Biomaterial-Assisted 3D In Vitro Tumor Models: From Organoid towards Cancer Tissue Engineering Approaches

: Cancers are a leading cause of death around the world, accounting for nearly 10 million deaths yearly [...]

An Osteosarcoma Model by 3D Printed Polyurethane Scaffold and In Vitro Generated Bone Extracellular Matrix

Osteosarcoma is a primary bone tumor characterized by a dismal prognosis, especially in the case of recurrent disease or metastases. Therefore, tools to understand in-depth osteosarcoma progression and ultimately develop new therapeutics are urgently required. 3D in vitro models can provide an optimal option, as they are highly reproducible, yet sufficiently complex, thus reliable alternatives to 2D in vitro and in vivo models. Here, we describe 3D in vitro osteosarcoma models prepared by printing polyurethane (PU) by fused deposition modeling, further enriched with human mesenchymal stromal cell (hMSC)-secreted biomolecules. We printed scaffolds with different morphologies by changing their design (i.e., the distance between printed filaments and printed patterns) to obtain different pore geometry, size, and distribution. The printed PU scaffolds were stable during in vitro cultures, showed adequate porosity (55–67%) and tunable mechanical properties (Young’s modulus ranging in 0.5–4.0 MPa), and resulted in cytocompatible. We developed the in vitro model by seeding SAOS-2 cells on the optimal PU scaffold (i.e., 0.7 mm inter-filament distance, 60 pattern), by testing different pre-conditioning factors: none, undifferentiated hMSC-secreted, and osteo-differentiated hMSC-secreted extracellular matrix (ECM), which were obtained by cell lysis before SAOS-2 seeding. Scaffolds pre-cultured with osteo-differentiated hMSCs, subsequently lysed, and seeded with SAOS-2 cells showed optimal colonization, thus disclosing a suitable biomimetic microenvironment for osteosarcoma cells, which can be useful both in tumor biology study and, possibly, treatment

The Impact of COVID-19 on Plastic Surgery Residency Training

Abstract: Nowadays didactic and surgical activities for residents in the surgery field are less and less due to an increasing burden of documentation and \u201cnon-educational work.\u201d Considering the current lockdown due to the COVID-19 pandemic, it has never been so important to find different ways to allow residents to improve their knowledge. We asked all plastic and esthetic surgery residents in our country to fill out a questionnaire to investigate changes in their didactical activity and analyze problems about their professional growth in the last few months. From the results of such questionnaires, we found that most of the residents feel the decrease in surgical activities during this time is a detrimental factor for their training and that even if all the schools have changed their didactical activities no school has introduced the use of virtual simulators to compensate for the decrease in surgical practice. Actually, the majority of residents use webinars to keep updated, stating that such technologies are useful but not sufficient to analyze plastic surgery topics in depth during COVID-19 lockdown. Virtual interactive tools are well known in different clinical and surgical specialties, and they are considered as a valid support, but it seems that in plastic surgery they are not so used. According to the most recent studies about residents\u2019 didactical program, we have investigated the potential of Anatomage Table in combination with Touch Surgery application as physical and mental aids to bypass the decreased number and kind of surgical interventions performed in this particular time. Anatomage is an academic user-friendly touch screen table; it is used by both medical students and residents to learn human anatomy and to master surgical anatomy. Touch Surgery is an application available on smartphones and tablets that gives the possibility to watch real and virtually designed surgical videos, accompanied by explanatory comments on the surgical phases; they are interactive and give the possibility to check what you have learned through tests administered after virtual classes. In our opinion, these tools represent reliable solutions to improve plastic residents\u2019 training, mostly during the COVID-19 pandemic. Level of Evidence V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266

An osteosarcoma model by 3D printed polyurethane scaffold and in vitro generated bone extracellular matrix

Osteosarcoma is a primary bone tumor characterized by a dismal prognosis, especially in the case of recurrent disease or metastases. Therefore, tools to understand in-depth osteosarcoma progression and ultimately develop new therapeutics are urgently required. 3D in vitro models can provide an optimal option, as they are highly reproducible, yet sufficiently complex, thus reliable alternatives to 2D in vitro and in vivo models. Here, we describe 3D in vitro osteosarcoma models prepared by printing polyurethane (PU) by fused deposition modeling, further enriched with human mesenchymal stromal cell (hMSC)-secreted biomolecules. We printed scaffolds with different morphologies by changing their design (i.e., the distance between printed filaments and printed patterns) to obtain different pore geometry, size, and distribution. The printed PU scaffolds were stable during in vitro cultures, showed adequate porosity (55–67%) and tunable mechanical properties (Young’s modulus ranging in 0.5–4.0 MPa), and resulted in cytocompatible. We developed the in vitro model by seeding SAOS-2 cells on the optimal PU scaffold (i.e., 0.7 mm inter-filament distance, 60° pattern), by testing different pre-conditioning factors: none, undifferentiated hMSC-secreted, and osteo-differentiated hMSC-secreted extracellular matrix (ECM), which were obtained by cell lysis before SAOS-2 seeding. Scaffolds pre-cultured with osteo-differentiated hMSCs, subsequently lysed, and seeded with SAOS-2 cells showed optimal colonization, thus disclosing a suitable biomimetic microenvironment for osteosarcoma cells, which can be useful both in tumor biology study and, possibly, treatment

Plant Tissues as 3D Natural Scaffolds for Adipose, Bone and Tendon Tissue Regeneration

Decellularized tissues are a valid alternative as tissue engineering scaffolds, thanks to the three-dimensional structure that mimics native tissues to be regenerated and the biomimetic microenvironment for cells and tissues growth. Despite decellularized animal tissues have long been used, plant tissue decellularized scaffolds might overcome availability issues, high costs and ethical concerns related to the use of animal sources. The wide range of features covered by different plants offers a unique opportunity for the development of tissue-specific scaffolds, depending on the morphological, physical and mechanical peculiarities of each plant. Herein, three different plant tissues (i.e., apple, carrot, and celery) were decellularized and, according to their peculiar properties (i.e., porosity, mechanical properties), addressed to regeneration of adipose tissue, bone tissue and tendons, respectively. Decellularized apple, carrot and celery maintained their porous structure, with pores ranging from 70 to 420 μm, depending on the plant source, and were stable in PBS at 37°C up to 7 weeks. Different mechanical properties (i.e., Eapple = 4 kPa, Ecarrot = 43 kPa, Ecelery = 590 kPa) were measured and no indirect cytotoxic effects were demonstrated in vitro after plants decellularization. After coating with poly-L-lysine, apples supported 3T3-L1 preadipocytes adhesion, proliferation and adipogenic differentiation; carrots supported MC3T3-E1 pre-osteoblasts adhesion, proliferation and osteogenic differentiation; celery supported L929 cells adhesion, proliferation and guided anisotropic cells orientation. The versatile features of decellularized plant tissues and their potential for the regeneration of different tissues are proved in this work

Tripolyphosphate-crosslinked chitosan/gelatin biocomposite ink for 3D printing of uniaxial scaffolds.

Chitosan is a natural polymer widely investigated and used due to its antibacterial activity, mucoadhesive, analgesic, and hemostatic properties. Its biocompatibility makes chitosan a favorable candidate for different applications in tissue engineering (TE), such as skin, bone, and cartilage tissue regeneration. Despite promising results obtained with chitosan 3D scaffolds, significant challenges persist in fabricating hydrogel structures with ordered architectures and biological properties to mimic native tissues. In this work, chitosan has been investigated aiming at designing and fabricating uniaxial scaffolds which can be proposed for the regeneration of anisotropic tissues (i.e., skin, skeletal muscle, myocardium) by 3D printing technology. Chitosan was blended with gelatin to form a polyelectrolyte complex in two different ratios, to improve printability and shape retention. After the optimization of the printing process parameters, different crosslinking conditions were investigated, and the 3D printed samples were characterized. Tripolyphosphate (TPP) was used as crosslinker for chitosan-based scaffolds. For the optimization of the printing temperature, the sol-gel temperature of the chitosan-gelatin blend was determined by rheological measurements and extrusion temperature was set to 20°C (i.e., below sol-gel temperature). The shape fidelity and surface morphology of the 3D printed scaffolds after crosslinking was dependent on crosslinking conditions. Interestingly, mechanical properties of the scaffolds were also significantly affected by the crosslinking conditions, nonetheless the stability of the scaffolds was strongly determined by the content of gelatin in the blend. Lastly, in vitro cytocompatibility test was performed to evaluate the interactions between L929 cells and the 3D printed samples. 2% w/v chitosan and 4% w/v gelatin hydrogel scaffolds crosslinked with 10% TPP, 30 min at 4°C following 30 min at 37°C have shown cytocompatible and stable characteristics, compared to all other tested conditions, showing suitable properties for the regeneration of anisotropic tissues

Tunable crosslinking and adhesion of gelatin hydrogels via bioorthogonal click chemistry

Engineering cytocompatible hydrogels with tunable physico-mechanical properties as a biomimetic three-dimensional extracellular matrix (ECM) is fundamental to guide cell response and target tissue regeneration or development of in vitro models. Gelatin represents an optimal choice given its ECM biomimetic properties; however, gelatin cross-linking is required to ensure structural stability at physiological temperature (i.e., T > Tsol–gel gelatin). Here, we use a previously developed cross-linking reaction between tetrazine (Tz)- and norbornene (Nb) modified gelatin derivatives to prepare gelatin hydrogels and we demonstrate the possible tuning of their properties by varying their degree of modification (DOM) and the Tz/Nb ratio (R). The percentage DOM of the gelatin derivatives was tuned between 5 and 15%. Hydrogels prepared with higher DOM cross-linked faster (i.e., 10–20 min) compared to hydrogels prepared with lower DOM (i.e., 60–70 min). A higher DOM and equimolar Tz/Nb ratio R resulted in hydrogels with lower weight variation after immersion in PBS at 37 °C. The mechanical properties of the hydrogels were tuned by varying DOM and R by 1 order of magnitude, achieving elastic modulus E values ranging from 0.5 (low DOM and nonequimolar Tz/Nb ratio) to 5 kPa (high DOM and equimolar Tz/Nb ratio). Human dental pulp stem cells were embedded in the hydrogels and successfully 3D cultured in the hydrogels (percentage viable cells >85%). An increase in metabolic activity and a more elongated cell morphology was detected for cells cultured in hydrogels with lower mechanical properties (E < 1 kPa). Hydrogels prepared with an excess of Tz or Nb were successfully adhered and remained in contact during in vitro cultures, highlighting the potential use of these hydrogels as compartmentalized coculture systems. The successful tuning of the gelatin hydrogel properties here developed by controlling their bioorthogonal cross-linking is promising for tissue engineering and in vitro modeling applications

Plant tissues as 3D natural scaffolds for adipose, bone and tendon tissue regeneration

Decellularized tissues are a valid alternative as tissue engineering scaffolds, thanks to the three-dimensional structure that mimics native tissues to be regenerated and the biomimetic microenvironment for cells and tissues growth. Despite decellularized animal tissues have long been used, plant tissue decellularized scaffolds might overcome availability issues, high costs and ethical concerns related to the use of animal sources. The wide range of features covered by different plants offers a unique opportunity for the development of tissue-specific scaffolds, depending on the morphological, physical and mechanical peculiarities of each plant. Herein, three different plant tissues (i.e., apple, carrot, and celery) were decellularized and, according to their peculiar properties (i.e., porosity, mechanical properties), addressed to regeneration of adipose tissue, bone tissue and tendons, respectively. Decellularized apple, carrot and celery maintained their porous structure, with pores ranging from 70 to 420 μm, depending on the plant source, and were stable in PBS at 37°C up to 7 weeks. Different mechanical properties (i.e., Eapple = 4 kPa, Ecarrot = 43 kPa, Ecelery = 590 kPa) were measured and no indirect cytotoxic effects were demonstrated in vitro after plants decellularization. After coating with poly-L-lysine, apples supported 3T3-L1 preadipocytes adhesion, proliferation and adipogenic differentiation; carrots supported MC3T3-E1 pre-osteoblasts adhesion, proliferation and osteogenic differentiation; celery supported L929 cells adhesion, proliferation and guided anisotropic cells orientation. The versatile features of decellularized plant tissues and their potential for the regeneration of different tissues are proved in this work

3D Bioprinting of Pectin-Cellulose Nanofibers Multicomponent Bioinks

Pectin has found extensive interest in biomedical applications, including wound dressing, drug delivery, and cancer targeting. However, the low viscosity of pectin solutions hinders their applications in 3D bioprinting. Here, we developed multicomponent bioinks prepared by combining pectin with TEMPO-oxidized cellulose nanofibers (TOCNFs) to optimize the inks’ printability while ensuring stability of the printed hydrogels and simultaneously print viable cell-laden inks. First, we screened several combinations of pectin (1%, 1.5%, 2%, and 2.5% w/v) and TOCNFs (0%, 0.5%, 1%, and 1.5% w/v) by testing their rheological properties and printability. Addition of TOCNFs allowed increasing the inks’ viscosity while maintaining shear thinning rheological response, and it allowed us to identify the optimal pectin concentration (2.5% w/v). We then selected the optimal TOCNFs concentration (1% w/v) by evaluating the viability of cells embedded in the ink and eventually optimized the writing speed to be used to print accurate 3D grid structures. Bioinks were prepared by embedding L929 fibroblast cells in the ink printed by optimized printing parameters. The printed scaffolds were stable in a physiological-like environment and characterized by an elastic modulus of E = 1.8 ± 0.2&nbsp;kPa. Cells loaded in the ink and printed were viable (cell viability &gt;80%) and their metabolic activity increased in time during the in vitro culture, showing the potential use of the developed bioinks for biofabrication and tissue engineering applications

Cross-linking strategies for electrospun gelatin scaffolds

Electrospinning is an exceptional technology to fabricate sub-micrometric fiber scaffolds for regenerative medicine applications and to mimic the morphology and the chemistry of the natural extracellular matrix (ECM). Although most synthetic and natural polymers can be electrospun, gelatin frequently represents a material of choice due to the presence of cell-interactive motifs, its wide availability, low cost, easy processability, and biodegradability. However, cross-linking is required to stabilize the structure of the electrospun matrices and avoid gelatin dissolution at body temperature. Different physical and chemical cross-linking protocols have been described to improve electrospun gelatin stability and to preserve the morphological fibrous arrangement of the electrospun gelatin scaffolds. Here, we review the main current strategies. For each method, the cross-linking mechanism and its efficiency, the influence of electrospinning parameters, and the resulting fiber morphology are considered. The main drawbacks as well as the open challenges are also discusse