89 cm2). Surrounding holders for micromoulds were designed for time-efficient fabrication of MAPs. The influence of needle densities on mechanical strength, insertion efficiency and in vitro permeation of ibuprofen sodium (IBU) was analysed. Insertion of both MAPs into an artificial skin model and neonatal porcine skin was comparable. No significant difference was observed in permeation studies of IBU (p > 0.05), with a delivery of 8.7 ± 1.7 mg for low-density and 9.5 ± 0.1 mg for high-density MAPs within 24 h. This highlights the potential of these novel micromoulds for manufacturing polymeric MAPs with a higher needle density for future applications.

Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes.

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1h from day 0 to day 3, every 2h from day 4 to day 7, and every 1h for 24h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

Effect of myofibre architecture on ventricular pump function by using a neonatal porcine heart model: from DT-MRI to rule-based methods.

Myofibre architecture is one of the essential components when constructing personalized cardiac models. In this study, we develop a neonatal porcine bi-ventricle model with three different myofibre architectures for the left ventricle (LV). The most realistic one is derived from ex vivo diffusion tensor magnetic resonance imaging, and other two simplifications are based on rule-based methods (RBM): one is regionally dependent by dividing the LV into 17 segments, each with different myofibre angles, and the other is more simplified by assigning a set of myofibre angles across the whole ventricle. Results from different myofibre architectures are compared in terms of cardiac pump function. We show that the model with the most realistic myofibre architecture can produce larger cardiac output, higher ejection fraction and larger apical twist compared with those of the rule-based models under the same pre/after-loads. Our results also reveal that when the cross-fibre contraction is included, the active stress seems to play a dual role: its sheet-normal component enhances the ventricular contraction while its sheet component does the opposite. We further show that by including non-symmetric fibre dispersion using a general structural tensor, even the most simplified rule-based myofibre model can achieve similar pump function as the most realistic one, and cross-fibre contraction components can be determined from this non-symmetric dispersion approach. Thus, our study highlights the importance of including myofibre dispersion in cardiac modelling if RBM are used, especially in personalized models.