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.

Cryogel-based Injectable 3D Microcarrier Co-culture for Support of Hematopoietic Progenitor Niches.

Although hematopoietic stem cell (HSC) transplantation can restore functional hematopoiesis upon immune or chemotherapy-induced bone marrow failure, complications often arise during recovery, leading to up to 25% transplant-related mortality in treated patients. In hematopoietic homeostasis and regeneration, HSCs in the bone marrow give rise to the entirety of cellular blood components. One of the challenges in studying hematopoiesis is the ability to successfully mimic the relationship between the stroma and hematopoietic stem and progenitor cells (HSPCs). This study and the described protocols propose an advantageous method for culturing and assessing stromal hematopoietic support in three dimensions, representing a simplified in vitro model of the bone marrow niche that can be transplanted in vivo by injection. By co-culturing OP9 bone marrow-derived stromal cells (BMSCs) and cKit+ Sca-1+ Lin- (KLS+ ) HSPCs on collagen-coated carboxymethylcellulose scaffolds for 2 weeks in the absence of cytokines, we established a methodology for in vivo subcutaneous transplantation. With this model we were able to detect early signs of extramedullary hematopoiesis. This work can be useful for studying various stromal cell populations in co-culture, as well as simple transfer by injection of these scaffolds in vivo for heterotopic regeneration of the marrow microenvironment. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of HSPCs from mice Basic Protocol 2: Co-seeding of HSPCs and BMSCs on collagen-coated CCMs Basic Protocol 3: Maintenance, real-time imaging, and analysis of co-seeded scaffolds Basic Protocol 4: End-point analysis of co-seeded scaffolds using flow cytometry and CFU assays Basic Protocol 5: Transplantation of scaffolds by subcutaneous injection Support Protocol: Preparation of custom scaffold drying device.

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.

Hydrogels for 3D Neural Tissue Models: Understanding Cell-Material Interactions at a Molecular Level.

The development of 3D neural tissue analogs is of great interest to a range of biomedical engineering applications including tissue engineering of neural interfaces, treatment of neurodegenerative diseases and in vitro assessment of cell-material interactions. Despite continued efforts to develop synthetic or biosynthetic hydrogels which promote the development of complex neural networks in 3D, successful long-term 3D approaches have been restricted to the use of biologically derived constructs. In this study a poly (vinyl alcohol) biosynthetic hydrogel functionalized with gelatin and sericin (PVA-SG), was used to understand the interplay between cell-cell communication and cell-material interaction. This was used to probe critical short-term interactions that determine the success or failure of neural network growth and ultimately the development of a useful model. Complex primary ventral mesencephalic (VM) neural cells were encapsulated in PVA-SG hydrogels and critical molecular cues that demonstrate mechanosensory interaction were examined. Neuronal presence was constant over the 10 day culture, but the astrocyte population decreased in number. The lack of astrocytic support led to a reduction in neural process outgrowth from 24.0 ± 1.3 µm on Day 7 to 7.0 ± 0.1 µm on Day 10. Subsequently, purified astrocytes were studied in isolation to understand the reasons behind PVA-SG hydrogel inability to support neural network development. It was proposed that the spatially restrictive nature (or tight mesh size) of PVA-SG hydrogels limited the astrocytic actin polymerization together with a cytoplasmic-nuclear translocation of YAP over time, causing an alteration in their cell cycle. This was confirmed by the evaluation of p27/Kip1 gene that was found to be upregulated by a twofold increase in expression at both Days 7 and 10 compared to Day 3, indicating the quiescent stage of the astrocytes in PVA-SG hydrogel. Cell migration was further studied by the quantification of MMP-2 production that was negligible compared to 2D controls, ranging from 2.7 ± 2.3% on Day 3 to 5.3 ± 2.9% on Day 10. This study demonstrates the importance of understanding astrocyte-material interactions at the molecular level, with the need to address spatial constraints in the 3D hydrogel environment. These findings will inform the design of future hydrogel constructs with greater capacity for remodeling by the cell population to create space for cell migration and neural process extension.

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.

Adaptive reorientation of endothelial collectives in response to strain.

Mature epithelial monolayers share the ability to coherently respond to external mechanical stimuli. Tissue remodeling requires cell shape changes and coordinated movements. Human endothelia provide an exquisite example of such emerging collective activities. As part of their function in maintaining body homeostasis under variable hemodynamic loadings, endothelial ensembles must dynamically adapt to wall shear stress and cyclic deformation. While the alignment of several types of cells, including fibroblasts, osteoblasts and epithelial tissues, in response to various flow conditions or wall shear stress levels has been described in detail, less is known about collective endothelial remodeling under pure wall deformation. Here, using a custom-developed bioreactor, we exposed mature human endothelia to two distinct physiological levels of cyclic loading, generating overlapping gradients of strain. Endothelial cells remodeled depending on the level of imposed strain yielding local variations of cell density. In particular, a collective cell orientation orthogonal to the main direction of strain was observed at low levels of wall deformation, while cells reoriented parallel to the main direction of strain at high levels of wall deformation. The tissue adaptation depended on the establishment of mature adherens junctions, which were reinforced by the polarized recruitment of the adaptor protein vinculin. The pivotal role of cell-to-cell junctions was confirmed by the biochemical inhibition of vascular endothelial cadherin homotypic contacts, which impaired the collective remodeling. Together, our data establish wall deformation as an independent determinant of endothelial architecture with direct implications in vascular physiopathology.