Validation and scalability of homemade polycaprolactone macrobeads grafted with thermo-responsive poly(N-isopropylacrylamide) for mesenchymal stem cell expansion and harvesting.

In this study, polycaprolactone (PCL) macrobeads were prepared by an oil-in-water (o/w) emulsion solvent evaporation method with poly(vinyl alcohol) (PVA) as an emulsifier and conjugated to poly(N-isopropylacrylamide) (PNIPAAm) to be used as cell carriers with noninvasive cell detachment properties (thermo-response). Following previous studies with PCL-PNIPAAm carriers, our objectives were to confirm the successful conjugation on homemade macrobeads and to show the advantages of homemade production over commercial beads to control morphological, biological, and fluidization properties. The effects of PCL concentration on the droplet formation and of flow rate and PVA concentration on the size of the beads were demonstrated. The size of the beads, all spherical, ranged from 0.5 to 3.7 mm with four bead categories based on production parameters. The morphology and size of the beads were observed by scanning electron microscopy to show surface roughness enhancing cell attachment and proliferation compared to commercial beads. The functionalization steps with PNIPAAm were then characterized and confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersion spectroscopy. PNIPAAm-grafted macrobeads allowed mesenchymal stem cells (MSCs) to spread and grow for up to 21 days. By reducing the temperature to 25°C, the MSCs were successfully detached from the PCL-PNIPAAm beads as observed with fluorescence microscopy. Furthermore, we validated the scalability potential of both macrobeads production and conjugation with PCL, to produce easily kilograms of thermo-responsive macrocarriers in a lab environment. This could help moving such approaches towards clinically and industrially relevant processes were cell expansion is needed at very large scale.

Biomimetic and Nonbiomimetic Approaches in Dura Substitutes: The Influence of Mechanical Properties.

The dura mater, the furthest and strongest layer of the meninges, is crucial for protecting the brain and spinal cord. Its biomechanical behavior is vital, as any alterations can compromise biological functions. In recent decades, interest in the dura mater has increased due to the need for hermetic closure of dural defects prompting the development of several substitutes. Collagen-based dural substitutes are common commercial options, but they lack the complex biological and structural elements of the native dura mater, impacting regeneration and potentially causing complications like wound/postoperative infection and cerebrospinal fluid (CSF) leakage. To face this issue, recent tissue engineering approaches focus on creating biomimetic dura mater substitutes. The objective of this review is to discuss whether mimicking the mechanical properties of native tissue or ensuring high biocompatibility and bioactivity is more critical in developing effective dural substitutes, or if both aspects should be systematically linked. After a brief description of the properties and architecture of the native cranial dura, we describe the advantages and limitations of biomimetic dura mater substitutes to better understand their relevance. In particular, we consider biomechanical properties' impact on dura repair's effectiveness. Finally, the obstacles and perspectives for developing the ideal dural substitute are explored.

Towards Ready-to-Use Iron-Crosslinked Alginate Beads as Mesenchymal Stem Cell Carriers.

Micro-carriers, thanks to high surface/volume ratio, are widely studied as mesenchymal stem cell (MSCs) in vitro substrate for proliferation at clinical rate. In particular, Ca-alginate-based biomaterials (sodium alginate crosslinked with CaCl2) are commonly investigated. However, Ca-alginate shows low bioactivity and requires functionalization, increasing labor work and costs. In contrast, films of sodium alginate crosslinked with iron chloride (Fe-alginate) have shown good bioactivity with fibroblasts, but MSCs studies are lacking. We propose a first proof-of-concept study of Fe-alginate beads supporting MSCs proliferation without functionalization. Macro- and micro-carriers were prepared (extrusion and electrospray) and we report for the first time Fe-alginate electrospraying optimization. FTIR spectra, stability with various mannuronic acids/guluronic acids (M/G) ratios and size distribution were analyzed before performing cell culture. After confirming literature results on films with human MSCs, we showed that Macro-Fe-alginate beads offered a better environment for MSCs adhesion than Ca-alginate. We concluded that Fe-alginate beads showed great potential as ready-to-use carriers.

Encapsulation and differentiation of adipose-derived mesenchymal stem cells in a biomimetic purine cross-linked chitosan sponge.

Mesenchymal stem cells derived from adipose tissue have become a widely investigated cell source to use in tissue engineering applications. However, an optimal delivery scaffold for these cells is still needed. A rapidly gelling, injectable chitosan sponge was proposed in this study as a potential candidate for a suitable delivery scaffold. The results demonstrated the ability to encapsulate the stem cells at a 97.6% encapsulation efficiency and that the cells maintain their viability within the sponge. With the potential of using this scaffold for bone tissue engineering, ALP activity assay and fluorescent imaging for osteocalcin proved the ability to differentiate the encapsulated cells into the osteogenic lineage. Furthermore, co-encapsulation of pyrophosphatase within the sponge was investigated as a method to overcome the inhibitory effects that the sponge degradation by-products have on mineralization. Alizarin Red S staining demonstrated the beneficial effects of adding pyrophosphatase, where a significant increase in mineralization levels was achieved.

A core-shell guanosine diphosphate crosslinked chitosan scaffold as a potential co-encapsulation platform.

Recent engineering strategies to better mimic native tissue architecture involve co-encapsulation of cell lineages and/or growth factors in multi-compartmental scaffolds. This study introduces a core-shell platform based on a rapidly gelling guanosine diphosphate cross-linked chitosan scaffold for co-culture. The core-shell sponge is fabricated through combination of chitosan and guanosine diphosphate in 3 steps with each shell layer deposited around the previous layer. Co-encapsulation of pre-osteoblastic MC-3T3 cells and growth factors in the core-shell sponge showed similar microstructure to the standard sponge with high pore connectivity and low closed porosity (<0.4 %). A viable cell population was maintained over time with enhanced cellular functionality when ascorbic acid was added in the same compartment. Co-culture was explored with a proof-of-concept study shown for MC-3T3 and endothelial cells showing homogeneous distribution of cells in their intended compartment. Overall, this core-shell scaffold shows potential as a platform for the regeneration of multiple tissues.

Objectives, benefits and challenges of bioreactor systems for the clinical-scale expansion of T lymphocyte cells.

Cell therapies based on T cell have gathered interest over the last decades for treatment of cancers, becoming recently the most investigated lineage for clinical trials. Although results of adoptive cell therapies are very promising, obtaining large batches of T cell at clinical scale is still challenging nowadays. We propose here a review study focusing on how bioreactor systems could increase expansion rates of T cell culture specifically towards efficient, reliable and reproducible cell therapies. After describing the specificities of T cell culture, in particular activation, phenotypical characterization and cell density considerations, we detail the main objectives of bioreactors in this context, namely scale-up, GMP-compliance and reduced time and costs. Then, we report recent advances on the different classes of bioreactor systems commonly investigated for non-adherent cell expansion, in comparison with the current "gold standard" of T cell culture (flasks and culture bag). Results obtained with hollow fibres, G-Rex® flasks, Wave bioreactor, multiple-step bioreactors, spinner flasks as well as original homemade designs are discussed to highlight advantages and drawbacks in regards to T cells' specificities. Although there is currently no consensus on an optimal bioreactor, overall, most systems reviewed here can improve T cell culture towards faster, easier and/or cheaper protocols. They also offer strong outlooks towards automation, process control and complete closed systems, which could be mandatory developments for a massive clinical breakthrough. However, proper controls are sometimes lacking to conclude clearly on the features leading to the progresses regarding cell expansion, and the field could benefit from process engineering methods, such as quality by design, to perform multi parameters studies and face these challenges.

Donor variability alters differentiation and mechanical cohesion of tissue-engineered constructs with human endothelial/MSC co-culture.

To move towards clinical applications, tissue engineering (TE) should be validated with human primary cells and offer easy connection to the native vascularisation. Based on a sheet-like bone substitute developed previously, we investigated a mesenchymal stem cells/endothelial cells (MSCs/ECs) coculture to enhance pre-vascularisation. Using MSCs from six independent donors whose differentiation potential was assessed towards two lineages, we focused on donor variability and cell crosstalk regarding bone differentiation. Coculture was performed on calcium phosphate granules in a specific chamber during 1 month. MSCs were seeded first then ECs were added after 2 weeks, with respective monocultures as control groups. Cell viability and organisation (fluorescence, electronic microscopy), differentiation (ALP staining/activity, RT-qPCR) and mechanical cohesion were analysed. Adaptation of the protocol to coculture was validated (high cell viability and proliferation). Activity and differentiation showed strong trends towards synergistic effects between cell types. MSCs reached early mineralisation stage of maturation. The delayed addition of ECs allowed for their attachment on developed MSCs' matrix. The main impact of donor variability could be here the lack of cell proliferation potential with some donors, leading to low differentiation and mechanical cohesion and therefore absence of sheet-like shape successfully obtained with others. We suggest therefore adapting protocols to cell proliferation potentials from one batch of cells to the other in a patient-specific approach.

In Vitro Bone Cell Response to Tensile Mechanical Solicitations: Is There an Optimal Protocol?

Bone remodeling is strongly linked to external mechanical signals. Such stimuli are widely used in vitro for bone tissue engineering by applying mechanical solicitations to cell cultures so as to trigger specific cell responses. However, the literature highlights considerable variability in devices and protocols. Here the major biological, mechanical, and technical parameters implemented for in vitro tensile loading applications are reviewed. The objective is to identify which values are used most, and whether there is an optimal protocol to obtain a functional tissue-engineering construct. First, a shift that occurred from fundamental comprehension of bone formation, to its application in rebuilt tissues and clinical fields is shown. Despite the lack of standardized protocols, consensual conditions relevant for in vitro bone development, in particular cell differentiation, could be highlighted. Culture processes are guided by physiological considerations, although out-of-range conditions are sometimes used without implying negative results for the development of rebuilt tissue. Consensus can be found on several parameters, such as strain frequency (1 Hz) or the use of rest periods, but other points have not yet been fully established, especially synergies with other solicitations. It is believed that the present work will be useful to develop new tissue-engineering processes based on stretching.

Development of thermo-responsive polycaprolactone macrocarriers conjugated with Poly(N-isopropyl acrylamide) for cell culture.

Poly(N-isopropyl acrylamide) (PNIPAAm) is a well-known 'smart' material responding to external stimuli such as temperature. PNIPAAm was successfully conjugated to polycaprolactone (PCL) bead surfaces through amidation reaction. Functionalization steps were characterized and confirmed by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and Energy Dispersion Spectroscopy. PNIPAAm-conjugated PCL allowed human dermal fibroblast cells (HDF) and mesenchymal stem cells (MSC) to adhere, spread, and grow successfully. By reducing the temperature to 30 °C, more than 70% of HDF were detached from PNIPAAm-conjugated PCL macrocarriers with 85% viability. The cell detachment ratio by trypsin treatment was slightly higher than that induced by reduced temperature, however, cell detachment from PNIPAAm-conjugated macrocarriers by lowering the temperature significantly reduced cell death and increased both cell viability and the recovery potential of the detached cells. HDF attachment and detachment were also observed by Live-Dead staining and phase contrast imaging. The expression of extracellular matrix proteins such as Laminin and Fibronectin was also affected by the trypsinization process but not by the reduced temperature process. Taken together, our results showed that thermo-responsive macrocarriers could be a promising alternative method for the non-invasive detachment of cells, in particular for tissue engineering, clinical applications and the use of bioreactors.

Multilineage Constructs for Scaffold-Based Tissue Engineering: A Review of Tissue-Specific Challenges.

There is a growing interest in the regeneration of tissue in interfacial regions, where biological, physical, and chemical attributes vary across tissue type. The simultaneous use of distinct cell lineages can help in developing in vitro structures, analogous to native composite tissues. This literature review gathers the recent reports that have investigated multiple cell types of various sources and lineages in a coculture system for tissue-engineered constructs. Such studies aim at mimicking the native organization of tissues and their interfaces, and/or to improve the development of complex tissue substitutes. This paper thus distinguishes itself from those focusing on technical aspects of coculturing for a single specific tissue. The first part of this review is dedicated to variables of cocultured tissue engineering such as scaffold, cells, and in vitro culture environment. Next, tissue-specific coculture methods and approaches are covered for the most studied tissues. Finally, cross-analysis is performed to highlight emerging trends in coculture principles and to discuss how tissue-specific challenges can inspire new approaches for regeneration of different interfaces to improve the outcomes of various tissue engineering strategies.

The bioconjugation mechanism of purine cross-linkers affects microstructure and cell response to ultra rapidly gelling purine-chitosan sponges.

As a cell carrier, cross-linking is one of the most common approaches used to provide chitosan with greater structural integrity. We introduced a cross-linking strategy by using two purines, guanosine 5'-diphosphate (GDP) or adenosine 5'-diphosphate (ADP), as cross-linkers. The rationale for this approach is that both GDP and ADP have an important physiological role and act as intercellular signaling molecules in numerous biological processes. The slight difference between the chemical structure of guanine and adenosine in GDP and ADP, respectively, affect the cross-linking mechanism. This resulted in a different scaffold microstructure and thus, altered the response of encapsulated cells to the scaffold. FTIR and solid-state 13C-NMR revealed the formation of a quadruplex structure among the four GDP molecules confined between the chitosan backbone. This resulted from the ability of guanine to form intermolecular hydrogen bonds, while adenosine in ADP lacks this capacity. The formation of a more organized structure in GDP-chitosan sponges also increased the crystallinity of the sponge as shown by X-ray diffraction data. Further, physicochemical analyses with SEM and µCT indicated a more open-pore architecture and increased porosity. Although an active population of encapsulated cells was maintained in all chitosan sponges overtime, the GDP-based sponges provided a 6-fold increase in the activity of MC-3T3 cells and significantly enhanced their proliferation due to a more appropriate microstructure. Overall, these findings suggest that slight changes in the chemical structure of the cross-linker in the preparation of chitosan-based biomaterials will have a significant impact on the structural properties of the chitosan. This important parameter can be utilized to modulate cell response and to understand the cell signaling pathway of chitosan-based biomaterials in the context of their applications in tissue engineering.

The Osteogenic and Tenogenic Differentiation Potential of C3H10T1/2 (Mesenchymal Stem Cell Model) Cultured on PCL/PLA Electrospun Scaffolds in the Absence of Specific Differentiation Medium.

The differentiation potential of mesenchymal stem cells (MSC) has been extensively tested on electrospun scaffolds. However, this potential is often assessed with lineage-specific medium, making it difficult to interpret the real contribution of the properties of the scaffold in the cell response. In this study, we analyzed the ability of different polycaprolactone/polylactic acid PCL/PLA electrospun scaffolds (pure or blended compositions, random or aligned fibers, various fiber diameters) to drive MSC towards bone or tendon lineages in the absence of specific differentiation medium. C3H10T1/2 cells (a mesenchymal stem cell model) were cultured on scaffolds for 96 h without differentiation factors. We performed a cross-analysis of the cell-scaffold interactions (spreading, organization, and specific gene expression) with mechanical (elasticity), morphological (porosity, fibers diameter and orientation) and surface (wettability) characterizations of the electrospun fibers. We concluded that (1) osteogenic differentiation can be initiated on pure PCL-based electrospun scaffolds without specific culture conditions; (2) fiber alignment modified cell organization in the short term and (3) PLA added to PCL with an increased fiber diameter encouraged the stem cells towards the tendon lineage without additional tenogenic factors. In summary, the differentiation potential of stem cells on adapted electrospun fibers could be achieved in factor-free medium, making possible future applications in clinically relevant situations.

Towards the development and characterization of an easy handling sheet-like biohybrid bone substitute.

We designed a sheet-like bone substitute capable of adapting to different geometries and becoming a standard tissue-engineered process for bone surgery. Preosteoblastic cells were seeded on to a monolayer of calcium phosphate granules and cultured in a flat parallelepipedic cell culture chamber for 1 month. From the various diameters of the granules examined, the 80-200 µm group exhibited the most homogeneous performances regarding both biological (cell morphology, viability, differentiation, and simple metabolic activity) and mechanical (cohesion and stress-strain behavior) properties. This sheet was easy to handle after extraction from the culture chamber and showed versatile geometry and flexibility, making it easy to use for surgeons, especially for small defects of the maxillofacial area.