Thermo-responsive hyaluronan-based hydrogels and FE002 human primary chondroprogenitor cell sources have both been previously proposed as modern therapeutic options for the management of osteoarthritis (OA). For the translational development of a potential orthopedic combination product based on both technologies, respective technical aspects required further optimization phases (e.g., hydrogel synthesis upscaling and sterilization, FE002 cytotherapeutic material stabilization). The first aim of the present study was to perform multi-step in vitro characterization of several combination product formulas throughout the established and the optimized manufacturing workflows, with a strong focus set on critical functional parameters. The second aim of the present study was to assess the applicability and the efficacy of the considered combination product prototypes in a rodent model of knee OA. Specific characterization results (i.e., spectral analysis, rheology, tribology, injectability, degradation assays, in vitro biocompatibility) of hyaluronan-based hydrogels modified with sulfo-dibenzocyclooctyne-PEG4-amine linkers and poly(N-isopropylacrylamide) (HA-L-PNIPAM) containing lyophilized FE002 human chondroprogenitors confirmed the suitability of the considered combination product components. Specifically, significantly enhanced resistance toward oxidative and enzymatic degradation was shown in vitro for the studied injectable combination product prototypes. Furthermore, extensive multi-parametric (i.e., tomography, histology, scoring) in vivo investigation of the effects of FE002 cell-laden HA-L-PNIPAM hydrogels in a rodent model revealed no general or local iatrogenic adverse effects, whereas it did reveal some beneficial trends against the development of knee OA. Overall, the present study addressed key aspects of the preclinical development process for novel biologically-based orthopedic combination products and shall serve as a robust methodological basis for further translational investigation and clinical work.

Hyaluronic acid (HA) constitutes a versatile chemical framework for the development of osteoarthritis pain treatment by means of injection in the joints, so-called viscosupplementation. Without appropriate physico-chemical tuning, such preparations are inherently hindered by prompt in vivo degradation, mediated by hyaluronidases and oxidative stress. To prolong hydrogel residence time and confer optimized product functionality, novel thermoresponsive nanoforming HA derivatives were proposed and characterized. Combined use of sulfo-dibenzocyclooctyne-PEG4-amine linkers and poly(N-isopropylacrylamide) in green chemistry process enabled the synthesis of HA-based polymers, with in situ obtention of appropriate viscoelastic properties. Spontaneous and reversible thermoformation of nanoparticles above 30 °C was experimentally confirmed. Lead formulations were compared to a commercially available HA-based product and shown significantly better in vitro resistance to enzymatic and oxidative degradation, required half the injection force with optimal viscoelastic hydrogel properties in equine synovial fluids. Results highlighted the vast potential of appropriately engineered HA-based systems as next-generation long-acting viscosupplementation products for osteoarthritic patients.

Cultured progenitor cells and derivatives have been used in various homologous applications of cutaneous and musculoskeletal regenerative medicine. Active pharmaceutical ingredients (API) in the form of progenitor cell derivatives such as lysates and lyophilizates were shown to retain function in controlled cellular models of wound repair. On the other hand, hyaluronan-based hydrogels are widely used as functional vehicles in therapeutic products for tendon tissue disorders. The aim of this study was the experimental characterization of formulations containing progenitor tenocyte-derived APIs and hyaluronan, for the assessment of ingredient compatibility and stability in view of eventual therapeutic applications in tendinopathies. Lyophilized APIs were determined to contain relatively low quantities of proteins and growth factors, while being physicochemically stable and possessing significant intrinsic antioxidant properties. Physical and rheological quantifications of the combination formulas were performed after hydrogen peroxide challenge, outlining significantly improved evolutive viscoelasticity values in accelerated degradation settings. Thus, potent effects of physicochemical protection or stability enhancement of hyaluronan by the incorporated APIs were observed. Finally, combination formulas were found to be easily injectable into ex vivo tendon tissues, confirming their compatibility with further translational clinical approaches. Overall, this study provides the technical bases for the development of progenitor tenocyte derivative-based injectable therapeutic products or devices, to potentially be applied in tendinous tissue disorders.

Mesenchymal stem cell (MSC) therapy shows potential in regenerative medicine, particularly in treating osteoarthritis (OA). MSCs injected into the joint can secrete growth factors and extracellular matrix molecules, contributing to paracrine communication and cartilage regeneration. However, in the non-vascularized joint environment, MSCs lacking nutrient supply, starve and die too quickly to efficiently deliver enough of these factors. We have recently synthesized a new hydrogel containing hyaluronic acid and glucose (HA-GLC). This hydrogel allows MSCs to survive and proliferate in an environment with otherwise low glucose levels. Furthermore, it releases glucose through enzymatic cleavage by ß-glucosidase, an enzyme which we have shown to be available and active in human bone marrow mesenchymal stem cells (BM-MSCs). In this study, we did incorporate MSCs to this HA-GLC hydrogel. Proteomic analysis of the MSC secretome revealed that glucose deprivation modified the profile of secreted factors, inducing changes in several key pathways, including extra-cellular matrix production. We then tested the effect of glucose deprivation in MSC secretome on human chondrocyte (hCH) proliferation and IL-6 secretion. Our results showed an increase in hCH proliferation and a significant decrease in IL-6 expression, when cells were exposed to the secretome of MSCs cultured in glucose-provided media rather than glucose-deprived conditions. These findings highlighted the ability of this new technology (HA-GLC hydrogel) to modulate the MSC secretome function, potentially enhancing cartilage regeneration in OA.