Progress in scaffold-free bioprinting for cardiovascular medicine

Biofabrication of tissue analogues is aspiring to become a disruptive technology capable to solve standing biomedical problems, from generation of improved tissue models for drug testing to alleviation of the shortage of organs for transplantation. Arguably, the most powerful tool of this revolution is bioprinting, understood as the assembling of cells with biomaterials in three-dimensional structures. It is less appreciated, however, that bioprinting is not a uniform methodology, but comprises a variety of approaches. These can be broadly classified in two categories, based on the use or not of supporting biomaterials (known as "scaffolds," usually printable hydrogels also called "bioinks"). Importantly, several limitations of scaffold-dependent bioprinting can be avoided by the "scaffold-free" methods. In this overview, we comparatively present these approaches and highlight the rapidly evolving scaffold-free bioprinting, as applied to cardiovascular tissue engineering

Of balls, inks and cages: Hybrid biofabrication of 3D tissue analogs

The overarching principle of three-dimensional (3D) bioprinting is the placing of cells or cell clusters in the 3D space to generate a cohesive tissue microarchitecture that comes close to in vivo characteristics. To achieve this goal, several technical solutions are available, generating considerable combinatorial bandwidth: (i) Support structures are generated first, and cells are seeded subsequently; (ii) alternatively, cells are delivered in a printing medium, so-called “bioink,” that contains them during the printing process and ensures shape fidelity of the generated structure; and (iii) a “scaffold-free” version of bioprinting, where only cells are used and the extracellular matrix is produced by the cells themselves, also recently entered a phase of accelerated development and successful applications. However, the scaffold-free approaches may still benefit from secondary incorporation of scaffolding materials, thus expanding their versatility. Reversibly, the bioink-based bioprinting could also be improved by adopting some of the principles and practices of scaffold-free biofabrication. Collectively, we anticipate that combinations of these complementary methods in a “hybrid” approach, rather than their development in separate technological niches, will largely increase their efficiency and applicability in tissue engineering

Of balls, inks and cages: Hybrid biofabrication of 3D tissue analogs

The overarching principle of three-dimensional (3D) bioprinting is the placing of cells or cell clusters in the 3D space to generate a cohesive tissue microarchitecture that comes close to in vivo characteristics. To achieve this goal, several technical solutions are available, generating considerable combinatorial bandwidth: (i) Support structures are generated first, and cells are seeded subsequently; (ii) alternatively, cells are delivered in a printing medium, so-called “bioink,” that contains them during the printing process and ensures shape fidelity of the generated structure; and (iii) a “scaffold-free” version of bioprinting, where only cells are used and the extracellular matrix is produced by the cells themselves, also recently entered a phase of accelerated development and successful applications. However, the scaffold-free approaches may still benefit from secondary incorporation of scaffolding materials, thus expanding their versatility. Reversibly, the bioink-based bioprinting could also be improved by adopting some of the principles and practices of scaffold-free biofabrication. Collectively, we anticipate that combinations of these complementary methods in a “hybrid” approach, rather than their development in separate technological niches, will largely increase their efficiency and applicability in tissue engineering

EMBODYING INEQUALITY: THREE PAPERS ON THE ROLE OF GENDER AND DISCRIMINATION IN THE LIVES OF WOMEN

Women experience different forms of discrimination throughout their lives such as unfair treatment in interpersonal interactions in the public spheres and in the private sphere, as prescribed by societal gender roles, women can also experience inequality and discrimination as a disproportionate share of household work and caregiving, limited participation in decision making, unequal access and share of financial resources and leisure, and as well as in cases, of emotional, and physical, or sexual abuse. Limited research explored the potential joint effect of these forms of discrimination. The questions explored in this dissertation were: (1) How does gendered intra-household inequality, experienced as a constraint to women’s agency, interact with post-migration factors like interpersonal discrimination and how does the combination of factors affect the mental health of immigrant women? (2) how does gendered intra-household inequality and discrimination interact to affect mental health among women and men in the U.S? And lastly, how do major stressful and traumatic events and discrimination interact to affect mental health among women and men in the U.S? In the first paper, I analysed data from the National Latino and Asian American Study and I found that first- and second-generation immigrant and refugee women experience intra-household inequality such as having no say in final decisions, experiencing excessive demands from their spouse and moderate or severe violence and that both discrimination and intrahousehold inequality made a separate and a significant contribution to increasing women’s risk for meeting criteria for depression and PTSD. The second paper had two small substudies. In the first I analyzed data from the 2011-2014 MIDUS Refresher study, and I examined the relationship between perceived everyday discrimination, intrahousehold inequality, and depression and anxiety among women and men. I found that everyday discrimination was associated with depression and perceived role strain with both health outcomes. In the second substudy I analyzed data from the 2012-2016 MIDUS Refresher Biomarker to explore a potential pathway between role strain and depression and anxiety symptoms and whether these processes were contingent upon the perception of discrimination in social interactions. I found that perceived stress mediated the relationship between role strain and depression and anxiety and that discrimination moderated that relationship such that in the presence of perceived discrimination in interpersonal interactions, intrahousehold inequality was associated with more psychological distress and more severe symptoms of depression and anxiety. In the last paper, I used data from the 2012-2016 MIDUS Refresher Biomarker and I examined the association between major stressful and traumatic events, perceived discrimination and perceived multiple reasons for discrimination and anxiety and depression among women and men. I first examined a potential pathway between adverse experiences and depression and anxiety symptoms, and I found that perceived stress mediates these relationships. In the second part, I tested the moderating role of discrimination and of perceived multiple forms of discrimination and found that individuals who reported a greater number of major stressful events reported higher levels of stress if they perceive higher levels of discrimination and, further, that higher levels of stress predicted higher levels of depressive symptoms if they perceive higher levels of discrimination. Perception of multiple forms of discrimination (two forms or there or more) was also associated with higher levels of perceived stress. The concluding chapter presents the main findings of these studies and recommendations for social work practice and future research

Modeling Stem/Progenitor Cell-Induced Neovascularization and\ud Oxygenation around Solid Implants

Tissue engineering constructs and other solid implants with biomedical applications, such as drug delivery devices or bioartificial organs, need oxygen (O2) to function properly. To understand better the vascular integration of such devices, we recently developed a novel model sensor containing O2-sensitive crystals, consisting of a polymeric capsule limited by a nano-porous filter. The sensor was implanted in mice with hydrogel alone (control) or hydrogel embedded with mouse CD117/c-kit+ bone marrow progenitor cells (BMPC) in order to stimulate peri-implant neovascularization. The sensor provided local partial O2 pressure (pO2) using non-invasive electron paramagnetic resonance (EPR) signal measurements. A consistently higher level of per-implant oxygenation was observed in the cell-treatment case as compared to the control over a 10-week period. In order to provide a mechanistic explanation of these experimental observations, we present in this paper a mathematical model, formulated as a system of coupled partial differential equations, that simulates peri-implant vascularization. In the control case, vascularization is considered to be the result of a Foreign Body Reaction (FBR) while in the cell-treatment case, adipogenesis in response to paracrine stimuli produced by the stem cells is assumed to induce neovascularization. The model is validated by fitting numerical predictions of local pO2 to measurements from the implanted sensor. The model is then used to investigate further the potential for using stem cell treatment to enhance the vascular integration of biomedical implants. We thus demonstrate how mathematical modeling combined with experimentation can be used to infer how vasculature develops around biomedical implants in control and stem celltreated cases

Comparison of Biomaterial-Dependent and -Independent Bioprinting Methods for Cardiovascular Medicine

There is an increasing need of human organs for transplantation, of alternatives to animal experimentation, and of better in vitro tissue models for drug testing. All these needs create unique opportunities for the development of novel and powerful tissue engineering methods, among which the 3D bioprinting is one of the most promising. However, after decades of incubation, ingenuous efforts, early success and much anticipation, biomaterial-dependent 3D bioprinting, although shows steady progress, is slow to deliver the expected clinical results. For this reason, alternative ‘scaffold-free’ 3D bioprinting methods are developing in parallel at an accelerated pace. In this opinion paper we discuss comparatively the two approaches, with specific examples drawn from the cardiovascular field. Moving the emphasis away from competition, we show that the two platforms have similar goals but evolve in complementary technological niches. We conclude that the biomaterial-dependent bioprinting is better suited for tasks requiring faster, larger, anatomically-true, cell-homogenous and matrix-rich constructs, while the scaffold-free biofabrication is more adequate for cell-heterogeneous, matrix-poor, complex and smaller constructs, but requiring longer preparation time

Labeling of endothelial cells with magnetic microbeads by angiophagy

Objectives Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism ‘angiophagy’. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. Results In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5 min, as compared to gravitational settling (p < 0.0001). Conclusion We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting

Bioprinting is a technology with the prospect to change the way many diseases are treated, by replacing the damaged tissues with live de novo created biosimilar constructs. However, after more than a decade of incubation and many proofs of concept, the field is still in its infancy. The current stagnation is the consequence of its early success: the first bioprinters, and most of those that followed, were modified versions of the three-dimensional printers used in additive manufacturing, redesigned for layer-by-layer dispersion of biomaterials. In all variants (inkjet, microextrusion, or laser assisted), this approach is material (“scaffold”) dependent and energy intensive, making it hardly compatible with some of the intended biological applications. Instead, the future of bioprinting may benefit from the use of gentler scaffold-free bioassembling methods. A substantial body of evidence has accumulated, indicating this is possible by use of preformed cell spheroids, which have been assembled in cartilage, bone, and cardiac muscle-like constructs. However, a commercial instrument capable to directly and precisely “print” spheroids has not been available until the invention of the microneedles-based (“Kenzan”) spheroid assembling and the launching in Japan of a bioprinter based on this method. This robotic platform laces spheroids into predesigned contiguous structures with micron-level precision, using stainless steel microneedles (“kenzans”) as temporary support. These constructs are further cultivated until the spheroids fuse into cellular aggregates and synthesize their own extracellular matrix, thus attaining the needed structural organization and robustness. This novel technology opens wide opportunities for bioengineering of tissues and organs

Prospects for the use of the laparoscopic transabdominal pre-peritoneal approach (TAPP) in groin hernia repair

Curs Chirurgie al Facultăţii Stomatologie, USMF “Nicolae Testemiţanu”, Spitalul Clinic Militar Central, Chisinău, Republica Moldova, Al XII-lea Congres al Asociației Chirurgilor „Nicolae Anestiadi” din Republica Moldova cu participare internațională 23-25 septembrie 2015Introducere: Abordul laparoscopic în cura herniilor inghinale devine o metodă de elecţie pe plan mondial. Rămîne actuală problema standardizării tehnicii chirurgicale şi optimizării rezultatelor acestui procedeu. Material şi metode: În perioada anilor 2008-2014 în Clinică a fost efectuată cura laparoscopică a herniei inghinale la 271 pacienţi (16 – bilateral). Repartiţia herniilor conform clasificării Nyhus: tip II (n=188), tip IIIa (n=64), tip IIIb (n=18), tip IIIc (n=9), tip IV (n=12). A fost utilizată tehnica transabdominală preperitoneală (TAPP). Rezultate: Durata intervenţiei a constituit în medie 47,8±25,07 min, fiind mai lungă pentru herniile recidivante – 95±48,99 min (60-180) şi bilaterale – 92,78±23,47 min (65-140). Mediana spitalizării – 3 zile, reîntoarcerea în cîmpul muncii – sub 10 zile. Incidentele intraoperatorii au fost corectate laparoscopic. Conversia a fost efectuată la un pacient. Nu au fost constatate cazuri de infecţie a plăgii postoperatorii. Aprecierea rezultatelor tratamentului chirurgical la distanţă a fost realizată la 223 pacienţi. Pentru evaluarea durerii la pacienţii cu diagnosticul hernie inghinală a fost utilizată scala de evaluare numerică NRS – 11. În perioada postoperatorie au prevalat pacienţi cu sindrom algic redus (NRS: 1-3). Algoparestezia postoperatorie persistentă a fost diagnosticată la 4 pacienţi. Recidiva herniei a fost înregistrată la 2 pacienţi, în ambele cazuri recidiva a fost corectată laparoscopic. Concluzii: Experienţa noastră confirmă posibilitatea utilizării procedeului TAPP la diferite tipuri de hernie inghinală. Acumularea experienţei permite de a lărgi indicaţiile pentru abordul laparoscopic la pacienţii cu hernii bilaterale, glisante şi recurente. Avantajele hernioplastiei laparoscopice sunt: micşorarea sindromului algic postoperator, reintegrarea socioprofesională rapidă şi numărul redus de complicaţii parietale.Introduction: The transabdominal pre-peritoneal procedure (TAPP) represents one of the most popular techniques used for inguinal hernia repair. The analysis of the reported cases helps to standardize the relatively new laparoscopic technique and to improve the overall results. Material and methods: The group of 271 patients underwent laparoscopic hernia repair (16 bilateral) for the period 2008-2014. According to Nyhus classification, the groin hernias were classified as type II (n=188), type IIIa (n=64), type IIIb (n=18), type IIIc (n=9), type IV (n=12). The TAPP procedure was utilized. Results: The mean operating time was 47.8±25.07 minutes, being statistically longer for recurrent hernias 95±48.99 min (range, 60-180) and bilateral hernias – 92.78±23.47 min (range, 65-140). The average length of hospital stay was 3 days. Patients returned to work in an average of 10 days. The postoperative morbidity rate was 2.2%. The majority of intraoperative incidents (intraoperative hemorrhage, n=4) were solved laparoscopically without sequelae. One case was converted to Lichtenstein repair. Patients were evaluated at a median follow up of 24 month (range, 12-36 month). A total of 223 patients were assesssed for long-term outcomes. Pain was assessed with Numerical Rating Scale (NRS – 11). The vast majority of post-operative patients had minor pain manifestation of pain (NRS: 1-3). We observed 4 cases of persistent inguinal pain. The hernia recurrence was developed in 2 patients and has been corrected via laparoscopic approach. Conclusions: While laparoscopic hernia repair requires a lengthy learning curve, it represents safe and valid alternative to open hernia repairs and could be effectively used for bilateral, recurrent and sliding hernias. The advantages of laparoscopic repair include less postoperative pain, faster return to normal activities and low wound infection rate