Decellularization of allogeneic and xenogeneic tissues for cardiovascular reconstruction; assessment of a NaOH based process and perspectives

Current biological cardiovascular substitutes are hindered by inability to grow and fast degeneration of these tissues occurs in the adult and more precociously in child. This tissue deterioration is probably related to host immune reaction and glutaraldehyde cytotoxicity. Tissue decellularization/desantigenization is an interesting way to reduce immune reaction and to avoid the need of glutaraldehyde to mask antigenicity. Clinical results with decellularized patches, vessels or valves are promising for some studies while poor results may be associated with inadequate decellularization. No Gold standard decellularization process exists. Most of used processes are detergent-based. Our primary experimental work aims to evaluate a sodium hydroxide-based process as a decellularization agent. This process possess an additional propriety which is to inactivate conventional and non-conventional pathogens (such as prions) that other processes do not include. We assessed the efficacy of this processing in xenogeneic and allogeneic patches (patch study), porcine vessels (conduit study) and porcine valves (valve study). In each study, the tissues were firstly investigated in vitro for decellularization (cellularity was assessed by Hematoxylin and eosin-DAPI-DNA; antigenicity by IHC for alpha-Gal/MHC-I) and for mechanical resistance (by elongation stress test or compression test). Secondly, tissues were assessed in in vivo models (subcutaneous and intravascular) for inflammation (IHC for CD3/CD68), calcifications (von Kossa/Ca2+ tissue content) and vascular remodeling (IHF asma and CD31). In the patch study, adequate decellularization of porcine, bovine pericardia, peritoneum and human pericardium was obtained. In vitro, our Decellularized bovine pericardium (DBP) showed better decellularization in comparison to otherpericardia (two commercially available pericardia: Synovis pericardium (decellularized with another NaOH based-process and fixed in glutaraldehyde) and the Edwards pericardium (not decellularized et glutaraldehyde-fixed). After 4 months, in a vascular rodent model (n=15), the DBP used as a patch on the abdominal aorta, showed lower postoperative complications, lower calcifications, lower inflammation and better vascular remodeling than Synovis pericardium. In the conduit study, the entire porcine carotid artery was well decellularized. After 1 month, in a rodent vascular model (n=5), its showed low inflammation, partial recellularization and vascular remodeling. In the porcine carotid interposition model (n=2), the decellularized carotid was partially recellularized and showed less calcification, less inflammation, less intimal hyperplasia and better remodeling than PTFE/ synthetic prosthesis (control). Eventually, in our valve study, the porcine pulmonary valve was better decellularized with the NaOH-based process in comparison to a detergent process. In vivo, in a growing orthotopic implantation porcine model (n=3), after 1 month (n=1) and 3 months (n=2), the processed valves showed lower inflammation, partial recellularization and absence of major structural deterioration. In conclusion, we demonstrated the sodium hydroxide-based process can be proposed in order to obtain a decellularization of a wide range of cardiovascular substitutes. This tissue processing must therefore, be considered as an effective tool in the field of vascular tissue reconstruction.</p

Efficacy of a Sodium Hydroxide-based process for the decellularization of allogeneic and xenogeneic tissues in cardiovascular reconstruction

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, 201

Regeneration of abdominal wall musculofascial defects by a human acellular collagen matrix

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

The influence of implantation site on the biocompatibility and survival of alginate encapsulated pig islets in rats.

This work investigated the impact of implantation sites on the biocompatibility of alginate encapsulated pig islets. Non-diabetic rats were implanted with adult pig islets encapsulated in alginate either intraperitoneally (IP; n=25), subcutaneously (SC; n=37) or under the kidney capsule (KC; n=34). Capsule biocompatibility (retrieval rate, capsule diameter, degree of capsule broken and cellular overgrowth, CD68/CD3 staining) as well as islets viability and functionality were assessed until 30 days after transplantation. Implantation site did not significantly influence the biocompatibility of empty alginate capsules after transplantation (n=48). Most of the empty capsules (>90%) were retrieved after harvesting and were free of cellular overgrowth until day 30 post-transplantation. Three days after implantation, no significant difference for encapsulated pig islets was observed in terms of capsule biocompatibility and islet functionality in peritoneum, KC or subcutaneously. However, between days 5 and 30 after transplantation, explanted capsules from IP demonstrated a higher degree of broken capsules (>13%) and capsules with severe cellular overgrowth (>50%, CD68+ infiltration) than capsules removed from SC and KC (p<0.05). This was associated with a significant reduction of islet viability, insulin content and insulin secretion. In rats, the peritoneum site seems not appropriate for promoting the engraftment of encapsulated pig islets. Kidney subcapsular and subcutaneous spaces represent an interesting alternative

Streptozotocin-induced diabetes in large animals (pigs/primates): role of GLUT2 transporter and beta-cell plasticity.

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

Enhanced Vascular Biocompatibility and Remodeling of Decellularized and Secured Xenogeneic/Allogeneic Matrices in a Porcine Model.

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

Short-term outcomes of aortic valve neocuspidization for various aortic valve diseases

Objectives: Bioprosthetic valve deterioration remains a major limitation following aortic valve replacement. Favorable results have been reported with an autologous pericardium aortic valve neocuspidization. Methods: Seventy patients (31 women and 39 men) (mean age, 62 ± 12 years) with aortic stenosis (n = 52 [74%]) or aortic regurgitation (n = 18 [26%]) underwent the aortic valve neocuspidization procedure. Thirty-four patients (49%) had a tricuspid valve, 35 (50%) had a bicuspid valve, and 1 (1%) had a monocuspid valve. European System for Cardiac Operative Risk Evaluation and Society of Thoracic Surgeons scores were, respectively, 2.2% ± 2% and 2.0% ± 1.8%. Four patients (6%) had active endocarditis and 2 (3%) had endocarditis sequelae. One patient (1%) had fibroelastoma. A combined procedure was performed in 33 patients (46%). Results: The follow-up period was 24 ± 12 months. One patient (1%) died in hospital and 1 patient (1%) underwent conventional valve replacement for significant aortic regurgitation. Postoperative peak and mean pressure gradients were respectively 14 ± 5 and 8 ± 3 mm Hg. Aortic valve area was 2.5 ± 0.6 cm2. During follow-up, no patients died. Reintervention occurred in 2 patients (3%). At last follow-up, peak pressure gradient was 13 ± 7 mm Hg, mean pressure gradient was 7 ± 4 mm Hg, and aortic valve area was 2.3 ± 0.7 cm2. There was 1 recurrence of moderate aortic stenosis (1%). All patients were in New York Heart Association functional class I (90%) or II (10%). Freedom from major valve-related events was 92.1%, (98.5% for death, 95.2% for reintervention, and 95.2% for endocarditis). Conclusions: In our experience, the midterm outcomes of the aortic valve neocuspidization procedure with autologous glutaraldehyde fixed pericardium were acceptable for survival, operative risk and valve-related complications, for our all-comer patient population with various aortic valve diseases.</p

Decellularized and Secured Porcine Arteries with NaOH-based Process: Proof of Concept.

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

Feasibility of transapical aortic valve replacement through a left ventricular apical diverticulum.

ABSTRACT: Transapical aortic valve replacement is an established technique performed in high-risk patients with symptomatic aortic valve stenosis and vascular disease contraindicating trans-vascular and trans-aortic procedures. The presence of a left ventricular apical diverticulum is a rare event and the treatment depends on dimensions and estimated risk of embolisation, rupture, or onset of ventricular arrhythmias. The diagnosis is based on standard cardiac imaging and symptoms are very rare. In this case report we illustrate our experience with a 81 years old female patient suffering from symptomatic aortic valve stenosis, respiratory disease, chronic renal failure and severe peripheral vascular disease (logistic euroscore: 42%), who successfully underwent a transapical 23 mm balloon-expandable stent-valve implantation through an apical diverticulum of the left ventricle. Intra-luminal thrombi were absent and during the same procedure were able to treat the valve disease and to successfully exclude the apical diverticulum without complications and through a mini thoracotomy. To the best of our knowledge, this is the first time that a transapical procedure is successfully performed through an apical diverticulum