Chiara Arrigoni

Rotating versus perfusion bioreactor for the culture of engineered vascular constructs based on hyaluronic acid.

It is generally accepted that dynamic culture conditions are required for vascular tissue engineering. We compared the effects of two dynamic culture systems, a perfusion and a rotating bioreactor, using tubular constructs based on hyaluronic acid seeded with porcine aortic smooth muscle cells (SMC), that we recently showed to be adequate for the generation of vascular tissue. In perfused constructs mechanical stimulation importantly affected cell morphology, increased the incidence of cell proliferation and reduced apoptosis. However, extracellular matrix deposition, cytoskeletal organization and mechanical properties were poor. In rotated constructs cell proliferation was also higher and apoptosis lower than in static controls. Rotated constructs showed the highest ultimate stress and the lowest elastic modulus. Our data indicate that the rotating bioreactor is more efficient than the perfusion bioreactor and we then suggest that this method can be considered a valid alternative to complex bioreactor systems described in the literature. Biotechnol. Bioeng. 2008;100: 988–997.

Vascular tissue engineering.

Reconstructive surgery using autologous vessels is the conventional approach for substitution of diseased vessels or for generation of bypass to improve blood supply downstream of stenosed vessels. In some circumstances the use of autologous material is not possible due to concomitant diseases or previous use, and artificial grafts must be used. Unfortunately, these grafts cannot substitute small-caliber arterial vessels because of thrombotic complications. The objective of tissue engineering at the vascular level is then to generate biological substitutes of arterial conduits with functional characteristics of native vessels, combining cellular components with biodegradable scaffolds. These research projects started in several laboratories, in the late 1990s, and have expanded in different directions using a number of experimental approaches. The objective of this review is to give an overview of the results so far obtained in this area of research, and to discuss the problems related to these investigations, at the experimental and clinical level. The article provides an overview of different biodegradable scaffolds used, experimental techniques for vessels maturation in vitro under mechanical stimulation, and of differentiated as well as precursors of vascular cells, which opens new opportunities for further development of this form of cell transplantation. Finally, the current available results in clinical research will be discussed.

Rational Design of Prevascularized Large 3D Tissue Constructs Using Computational Simulations and Biofabrication of Geometrically Controlled Microvessels.

A major challenge in the development of clinically relevant 3D tissue constructs is the formation of vascular networks for oxygenation, nutrient supply, and waste removal. To this end, this study implements a multimodal approach for the promotion of vessel-like structures formation in stiff fibrin hydrogels. Computational simulations have been performed to identify the easiest microchanneled configuration assuring normoxic conditions throughout thick cylindrical hydrogels (8 mm height, 6 mm ∅), showing that in our configuration a minimum of three microchannels (600 μm ∅), placed in a non-planar disposition, is required. Using small hydrogel bricks with oxygen distribution equal to the microchanneled configuration, this study demonstrates that among different culture conditions, co-culture of mesenchymal and endothelial cells supplemented with ANG-1 and VEGF leads to the most developed vascular network. Microchanneled hydrogels have been then cultured in the same conditions both statically and in a bioreactor for 7 d. Unexpectedly, the combination between shear forces and normoxic conditions is unable to promote microvascular networks formation in three-channeled hydrogels. Differently, application of either shear forces or normoxic conditions alone results in microvessels outgrowth. These results suggest that to induce angiogenesis in engineered constructs, complex interactions between several biochemical and biophysical parameters have to be modulated.

Direct but not indirect co-culture with osteogenically differentiated human bone marrow stromal cells increases RANKL/OPG ratio in human breast cancer cells generating bone metastases

BackgroundBone metastases arise in nearly 70% of patients with advanced breast cancer, but the complex metastatic process has not been completely clarified yet. RANKL/RANK/OPG pathway modifications and the crosstalk between metastatic cells and bone have been indicated as potential drivers of the process. Interactions between tumor and bone cells have been studied in vivo and in vitro, but specific effects of the direct contact between human metastatic cells and human bone cells on RANKL/RANK/OPG pathway have not been investigated.FindingsWe directly co-cultured bone metastatic human breast cancer cells (BOKL) with osteo-differentiated human mesenchymal cells (BMSCs) from 3 different donors. BMSCs and BOKL were then enzymatically separated and FACS sorted. We found a significant increase in the RANKL/OPG ratio as compared to control, which was not observed in BOKL cultured in medium conditioned by BMSCs, neither in BOKL directly cultured with fibroblasts or medium conditioned by fibroblasts. Direct co-culture with osteo-differentiated BMSCs caused BOKL aggregation while proliferation was not affected by co-culture. To more specifically associate RANKL expression to osteogenic differentiation degree of BMSCs, we determined their osteogenic markers expression and matrix calcification relative to osteoblasts and fibroblasts.ConclusionsIn conclusion, our co-culture model allowed to demonstrate for the first time that direct contact but not paracrine interactions between human metastatic breast cancer cells and bone cells has a significant effect on RANKL/OPG expression in bone metastatic cells. Furthermore, only direct contact with the bone microenvironment induced BOKL clustering without however significantly influencing their proliferation and migration.

In Vitro Co-Culture Models of Breast Cancer Metastatic Progression towards Bone

Advanced breast cancer frequently metastasizes to bone through a multistep process involving the detachment of cells from the primary tumor, their intravasation into the bloodstream, adhesion to the endothelium and extravasation into the bone, culminating with the establishment of a vicious cycle causing extensive bone lysis. In recent years, the crosstalk between tumor cells and secondary organs microenvironment is gaining much attention, being indicated as a crucial aspect in all metastatic steps. To investigate the complex interrelation between the tumor and the microenvironment, both in vitro and in vivo models have been exploited. In vitro models have some advantages over in vivo, mainly the possibility to thoroughly dissect in controlled conditions and with only human cells the cellular and molecular mechanisms underlying the metastatic progression. In this article we will review the main results deriving from in vitro co-culture models, describing mechanisms activated in the crosstalk between breast cancer and bone cells which drive the different metastatic steps.

Engineered miniaturized models of musculoskeletal diseases.

The musculoskeletal system is an incredible machine that protects, supports and moves the human body. However, several diseases can limit its functionality, compromising patient quality of life. Designing novel pathological models would help to clarify the mechanisms driving such diseases, identify new biomarkers and screen potential drug candidates. Miniaturized models in particular can mimic the structure and function of basic tissue units within highly controlled microenvironments, overcoming the limitations of traditional macroscale models and complementing animal studies, which despite being closer to the in vivo situation, are affected by species-specific differences. Here, we discuss the miniaturized models engineered over the past few years to analyze osteochondral and skeletal muscle pathologies, demonstrating how the rationale design of novel systems could provide key insights into the pathological mechanisms behind diseases of the musculoskeletal system.

Bioprinting and Organ-on-Chip Applications Towards Personalized Medicine for Bone Diseases

The skeleton supports and confers structure to the whole body but several pathological and traumatic conditions affect the bone tissue. Most of those pathological conditions are specific and different among different patients, such as bone defects due to traumatic injuries or bone remodeling alterations due to congenital diseases. In this context, the development of personalized therapies would be highly desirable. In recent years the advent of innovative techniques like bioprinting and microfluidic organ-on-chip raised hopes of achieving key tools helping the application of personalized therapies for bone diseases. In this review we will illustrate the latest progresses in the bioprinting of personalized bone grafts and generation of patient-specific bone-on-chip devices, describing current approaches and limitations and possible future improvements for more effective personalized bone grafts and disease models.

Catch-and-Release of Target Cells Using Aptamer-Conjugated Electroactive Zwitterionic Oligopeptide SAM

Nucleic acid aptamers possess attractive features such as specific molecular recognition, high-affinity binding, and rapid acquisition and replication, which could be feasible components for separating specific cells from other cell types. This study demonstrates that aptamers conjugated to an oligopeptide self-assembled monolayer (SAM) can be used to selectively trap human hepatic cancer cells from cell mixtures containing normal human hepatocytes or human fibroblasts. Molecular dynamics calculations have been performed to understand how the configurations of the aptamers are related to the experimental results of selective cell capture. We further demonstrate that the captured hepatic cancer cells can be detached and collected along with electrochemical desorption of the oligopeptide SAM, and by repeating these catch-and-release processes, target cells can be enriched. This combination of capture with aptamers and detachment with electrochemical reactions is a promising tool in various research fields ranging from basic cancer research to tissue engineering applications.

The effect of polyurethane scaffold surface treatments on the adhesion of chondrocytes subjected to interstitial perfusion culture

The purpose of this study was to measure chondrocytes detachment from cellularized constructs cultured in a perfusion bioreactor, and to evaluate the effect of different scaffold coatings on cell adhesion under a fixed flow rate. The scaffolds were polyurethane foams, treated to promote cell attachment and seeded with human chondrocytes. In a preliminary static culture experiment, the scaffolds were imbibed with fetal bovine serum (FBS) and then cultured for 4 weeks. To quantify cell detachment, the number of detached cells from the scaffold treated with FBS was estimated under different interstitial perfusion flow rates and shear stress levels (0.005 mL/min equivalent to 0.05 mPa, 0.023 mL/min equivalent to 0.23 mPa, and 0.045 mL/min equivalent to 0.45 mPa). Finally, groups of scaffolds differently treated (FBS, plasma plus FBS, plasma plus collagen type I) were cultured under a fixed perfusion rate of 0.009 mL/min, equivalent to a shear stress of 0.09 mPa, and the detached cells were counted. Static cultivation showed that cell proliferation increased with time and matrix biosynthesis decreased after the first week of culture. Perfused culture showed that the number of detached cells increased with the perfusion rate on FBS-treated constructs. The plasma-treated/collagen-coated scaffolds showed the highest resistance to cell detachment. To minimize cell detachment, the perfusion rate must be maintained in the order of 0.02 mL/min, giving a shear stress of 0.2 mPa. Our set-up allowed estimating the resistance to cell detachment under interstitial perfusion in a repeatable manner, to test other scaffold coatings and cell types.

Industrialization of a perfusion bioreactor: prime example of a non-straightforward process

Bioreactors are essential enabling technologies for the translation of advanced therapies medicinal products from the research field towards a successful clinical application. In order to speed up the translation and the spread of novel tissue engineering products into the clinical routine, tissue engineering bioreactors should evolve from laboratory prototypes towards industrialized products. In this work, we thus challenged the industrialization process of a novel technological platform, based on an established research prototype of perfusion bioreactor, following a GMP‐driven approach. We describe how the combination of scientific background, intellectual property, start‐up factory environment, wise industrial advice in the biomedical field, design, and regulatory consultancy allowed us to turn a previously validated prototype technology into an industrial product suitable for serial production with improved replicability and user‐friendliness. The solutions implemented enhanced aesthetics, ergonomics, handling, and safety of the bioreactor, and they allowed compliance with the fundamental requirements in terms of traceability, reproducibility, efficiency, and safety of the manufacturing process of advanced therapies medicinal products. The result is an automated incubator‐compatible device, housing 12 disposable independent perfusion chambers for seeding and culture of any perfusable tissue. We validated the cell seeding process of the industrialized bioreactor by means of the Design of Experiment approach, whilst the effectiveness of perfusion culture was evaluated in the context of bone tissue engineering.