Tunable mechanical properties of gellan gum/poly (ethylene glycol) diacrylate hydrogels for articular cartilage engineering

Articular cartilage (AC) is a complex multi-layered structure organized into four zones, namely superficial, middle, deep and calcified layers [1]. Its mechanical properties are varying with depth, and the compression modulus of the superficial and middle zone can achieve the value of 0.28 ± 0.16 MPa and 0.73 ± 0.26 MPa, respectively [2]. This paper reports the preliminary results concerning mechanically tunable gellan gum/poly (ethylene glycol) diacrylate hydrogels for mimicking the compression mechanical properties of the first two layers of the AC tissue

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

A Novel H2S-releasing Amino-Bisphosphonate which combines bone anti-catabolic and anabolic functions

Bisphosphonates (BPs) are the first-line treatment of bone loss resulting from various pathological conditions. Due to their high affinity to bone they have been used to develop conjugates with pro-Anabolic or anti-catabolic drugs. We recently demontrated that hydrogen sulfide (H2S), promotes osteogenesis and inhibits osteoclast differentiation. Here we developed an innovative molecule, named DM-22, obtained from the combination of alendronate (AL) and the H2S-releasing moiety aryl-isothiocyanate. DM-22 and AL were assayed in vitro in the concentration range 1-33 μM for effects on viability and function of human osteoclasts (h-OCs) and mesenchymal stromal cells (h-MSCs) undergoing osteogenic differentiation. Amperometric measures revealed that DM-22 releases H2S at a slow rate with a thiol-dependent mechanism. DM-22 significantly inhibited h-OCs differentiation and function, maintaining a residual h-OCs viability even at the high dose of 33 μM. Contrary to AL, in h-MSCs DM-22 did not induce cytotoxicity as revealed by LDH assay, significantly stimulated mineralization as measured by Alizarin Red staining and increased mRNA expression of Collagen I as compared to control cultures. In conclusion, DM-22 is a new BP which inhibits h-OCs function and stimulate osteogenic differentiation of h-MSCs, without cytotoxicity. DM-22 is an ideal candidate for a novel family of osteoanabolic drugs

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

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

Hypoxia Preconditioning of Human MSCs: a Direct Evidence of HIF-1α and Collagen Type XV Correlation

Background/Aims: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. Methods: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo – Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. Results: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. Conclusion: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied