DTA

Dottorato

TRUCCO, DIEGO

etd-03202023-175520

Acellular and cell-laden biomaterials for the substitution and regeneration of osteoarticular tissues

ING-IND/34

Istituto di Biorobotica - PHD IN BIOROBOTICA

relatore Prof. RICOTTI, LEONARDO
Tutor Dott. VANNOZZI, LORENZO
Membro Dott.ssa LISIGNOLI, GINA

• acellular biomaterials
• cell-laden biomaterials
• articular cartilage
• tissue substitution
• tissue engineering
• cartilage diseases
• injectable hydrogels
• hydrogels
• hydrogel adhesion
• delivery devices

09/06/2023;

parziale

Articular cartilage (AC) is an anisotropic and viscoelastic connective tissue devoid of blood vessels, lymphatics, and nerves [1], that covers the diarthrodial joints and at the end of long bones, such as hips, elbows, shoulders and knee joints [2, 3]. Due to its position, it is subject to a harsh biomechanical environment. Nonetheless, it provides a low-friction surface for joints to move against each other, supports shock absorption, distributes loads, reduces stresses on the subchondral bone, and guarantees wear resistance [2, 4]. Structurally, AC comprises four zones (superficial, middle, deep, calcified) with particular features laid down by extracellular matrix (ECM) composition, density, and collagen fibers assembling[4]. The cells featuring AC are chondrocytes that vary through the tissue thickness in terms of disposition, number, activity, and phenotype [5-7].
In the orthopedic field, cartilage-related diseases represent a challenge, still to be solved [8]. The pathologies linked to AC are mainly caused by mechanical injuries (i.e., adventitious or sport-related traumas) or age-related degeneration (i.e., osteoarthritis)[9, 10]. In fact, middle-aged and elderly people are prone to pain and disability and progressive cartilage degeneration (chronic condition) due to a widespread state of inflammation [11]. As a consequence, the main pathological effects are the formation of defects that alter the tissue surfaces and functionalities within diarthrodial joints. The defects can be mainly classified as chondral (partial and full-thickness) and osteochondral [12]. Chondral defects only affect AC and do not extend to the underlying subchondral bone, while osteochondral lesions are joint damages that affect the cartilage and underlying bone [13, 14]. Different therapies can be applied to resolve the damage depending on the AC defect grade of severity [15]. A full-thickness cartilage injury can be surgically restored by different clinical strategies, such as microfracture, mosaicplasty, autologous chondrocyte or stem cell implantation alone or in combination with inducing factors or within a biomaterial [15]. However, a few upsides and many downsides are associated with each operation. Summarizing, the main drawbacks refer to the difficulty of achieving a complete restoration of the tissue in terms of mechanical and biological properties, the mismatch of implanted constructs due to the defect debridement, the need for surgical and invasive open surgery with correlated long-term post-operation recovery time, and extensive rehabilitation as well as the considerable cost of each part of the procedures.
For these reasons, the cartilage tissue engineering (CTE) field is rapidly evolving, with the aim to regenerate or replace damaged or diseased cartilaginous tissues. CTE involves the development of engineered tissue constructs through the combination of cells, biomaterials, and engineering approaches [16-18]. However, an acellular strategy can also be pursued to restore damages, thus avoiding cell-related issues (i.e., patient-specific cell availability, cell isolation cost), and may boost and potentially simplify a further clinical translation.
Nowadays, the investigation of hydrogels represents a key research line within the CTE field. Hydrogels are hydrophilic 3D networks made of natural (e.g., polysaccharides and proteins) and/or synthetic polymers crosslinked by chemical or physical methods to form a water-insoluble matrix [19]. They show interesting features for being placed in the joint in form of 3D structures or, otherwise, directly injected [20]. Indeed, injectable hydrogels have recently received a higher attention because they may facilitate the treatment of irregular AC defects filling the implantation site of the tissues [21, 22]. However, several specifications must be considered for the use of injectable hydrogels. First, the shear-tinning behavior that inevitably influences the shear stress acting on cells during the extrusion of cell-laden hydrogels and injection force. As reported in the EU ISO 7886-1:2018, the injection force during the extrusion from syringes should be less than 10 N. Another disadvantage in the use of hydrogel in CTE is related to the mechanical properties achieving by the hydrophilic network even after the crosslinking step. Indeed, recently the hydrogel’s reinforcements means to improve the hydrogel mechanical features by embedding nanomaterials [23] or fibers [24] and by deposing hydrogels on fibrous structures to fill them [25, 26].
In parallel to the material properties, the delivery system plays a relevant role to release injectable hydrogels directly on the site of interest, without harmful effects on cells [27, 28]. A few approaches and tools have been developed for a reliable and localized deposition of hydrogels and cells into AC lesions, mainly related to hand-held systems. Even though the enormous thrill for these strategies, they still need an open surgery procedure [29-31], and the development of arthroscopic delivery systems to enable mini-invasive procedures represents still a challenge [32]. After the extrusion of injectable hydrogel directly on the target site, an important but still relevant and under-investigated aspect is represented by the investigation of the hydrogel adhesion to the surrounding tissue. Indeed, a weak integration between the delivered material and the AC tissue may produce possible detachment over time, which means a low efficiency in the regenerative or substitution process of the tissue. For that reason, and due to sub-optimal transmissions of mechanical loads and other undesired phenomena, CTE implant may fail. To overcome this common challenge to both acellular and cellular strategies, the adhesion of the delivered hydrogels can be improved by delivering a priori adhesive primers on the targeted tissue or enhancing the hydrogel adhesiveness to reach a clinically acceptable adhesion force equal to 10 kPa [33].

After highlighting the AC’s properties and the conventional strategies limitations in treating cartilage diseases, the thesis work got ahead of the CTE purposes presenting two macro-strategies to replace damaged or lost tissues: tissue substitution and tissue regeneration. The tissue substitution approach refers to the replacement of damaged or lost tissue with a functional acellular replacement construct that mimics the original tissue structure and functions. On the other hand, tissue regeneration strategy promotes the body natural regenerative capacity by providing a suitable environment offered by biomaterials and growth factors for the proliferation and differentiation of exogenous and/or endogenous cells into damaged tissues also including external stimuli [34].
To address the first strategy, an acellular bilayered hydrogel was designed to mimic both mechanical and lubrification properties of AC. The bilayered structure was composed of layers that replicate the superficial and deep zones' compressive modulus of the AC, while the superficial layer hydrogel exhibited a coefficient of friction similar to AC thanks to the addition of graphene oxide nanoflakes. Then, a deep characterization was carried out on the same type of hydrogels to investigate a characterization protocol to pursue a material composition optimization. The analyses included advanced techniques, such as micro-computer tomography, analysis at optic profilometer, wear test and Raman mass spectroscopy. Finally, a new kind of hydrogel reinforcement was pursued to improve the hydrogel's mechanical properties, make them more suitable to withstand loads, while keeping acceptable the material injectability. Indeed, melt electrowritten structures have been embedded into natural-based hydrogel to be injected through stainless-steel needles with different diameters compatible with in vivo applications and with the aim to be further delivery in situ via minimally invasive procedures and tools (i.e., arthroscopic devices). As a result, the reinforced-hydrogel after injection reached improved mechanical properties than the bare one.
To address the second strategy, the thesis work reported the evaluation of three different cell-laden fully natural hydrogels that were crosslinked by using three different strategies (enzymatically, physically, and ionically). Firstly, a silk fibroin-gelatin-based hydrogel was handled to be deposed by an extrusion-based 3D bioprinting technique to drive bone marrow-derived mesenchymal stem cells towards a chondrogenic phenotype. In addition, an analytical model was employed to predict the deposited filament width during printing based on the hydrogel rheological properties (i.e., viscosity), the printing parameters (i.e., pressure, speed) and the printing set-up (i.e., needle lengths and inner diameters) to overcome the challenge of poor printing resolution with natural-based materials. Furthermore, the thesis work concerned a deep evaluation of biological properties showed by VitroGel® hydrogels at two different concentrations and with or without linking the arginyl-glycyl-aspartic acid (RGD)–motifs to boost the metabolic activity and the chondrogenic differentiation of embedded human adipose-derived stem cells. The best hydrogel formulation resulted from the first assessment was further investigated to be loaded with graphene oxide nanoflakes and barium titanate nanoparticles to explore how a nanocomposite hydrogel can support chondrogenesis in human adipose-derived stem cells coupled with dose-controlled ultrasound (US) stimulations. Here, the use of US as external stimuli was investigated performing a screening of the stimulation parameters. Finally, a methacrylation procedure was performed to obtain a light-responsive methacrylated gellan gum which tunable properties and structure similar to the glycosaminoglycans on the cartilage- An optimization process to find the best formulation to be photo-crosslinked with visible light was carried out by varying the photo-initiator concentration and the exposure time to the light. Then, biological analysis of the optimized formulations was performed to evaluate the capability to safely host human chondrocytes and the upregulation of typical markers of the cartilage (i.e., collagen type II).
Furthermore, this thesis work involved the validation of new delivery systems invented for the minimally invasive extrusion of injectable hydrogels. Indeed, a tri-axial system was implemented to deliver three different materials (core, shell, primer), simultaneously, whereas volumetric- and pneumatic-based extrusion systems were compared and evaluated to obtain highly precise material depositions.
Finally, the thesis reported for the first time the evaluation of the adhesion of injectable hydrogels to the cartilage tissue to provide a solution to such a challenging aspect, often under-investigated during material development. Here, the use of adhesive primers between the surrounding tissue and the above hydrogel and the embedding of nanomaterials were evaluated to enhance the adhesion and then, also the engineered tissue integration.
To sum up, the present thesis showcases a wide range of possible solutions based on both tissue substitution and regeneration strategies. The results underline the possibility to go more in deep with the substitution strategy to overcome some limitations that occur in the regeneration one, such as poor mechanical properties of natural-based materials and the long regulatory validation of cell-laden engineered materials to arrive in clinic. On the other hand, the combination of nanomaterials and optimized ultrasound stimulation parameters boosted the stem cell chondrogenic differentiation to obtain a proper cartilaginous extracellular matrix formation in natural-based injectable hydrogels. Thus, the validated new arthroscopic tools for delivering injectable hydrogels open the way to optimized material deposition directly on the target site previously treated with adhesive primers as a suitable approach for the repairment of osteoarticular cartilage tissues via minimally invasive procedures.

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