Chronic and acute tendon injuries are frequent afflictions, for which treatment is often long and unsatisfactory. When facing extended injuries, matrices and scaffolds with sufficient biomechanical properties are required for surgical repair and could additionally serve as supports for cellular therapies to improve healing. In this study, protocols of either commonly used detergents only (SDS 1%, Triton 1%, TBP 1%, and Tween‐20 1%) or a combination of freeze/thaw (F/T) cycles with decellularization agents (NaCl 1M, ddH2O) were evaluated for the decellularization of horse equine superficial digital flexor tendon (SDFT) for hand flexor or extensor tendon reconstruction. Decellularization efficiency was assessed microscopically by histological staining (HE, DAPI) and DNA quantification. Macroscopical structure and biomechanical integrity of the tendon matrices were further assessed by gross observation, histological staining (SR), and mechanical testing (ultimate strain and stress, Young’s modulus, energy to failure) for select protocols. Decellularization with hypertonic NaCl 1M in association with F/T cycles produced the most robust tendon matrices, which were nontoxic after 10 days for subsequent recellularization with human fetal progenitor tendon cells (hFPTs). This standardized protocol uses a less aggressive decellularization agent than current practice, which allows subsequent reseeding with allogenic cells, therefore making them very suitable and bioengineered tendon matrices for human tendon reconstruction in the clinic.

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.

Cultured primary progenitor tenocytes in lyophilized form were previously shown to possess intrinsic antioxidant properties and hyaluronan-based hydrogel viscosity-modulating effects in vitro. The aim of this study was to prepare and functionally characterize several stabilized (lyophilized) cell-free progenitor tenocyte extracts for inclusion in cytotherapy-inspired complex injectable preparations. Fractionation and sterilization methods were included in specific biotechnological manufacturing workflows of such extracts. Comparative and functional-oriented characterizations of the various extracts were performed using several orthogonal descriptive, colorimetric, rheological, mechanical, and proteomic readouts. Specifically, an optimal sugar-based (saccharose/dextran) excipient formula was retained to produce sterilizable cytotherapeutic derivatives with appropriate functions. It was shown that extracts containing soluble cell-derived fractions possessed conserved and significant antioxidant properties (TEAC) compared to the freshly harvested cellular starting materials. Progenitor tenocyte extracts submitted to sub-micron filtration (0.22 µm) and 60Co gamma irradiation terminal sterilization (5–50 kGy) were shown to retain significant antioxidant properties and hyaluronan-based hydrogel viscosity modulating effects. Hydrogel combination products displayed important efficacy-related characteristics (friction modulation, tendon bioadhesivity) with significant (p < 0.05) protective effects of the cellular extracts in oxidative environments. Overall, the present study sets forth robust control methodologies (antioxidant assays, H2O2-challenged rheological setups) for stabilized cell-free progenitor tenocyte extracts. Importantly, it was shown that highly sensitive phases of cytotherapeutic derivative manufacturing process development (purification, terminal sterilization) allowed for the conservation of critical biological extract attributes.

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products.

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.