3D Niche-Inspired Scaffolds as a Stem Cell Delivery System for the Regeneration of the Osteochondral Interface.

The regeneration of the osteochondral unit represents a challenge due to the distinct cartilage and bone phases. Current strategies focus on the development of multiphasic scaffolds that recapitulate features of this complex unit and promote the differentiation of implanted bone-marrow derived stem cells (BMSCs). In doing so, challenges remain from the loss of stemness during in vitro expansion of the cells and the low control over stem cell activity at the interface with scaffolds in vitro and in vivo. Here, this work scaffolds inspired by the bone marrow niche that can recapitulate the natural healing process after injury. The construct comprises an internal depot of quiescent BMSCs, mimicking the bone marrow cavity, and an electrospun (ESP) capsule that "activates" the cells to migrate into an outer "differentiation-inducing" 3D printed unit functionalized with TGF-ß and BMP-2 peptides. In vitro, niche-inspired scaffolds retained a depot of nonproliferative cells capable of migrating and proliferating through the ESP capsule. Invasion of the 3D printed cavity results in location-specific cell differentiation, mineralization, secretion of alkaline phosphatase (ALP) and glycosaminoglycans (GAGs), and genetic upregulation of collagen II and collagen I. In vivo, niche-inspired scaffolds are biocompatible, promoted tissue formation in rat subcutaneous models, and regeneration of the osteochondral unit in rabbit models.

Assessing the Genotoxicity of Cellulose Nanomaterials in a Co-Culture of Human Lung Epithelial Cells and Monocyte-Derived Macrophages.

Cellulose micro/nanomaterials (CMNMs) are innovative materials with a wide spectrum of industrial and biomedical applications. Although cellulose has been recognized as a safe material, the unique properties of its nanosized forms have raised concerns about their safety for human health. Genotoxicity is an endpoint that must be assessed to ensure that no carcinogenic risks are associated with exposure to nanomaterials. In this study, we evaluated the genotoxicity of two types of cellulose micro/nanofibrils (CMF and CNF) and one sample of cellulose nanocrystals (CNC), obtained from industrial bleached Eucalyptus globulus kraft pulp. For that, we exposed co-cultures of human alveolar epithelial A549 cells and THP-1 monocyte-derived macrophages to a concentration range of each CMNM and used the micronucleus (MN) and comet assays. Our results showed that only the lowest concentrations of the CMF sample were able to induce DNA strand breaks (FPG-comet assay). However, none of the three CMNMs produced significant chromosomal alterations (MN assay). These findings, together with results from previous in vitro studies using monocultures of A549 cells, indicate that the tested CNF and CNC are not genotoxic under the conditions tested, while the CMF display a low genotoxic potential.

A Humanized In Vitro Model of Innervated Skin for Transdermal Analgesic Testing.

Sensory innervation of the skin is essential for its function, homeostasis, and wound healing mechanisms. Thus, to adequately model the cellular microenvironment and function of native skin, in vitro human skin equivalents (hSE) containing a sensory neuron population began to be researched. In this work, a fully human 3D platform of hSE innervated by induced pluripotent stem cell-derived nociceptor neurospheres (hNNs), mimicking the native mode of innervation, is established. Both the hSE and nociceptor population exhibit morphological and phenotypical characteristics resembling their native counterparts, such as epidermal and dermal layer formation and nociceptor marker exhibition, respectively. In the co-culture platform, neurites develop from the hNNs and navigate in 3D to innervate the hSE from a distance. To probe both skin and nociceptor functionality, a clinically available capsaicin patch (Qutenza) is applied directly over the hSE section and neuron reaction is analyzed. Application of the patch causes an exposure time-dependent neurite regression and degeneration. In platforms absent of hSE, axonal degeneration is further increased, highlighting the role of the skin construct as a barrier. In sum, an in vitro tool of functional innervated skin with high interest for preclinical research is established.

Installation of click-type functional groups enable the creation of an additive manufactured construct for the osteochondral interface.

Melt extrusion-based additive manufacturing (AM) is often used to fabricate scaffolds for osteochondral (OC) regeneration. However, there are two shortcomings associated with this scaffold manufacturing technique for engineering of tissue interfaces: (a) most polymers used in the processing are bioinert, and (b) AM scaffolds often contain discrete (material) gradients accompanied with mechanically weak interfaces. The inability to mimic the gradual transition from cartilage to bone in OC tissue leads to poor scaffold performance and even failure. We hypothesized that introducing peptide gradients on the surface could gradually guide human mesenchymal stromal cell (hMSC) differentiation, from a chondrogenic towards on osteogenic phenotype. To work towards this goal, we initially manufactured poly(ϵ-caprolactone)-azide (PCLA) and PCL-maleimide (PCLM) scaffolds. The surface exposed click-type functional groups, with a surface concentration in the 102pmol cm-2regime, were used to introduce bone morphogenic protein-2 or transforming growth factor-beta binding peptide sequences to drive hMSC differentiation towards osteogenic or chondrogenic phenotypes, respectively. After 3 weeks of culture in chondrogenic medium, we observed differentiation towards hypertrophic chondrogenic phenotypes with expression of characteristic markers such as collagen X. In osteogenic medium, we observed the upregulation of mineralization markers. In basic media, the chondro-peptide displayed a minor effect on chondrogenesis, whereas the osteo-peptide did not affect osteogenesis. In a subcutaneous rat model, we observed a minimal foreign body response to the constructs, indicating biocompatibility. As proof-of-concept, we finally used a novel AM technology to showcase its potential to create continuous polymer gradients (PCLA and PCLM) across scaffolds. These scaffolds did not display delamination and were mechanically stronger compared to discrete gradient scaffolds. Due to the versatility of the orthogonal chemistry applied, this approach provides a general strategy for the field; we could anchor other tissue specific cues on the clickable groups, making these gradient scaffolds interesting for multiple interfacial tissue applications.

Analysis of the In Vitro Toxicity of Nanocelluloses in Human Lung Cells as Compared to Multi-Walled Carbon Nanotubes.

Cellulose micro/nanomaterials (CMNM), comprising cellulose microfibrils (CMF), nanofibrils (CNF), and nanocrystals (CNC), are being recognized as promising bio-nanomaterials due to their natural and renewable source, attractive properties, and potential for applications with industrial and economical value. Thus, it is crucial to investigate their potential toxicity before starting their production at a larger scale. The present study aimed at evaluating the cell internalization and in vitro cytotoxicity and genotoxicity of CMNM as compared to two multi-walled carbon nanotubes (MWCNT), NM-401 and NM-402, in A549 cells. The exposure to all studied NM, with the exception of CNC, resulted in evident cellular uptake, as analyzed by transmission electron microscopy. However, none of the CMNM induced cytotoxic effects, in contrast to the cytotoxicity observed for the MWCNT. Furthermore, no genotoxicity was observed for CNF, CNC, and NM-402 (cytokinesis-block micronucleus assay), while CMF and NM-401 were able to significantly raise micronucleus frequency. Only NM-402 was able to induce ROS formation, although it did not induce micronuclei. Thus, it is unlikely that the observed CMF and NM-401 genotoxicity is mediated by oxidative DNA damage. More studies targeting other genotoxicity endpoints and cellular and molecular events are underway to allow for a more comprehensive safety assessment of these nanocelluloses.

Evaluating the genotoxicity of cellulose nanofibrils in a co-culture of human lung epithelial cells and monocyte-derived macrophages.

Cellulose nanofibrils (CNF) are manufactured nanofibres that hold impressive expectations in forest, food, pharmaceutical, and biomedical industries. CNF production and applications are leading to an increased human exposure and thereby it is of utmost importance to assess its safety to health. In this study, we screened the cytotoxic, immunotoxic and genotoxic effects of a CNF produced by TEMPO-mediated oxidation of an industrial bleached Eucalyptus globulus kraft pulp on a co-culture of lung epithelial alveolar (A549) cells and monocyte-derived macrophages (THP-1 cells). The results indicated that low CNF concentrations can stimulate A549 cells proliferation, whereas higher concentrations are moderately toxic. Moreover, no proinflammatory cytokine IL-1ß was detected in the co-culture medium suggesting no immunotoxicity. Although CNF treatment did not induce sizable levels of DNA damage in A549 cells, it leaded to micronuclei formation at 1.5 and 3 µg/cm2. These findings suggest that this type of CNF is genotoxic through aneugenic or clastogenic mechanisms. Noteworthy, cell overgrowth and genotoxicity, which are events relevant for cell malignant transformation, were observed at low CNF concentration levels, which are more realistic and relevant for human exposure, e.g., in occupational settings.