Comparison of Two Percutaneous Atrial Septal Defect Occluders for Device Healing and Nickel Release in a Chronic Porcine Model.

AIMS: To investigate the healing process and nickel release of the Hyperion occluder (Comed BV, Netherlands), as compared to the Amplatzer septal occluder (ASO) (St. Jude Medical Inc., St. Paul, MN, USA) in a chronic swine model. BACKGROUND: Some long-term complications occurring after percutaneous atrial septal defect (ASD) closure may be partially associated with an inappropriate healing of the device and increased nickel release. There is no direct comparative study of different occluders for healing and nickel release. METHODS: After percutaneous ASD creation, 12 pigs were implanted with 15 mm Hyperion (n = 6) and 15 mm ASO (n = 6) devices. After 1 month (n = 3 for each device) and 3 months (n = 3 for each device) of follow-up, device explantation was performed and healing was assessed using histopathological workup. Systemic and tissular nickel release was performed. RESULTS: Implantation was successful in 100% without complications. Device coverage was observed as early as 1 month after implantation and was almost complete after 3 months. A granulation tissue with a predominantly mononuclear inflammatory reaction was observed in contact with nitinol wires while an inflammatory reaction was seen in contact with textile fibers. We found no statistically significant difference between the 2 devices whether for histological grading scores or systemic nickel release, regardless to follow-up duration. CONCLUSIONS: In this preclinical study, we demonstrated that Amplatzer septal occluder and Hyperion occluder were not significantly different for device healing and nickel release processes.

Comparison of the impact of preservation methods on amniotic membrane properties for tissue engineering applications.

Human amniotic membrane (hAM) is considered as an attractive biological scaffold for tissue engineering. For this application, hAM has been mainly processed using cryopreservation, lyophilization and/or decellularization. However, no study has formally compared the influence of these treatments on hAM properties. The aim of this study was to develop a new decellularization-preservation process of hAM, and to compare it with other conventional treatments (fresh, cryopreserved and lyophilized). The hAM was decellularized (D-hAM) using an enzymatic method followed by a detergent decellularization method, and was then lyophilized and gamma-sterilized. Decellularization was assessed using DNA staining and quantification. D-hAM was compared to fresh (F-hAM), cryopreserved (C-hAM) and lyophilized/gamma-sterilized (L-hAM) hAM. Their cytotoxicity on human bone marrow mesenchymal stem cells (hBMSCs) and their biocompatibility in a rat subcutaneous model were also evaluated. The protocol was effective as judged by the absence of nuclei staining and the residual DNA lower than 50 ng/mg. Histological staining showed a disruption of the D-hAM architecture, and its thickness was 84% lower than fresh hAM (p < 0.001). Despite this, the labeling of type IV and type V collagen, elastin and laminin were preserved on D-hAM. Maximal force before rupture of D-hAM was 92% higher than C-hAM and L-hAM (p < 0.01), and D-hAM was 37% more stretchable than F-hAM (p < 0.05). None of the four hAM were cytotoxic, and D-hAM was the most suitable scaffold for hBMSCs proliferation. Finally, D-hAM was well integrated in vivo. In conclusion, this new hAM decellularization process appears promising for tissue engineering applications.

In situ prevascularization designed by laser-assisted bioprinting: effect on bone regeneration.

Vascularization plays a crucial role in bone formation and regeneration process. Development of a functional vasculature to improve survival and integration of tissue-engineered bone substitutes remains a major challenge. Biofabrication technologies, such as bioprinting, have been introduced as promising alternatives to overcome issues related to lack of prevascularization and poor organization of vascular networks within the bone substitutes. In this context, this study aimed at organizing endothelial cells in situ, in a mouse calvaria bone defect, to generate a prevascularization with a defined architecture, and promote in vivo bone regeneration. Laser-assisted bioprinting (LAB) was used to pattern Red Fluorescent Protein-labeled endothelial cells into a mouse calvaria bone defect of critical size, filled with collagen containing mesenchymal stem cells and vascular endothelial growth factor. LAB technology allowed safe and controlled in vivo printing of different cell patterns. In situ printing of endothelial cells gave rise to organized microvascular networks into bone defects. At two months, vascularization rate (vr) and bone regeneration rate (br) showed statistically significant differences between the 'random seeding' condition and both 'disc' pattern (vr = +203.6%; br = +294.1%) and 'crossed circle' pattern (vr = +355%; br = +602.1%). These results indicate that in vivo LAB is a valuable tool to introduce in situ prevascularization with a defined configuration and promote bone regeneration.

Cell and tissue responses at the interface with a chitosan hydrogel intended for vascular applications: in vitro and in vivo exploration.

AIMS: The need for small caliber vessels to treat cardiovascular diseases has grown. However, synthetic polymers perform poorly in small-diameter applications. Chitosan hydrogels can provide a novel biological scaffold for vascular engineering. The goal of this study was to explore host cell and tissue behavior at the interface with chitosan-based scaffolds in vitro and in vivo. METHODS AND RESULTS: in vitro, we assessed the ability of endothelial cells lining chitosan hydrogels to produce tissue factor (TF), thrombomodulin (TM) and nitric oxide. We showed that endothelial cells behave as a native endothelium since under stimulation, TF and TM expression increased and decreased, respectively. Endothelial cells seeded on chitosan produced nitric oxide, but no change was observed under stimulation. After in vivo subcutaneous implantation of chitosan hydrogels in rats, macrophage activation phenotypes, playing a crucial role in biomaterial/tissue, were explored by immunohistochemistry. Our results suggested a balance between pro- and anti-inflammatory signals since we observed an inflammatory response in favor of macrophage M2 phenotype. CONCLUSION: in vitro exploration of endothelial cell response at the interface with chitosan hydrogel showed a functional endothelium and in vivo exploration of tissue response revealed a biointegration of chitosan hydrogels.

Colorectal wall regeneration resulting from the association of chitosan hydrogel and stromal vascular fraction from adipose tissue.

Chitosan hydrogel and adipose derived stem cells (ADCS) have been reported as the optimal partnership for colorectal tissue engineering. In that field, the aim of the current experiment was to assess the interest of seeding ADSC on chitosan hydrogel patches in an in vivo comparative study and on a tube intended replace a colonic segment in an in vivo feasibility study. In the comparative study, a 2 × 3 cm colonic wall defect was performed in 20 swine and repaired by suturing a chitosan hydrogel patch: acellular matrix (group A, n = 10) versus matrix seeded with autologous stromal vascular fraction (SVF) (group B, n = 10). In the feasibility study, a circular colonic section was performed and a 2-cm-length chitosan hydrogel tube (seeded with autologous SVF) was implanted between the two edges of the colon in 3 pigs. Graft areas were explanted at 8 weeks for examinations. Endpoints were technical feasibility, fibrosis ratio, and smooth muscle layer regeneration. A complete coverage of the patched area was observed with an ad integrum regeneration of the colonic wall including smooth muscle cells layer around a thin fibrosis scare. Fibrosis ratio was significantly lower group B: 13% versus 55% (p = 0.013). Segmental colonic replacement appeared accurate. Our data confirmed in a large animal model the healing effect of chitosan on colorectal tissue. The very low rate of the fibrosis ratio in the cellularized group emphasizes inflammatory control effect of the chitosan hydrogel and SVF association. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 460-467, 2018.

In situ printing of mesenchymal stromal cells, by laser-assisted bioprinting, for in vivo bone regeneration applications.

Bioprinting has emerged as a novel technological approach with the potential to address unsolved questions in the field of tissue engineering. We have recently shown that Laser Assisted Bioprinting (LAB), due to its unprecedented cell printing resolution and precision, is an attractive tool for the in situ printing of a bone substitute. Here, we show that LAB can be used for the in situ printing of mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite, in order to favor bone regeneration, in a calvaria defect model in mice. Also, by testing different cell printing geometries, we show that different cellular arrangements impact on bone tissue regeneration. This work opens new avenues on the development of novel strategies, using in situ bioprinting, for the building of tissues, from the ground up.

Unexpected Internalization of a Pulmonary Artery Band in a Porcine Model of Tetralogy of Fallot.

BACKGROUND: We report our experience of an unexpected complication of internalization of a pulmonary artery (PA) band in the vascular lumen, which occurred in a chronic porcine model of repaired tetralogy of Fallot (TOF). METHODS: Twelve piglets were divided into 3 groups: (1) TOF model animals (PA band plus pulmonary valvotomy, n = 4), (2) pulmonary insufficiency (PI) animals (pulmonary valvotomy, n = 4), and (3) control animals (n = 4). A nonabsorbable, coated braided polyester tape was used to perform the main pulmonary artery banding. Echocardiography was performed 4 months postoperatively. After each animal was euthanized, PA histological analysis was performed in animals with band internalization. RESULTS: Significant postsurgical pulmonary regurgitation and right ventricular enlargement were present in the TOF and PI, compared with control animals, whereas no significant pulmonary stenosis was observed in TOF animals when compared with PI group. Postmortem examination of all TOF animals revealed the constricting band to be intact but partially internalized into the PA lumen, allowing blood flow around the stenosis. Histological sections of the banded PA in the area of internalization showed a significant disorganization of the medial layer, with significant scarring and fibrotic reaction surrounding the outside of the band and the presence of inflammatory cells suggesting a significant inflammatory response during band internalization. CONCLUSIONS: Band internalization may occur after PA banding using a nonabsorbable, coated braided polyester tape in a chronic porcine model of repaired TOF. This unusual complication was likely due to the type of material used for banding.

Colorectal tissue engineering: A comparative study between porcine small intestinal submucosa (SIS) and chitosan hydrogel patches.

OBJECTIVE: Tissue engineering may provide new operative tools for colorectal surgery in elective indications. The aim of this study was to define a suitable bioscaffold for colorectal tissue engineering. METHODS: We compared 2 bioscaffolds with in vitro and in vivo experiments: porcine small intestinal submucosa (SIS) versus chitosan hydrogel matrix. We assessed nontoxicity of the scaffold in vitro by using human adipose-derived stem cells (hADSC). In vivo, a 1 × 2-cm colonic wall defect was created in 16 rabbits. Animals were divided randomly into 2 groups according to the graft used, SIS or chitosan hydrogel. Graft area was explanted at 4 and 8 weeks. The end points of in vivo experiments were technical feasibility, behavior of the scaffold, in situ putative inflammatory effect, and the quality of tissue regeneration, in particular smooth muscle layer regeneration. RESULTS: In vitro, hADSC attachment and proliferation occurred on both scaffolds without a substantial difference. After proliferation, hADSCs kept their mesenchymal stem cell characteristics. In vivo, one animal died in each group. Eight weeks after implantation, the chitosan scaffold allowed better wound healing compared with the SIS scaffold, with more effective control of inflammatory activity and an integral regeneration of the colonic wall including the smooth muscle cell layer. CONCLUSION: The outcomes of in vitro experiments did not differ greatly between the 2 groups. Macroscopic and histologic findings, however, revealed better wound healing of the colonic wall in the chitosan group suggesting that the chitosan hydrogel could serve as a better scaffold for colorectal tissue engineering.

Bioresorption mechanisms of chitosan physical hydrogels: a scanning electron microscopy study.

Tissue-engineered biodegradable medical devices are widely studied and systems must present suitable balance between versatility and elaboration simplicity. In this work, we aim at illustrating that such equilibrium can be found by processing chitosan physical hydrogels without external cross-linker. Chitosan concentration, degree of acetylation, solvent composition, and neutralization route were modulated in order to obtain hydrogels exhibiting different physico-chemical properties. The resulting in vivo biological response was investigated by scanning electron microscopy. "Soft" hydrogels were obtained from chitosan of high degree of acetylation (35%) and by the neutralization with gaseous ammonia of a chitosan acetate aqueous solutions presenting low polymer concentration (Cp=1.6% w/w). "Harder" hydrogels were obtained from chitosan with lower degree of acetylation (5%) and after neutralization in sodium hydroxide bath (1M) of hydro-alcoholic chitosan solutions (50/50 w/w water/1,2-propanediol) with a polymer concentration of 2.5% w/w. Soft and hard hydrogels exhibited bioresorption times from below 10 days to higher than 60 days, respectively. We also evidenced that cell colonization and neo-vascularization mechanisms depend on the hydrogel-aggregated structure that is controlled by elaboration conditions and possibly in relation with mechanical properties. Specific processing conditions induced micron-range capillary formation, which can be assimilated to colonization channels, also acting on the resorption scenario.

Physicochemical modulation of chitosan-based hydrogels induces different biological responses: interest for tissue engineering.

Polysaccharide-based hydrogels are remarkable materials for the development of tissue engineering strategies as they meet several critical requirements for such applications and they may partly mimic the extracellular matrix. Chitosan is widely envisioned as hydrogel in biomedical fields for its bioresorbability, biocompatibility, and fungistatic and bacteriostatic properties. In this study, we report that the modulation of the polymer concentration, the degree of acetylation, the gelation processes [or neutralization routes (NR)] in the preparation of different chitosan-based hydrogels lead to substantially and significantly different biological responses. We show that it is possible to tune the physicochemical characteristics, mechanical properties, and biological responses of such matrices. Physical hydrogels prepared from highly acetylated chitosan were softer, degraded quickly in vivo, and were not suitable for in vitro culture of human mesenchymal stem and progenitor derived endothelial cells. In contrast, for a same chitosan concentration and obtained by the same processing route, a low degree of acetylation chitosan hydrogel provided a more elastic material, better cell adhesion on its surface and tissue regeneration, and restored tissue neo-vascularization as well. This work offers promising and innovative perspectives for the design of hydrogel materials with tunable properties for tissue engineering and regenerative medicine.

Assessment of biphasic calcium phosphate to repair nasal septum defects in sheep.

BACKGROUND: Saddle nose and septal perforations are among the most surgically challenging situations in nasal reconstruction. They require a significant volume of autologous graft and a complex surgical procedure. The aim of this study was to evaluate the biocompatibility of the biphasic calcium phosphate implant in the nasal septum and its ability to replace septal skeleton with unilateral or bilateral exposure. METHODS: Thirty sheep underwent anterior nasal septum perforation. Only 20 septa were repaired with the implant exposed to nasal content on bilateral (group 2) and unilateral (group 3) sides. After 45 days of spontaneous cicatrization, the surface of new airway mucosa covering implants and the amount of closure were evaluated macroscopically. Light microscopy, histomorphometry, immunohistochemistry, and transmission electron microscopy were performed to assess soft-tissue growth and differentiation. Statistical analysis was performed by means of the Mann-Whitney test. RESULTS: The mean rate of mucoperichondrial flap recovery of the implant was 66 percent in group 2 and 82 percent in group 3, and was significantly different from that of the control group (p < 10(-4)). The mean amount of closure was 32 and 64 percent, respectively (p < 10(-3)). The thickness of the perichondrium was greater than the control on both sides (p < 10(-4)). Vascularized soft tissues and bone formation invaded pores of implants. No pathologic inflammation was observed in submucosa. Moderately differentiated and well differentiated newly formed epithelium were the most frequent types observed, with good correlation between immunostaining and morphologic features. CONCLUSION: These data suggest a good biocompatibility of biphasic calcium phosphate and its ability to repair the nasal septum in sheep.