Ultrasound Stimulation of Piezoelectric Nanocomposite Hydrogels Boosts Chondrogenic Differentiation in Vitro, in Both a Normal and Inflammatory Milieu.

The use of piezoelectric nanomaterials combined with ultrasound stimulation is emerging as a promising approach for wirelessly triggering the regeneration of different tissue types. However, it has never been explored for boosting chondrogenesis. Furthermore, the ultrasound stimulation parameters used are often not adequately controlled. In this study, we show that adipose-tissue-derived mesenchymal stromal cells embedded in a nanocomposite hydrogel containing piezoelectric barium titanate nanoparticles and graphene oxide nanoflakes and stimulated with ultrasound waves with precisely controlled parameters (1 MHz and 250 mW/cm2, for 5 min once every 2 days for 10 days) dramatically boost chondrogenic cell commitment in vitro. Moreover, fibrotic and catabolic factors are strongly down-modulated: proteomic analyses reveal that such stimulation influences biological processes involved in cytoskeleton and extracellular matrix organization, collagen fibril organization, and metabolic processes. The optimal stimulation regimen also has a considerable anti-inflammatory effect and keeps its ability to boost chondrogenesis in vitro, even in an inflammatory milieu. An analytical model to predict the voltage generated by piezoelectric nanoparticles invested by ultrasound waves is proposed, together with a computational tool that takes into consideration nanoparticle clustering within the cell vacuoles and predicts the electric field streamline distribution in the cell cytoplasm. The proposed nanocomposite hydrogel shows good injectability and adhesion to the cartilage tissue ex vivo, as well as excellent biocompatibility in vivo, according to ISO 10993. Future perspectives will involve preclinical testing of this paradigm for cartilage regeneration.

A Novel Approach for Multiple Material Extrusion in Arthroscopic Knee Surgery.

Articular cartilage defects and degenerative diseases are pathological conditions that cause pain and the progressive loss of joint functionalities. The most severe cases are treated through partial or complete joint replacement with prostheses, even if the interest in cartilage regeneration and re-growth methods is steadily increasing. These methods consist of the targeted deposition of biomaterials. Only a few tools have been developed so far for performing these procedures in a minimally invasive way. This work presents an innovative device for the direct deposition of multiple biomaterials in an arthroscopic scenario. The tool is easily handleable and allows the extrusion of three different materials simultaneously. It is also equipped with a flexible tip to reach remote areas of the damaged cartilage. Three channels are arranged coaxially and a spring-based dip-coating approach allows the fabrication and assembly of a bendable polymeric tip. Experimental tests were performed to characterize the tip, showing the ability to bend it up to 90° (using a force of ~ 1.5 N) and to extrude three coaxial biomaterials at the same time with both tip straight and tip fully bent. Rheometric analysis and fluid-dynamic computational simulations were performed to analyze the fluids' behavior; the maximum shear stresses were observed in correspondence to the distal tip and the channel convergence chamber, but with values up to ~ 1.2 kPa, compatible with a safe extrusion of biomaterials, even laden with cells. The cells viability was assessed after the extrusion with Live/Dead assay, confirming the safety of the extrusion procedures. Finally, the tool was tested arthroscopically in a cadaveric knee, demonstrating its ability to deliver the biomaterial in different areas, even ones that are typically hard-to-reach with traditional tools.

Primers for the Adhesion of Gellan Gum-Based Hydrogels to the Cartilage: A Comparative Study.

A stable adhesion to the cartilage is a crucial requisite for hydrogels used for cartilage regeneration. Indeed, a weak interface between the tissue and the implanted material may produce a premature detachment and thus the failure of the regeneration processes. Fibrin glue, cellulose nanofibers and catecholamines have been proposed in the state-of-the-art as primers to improve the adhesion. However, no studies focused on a systematic comparison of their performance. This work aims to evaluate the adhesion strength between ex vivo cartilage specimens and polysaccharide hydrogels (gellan gum and methacrylated gellan gum), by applying the mentioned primers as intermediate layer. Results show that the fibrin glue and the cellulose nanofibers improve the adhesion strength, while catecholamines do not guarantee reaching a clinically acceptable value. Stem cells embedded in gellan gum hydrogels reduce the adhesion strength when fibrin glue is used as a primer, being anyhow still sufficient for in vivo applications.

RGD-Functionalized Hydrogel Supports the Chondrogenic Commitment of Adipose Mesenchymal Stromal Cells.

Articular cartilage is known to have limited intrinsic self-healing capacity when a defect or a degeneration process occurs. Hydrogels represent promising biomaterials for cell encapsulation and injection in cartilage defects by creating an environment that mimics the cartilage extracellular matrix. The aim of this study is the analysis of two different concentrations (1:1 and 1:2) of VitroGel® (VG) hydrogels without (VG-3D) and with arginine-glycine-aspartic acid (RGD) motifs, (VG-RGD), verifying their ability to support chondrogenic differentiation of encapsulated human adipose mesenchymal stromal cells (hASCs). We analyzed the hydrogel properties in terms of rheometric measurements, cell viability, cytotoxicity, and the expression of chondrogenic markers using gene expression, histology, and immunohistochemical tests. We highlighted a shear-thinning behavior of both hydrogels, which showed good injectability. We demonstrated a good morphology and high viability of hASCs in both hydrogels. VG-RGD 1:2 hydrogels were the most effective, both at the gene and protein levels, to support the expression of the typical chondrogenic markers, including collagen type 2, SOX9, aggrecan, glycosaminoglycan, and cartilage oligomeric matrix protein and to decrease the proliferation marker MKI67 and the fibrotic marker collagen type 1. This study demonstrated that both hydrogels, at different concentrations, and the presence of RGD motifs, significantly contributed to the chondrogenic commitment of the laden hASCs.

Modeling and Fabrication of Silk Fibroin-Gelatin-Based Constructs Using Extrusion-Based Three-Dimensional Bioprinting.

Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure's fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs' fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.

Graphene Oxide-Doped Gellan Gum-PEGDA Bilayered Hydrogel Mimicking the Mechanical and Lubrication Properties of Articular Cartilage.

Articular cartilage (AC) is a specialized connective tissue able to provide a low-friction gliding surface supporting shock-absorption, reducing stresses, and guaranteeing wear-resistance thanks to its structure and mechanical and lubrication properties. Being an avascular tissue, AC has a limited ability to heal defects. Nowadays, conventional strategies show several limitations, which results in ineffective restoration of chondral defects. Several tissue engineering approaches have been proposed to restore the AC's native properties without reproducing its mechanical and lubrication properties yet. This work reports the fabrication of a bilayered structure made of gellan gum (GG) and poly (ethylene glycol) diacrylate (PEGDA), able to mimic the mechanical and lubrication features of both AC superficial and deep zones. Through appropriate combinations of GG and PEGDA, cartilage Young's modulus is effectively mimicked for both zones. Graphene oxide is used as a dopant agent for the superficial hydrogel layer, demonstrating a lower friction than the nondoped counterpart. The bilayered hydrogel's antiwear properties are confirmed by using a knee simulator, following ISO 14243. Finally, in vitro tests with human chondrocytes confirm the absence of cytotoxicity effects. The results shown in this paper open the way to a multilayered synthetic injectable or surgically implantable filler for restoring AC defects.

Wear Behavior Characterization of Hydrogels Constructs for Cartilage Tissue Replacement.

This paper aims to characterize the wear behavior of hydrogel constructs designed for human articular cartilage replacement. To this purpose, poly (ethylene glycol) diacrylate (PEGDA) 10% w/v and gellan gum (GG) 1.5% w/v were used to reproduce the superior (SUP) cartilage layer and PEGDA 15% w/v and GG 1.5% w/v were used to reproduce the deep (DEEP) cartilage layer, with or without graphene oxide (GO). These materials (SUP and DEEP) were analyzed alone and in combination to mimic the zonal architecture of human articular cartilage. The developed constructs were tested using a four-station displacement control knee joint simulator under bovine calf serum. Roughness and micro-computer tomography (µ-CT) measurements evidenced that the hydrogels with 10% w/v of PEGDA showed a worse behavior both in terms of roughness increase and loss of uniformly distributed density than 15% w/v of PEGDA. The simultaneous presence of GO and 15% w/v PEGDA contributed to keeping the hydrogel construct's characteristics. The Raman spectra of the control samples showed the presence of unreacted C=C bonds in all the hydrogels. The degree of crosslinking increased along the series SUP < DEEP + SUP < DEEP without GO. The Raman spectra of the tested hydrogels showed the loss of diacrylate groups in all the samples, due to the washout of unreacted PEGDA in bovine calf serum aqueous environment. The loss decreased along the series SUP > DEEP + SUP > DEEP, further confirming that the degree of photo-crosslinking of the starting materials plays a key role in determining their wear behavior. µ-CT and Raman spectroscopy proved to be suitable techniques to characterize the structure and composition of hydrogels.

Impact of Isolation Procedures on the Development of a Preclinical Synovial Fibroblasts/Macrophages in an In Vitro Model of Osteoarthritis.

There is a lack ofin vitromodels able to properly represent osteoarthritis (OA) synovial tissue (ST). We aimed to characterize OA ST and to investigate whether a mechanical or enzymatic digestion procedures influence synovial cell functional heterogeneity in vitro. Procedures using mechanical nondigested fragments (NDF), synovial digested fragments (SDF), and filtrated synovial digested cells (SDC) were compared. An immunophenotypic profile was performed to distinguish synovial fibroblasts (CD55, CD73, CD90, CD106), macrophages (CD14, CD68), M1-like (CD80, CD86), and M2-like (CD163, CD206) synovial macrophages. Pro-inflammatory (interleukin 6 IL6), tumor necrosis factor alpha (TNFα), chemokine C-C motif ligand 3 (CCL3/MIP1α), C-X- motif chemokine ligand 10 (CXCL10/IP10) and anti-inflammatory (interleukin 10 (IL10)), transforming growth factor beta 1 (TGFß1), C-C motif chemokine ligand 18 (CCL18) cytokines were evaluated. CD68 and CD163 markers were higher in NDF and SDF compared to the SDC procedure, while CD80, CD86, and CD206 were higher only in NDF compared to the SDC procedure. Synovial fibroblast markers showed similar percentages. TNFα, CCL3/MIP1α, CXCL10/IP10, and CCL18 were higher in NDF compared to SDC, but not compared to SDF. IL10 and TGFß1 were higher in NDF than SDC at the molecular level, while IL6 did not show differences among procedures. We demonstrated that NDF isolation procedures better preserved the heterogeneity of specific OA synovial populations (fibroblasts, macrophages), fostering their use for testing new cell therapies or drugs for OA, reducing or avoiding the use of animal models.

Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells.

Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as ß-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.