Hypoglossal nerve stimulation (HNS) is indicated in the treatment of patients with moderate to severe obstructive sleep apnea (OSA) who fail or cannot tolerate positive airway pressure treatments. However, the availability of preclinical models is limiting further optimization of the HNS. This study aimed to develop a preclinical model to evaluate contraction force of genioglossus muscle following HNS. Male anesthetized pigs were surgically dissected to locate the genioglossus muscles and hypoglossal nerves. Muscle contraction force following electrical stimulation was studied using two protocols including single twitch and pulse train. Single twitch experiments demonstrated that muscle contraction force increased with amplitude until reaching a plateau, with strong fits to sigmoidal curves. The more gradual slope observed at shorter pulse durations suggests that amplitude titration at these pulse durations enables finer adjustment of treatment intensity, potentially improving patient tolerability. Pulse train experiments showed increase in force generation and fusion index with increasing frequency. The findings were reproducible across the animals with acceptable variations between animals. This HNS model in pigs can be a reliable and effective tool in evaluating activation of genioglossus muscle with variations in stimulation parameters and complement existing models of upper airway expansion.

Investigations on acute carbon monoxide (CO) poisoning struggle to highlight a relevant discriminant criterion related to CO poisoning severity for predicting complications, such as delayed neurological syndromes. In this context, it remains difficult to demonstrate the superiority of one method of oxygen (O2) administration over others or to identify the optimal duration of normobaric 100% oxygen (NBO) treatment. Myoglobin, as hemoglobin, are a potential binding site for CO, which could be a source of extravascular CO storage that impacts the severity of CO poisoning. It is not possible in routine clinical practice to estimate this potential extravascular CO storage. Indirect means of doing so that are available in the first few hours of poisoning could include, for example, the carboxyhemoglobin half-life (COHbt1/2), which seems to be influenced itself by the level and duration of CO exposure affecting this store of CO within the body. However, before the elimination of CO can be assessed, the COHbt1/2 toxicokinetic model must be confirmed: research still debates whether this model mono- or bi-compartmental. The second indirect mean could be the assessment of a potential COHb rebound after COHb has returned to 5% and NBO treatment has stopped. Moreover, a COHb rebound could be considered to justify the duration of NBO treatment. On an experimental swine model exposed to moderate CO poisoning (940 ppm for ±118 min until COHb reached 30%), we first confirm that the COHb half-life follows a bi-compartmental model. Secondly, we observe for the first time a slight COHb rebound when COHb returns to 5% and oxygen therapy is stopped. On the basis of these two toxicokinetic characteristics in favor of extravascular CO storage, we recommend that COHbt1/2 is considered using the bi-compartmental model in future clinical studies that compare treatment effectiveness as a potential severity criterion to homogenize cohorts of the same severity. Moreover, from a general toxicokinetic point of view, we confirm that a treatment lasting less than 6 hours appears to be insufficient for treating moderate CO poisoning.

Clinical pancreatic islet xenotransplantation will most probably rely on genetically modified pigs as donors. Several lines of transgenic pigs carrying one and more often, multiple modifications already exist. The vast majority of these modifications aim to mitigate the host immune response by suppressing major xeno-antigens, or expressing immunomodulatory molecules that act locally at the graft site. While these modifications are essential and have proven beneficial in preclinical trials, ensuring good intrinsic islet secretory function is equally important to achieve normoglycemia in recipients. Neonatal and even adult porcine islets are known for their low secretory response to physiological stimulation, a shortcoming that is often overcome by implanting extremely large numbers of such islets to compensate for insulin requirement incompatibilities between donor pigs and rodent, non-human primate or human recipients. Recent studies have revealed the existence of secretory amplifying pathways in porcine beta-cells previously identified in murine and human cells. Building upon these findings, a new line of transgenic pigs where these pathways are activated specifically in beta-cells has been created. Compared to their wild-type counterparts, islets from these transgenic pigs have proven to be better insulin secretors in their native pancreas environment, in vitro after isolation and most importantly in vivo after transplantation to diabetic mice.

XenotransplantationVolume 31, Issue 3 e12853 COMMENTARY In Memoriam – Guy Alexandre (1934–2024) David K. C. Cooper, Corresponding Author David K. C. Cooper [email protected] orcid.org/0000-0002-8899-9431 Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA Correspondence David K. C. Cooper, Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital Department of Surgery, Boston, USA. Email: [email protected]Search for more papers by this authorPierre Gianello, Pierre Gianello orcid.org/0000-0002-5326-0864 Institute of Experimental Medicine, Laboratory of Experimental Surgery and Transplantation, University of Louvain, Woluwe, Brussels, BelgiumSearch for more papers by this author David K. C. Cooper, Corresponding Author David K. C. Cooper [email protected] orcid.org/0000-0002-8899-9431 Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA Correspondence David K. C. Cooper, Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital Department of Surgery, Boston, USA. Email: [email protected]Search for more papers by this authorPierre Gianello, Pierre Gianello orcid.org/0000-0002-5326-0864 Institute of Experimental Medicine, Laboratory of Experimental Surgery and Transplantation, University of Louvain, Woluwe, Brussels, BelgiumSearch for more papers by this author First published: 23 June 2024 https://doi.org/10.1111/xen.12853Read the full textAboutPDF 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 onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1Alexandre GPJ. From the early days of human kidney allotransplantation to prospective xenotransplantation. In: PI, Terasaki ed. History of Transplantation: Thirty-five Recollections. IUCLA Tissue Typing Laboratory; 1991: 337-348. Google Scholar 2Cooper DKC. Guy Alexandre – an appreciation of his innovative contributions to organ transplantation. Transpl Immunol. 2020; 61:101309. doi:10.1016/j.trim.2020.101309 10.1016/j.trim.2020.101309 CASPubMedWeb of Science®Google Scholar 3Murray JE, Merrill JP, Dammin GJ, Dealy JB Jr, Alexandre GPJ, Harrison JH. Kidney transplantation in modified recipients. Ann Surg. 1962; 156: 337-355. 10.1097/00000658-196209000-00002 CASPubMedWeb of Science®Google Scholar 4Mollaret P, Goulon M. Coma depassé (in French). Rev Neurol. 1959; 101: 3-15. CASPubMedGoogle Scholar 5 GE Wolstenholme, M O'Connor, eds. Ethics in Medical Progress: With Special Reference to Transplantation. Churchill; 1966. 10.1002/9780470719480 Google Scholar 6 Ad Hoc Committee of the Harvard Medical School. A definition of irreversible coma. Report of the Ad Hoc Committee of the Harvard Medical School to Examine the Definition of Brain Death. JAMA. 1968; 205: 337-340 10.1001/jama.1968.03140320031009 PubMedWeb of Science®Google Scholar 7Alexandre GP, De Bruyere M, Squifflet JP, Moriau M, Latinne D, Pirson Y. Human ABO-Incompatible living donor renal homografts. Neth J Med. 1985; 28: 231-234. CASPubMedWeb of Science®Google Scholar 8Squifflet JP, De Meyer M, Malaise J, Latinne D, Pirson Y, Alexandre GPJ. Lessons learned from ABO-incompatible living donor kidney transplantation:years later. Clin Transplantation. 2004; 2: 208-213. Google Scholar 9Alexandre GPJ, Gianello P, Latinne D, et al. Plasmapheresis and splenectomy in experimental renal xenotransplantation. In: MA Hardy, ed. Xenograft 25. Elsevier Sciences (Biomedical Division); 1989: 259-266. Web of Science®Google Scholar 10Alexandre GPJ. From ABO-incompatible human kidney transplantation to xenotransplantation. Xenotransplantation. 2004; 11: 233-236. 10.1111/j.1399-3089.2004.00105.x PubMedWeb of Science®Google Scholar 11Lambrigts D, Franssen C, Martens H, et al. Development of thymus autografts under the kidney capsule in the pig. A new "organ" for xenotransplantation. Xenotransplantation. 1996; 3: 296-303. 10.1111/j.1399-3089.1996.tb00151.x Web of Science®Google Scholar 12Lambrigts D, Menard MY, Alexandre GPJ, et al. Creation of the "thymoheart" allograft. Implantation of autologous thymus into the heart prior to procurement. Transplantation. 1998; 66: 810-814. 10.1097/00007890-199809270-00019 CASPubMedWeb of Science®Google Scholar Volume31, Issue3May/June 2024e12853 ReferencesRelatedInformation

Introduction: Nipple–areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization–recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast.Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30 days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7 days, while human keratinocytes were seeded on the scaffold epidermal side for 10 days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis.Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30 days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7 days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7 days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10 days of culture.Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

Background and aimsHuman islet preparations designated for research exhibit diverse insulin-secretory profiles. This study aims to assess the impact of donor- and isolation-related factors on in vitro islet secretory function.MethodsA retrospective analysis of 46 isolations from 23 pancreata discarded for clinical transplantation was conducted. In vitro islet secretory function tests were performed on Day 1 and Day 7 of culture. Linear mixed-effects models (LMMs) were employed to investigate the relationships between various predictors characterizing the patient and donor characteristics as well as the isolation effectiveness and two functional outcomes including the islet stimulation index (SI) and area under the insulin curve (AUC). Fixed effects were introduced to represent the main effects of each predictor, and backward elimination was utilized to select the most significant fixed effects for the final model. Interaction effects between the timepoint (Day 7 vs. Day 1) and the predictors were also evaluated to assess whether predictors were associated with the temporal evolution of SI and AUC. Fold-change (Fc) values associated with each predictor were obtained by exponentiating the corresponding coefficients of the models, which were built on log-transformed outcomes.ResultsAnalysis using LMMs revealed that donor body mass index (BMI) (Fc = 0.961, 95% CI = 0.927–0.996, p = 0.05), donor gender (female vs. male, Fc = 0.702, 95% CI = 0.524–0.942, p = 0.04), and donor hypertension (Fc = 0.623, 95% CI = 0.466–0.832, p= <0.01) were significantly and independently associated with SI. Moreover, donor gender (Fc = 0.512, 95% CI = 0.302–0.864, p = 0.02), donor cause of death (cerebrovascular accident vs. cardiac arrest, Fc = 2.129, 95% CI = 0.915–4.946, p = 0.09; trauma vs. cardiac arrest, Fc = 2.129, 95% CI = 1.112–7.106, p = 0.04), pancreas weight (Fc = 1.01, 95% CI = 1.001–1.019, p = 0.03), and islet equivalent (IEQ)/mg (Fc = 1.277, 95% CI = 1.088–1.510, p ≤ 0.01) were significantly and independently associated with AUC. There was no predictor significantly associated with the temporal evolution between Day 1 and Day 7 for both SI and AUC outcomes.ConclusionThis study identified donor- and isolation-related factors influencing in vitro islet secretory function. Further investigations are essential to validate the applicability of these results in clinical practice.

To be clinically efficient, beta cell replacement therapies such as pig islet xenotransplantation must ensure sufficient insulin secretion from grafted islets. While protection from host immune reaction is essential for islet engraftment and their subsequent functioning, intrinsic physiological properties of used cells are also a key factor. We have previously shown that islets with adenoviral-mediated expression of a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP-1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in their beta cells have greatly improved insulin secretory response to glucose stimulation that is otherwise 4 to 10 times lower than human islets. Here, we describe in vitro characterization of the secretory function of pancreatic islets, derived from transgenic pigs expressing the GLP-1M3R cassette under the porcine insulin promoter (InsGLP-1M3R), and their usage to treat insulin-dependent diabetes in an immunodeficient mouse model. Our results show that InsGLP-1M3R islets isolated from neonatal and adult pigs secrete up to 15-fold more insulin in response to glucose stimulation compared to wild-type (WT) islets. They also proved to be more efficient in treating diabetes in a preclinical model as shown by a significantly higher percentage of normoglycemic recipients and higher porcine C-peptide levels up to 9 mo post implantation.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes, but the survival and function of transplanted islets are hindered by the loss of extracellular matrix (ECM) during islet isolation and by low oxygenation upon implantation. This study aimed to evaluate the impact of hypoxia on ECM using a cutting-edge imaging approach based on tissue clearing and 3D microscopy. Human and rat islets were cultured under normoxic (O2 21%) or hypoxic (O2 1%) conditions. Immunofluorescence staining targeting insulin, glucagon, CA9 (a hypoxia marker), ECM proteins (collagen 4, fibronectin, laminin), and E-cadherin (intercellular adhesion protein) was performed on fixed whole islets. The cleared islets were imaged using Light Sheet Fluorescence Microscopy (LSFM) and digitally analyzed. The volumetric analysis of target proteins did not show significant differences in abundance between the experimental groups. However, 3D projections revealed distinct morphological features that differentiated normoxic and hypoxic islets. Under normoxic conditions, ECM could be found throughout the islets. Hypoxic islets exhibited areas of scattered nuclei and central clusters of ECM proteins, indicating central necrosis. E-cadherin was absent in these areas. Our results, demonstrating a diminution of islets’ functional mass in hypoxia, align with the functional decline observed in transplanted islets experiencing low oxygenation after grafting. This study provides a methodology combining tissue clearing, multiplex immunofluorescence, Light Sheet Fluorescence Microscopy, and digital image analysis to investigate pancreatic islet morphology. This 3D approach allowed us to highlight ECM organizational changes during hypoxia from a morphological perspective.

Background.In islet transplantation, the use of dynamic hypothermic preservation techniques is a current challenge. This study compares the efficacy of 3 pancreas preservation methods: static cold storage, hypothermic machine perfusion (HMP), and oxygenated HMP. Methods.A standardized human pancreas split model was employed using discarded organs from both donation after brain death (n = 15) and donation after circulatory death (DCD) (n = 9) donors. The pancreas head was preserved using static cold storage (control group), whereas the tail was preserved using the 3 different methods (study group). Data on donor characteristics, pancreas histology, isolation outcomes, and functional tests of isolated islets were collected. Results.Insulin secretory function evaluated by calculating stimulation indices and total amount of secreted insulin during high glucose stimulation (area under the curve) through dynamic perifusion experiments was similar across all paired groups from both DCD and donation after brain death donors. In our hands, islet yield (IEQ/g) from the pancreas tails used as study groups was higher than that of the pancreas heads as expected although this difference did not always reach statistical significance because of great variability probably due to suboptimal quality of organs released for research purposes. Moreover, islets from DCD organs had greater purity than controls (P <= 0.01) in the HMP study group. Furthermore, our investigation revealed no significant differences in pancreas histology, oxidative stress markers, and apoptosis indicators. Conclusions.For the first time, a comparative analysis was conducted, using a split model, to assess the effects of various preservation methods on islets derived from pancreas donors. Nevertheless, no discernible variances were observed in terms of islet functionality, histological attributes, or isolation efficacy. Further investigations are needed to validate these findings for clinical application.

Short bubble and subsequent surface oxygenation is an innovative oxygenation technique and alternative for membrane oxygenation during hypothermic machine perfusion (HMP). The metabolic effect of the interruption of surface oxygenation for 4 h (mimicking organ transport) during HMP was compared to continuous surface and membrane oxygenation in a pig kidney ex situ preservation model. After 30 min of warm ischemia by vascular clamping, a kidney of a ±40 kg pig was procured and subsequently preserved according to one of the following groups: (1) 22-h HMP + intermittent surface oxygenation (n = 12); (2) 22-h HMP + continuous membrane oxygenation (n = 6); and (3) 22-h HMP + continuous surface oxygenation (n = 7). Brief perfusate O2 uploading before kidney perfusion was either obtained by direct bubble (groups 1, 3) or by membrane (group 2) oxygenation. Bubble oxygenation during minimum 15 min was as efficient as membrane oxygenation in achieving supraphysiological perfusate pO2 levels before kidney perfusion. Metabolic tissue analysis (i.e., lactate, succinate, ATP, NADH, and FMN) during and at the end of the preservation period demonstrated similar mitochondrial protection between all study groups. Short bubble and subsequent intermittent surface oxygenation of the perfusate of an HMP-kidney might be an effective and cheap preservation strategy to protect mitochondria, eliminating the need/costs of a membrane oxygenator and oxygen source during transport.

Background & aims: To be clinically efficient, beta-cell replacement therapies such as pig islet xenotransplantation must ensure sufficient insulin secretion from grafted islets. While protection from host immune reactions is essential for islet engraftment and their subsequent functioning, intrinsic physiological properties of used cells are also a key factor. We have previously shown that islets from transgenic pigs expressing a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP-1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in their beta-cells have greatly improved insulin secretory response to glucose stimulation that is otherwise 4-10 times lower than human islets. Our aim in the current study was to use such modified islets to treat insulin-dependent diabetes in a xenotransplantation model. Material & methods: Pancreatic islets were isolated from transgenic and wild-type neonatal piglets and kept in culture in a maturation medium for 8 days. Glucose-stimulated insulin secretion from mature islets was evaluated in vitro during dynamic perifusion experiments. Streptozotocin-induced diabetic immunodeficient NSG mice received 2500 IEQ transgenic or control islets under the left kidney capsule. Blood glucose was monitored weekly and porcine c-peptide was measured monthly to evaluate graft function. Normoglycemic mice had their graft-bearing kidney removed 9 months post-implantation and follow-up continued for hyperglycemia to occur. Sections of graft bearing kidneys and of recipient micee pancreas were stained for insulin and glucagon to evaluate graft maturation and exclude endogenous beta-cell regeneration. Results: Islets isolated from transgenic InsGLP-1M3R piglets secreted 5.5 to 7.5-fold more insulin compared to controls upon stimulation with 15 mM glucose and had G15/G1 stimulation ratio of 3.7 to 7.5 compared to 2.6 for wild type islets. 80% of InsGLP-1M3R islet mice recipients became normoglycemic within 4 weeks vs. 11% of wild-type islet recipients. Porcine c-peptide could be detected in mice blood by the 3rd week post-implantation and gradually increased throughout the 9-month follow-up period with the highest values recorded in recipients of transgenic islets. Graft removal resulted in hyperglycemia in all normoglycemic mice. Immunohistological analysis showed a significant increase of insulin-positive cells within grafted islets whereas no insulin could be detected in the pancreas 9 months following streptozotocin treatment. Conclusion & perspectives: InsGLP-1M3R neonatal pig islets showed significantly improved insulin secretion both in vitro and in vivo. They were found to be more efficient in treating insulin-dependent diabetes in a xenotransplantation immunodeficient model compared to non-modified islets. These islets sourced from a specific pathogen-free herd represent a very promising beta-cell replacement therapy alternative for use in research and in the clinic. Service public de Wallonie, SPW Economie, Emploi, Recherche: Grant ‘‘8318 SPW’’.

The purpose of this study is to enhance massive bone allografts osseointegration used to reconstruct large bone defects. These allografts show >50% complication rate requiring surgical revision in 20% cases. A new protocol for total bone decellularisation exploiting the vasculature can offer a reduction of postoperative complication by annihilating immune response and improving cellular colonization/ osseointegration. The nutrient artery of 18 porcine bones - humerus/femur/radius/ulna - was cannulated. The decellularization process involved immersion and sequential perfusion with specific solvents over a course of one week. Perfusion was realized by a peristaltic pump (mean flow rate: 6ml/min). The benefit of arterial perfusion was compared to a control group kept in immersion baths without perfusion. Bone samples were processed for histology (HE, Masson's trichrome and DAPI for cell detection), immunohistochemistry (IHC : Collagen IV/elastin for intraosseous vascular system evaluation, Swine Leukocyte Antigen – SLA for immunogenicity in addition to cellular clearance) and DNA quantification. Sterility and solvent residues in the graft were also evaluated with thioglycolate test and pH test respectively. Compared to native bones, no cells could be detected and residual DNA was <50ng/mg dry weight. Intramedullary spaces were completely cleaned. IHC showed the preservation of intracortical vasculature with channels bounded by Collagen IV and elastin within Haversian systems. IHC also showed a significant decrease in SLA signaling. All grafts were sterile at the last decellularization step and showed no solvent residue. The control group kept in immersion baths, paired with 6 perfused radii/ulnae, showed that the perfusion is mandatory to ensure complete decellularisation. Our results prove the effectiveness of a new concept of total bone decellularisation by perfusion. These promising results could lead to a new technique of Vascularized Composite Allograft transposable to pre-clinical and clinical models.

The lack of viability of massive bone allografts for critical-size bone defect treatment remains a challenge in orthopedic surgery. The literature has reviewed the advantages of a multi-combined treatment with the synergy of an osteoconductive extracellular matrix (ECM), osteogenic stem cells, and growth factors (GFs). Questions are still open about the need for ECM components, the influence of the decellularization process on the latter, the related potential loss of function, and the necessity of using pre-differentiated cells. In order to fill in this gap, a bone allograft surrounded by an osteogenic membrane made of a decellularized collagen matrix from human fascia lata and seeded with periosteal mesenchymal stem cells (PMSCs) was analyzed in terms of de-/recellularization, osteogenic properties, PMSC self-differentiation, and angiogenic potential. While the decellularization processes altered the ECM content differently, the main GF content was decreased in soft tissues but relatively increased in hard bone tissues. The spontaneous osteogenic differentiation was necessarily obtained through contact with a mineralized bone matrix. Trying to deepen the knowledge on the complex matrix-cell interplay could further propel these tissue engineering concepts and lead us to provide the biological elements that allow bone integration in vivo.

In terms of large bone defect reconstructions, massive bone allografts may sometimes be the only solution. However, they are still burdened with a high postoperative complication rate. Our hypothesis is that the immunogenicity of residual cells in the graft is involved in this issue. Decellularization by perfusion might therefore be the answer to process and create more biologically effective massive bone allografts. Seventy-two porcine bones were used to characterize the efficiency of our sodium hydroxide-based decellularization protocol. A sequence of solvent perfusion through each nutrient artery was set up to ensure the complete decellularization of whole long bones. Qualitative (histology and immunohistochemistry [IHC]) and quantitative (fluoroscopic absorbance and enzyme-linked immunosorbent assay) evaluations were performed to assess the decellularization and the preservation of the extracellular matrix in the bone grafts. Cytotoxicity and compatibility were also tested. Comparatively to nontreated bones, our experiments showed a very high decellularization quality, demonstrating that perfusion is mandatory to achieve an entire decellularization. Moreover, results showed a good preservation of the bone composition and microarchitecture, Haversian systems and vascular network included. This protocol reduces the human leukocyte antigen antigenic load of the graft by >50%. The majority of measured growth factors is still present in the same amount in the decellularized bones compared to the nontreated bones. Histology and IHC show that the bones were cell compatible, noncytotoxic, and capable of inducing osteoblastic differentiation of mesenchymal stem cells. Our decellularization/perfusion protocol allowed to create decellularized long bone graft models, thanks to their inner vascular network, ready for in vivo implantation or to be further used as seeding matrices.

Background The COBE 2991 cell processor, commonly used for pancreatic islet isolation, is no longer distributed in Europe, leading to a search for alternative purification procedures with equivalent efficacy. The aim of this study was to evaluate the efficacy of an alternative method based on the discontinuous purification of islets. Methods The conventional isolation procedure using a standard continuous islet purification with COBE 2991 of n = 4 human pancreas was compared to n = 8 procedures using a discontinuous purification with a “bottle” method from donors of similar characteristics. Islet equivalents, purity, and dynamic glucose-stimulated insulin secretion were evaluated. Results A similar islet yield was obtained using continuous vs. discontinuous purification methods (76,292.5 ± 40,550.44 vs. 79,625 ± 41,484.46 islet equivalents, p = 0.89). Islets from both groups had similar purity (78.75% ± 19.73% vs. 55% ± 18.16%, p = 0.08) and functionality both in terms of stimulation index (3.31 ± 0.83 vs. 5.58 ± 3.38, p = 0.22) and insulin secretion (1.26 ± 0.83 vs. 1.53 ± 1.40 mean AUC, p = 0.73). Moreover, the size of the islets was significantly larger in the discontinuous vs. continuous purification group (19.2% ± 10.3% vs. 45.4% ± 18.8% of islets less than 100 µm, p = 0.0097 and 23.7% ± 5.3% vs. 15.6% ± 5.8% of 200–250 µm islet size, p = 0.03). Conclusion Compared to the conventional purification procedure, discontinuous purification with a bottle method shows similar results with regard to isolation yield and islet secretory function. Furthermore, this alternative technique allows for obtaining larger islets.

Transplantation of heart following donation after circulatory death (DCD) was recently introduced into clinical practice. Ex vivo reperfusion following DCD and retrieval is deemed necessary in order to evaluate the recovery of cardiac viability after the period of warm ischemia. We tested the effect of four different temperatures (4 °C-18 °C-25 °C-35 °C) on cardiac metabolism during 3-h ex vivo reperfusion in a porcine model of DCD heart. We observed a steep fall in high-energy phosphate (ATP) concentrations in the myocardial tissue at the end of the warm ischemic time and only limited regeneration during reperfusion. Lactate concentration in the perfusate increased rapidly during the first hour of reperfusion and slowly decreased afterward. However, the temperature of the solution does not seem to have an effect on either ATP or lactate concentration. Furthermore, all cardiac allografts showed a significant weight increase due to cardiac edema, regardless of the temperature.

Introduction: Durable reconstruction of critical size bone defects is still a surgical challenge despite the availability of numerous autologous and substitute bone options. In this paper, we have investigated the possibility of creating a living bone allograft, using the perfusion/decellularization/recellularization (PDR) technique, which was applied to an original model of vascularized porcine bone graft.Materials and Methods: 11 porcine bone forelimbs, including radius and ulna, were harvested along with their vasculature including the interosseous artery and then decellularized using a sequential detergent perfusion protocol. Cellular clearance, vasculature, extracellular matrix (ECM), and preservation of biomechanical properties were evaluated. The cytocompatibility and in vitro osteoinductive potential of acellular extracellular matrix were studied by static seeding of NIH-3T3 cells and porcine adipose mesenchymal stem cells (pAMSC), respectively.Results: The vascularized bone grafts were successfully decellularized, with an excellent preservation of the 3D morphology and ECM microarchitecture. Measurements of DNA and ECM components revealed complete cellular clearance and preservation of ECM’s major proteins. Bone mineral density (BMD) acquisitions revealed a slight, yet non-significant, decrease after decellularization, while biomechanical testing was unmodified. Cone beam computed tomography (CBCT) acquisitions after vascular injection of barium sulphate confirmed the preservation of the vascular network throughout the whole graft. The non-toxicity of the scaffold was proven by the very low amount of residual sodium dodecyl sulfate (SDS) in the ECM and confirmed by the high live/dead ratio of fibroblasts seeded on periosteum and bone ECM-grafts after 3, 7, and 16 days of culture. Moreover, cell proliferation tests showed a significant multiplication of seeded cell populations at the same endpoints. Lastly, the differentiation study using pAMSC confirmed the ECM graft’s potential to promote osteogenic differentiation. An osteoid-like deposition occurred when pAMSC were cultured on bone ECM in both proliferative and osteogenic differentiation media.Conclusion: Fully decellularized bone grafts can be obtained by perfusion decellularization, thereby preserving ECM architecture and their vascular network, while promoting cell growth and differentiation. These vascularized decellularized bone shaft allografts thus present a true potential for future in vivo reimplantation. Therefore, they may offer new perspectives for repairing large bone defects and for bone tissue engineering.

Microencapsulating pancreatic islets in immunoprotective alginate hydrogels is a promising strategy for treatment of type 1 diabetes. However, this strategy is limited by inflammation and hypoxia mediated oxidative stress, due to encapsulation and the hydrogel itself, leading to impaired insulin secretion and limited short and long term cell survival. Herein, the antioxidant effect of fucoidan, an algae derived polysaccharide, on beta cells, and its positive effects on encapsulated beta cell viability and function is presented. Fucoidan from Fucus vesiculosus (FF) exhibits a high total antioxidant capacity, and free radical scavenging activity, and is able to significantly alleviate intracellular oxidative stress in rat insolinoma beta cells (INS1E). In addition, FF significantly increases insulin secretion in a dose‐ and time‐dependent manner. When FF is incorporated in ultrapure alginate used for microencapsulation of primary rat islets, both viability and glucose responsiveness of rat islets in these socalled Fucogel microcapsules (Fucocaps) are found to be significantly higher compared to islets encapsulated in alginate alone. Similar results are obtained with INS1E pseudoislets and neonatal pig islets. Fucocaps can provide a redox‐modulatory niche and an immune barrier for islets and beta cells in the same time leading to significantly improved survival and endocrine function by mitigating oxidative stress.