The Role of Interstitial Fluid Pressure in Cerebral Porous Biomaterial Integration.

Recent advances in biomaterials offer new possibilities for brain tissue reconstruction. Biocompatibility, provision of cell adhesion motives and mechanical properties are among the present main design criteria. We here propose a radically new and potentially major element determining biointegration of porous biomaterials: the favorable effect of interstitial fluid pressure (IFP). The force applied by the lymphatic system through the interstitial fluid pressure on biomaterial integration has mostly been neglected so far. We hypothesize it has the potential to force 3D biointegration of porous biomaterials. In this study, we develop a capillary hydrostatic device to apply controlled in vitro interstitial fluid pressure and study its effect during 3D tissue culture. We find that the IFP is a key player in porous biomaterial tissue integration, at physiological IFP levels, surpassing the known effect of cell adhesion motives. Spontaneous electrical activity indicates that the culture conditions are not harmful for the cells. Our work identifies interstitial fluid pressure at physiological negative values as a potential main driver for tissue integration into porous biomaterials. We anticipate that controlling the IFP level could narrow the gap between in vivo and in vitro and therefore decrease the need for animal screening in biomaterial design.

Design of an elastic porous injectable biomaterial for tissue regeneration and volume retention.

Soft tissue reconstruction currently relies on two main approaches, one involving the implantation of external biomaterials and the second one exploiting surgical autologous tissue displacement. While both methods have different advantages and disadvantages, successful long-term solutions for soft tissue repair are still limited. Specifically, volume retention over time and local tissue regeneration are the main challenges in the field. In this study the performance of a recently developed elastic porous injectable (EPI) biomaterial based on crosslinked carboxymethylcellulose is analyzed. Nearly quantitative volumetric stability, with over 90% volume retention at 6 months, is observed, and the pore space of the material is effectively colonized with autologous fibrovascular tissue. A comparative analysis with hyaluronic acid and collagen-based clinical reference materials is also performed. Mechanical stability, evidenced by a low-strain elastic storage modulus (G') approaching 1kPa and a yield strain of several tens of percent, is required for volume retention in-vivo. Macroporosity, along with in-vivo persistence of at least several months, is instead needed for successful host tissue colonization. This study demonstrates the importance of understanding material design criteria and defines the biomaterial requirements for volume retention and tissue colonization in soft tissue regeneration. STATEMENT OF SIGNIFICANCE: We present the design of an elastic, porous, injectable (EPI) scaffold suspension capable of inducing a precisely defined, stable volume of autologous connective tissue in situ. It combines volume stability and vascularized tissue induction capacity known from bulk scaffolds with the ease of injection in shear yielding materials. By comparative study with a series of clinically established biomaterials including a wound healing matrix and dermal fillers, we establish design rules regarding rheological and compressive mechanical properties as well as degradation characteristics that rationally underpin the volume stability and tissue induction in a high-performance biomaterial. These design rules should allow to streamline the development of new colonizable injectables.

An Injectable Meta-Biomaterial: From Design and Simulation to In Vivo Shaping and Tissue Induction.

A novel type of injectable biomaterial with an elastic softening transition is described. The material enables in vivo shaping, followed by induction of 3D stable vascularized tissue. The synthesis of the injectable meta-biomaterial is instructed by extensive numerical simulation as a suspension of irregularly fragmented, highly porous sponge-like microgels. The irregular particle shape dramatically enhances yield strain for in vivo stability against deformation. Porosity of the particles, along with friction between internal surfaces, provides the elastic softening transition. This emergent metamaterial property enables the material to reversibly change stiffness during deformation, allowing native tissue properties to be matched over a wide range of deformation amplitudes. After subcutaneous injection in mice, predetermined shapes can be sculpted manually. The 3D shape is maintained during excellent host tissue integration, with induction of vascular connective tissue that persists to the end of one-year follow-up. The geometrical design is compatible with many hydrogel materials, including cell-adhesion motives for cell transplantation. The injectable meta-biomaterial therefore provides new perspectives in soft tissue engineering and regenerative medicine.

Neurothreads: Development of supportive carriers for mature dopaminergic neuron differentiation and implantation.

In this study we present the use of elastic macroporous cryogels for differentiation and transplantation of mature neurons. We develop a coating suitable for long-term neuronal culture, including stem cell differentiation, by covalent immobilization of neural adhesion proteins. In the context of cell therapy for Parkinson's disease, we show compatibility with established dopaminergic differentiation of both immortalized mesencephalic progenitors - LUHMES - and human embryonic stem cells (hESCs). We adjust structural properties of the biomaterial to create carriers - Neurothreads - favourable for cell viability during transplantation. Finally, we show feasibility of preservation of mature neurons, supported by Neurothreads, one month after in-vivo transplantation. Preliminary data suggests that the Neurothread approach could provide more mature and less proliferative cells in vivo.

Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis.

Modeling the interaction between the supportive stroma and the hematopoietic stem and progenitor cells (HSPC) is of high interest in the regeneration of the bone marrow niche in blood disorders. In this work, we present an injectable co-culture system to study this interaction in a coherent in vitro culture and in vivo transplantation model. We assemble a 3D hematopoietic niche in vitro by co-culture of supportive OP9 mesenchymal cells and HSPCs in porous, chemically defined collagen-coated carboxymethylcellulose microscaffolds (CCMs). Flow cytometry and hematopoietic colony forming assays demonstrate the stromal supportive capacity for in vitro hematopoiesis in the absence of exogenous cytokines. After in vitro culture, we recover a paste-like living injectable niche biomaterial from CCM co-cultures by controlled, partial dehydration. Cell viability and the association between stroma and HSPCs are maintained in this process. After subcutaneous injection of this living artificial niche in vivo, we find maintenance of stromal and hematopoietic populations over 12 weeks in immunodeficient mice. Indeed, vascularization is enhanced in the presence of HSPCs. Our approach provides a minimalistic, scalable, biomimetic in vitro model of hematopoiesis in a microcarrier format that preserves the HSPC progenitor function, while being injectable in vivo without disrupting the cell-cell interactions established in vitro.

Regenerative potential of primary adult human neural stem cells on micropatterned bio-implants boosts motor recovery.

BACKGROUND: The adult brain is unable to regenerate itself sufficiently after large injuries. Therefore, hopes rely on therapies using neural stem cell or biomaterial transplantation to sustain brain reconstruction. The aim of the present study was to evaluate the improvement in sensorimotor recovery brought about by human primary adult neural stem cells (hNSCs) in combination with bio-implants. METHODS: hNSCs were pre-seeded on implants micropatterned for neurite guidance and inserted intracerebrally 2 weeks after a primary motor cortex lesion in rats. Long-term behaviour was significantly improved after hNSC implants versus cell engraftment in the grip strength test. MRI and immunohistological studies were conducted to elucidate the underlying mechanisms of neuro-implant integration. RESULTS: hNSC implants promoted tissue reconstruction and limited hemispheric atrophy and glial scar expansion. After 3 months, grafted hNSCs were detected on implants and expressed mature neuronal markers (NeuN, MAP2, SMI312). They also migrated over a short distance to the reconstructed tissues and to the peri-lesional tissues, where 26% integrated as mature neurons. Newly formed host neural progenitors (nestin, DCX) colonized the implants, notably in the presence of hNSCs, and participated in tissue reconstruction. The microstructured bio-implants sustained the guided maturation of both grafted hNSCs and endogenous progenitors. CONCLUSIONS: These immunohistological results are coherent with and could explain the late improvement observed in sensorimotor recovery. These findings provide novel insights into the regenerative potential of primary adult hNSCs combined with microstructured implants.

Detection of Alzheimer's disease amyloid-beta plaque deposition by deep brain impedance profiling.

OBJECTIVE: Alzheimer disease (AD) is the most common form of neurodegenerative disease in elderly people. Toxic brain amyloid-beta (Aß) aggregates and ensuing cell death are believed to play a central role in the pathogenesis of the disease. In this study, we investigated if we could monitor the presence of these aggregates by performing in situ electrical impedance spectroscopy measurements in AD model mice brains. APPROACH: In this study, electrical impedance spectroscopy measurements were performed post-mortem in APPPS1 transgenic mice brains. This transgenic model is commonly used to study amyloidogenesis, a pathological hallmark of AD. We used flexible probes with embedded micrometric electrodes array to demonstrate the feasibility of detecting senile plaques composed of Aß peptides by localized impedance measurements. MAIN RESULTS: We particularly focused on deep brain structures, such as the hippocampus. Ex vivo experiments using brains from young and old APPPS1 mice lead us to show that impedance measurements clearly correlate with the percentage of Aß plaque load in the brain tissues. We could monitor the effects of aging in the AD APPPS1 mice model. SIGNIFICANCE: We demonstrated that a localized electrical impedance measurement constitutes a valuable technique to monitor the presence of Aß-plaques, which is complementary with existing imaging techniques. This method does not require prior Aß staining, precluding the risk of variations in tissue uptake of dyes or tracers, and consequently ensuring reproducible data collection.

A compressible scaffold for minimally invasive delivery of large intact neuronal networks.

Millimeter to centimeter-sized injectable neural scaffolds based on macroporous cryogels are presented. The polymer-scaffolds are made from alginate and carboxymethyl-cellulose by a novel simple one-pot cryosynthesis. They allow surgical sterility by means of autoclaving, and present native laminin as an attachment motive for neural adhesion and neurite development. They are designed to protect an extended, living neuronal network during compression to a small fraction of the original volume in order to enable minimally invasive delivery. The scaffolds behave as a mechanical meta-material: they are soft at the macroscopic scale, enabling injection through narrow-bore tubing and potentially good cellular scaffold integration in soft target tissues such as the brain. At the same time, the scaffold material has a high local Young modulus, allowing protection of the neuronal network during injection. Based on macroscopic and nanomechanical characterization, the generic geometrical and mechanical design rules are presented, enabling macroporous cellular scaffold injectability.

Accurate resistivity mouse brain mapping using microelectrode arrays.

Electrical impedance spectroscopy measurements were performed in post-mortem mice brains using a flexible probe with an embedded micrometric electrode array. Combined with a peak resistance frequency method this allowed obtaining intrinsic resistivity values of brain tissues and structures with submillimetric resolution. Reproducible resistivity measurements are reported, which allows the resistivity in the cortex, ventricle, fiber tracts, thalamus and basal ganglia to be differentiated. Measurements of brain slices revealed resistivity profiles correlated with the local density of cell bodies hence allowing to discriminate between the different cortical layers. Finally, impedance measurements were performed on a model of cauterized mouse brain evidencing the possibility to measure the spatial extent and the degree of the tissue denaturation due to the cauterization.

Investigation of the competition between cell/surface and cell/cell interactions during neuronal cell culture on a micro-engineered surface.

To investigate the respective roles of topography and cell/cell interactions in the development of a guided neuronal network on an engineered surface, micropatterned PDMS substrates were generated with different microgrooves geometry and investigated for the influence of cell density on neurite outgrowth and alignment. Through this systematic investigation, using a human neuronal stem cell line, the rules of neuronal network development and guiding could be learned. The results show that when cell density is increased the influence on neuritic outgrowth and alignment is very different for the various grooves geometries. The data emphasized the competition, in neurite development, between physical cues brought by surface topographical features and cell to cell communications. These results can be of particular interest for designing functional neuronal networks with a controlled architecture.

[Micro/nano-engineering to control growth of neuronal cells and tissue engineering applied to the central nervous system]. / Micro/nano-ingénierie pour le contrôle de la croissance de cellules neuronales et ingénierie tissulaire appliquée au système nerveux central.

Central nervous system pathologies are often characterized by the loss of cell populations. A promising therapy now being developed consists in using bioactive materials, associating grafted cells to biopolymers which provide a scaffold for the in vitro building of new tissues, to be implanted in vivo. In the present article, the state of the art of this field, at crossroads between microtechnology and neuroscience, is described in detail; thereafter our own approach and results about interactions between adult human neural stem cells and microstructured polymers are summarized and discussed. In a second part, some central nervous system repair strategies, based on cerebral tissue engineering, are presented. We will report the main results of our studies to work out and characterize in vivo a cerebral bioprosthesis.

Elucidation of the role of carbon nanotube patterns on the development of cultured neuronal cells.

Carbon nanotubes (CNTs) promise various novel neural biomedical applications for interfacing neurons with electronic devices or to design appropriate biomaterials for tissue regeneration. In this study, we use a new methodology to pattern SiO(2) cell culture surfaces with double-walled carbon nanotubes (DWNTs). In contrast to homogeneous surfaces, patterned surfaces allow us to investigate new phenomena about the interactions between neural cells and CNTs. Our results demonstrate that thin layers of DWNTs can serve as effective substrates for neural cell culture. Growing neurons sense the physical and chemical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. Cells exhibit comparable adhesion and differentiation scores on homogeneous CNT layers and on a homogeneous control SiO(2) surface. Conversely, on patterned surfaces, it is found that cells preferentially grow on CNT patterns and that neurites are guided by micrometric CNT patterns. To further elucidate this observation, we investigate the interactions between CNTs and proteins that are contained in the cell culture medium by using quartz crystal microbalance measurements. Finally, we show that protein adsorption is enhanced on CNT features and that this effect is thickness dependent. CNTs seem to act as a sponge for culture medium elements, possibly explaining the selectivity in cell growth localization and differentiation.

Engineering of adult human neural stem cells differentiation through surface micropatterning.

Interaction between differentiating neural stem cells and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. To restore damage brain area by tissue engineering, the biomaterial scaffold has to mimic this microenvironment to allow organized tissue regeneration. To establish the validity of using microgrooved surfaces in order to simultaneously provide to primary adult human neural stem cells a permissive growth environment and a guide for neurite outgrowth in a pre-established direction, we have studied the long-term culture of adult human neural stem cells from patient biopsies on microgrooved polymers. By exploiting polymer moulding techniques, we engineered non-cytotoxic deep microstructured surfaces of polydimethylsiloxane (PDMS) exhibiting microchannels of various widths. Our results demonstrate that precoated micropatterned PDMS surfaces can serve as effective neurite guidance surfaces for human neural stem cells. Immunocytochemistry analysis show that channel width can impact strongly development and differentiation. In particular we found an optimal microchannel width, that conciliates a high differentiation rate with a pronounced alignment of neurites along the edges of the microchannels. The impact of the microstructures on neurite orientation turned out to be strongly influenced by cell density, attesting that cell/surface interactions at the origin of the alignment effect, are in competition with cell/cell interactions tending to promote interconnected networks of cells. Considering all these effects, we have been able to design appropriate structures allowing to obtain neuron development and differentiation rate comparable to a plane unpatterned surface, with an efficient neurite guidance and a long-term cell viability.

Frustrated phagocytosis on micro-patterned immune complexes to characterize lysosome movements in live macrophages.

Lysosome mobilization is a key cellular process in phagocytes for bactericidal activities and trans-matrix migration. The molecular mechanisms that regulate lysosome mobilization are still poorly known. Lysosomes are hard to track as they move toward phagosomes throughout the cell volume. In order to anticipate cell regions where lysosomes are recruited to, human and RAW264.7 macrophages were seeded on surfaces that were micro-patterned with immune complexes (ICs) as 4 µm-side squares. Distances between IC patterns were adapted to optimize cell spreading in order to constrain lysosome movements mostly in two dimensions. FcΓ receptors triggered local frustrated phagocytosis, frustrated phagosomes appeared as rings of F-actin dots around the IC patterns as early as 5 min after cells made contact with the substratum. Frustrated phagosomes recruited actin-associated proteins (vinculin, paxillin, and gelsolin). The fusion of lysosomes with frustrated phagosomes was shown by the release of beta-hexosaminidase and the recruitment of Lamp1 to frustrated phagosomes. Lysosomes of RAW264.7 macrophages were labeled with cathepsin-D-mCherry to visualize their movements toward frustrated phagosomes. Lysosomes saltatory movements were markedly slowed down compared to cells layered on non-opsonized patterns. In addition, the linearity of the trajectories and the frequency and duration of contacts of lysosomes with frustrated phagosomes were measured. Our experimental set-up is the first step toward deciphering molecular mechanisms which are involved in lysosome movements in the cytoplasm (speed, directionality, and interaction with phagosomes), and opens the door to approaches such as RNA interference, pharmacological inhibition, or mutant expression.