Giulia Cerino

A Survey of Methods for the Evaluation of Tissue Engineering Scaffold Permeability

The performance of porous scaffolds for tissue engineering (TE) applications is evaluated, in general, in terms of porosity, pore size and distribution, and pore tortuosity. These descriptors are often confounding when they are applied to characterize transport phenomena within porous scaffolds. On the contrary, permeability is a more effective parameter in (1) estimating mass and species transport through the scaffold and (2) describing its topological features, thus allowing a better evaluation of the overall scaffold performance. However, the evaluation of TE scaffold permeability suffers of a lack of uniformity and standards in measurement and testing procedures which makes the comparison of results obtained in different laboratories unfeasible. In this review paper we summarize the most important features influencing TE scaffold permeability, linking them to the theoretical background. An overview of methods applied for TE scaffold permeability evaluation is given, presenting experimental test benches and computational methods applied (1) to integrate experimental measurements and (2) to support the TE scaffold design process. Both experimental and computational limitations in the permeability evaluation process are also discussed.

Bioreactors as Engineering Support to Treat Cardiac Muscle and Vascular Disease

Cardiovascular disease is the leading cause of morbidity and mortality in the Western World. The inability of fully differentiated, load-bearing cardiovascular tissues to in vivo regenerate and the limitations of the current treatment therapies greatly motivate the efforts of cardiovascular tissue engineering to become an effective clinical strategy for injured heart and vessels. For the effective production of organized and functional cardiovascular engineered constructs in vitro, a suitable dynamic environment is essential, and can be achieved and maintained within bioreactors. Bioreactors are technological devices that, while monitoring and controlling the culture environment and stimulating the construct, attempt to mimic the physiological milieu. In this study, a review of the current state of the art of bioreactor solutions for cardiovascular tissue engineering is presented, with emphasis on bioreactors and biophysical stimuli adopted for investigating the mechanisms influencing cardiovascular tissue development, and for eventually generating suitable cardiovascular tissue replacements.

Three dimensional multi‐cellular muscle‐like tissue engineering in perfusion‐based bioreactors

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi‐cellular environments can better resemble the complexity of native tissues. Previous described co‐culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue‐derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two‐dimensional culture conditions. On the contrary, three‐dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm‐thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion‐based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion‐based culture. We tested percentages of serum promoting monolayer skeletal myoblast‐proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co‐culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co‐culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi‐cellular environment. Biotechnol. Bioeng. 2016;113: 226–236.

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

Engineered mesenchymal cell-based patches as controlled VEGF delivery systems to induce extrinsic angiogenesis

UNLABELLED Therapeutic over-expression of Vascular Endothelial Growth Factor (VEGF) by transduced progenitors is a promising strategy to efficiently induce angiogenesis in ischemic tissues (e.g. limb muscle and myocardium), but tight control over the micro-environmental distribution of the dose is required to avoid induction of angioma-like tumors. Therapeutic VEGF release was achieved by purified transduced adipose mesenchymal stromal cells (ASC) that homogeneously produce specific VEGF levels, inducing only normal angiogenesis after injection in non-ischemic tissues. However, the therapeutic potential of this approach mostly in the cardiac field is limited by the poor cell survival and the restricted area of effect confined to the cell-injection site. The implantation of cells previously organized in vitro in 3D engineered tissues could overcome these issues. Here we hypothesized that collagen sponge-based construct (patch), generated by ASC expressing controlled VEGF levels, can function as delivery device to induce angiogenesis in surrounding areas (extrinsic vascularization). A 7-mm-thick acellular collagen scaffold (empty), sutured beneath the patch, provided a controlled and reproducible model to clearly investigate the ongoing angiogenesis in subcutaneous mice pockets. VEGF-expressing ASC significantly increased the capillary in-growth inside both the patch itself and the empty scaffold compared to naïve cells, leading to significantly improved survival of implanted cells. These data suggest that this strategy confers control (i) on angiogenesis efficacy and safety by means of ASC expressing therapeutic VEGF levels and (ii) over the treated area through the specific localization in an engineered collagen sponge-based patch. STATEMENT OF SIGNIFICANCE Development of efficient pro-angiogenic therapies to restore the micro-vascularization in ischemic tissues is still an open issue. Although extensively investigated, the promising approach based on injections of progenitors transduced to over-express Vascular Endothelial Growth Factor (VEGF) has still several limitations: (i) need of a tight control over the microenvironmental VEGF dose to avoid angioma-like tumor growth; (ii) poor implanted cell survival; (iii) effect area restricted mainly to the injection sites. Here, we aimed to overcome these drawbacks by generating a novel cell-based controlled VEGF delivery device. In particular, transduced mesenchymal cells, purified to release a sustained, safe and efficient VEGF dose, were organized in three-dimensional engineered tissues to improve cell survival and provide a uniform vascularization throughout both the mm-thick implanted constructs themselves and the surrounding area.

Engineering of an angiogenic niche by perfusion culture of adipose-derived stromal vascular fraction cells

In vitro recapitulation of an organotypic stromal environment, enabling efficient angiogenesis, is crucial to investigate and possibly improve vascularization in regenerative medicine. Our study aims at engineering the complexity of a vascular milieu including multiple cell-types, a stromal extracellular matrix (ECM), and molecular signals. For this purpose, the human adipose stromal vascular fraction (SVF), composed of a heterogeneous mix of pericytes, endothelial/stromal progenitor cells, was cultured under direct perfusion flow on three-dimensional (3D) collagen scaffolds. Perfusion culture of SVF-cells reproducibly promoted in vitro the early formation of a capillary-like network, embedded within an ECM backbone, and the release of numerous pro-angiogenic factors. Compared to static cultures, perfusion-based engineered constructs were more rapidly vascularized and supported a superior survival of delivered cells upon in vivo ectopic implantation. This was likely mediated by pericytes, whose number was significantly higher (4.5-fold) under perfusion and whose targeted depletion resulted in lower efficiency of vascularization, with an increased host foreign body reaction. 3D-perfusion culture of SVF-cells leads to the engineering of a specialized milieu, here defined as an angiogenic niche. This system could serve as a model to investigate multi-cellular interactions in angiogenesis, and as a module supporting increased grafted cell survival in regenerative medicine.

Scaffold Composition Determines the Angiogenic Outcome of Cell-Based Vascular Endothelial Growth Factor Expression by Modulating Its Microenvironmental Distribution

Delivery of genetically modified cells overexpressing Vascular Endothelial Growth Factor (VEGF) is a promising approach to induce therapeutic angiogenesis in ischemic tissues. The effect of the protein is strictly modulated by its interaction with the components of the extracellular matrix. Its therapeutic potential depends on a sustained but controlled release at the microenvironmental level in order to avoid the formation of abnormal blood vessels. In this study, it is hypothesized that the composition of the scaffold plays a key role in modulating the binding, hence the therapeutic effect, of the VEGF released by 3D-cell constructs. It is found that collagen sponges, which poorly bind VEGF, prevent the formation of localized hot spots of excessive concentration, therefore, precluding the development of aberrant angiogenesis despite uncontrolled expression by a genetically engineered population of adipose tissue-derived stromal cells. On the contrary, after seeding on VEGF-binding egg-white scaffolds, the same cell population caused aberrantly enlarged vascular structures after 14 d. Collagen-based engineered tissues also induced a safe and efficient angiogenesis in both the patch itself and the underlying myocardium in rat models. These findings open new perspectives on the control and the delivery of proangiogenic stimuli, and are fundamental for the vascularization of engineered tissues/organs.

Myocardial infarction stabilization by cell-based expression of controlled Vascular Endothelial Growth Factor levels

Vascular Endothelial Growth Factor (VEGF) can induce normal or aberrant angiogenesis depending on the amount secreted in the microenvironment around each cell. Towards a possible clinical translation, we developed a Fluorescence Activated Cell Sorting (FACS)‐based technique to rapidly purify transduced progenitors that homogeneously express a desired specific VEGF level from heterogeneous primary populations. Here, we sought to induce safe and functional angiogenesis in ischaemic myocardium by cell‐based expression of controlled VEGF levels. Human adipose stromal cells (ASC) were transduced with retroviral vectors and FACS purified to generate two populations producing similar total VEGF doses, but with different distributions: one with cells homogeneously producing a specific VEGF level (SPEC), and one with cells heterogeneously producing widespread VEGF levels (ALL), but with an average similar to that of the SPEC population. A total of 70 nude rats underwent myocardial infarction by coronary artery ligation and 2 weeks later VEGF‐expressing or control cells, or saline were injected at the infarction border. Four weeks later, ventricular ejection fraction was significantly worsened with all treatments except for SPEC cells. Further, only SPEC cells significantly increased the density of homogeneously normal and mature microvascular networks. This was accompanied by a positive remodelling effect, with significantly reduced fibrosis in the infarcted area. We conclude that controlled homogeneous VEGF delivery by FACS‐purified transduced ASC is a promising strategy to achieve safe and functional angiogenesis in myocardial ischaemia.

Paracrine potential of adipose stromal vascular fraction cells to recover hypoxia-induced loss of cardiomyocyte function: MYTSYK et al.

Cell‐based therapies show promising results in cardiac function recovery mostly through paracrine‐mediated processes (as angiogenesis) in chronic ischemia. In this study, we aim to develop a 2D (two‐dimensional) in vitro cardiac hypoxia model mimicking severe cardiac ischemia to specifically investigate the prosurvival paracrine effects of adipose tissue‐derived stromal vascular fraction (SVF) cell secretome released upon three‐dimensional (3D) culture. For the 2D‐cardiac hypoxia model, neonatal rat cardiomyocytes (CM) were cultured for 5 days at < 1% (approaching anoxia) oxygen (O2) tension. Typical cardiac differentiation hallmarks and contractile ability were used to assess both the cardiomyocyte loss of functionality upon anoxia exposure and its possible recovery following the 5‐day‐treatment with SVF‐conditioned media (collected following 6‐day‐perfusion‐based culture on collagen scaffolds in either normoxia or approaching anoxia). The culture at < 1% O 2 for 5 days mimicked the reversible condition of hibernating myocardium with still living and poorly contractile CM (reversible state). Only SVF‐medium conditioned in normoxia expressing a high level of the prosurvival hepatocyte‐growth factor (HGF) and insulin‐like growth factor (IGF) allowed the partial recovery of the functionality of damaged CM. The secretome generated by SVF‐engineered tissues showed a high paracrine potential to rescue the nonfunctional CM, therefore resulting in a promising patch‐based treatment of specific low‐perfused areas after myocardial infarction.

Engineering of a contractile cardiac patch with an intrinsic vasculogenic potential

Introduction: Decellularized engineered extracellular matrices (ECM) are used in a variety of regenerative medicine applications. Existing decellularization strategies rely on cell lysis and generally result in a variable but significant impairment of the ECM structure/composition. As an alternative, we aimed at activating the apoptotic pathway in order to decellularize engineered matrices while preserving their osteo-inductive properties [1]. Materials and methods: We generated a death-inducible, immortalized human Mesenchymal Stromal Cell (hMSC) line [2]. Cells were seeded on ceramic scaffolds and cultured for 4 weeks in osteogenic medium in a 3D perfusion bioreactor system (U-cup, Cellec). The ECM was decellularized by direct supply of the apoptotic inducer in the 3D culture system. Grafts were implanted in a rat cranial defect model to assess their regenerative potential after 12 weeks. Results: Cells were successfully seeded and differentiated, leading to deposition of a dense ECM during 3D culture. The apoptosis induction allowed for efficient decellularization while preserving the secreted matrix. These “apoptized” cell-free ECM coated constructs induced superior bone regeneration than control materials (Fig. 2). Areas of de novo bone formation not connected with surrounding bone suggest osteoinductive properties of the grafts.