A standalone bioreactor system to deliver compressive load under perfusion flow to hBMSC-seeded 3D chitosan-graphene templates

The availability of engineered biological tissues holds great potential for both clinical applications and basic research in a life science laboratory. A prototype standalone perfusion/compression bioreactor system was proposed to address the osteogenic commitment of stem cells seeded onboard of 3D chitosan-graphene (CHT/G) templates. Testing involved the coordinated administration of a 1 mL/min medium flow rate together with dynamic compression (1% strain at 1 Hz; applied twice daily for 30 min) for one week. When compared to traditional static culture conditions, the application of perfusion and compression stimuli to human bone marrow stem cells using the 3D CHT/G template scaffold induced a sizable effect. After using the dynamic culture protocol, there was evidence of a larger number of viable cells within the inner core of the scaffold and of enhanced extracellular matrix mineralization. These observations show that our novel device would be suitable for addressing and investigating the osteogenic phenotype commitment of stem cells, for both potential clinical applications and basic research

Design of a custom-made device for real-time optical measurement of differential mineral concentrations in three-dimensional scaffolds for bone tissue engineering

Monitoring bone tissue engineered (TEed) constructs during their maturation is important to ensure the quality of applied protocols. Several destructive, mainly histochemical, methods are conventionally used to this aim, requiring the sacrifice of the investigated samples. This implies (i) to plan several scaffold replicates, (ii) expensive and time consuming procedures and (iii) to infer the maturity level of a given tissue construct from a cognate replica. To solve these issues, non-destructive techniques such as light spectroscopy-based methods have been reported to be useful. Here, a miniaturized and inexpensive custom-made spectrometer device is proposed to enable the non-destructive analysis of hydrogel scaffolds. Testing involved samples with a differential amount of calcium salt. When compared to a reference standard device, this custom-made spectrometer demonstrates the ability to perform measurements without requiring elaborate sample preparation and/or a complex instrumentation. This preliminary study shows the feasibility of light spectroscopy-based methods as useful for the non-destructive analysis of TEed constructs. Based on these results, this custom-made spectrometer device appears as a useful option to perform real-time/in-line analysis. Finally, this device can be considered as a component that can be easily integrated on board of recently prototyped bioreactor systems, for the monitoring of TEed constructs during their conditioning

Development of an electrical impedance tomography set-up for the quantification of mineralization in biopolymer scaffolds

Objective. 3D cell cultures are becoming a fundamental resource for in-vitro studies, as they mimic more closely in-vivo behavior. The analysis of these constructs, however, generally rely on destructive techniques, that prevent the monitoring over time of the same construct, thus increasing the results variability and the resources needed for each experiment. Approach. In this work, we focus on mineralization, a crucial process during maturation of artificial bone models, and propose electrical impedance tomography (EIT) as an alternative non-destructive approach. In particular, we discuss the development of an integrated hardware/software system capable of acquiring experimental data from 3D scaffolds and reconstructing the corresponding conductivity maps. We also show how the same software can test how the measurement is affected by biological features such as scaffold shrinking during the culture. Main results. An initial validation, comprising the acquisition of both a non-conductive phantom and alginate/gelatin scaffolds with known calcium content will be presented, together with the in-silico study of a cell-induced mineralization process. This analysis will allow for an initial verification of the systems functionality while limiting the effects of biological variability due to cell number and activity. Significance. Our results show the potential of EIT for the non-destructive quantification of matrix mineralization in 3D scaffolds, and open to the possible long term monitoring of this fundamental hallmark of osteogenic differentiation in hybrid tissue engineered constructs

Human Bone-Marrow-Derived Stem-Cell-Seeded 3D Chitosan–Gelatin–Genipin Scaffolds Show Enhanced Extracellular Matrix Mineralization When Cultured under a Perfusion Flow in Osteogenic Medium

Tissue-engineered bone tissue grafts are a promising alternative to the more conventional use of natural donor bone grafts. However, choosing an appropriate biomaterial/scaffold to sustain cell survival, proliferation, and differentiation in a 3D environment remains one of the most critical issues in this domain. Recently, chitosan/gelatin/genipin (CGG) hybrid scaffolds have been proven as a more suitable environment to induce osteogenic commitment in undifferentiated cells when doped with graphene oxide (GO). Some concern is, however, raised towards the use of graphene and graphene-related material in medical applications. The purpose of this work was thus to check if the osteogenic potential of CGG scaffolds without added GO could be increased by improving the medium diffusion in a 3D culture of differentiating cells. To this aim, the level of extracellular matrix (ECM) mineralization was evaluated in human bone-marrow-derived stem cell (hBMSC)-seeded 3D CGG scaffolds upon culture under a perfusion flow in a dedicated custom-made bioreactor system. One week after initiating dynamic culture, histological/histochemical evaluations of CGG scaffolds were carried out to analyze the early osteogenic commitment of the culture. The analyses show the enhanced ECM mineralization of the 3D perfused culture compared to the static counterpart. The results of this investigation reveal a new perspective on more efficient clinical applications of CGG scaffolds without added GO

Myogenic commitment of human stem cells by myoblasts Co-culture: a static vs. a dynamic approach

An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1 beta (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture

Analysis of intracellular magnesium and mineral depositions during osteogenic commitment of 3d cultured saos2 cells

In this study, we explore the behaviour of intracellular magnesium during bone phenotype modulation in a 3D cell model built to mimic osteogenesis. In addition, we measured the amount of magnesium in the mineral depositions generated during osteogenic induction. A two-fold increase of intracellular magnesium content was found, both at three and seven days from the induction of differentiation. By X-ray microscopy, we characterized the morphology and chemical composition of the mineral depositions secreted by 3D cultured differentiated cells finding a marked co-localization of Mg with P at seven days of differentiation. This is the first experimental evidence on the presence of Mg in the mineral depositions generated during biomineralization, suggesting that Mg incorporation occurs during the bone forming process. In conclusion, this study on the one hand attests to an evident involvement of Mg in the process of cell differentiation, and, on the other hand, indicates that its multifaceted role needs further investigation

Chemistry courses as the turning point for premedical students

Previous research has documented that negative experiences in chemistry courses are a major factor that discourages many students from continuing in premedical studies. This adverse impact affects women and students from under-represented minority (URM) groups disproportionately. To determine if chemistry courses have a similar effect at a large public university, we surveyed 1,036 students from three entering cohorts at the University of California, Berkeley. We surveyed students at the beginning of their first year at the university and again at the end of their second year. All subjects had indicated an interest in premedical studies at the time they entered the university. We conducted follow-up interviews with a stratified sub-set of 63 survey respondents to explore the factors that affected their level of interest in premedical studies. Using a 10-point scale, we found that the strength of interest in premedical studies declined for all racial/ethnic groups. In the follow-up interviews, students identified chemistry courses as the principal factor contributing to their reported loss of interest. URM students especially often stated that chemistry courses caused them to abandon their hopes of becoming a physician. Consistent with reports over more than 50 years, it appears that undergraduate courses in chemistry have the effect of discouraging otherwise qualified students, as reflected in their admission to one of the most highly selective public universities in the US, from continuing in premedical studies, especially in the case of URM students. Reassessment of this role for chemistry courses may be overdue

Chondrogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells in a Perfused Collagen Hydrogel Functionalized with hTGF-β1-Releasing PLGA Microcarrier

Tissue engineering strategies can be relevant for cartilage repair and regeneration. A collagen matrix was functionalized with the addition of poly-lactic-co-glycolic acid microcarriers (PLGA-MCs) carrying a human Transforming Growth Factor β1 (hTFG-β1) payload, to provide a 3D biomimetic environment with the capacity to direct stem cell commitment towards a chondrogenic phenotype. PLGA-MCs (mean size 3 ± 0.9 μm) were prepared via supercritical emulsion extraction technology and tailored to sustain delivery of payload into the collagen hydrogel for 21 days. PLGA-MCs were coseeded with human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) in the collagen matrix. Chondrogenic induction was suggested when dynamic perfusion was applied as indicated by transcriptional upregulation of COL2A1 gene (5-fold; p < 0.01) and downregulation of COL1A1 (0.07-fold; p < 0.05) and COL3A1 (0.11-fold; p < 0.05) genes, at day 16, as monitored by qRT-PCR. Histological and quantitative-immunofluorescence (qIF) analysis confirmed cell activity by remodeling the synthetic extracellular matrix when cultured in perfused conditions. Static constructs lacked evidence of chondrogenic specific gene overexpression, which was probably due to a reduced mass exchange, as determined by 3D system Finite Element Modelling (FEM) analysis. Proinflammatory (IL-6, TNF, IL-12A, IL-1β) and anti-inflammatory (IL-10, TGF-β1) cytokine gene expression by hBM-MSC was observed only in dynamic culture (TNF and IL-1β 10-fold, p < 0.001; TGF-β1 4-fold, p < 0.01 at Day 16) confirming the cells' immunomodulatory activity mainly in relation to their commitment and not due to the synthetic environment. This study supports the use of 3D hydrogel scaffolds, equipped for growth factor controlled delivery, as tissue engineered models for the study of in vitro chondrogenic differentiation and opens clinical perspectives for injectable collagen-based advanced therapy systems

3D Biomimetic Scaffold for Growth Factor Controlled Delivery: An In-Vitro Study of Tenogenic Events on Wharton's Jelly Mesenchymal Stem Cells.

The present work described a bio-functionalized 3D fibrous construct, as an interactive teno-inductive graft model to study tenogenic potential events of human mesenchymal stem cells collected from Wharton's Jelly (hWJ-MSCs). The 3D-biomimetic and bioresorbable scaffold was functionalized with nanocarriers for the local controlled delivery of a teno-inductive factor, i.e., the human Growth Differentiation factor 5 (hGDF-5). Significant results in terms of gene expression were obtained. Namely, the up-regulation of Scleraxis (350-fold, p ≤ 0.05), type I Collagen (8-fold), Decorin (2.5-fold), and Tenascin-C (1.3-fold) was detected at day 14; on the other hand, when hGDF-5 was supplemented in the external medium only (in absence of nanocarriers), a limited effect on gene expression was evident. Teno-inductive environment also induced pro-inflammatory, (IL-6 (1.6-fold), TNF (45-fold, p ≤ 0.001), and IL-12A (1.4-fold)), and anti-inflammatory (IL-10 (120-fold) and TGF-β1 (1.8-fold)) cytokine expression upregulation at day 14. The presented 3D construct opens perspectives for the study of drug controlled delivery devices to promote teno-regenerative events