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Calcifications and absence of growth potential are the major drawbacks of glutaraldehyde-treated prosthesis. Decellularized and secured xeno-/allogeneic matrices were assessed in a preclinical porcine model for biocompatibility and vascular remodeling in comparison to glutaraldehyde-fixed bovine pericardium (GBP; control). Native human (fascia lata, pericardium) and porcine tissues (peritoneum) were used and treated. In vitro, biopsies were performed before and after treatment to assess decellularization (hematoxylin and eosin/DAPI). In vivo, each decellularized and control tissue sample was implanted subcutaneously in 4 mini-pigs. In addition, 9 mini-pigs received a patch or a tubularized prosthesis interposition on the carotid artery or abdominal aorta of decellularized (D) human fascia lata (DHFL; n = 4), human pericardium (DHP; n = 9), porcine peritoneum (DPPt; n = 7), and control tissue (GBP: n = 3). Arteries were harvested after 1 month and subcutaneous samples after 15-30 days. Tissues were processed for hematoxylin and eosin/von Kossa staining and immunohistochemistry for CD31, alpha-smooth muscle actin, CD3, and CD68. Histomorphometry was achieved by point counting. A 95% decellularization was confirmed for DHP and DPPt, and to a lower degree for DHFL. In the subcutaneous protocol, CD3 infiltration was significantly higher at day 30 in GBP and DHFL, and CD68 infiltration was significantly higher for GBP (p < 0.05). In intravascular study, no deaths, aneurysms, or pseudoaneurysms were observed. Inflammatory reaction was significantly higher for DHFL and GBP (p < 0.05), while it was lower and comparable for DHP/DPPt. DHP and DPPt showed deeper recellularization, and a new arterial wall was characterized. In a preclinical model, DPPt and DHP offered better results than conventional commercialized GBP for biocompatibility and vascular remodeling.

Glutaraldehyde fixed xenogeneic heart valve prosthesis are hindered by calcification and lack of growth potential. The aim of tissue decellularization is to remove tissue antigenicity, avoiding the use of glutaraldehyde and improve valve integration with low inflammation and host cell recolonization. In this preliminary study, we investigated the efficacy of a NaOH-based process for decellularization and biocompatibility improvement of porcine pulmonary heart valves in comparison to a detergent-based process (SDS-SDC0, 5%). Native cryopreserved porcine pulmonary heart valves were treated with detergent and NaOH-based processes. Decellularization was assessed by Hematoxylin and eosin/DAPI/alpha-gal/SLA-I staining and DNA quantification of native and processed leaflets, walls and muscles. Elongation stress test investigated mechanical integrity of leaflets and walls (n = 3 tests/valve component) of valves in the native and treated groups (n = 4/group). Biochemical integrity (collagen/elastin/glycosaminoglycans content) of leaflet-wall and muscle of the valves (n = 4/group) was assessed and compared between groups with trichrome staining (Sirius Red/Miller/Alcian blue). Secondly, a preliminary in vivo study assessed biocompatibility (CD3 and CD68 immunostaining) and remodeling (Hematoxylin and eosin/CD31 and ASMA immunofluorescent staining) of NaOH processed valves implanted in orthotopic position in young Landrace pigs, at 1 (n = 1) and 3 months (n = 2). Decellularization was better achieved with the NaOH-based process (92% vs 69% DNA reduction in the wall). Both treatments did not significantly alter mechanical properties. The detergent-based process induced a significant loss of glycosaminoglycans (p < 0,05). In vivo, explanted valves exhibited normal morphology without any sign of graft dilatation, degeneration or rejection. Low inflammation was noticed at one and three months follow-up (1,8 +/- 3,03 and 0,9836 +/- 1,3605 CD3 cells/0,12 mm in the leaflets). In one

BACKGROUND: There is a need for small caliber vascular prosthesis. Synthetic grafts are hindered by thrombogenicity and rapid occlusion. Decellularized matrices could be an alternative. We assessed in vitro and in vivo the biocompatibility of porcine artery treated with a chemical/physical process for decellularization and graft securitization with non/conventional pathogens inactivation. METHODS: Porcine carotid arteries (PCA) were treated. First, biopsies (n = 4/tissue) were performed before/after treatment to assess decellularization (hematoxylin and eosin/-4',6-diamidino-2-phenylindole/DNA/Miller). Second, 5 rats received an abdominal aortic patch of decellularized PCA (DPCA). Four pigs received subcutaneous DPCA implants (n = 2/pig). Half were explanted at day 15 and half at day 30. Finally, 2 pigs received DPCA (n = 2) and polytetrafluoroethylene prosthesis (n = 1), respectively, as carotid interposition. Implants were removed at day 30. Inflammation (CD3 and CD68 immunostaining) calcifications (von Kossa staining), remodeling (hematoxylin and eosin), and vascular characterization (CD31 and alpha-smooth muscle actin immunofluorescent staining) were investigated. RESULTS: Ninety-five percentage of decellularization was obtained without structural deterioration. No death occurred. Low inflammatory reaction was found in the 2 models for DPCA. Acquisition of vascular identity was confirmed in the rodent and porcine models. Similarity between native PCA and DPCA was observed after 30 days. In contrast, polytetrafluoroethylene graft showed severe calcifications, higher CD3 reaction, and higher intimal hyperplasia (P < 0.05). CONCLUSIONS: The physical and chemical process ensures decellularization of carotid porcine arteries and their in vivo remodeling with the presence of an endothelium and smooth-muscle-like cells as well as a low level of inflammatory cells.

Current biological cardiovascular substitutes are hindered by inability to grow and rapid degeneration in the adult and even more faster in child. Tissue decellularization is a way to reduce host immune reaction and to avoid the need of cytotoxic glutaraldehyde that are both incriminated. Clinical results with decellularized prostheses are promising for some while poor results are associated with inadequate decellularization. No standard process exists. Our work aim was to test a sodium hydroxide-based process as a decellularization agent for xenogeneic and allogeneic patches, porcine vessel and porcine valve. We demonstrated the efficacy and superiority of the process in comparison to others. In in vivo studies, the processed tissues showed better biocompatibility and remodeling than currently used grafts. Our work suggests high hopes for cardiovascular tissue regeneration with our processed tissues and we have great confidence for future clinical translation., (MED - Sciences médicales) -- UCL, 2018

BACKGROUND: Reconstruction of muscle defects remains a challenge. Our work assessed the potential of an engineered construct made of a human acellular collagen matrix (HACM) seeded with porcine mesenchymal stem cells (MSCs) to reconstruct abdominal wall muscle defects in a rodent model. METHODS: This study compared 2 sources of MSCs (bone-marrow, BMSCs, and adipose, ASCs) in vitro and in vivo for parietal defect reconstruction. Cellular viability and growth factor release (VEGF, FGF-Beta, HGF, IGF-1, TGF-Beta) were investigated under normoxic/hypoxic culture conditions. Processed and recellularized HACMs were mechanically assessed. The construct was tested in vivo in full thickness abdominal wall defect treated with HACM alone vs. HACM+ASCs or BMSCs (n=14). Tissue remodeling was studied at day 30 for neo-angiogenesis and muscular reconstruction. RESULTS: A significantly lower secretion of IGF was observed with ASCs vs. BMSCs under hypoxic conditions (-97.6%, p<0.005) whereas significantly higher VEGF/FGF secretions were found with ASCs (+92%, p<0.001 and +72%, p<0.05, respectively). Processing and recellularization did not impair the mechanical properties of the HACM. In vivo, angiogenesis and muscle healing were significantly improved by the HACM+ASCs in comparison to BMSCs (p<0.05) at day 30. CONCLUSION: A composite graft made of an HACM seeded with ASCs can improve muscle repair by specific growth factor release in hypoxic conditions and by in vivo remodeling (neo-angiogenesis/graft integration) while maintaining mechanical properties.

Glutaraldehyde preservation is the gold standard for cardiovascular biological prosthesis. However, secondary calcifications and the absence of tissue growth remain major limitations. Our study assessed in vitro and in vivo the biocompatibility of human (fascia lata, pericardium) and porcine tissues (pericardium, peritoneum) treated with a physicochemical procedure for decellularization and non-conventional pathogens inactivation. Biopsies were performed before and after treatment to assess decellularization (HE/Dapi staining/DNA quantification/MHC I/alpha gal immunostaining) and mechanical integrity. Forty-five rats received an abdominal aortic patch of native cryopreserved tissues (n = 20), treated tissues (n = 20) or glutaraldehyde-preserved bovine pericardium (GBP, control, n = 5). Grafts were explanted at 4 weeks and processed for HE/von Kossa staining and immunohistochemistries for lymphocytes (CD3)/macrophages (CD68) histomorphometry. 95% of decellularization was obtained for all tissues except for fascia lata (75%). Mechanical properties were slightly altered. In the in vivo model, a significant increase of CD3 and CD68 infiltrations was found in native and control implants in comparison with decellularized tissues (p < 0.05). Calcifications were found in 3 controls. Decellularized tissues were recolonized. GBP showed the most inflammatory response. This physicochemical treatment improves the biocompatibility of selected xeno/allogeneic tissues in comparison with their respective native cryopreserved tissues and with GBP. Incomplete decellularization is associated with a significantly higher inflammatory response. Our treatment is a promising tool in the field of tissue decellularization and tissue banking.

Although normothermia and warm blood cardioplegia are widely used in adults, cold crystalloids and hypothermia remain routinely used in pediatric cardiac surgery. The superiority of either technique in both brain and myocardial protection remains controversial. We designed a prospective randomized study to compare both approaches in terms of early myocardial protection and late neurodevelopmental status.

This work studied the reconstruction of an abdominal wall defect by a human acellular collagen matrix. The abdominal wall defect was cured in 40 rats by implanting (i) polypropylene (Pro), (ii) polyester (Mers) meshes, and (iii) human acellular collagen matrix with two orientations: fibres in parallel (fascia lata longitudinal [FLL]) or perpendicular (fascia lata transversal [FLT]) to native rats' abdominal walls. Hernia recurrence, adhesions, and histology (for inflammation and remodelling) were assessed at 4 and 8 weeks after implantation. Two large abdominal eventrations were cured by a human acellular matrix in human patients. A higher hernia recurrence rate was observed for rats transplanted with FLL than with FLT/Pro/Mers at 4 and 8 weeks after implantation. A lower adhesion rate was achieved for FLL/FLT than for Pro/Mers meshes (p<0.05). A decrease in immunologic cell infiltrations in FLL/FLT was observed between day 30 and day 60 (p<0.05). Collagen, elastin, and muscular tissues were found only in FLL/FLT matrix; a weaker muscular cell infiltration for FLL occurred at 8 weeks. Human abdominal eventrations were totally cured by using FLT as confirmed by computed tomography scanning at 12 and 16 months after implantation. In conclusion, human acellular collagen matrix, placed in an FLT position, can induce an abdominal wall reconstitution without adhesions and hernia recurrence.

BACKGROUND: To induce irreversible diabetes in large animals, the efficiency of streptozotocin (STZ) was evaluated in pigs, primates and compared to the gold standard model in rats. METHODS: Low (50 mg/kg) and high (150 mg/kg) doses of STZ were tested. Hepatic/renal function, glucose metabolism (intravenous glucose tolerance tests, fasting blood glucose) and histomorphometry were evaluated prior to, 1, and 4 weeks after STZ treatment. RESULTS: In rats and primates, expressing a high level of GLUT2 expression on beta cells, a dose of 50 mg/kg STZ induced irreversible diabetes (due to the 97% destruction of beta cell mass) without provoking liver or renal failure. In pigs, despite the use of high STZ dose, partial correction of hyperglycaemia was observed four weeks after STZ injection (decreased fasting blood glucose and intravenous glucose tolerance tests; increased insulin production). The correction of hyperglycaemia was associated with significant hypertrophy of immature pig beta-cell clusters (+30%, P<0.05), whereas no hypertrophy was observed in rats/primates. CONCLUSION: These results demonstrated that STZ might be used to induce irreversible diabetes in rats and primates. In contrast, the low STZ sensitivity in pigs related to a low expression of GLUT2, higher number of immature beta cells and compensatory beta-cell hypertrophy, renders STZ-induced diabetes inappropriate for studying islet allografts in swine.