3D bioprinting of gellan gum-based hydrogels tethered with laminin-derived peptides for improved cellular behavior

The treatment of skeletal muscle defects is still a topic of noteworthy concern since surgical intervention is not capable of recovering muscle function. Herein, we propose myoblasts laden in laminin-inspired biofunctionalized gellan gum hydrogels as promising tissue-engineered skeletal muscle surrogates. Gellan gum-based hydrogels were developed by combining native gellan gum (GG) and GG tethered with laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T) or RKRLQVQLSIRTC (Q)), using different polymer content (0.75%â 1.875%). Hydrogels were characterized in terms of compressive modulus, molecules trafficking, and C2C12 adhesion. Hydrogels with higher polymeric content (1.125%â 1.875%) showed higher stiffness whereas hydrogels with lower polymer content (0.75%â 1.125%) showed higher fluorescein isothiocyanate-dextran molecules diffusion. Cell spreading was achieved regardless of the laminin-derived peptide but preferred in hydrogels with higher polymer content (1.125%â 1.875%). Taken together, hydrogels with 1.125% of polymer content were selected for printability analysis. GG-based inks showed a non-newtonian, shear-thinning, and thixotropic behavior suitable for printing. Accordingly, all inks were printable, but inks tethered with T and Q peptides presented some signs of clogging. Cell viability was affected after printing but increased after 7â days of culture. After 7â days, cells were spreading but not showing significant signs of cellâ cell communications. Therefore, cell density was increased, thus, myocytes loaded in V-tethered GG-based inks showed higher cellâ cell communication, spreading morphology, and alignment 7, 14â days post-printing. Overall, myoblasts laden in laminin-inspired biofunctionalized GG-based hydrogels are a promising skeletal muscle surrogate with the potential to be used as in vitro model or explored for further in vivo applications.CEEC Individual, Grant/Award Number: 2020.01541.CEECIND/CP1600/CT0024; Fundacao para a Ciencia e a Tecnologia, Grant/Award Number: PD/BD/128090/201

Estratégias de engenharia de tecidos para o tratamento de lesões do músculo esquelético

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisAté à data, não existem tratamentos eficazes para lesões musculares. Atualmente, os tratamentos existents para lesões musculares são limitados a cuidados paliativos ou cirurgias. Portanto, há uma necessidade premente de encontrar um tratamento eficaz para as lesões musculares esqueléticas. O primeiro objectivo desta tese foi investigar o potencial de células estaminais derivadas do tecido adiposo humano (hASCs) para a regeneração do músculo. Deste modo, lesões musculares em murganhos foram tratadas com hidrogéis esponjosos (SLH) à base de goma gelana (GG) e biofuncionalizados com RGD contendo hASCs. Esta estratégia demonstrarou ser uma abordagem confiável, dado que restabeleceu a rede vascular e os filamentos neurais, assim como promoveu a miogénese. O segundo objectivo baseou se na investigação do potencial de análogos do músculo esquelético para terapias de substituição ou para modelo de músculo esquelético para estudar a fisiologia do músculo esquelético ou para triagem de medicamentos. Portanto, hidrogéis à base de goma gelana foram biofuncionalizados com oligopeptídeos inspirados em laminina (isto é, CIKVAVS (V), KNRLTIELEVRTC (T), RKRLQVQLSIRTC (Q) e cRGD) e preparados com diferentes conteúdos polimérico. Miócitos foram adicionados no topo ou dentro dos hidrogéis e o seu comportamento ao longo do tempo. A adesão celular foi favorecida em hidrogéis com rigidez semelhante à do tecido muscular nativo (ou seja, 5-20 kPa). Além disso, os miócitos foram capazes de aderir, crescer, diferenciar e alinhar sobre hidrogéis à base de Q micropadronizados. No entanto, apesar de os miócitos aderirem, proliferarem e alinharem após bio impressão dentro das diferentes formulações de hidrogel, a diferenciação só foi alcançada quando impressos em hidrogéis baseados em cRGD. De modo a preparar análogos de músculo inervados, células neuronais foram bio-impressas lado a lado com células do músculo esquelético. Estes análogos demonstraram superior funcionalidade, como foi detectado pela descarga de cálcio e contratilidade. Por fim, o potencial dos miócitos encapsulados em hidrogéis à base de Q foi explorado para tratamento lesões musculares localizadas, por meio de injeção direta in-situ. Após a injeção, este tratamento promoveu uma miogénese, restabelecendo uma rede vascular profunda e filamentos neurais.Ongoing treatment procedures for skeletal muscle injuries are limited to palliative care therapy or surgery in severe cases. The main drawback of these approaches is the absence of functional recovery portrayed with a prolonged recovery scheme that may last months. Hence, there is an urgent need to find an effective treatment for skeletal muscle injuries. In this thesis, we aimed to explore the potential of human adipose-derived stem cells (hASCs) for the regeneration of volumetric muscle loss (VML). To this aim, we used gellan gum (GG)-based spongy-like hydrogels (SLH) biofunctionalized with RGD. hASCs-laden spongy-like hydrogels demonstrated a reliable approach by re-establishing a vascular network, neural filaments and promoting myogenesis. Moreover, we explored the potential of the skeletal muscle-analogue model for drug screening applications, pathophysiological studies, or to be used for in-vivo replacement therapies. Hence, we developed gellan gum-based hydrogels biofunctionalized with laminin-inspired oligopeptides (i.e. CIKVAVS (V), KNRLTIELEVRTC (T), RKRLQVQLSIRTC (Q), and cRGD) with different polymeric contents. Thus, we analyzed the behavior of C2C12 within and on top of these hydrogels. Cell adhesion was favored in hydrogels with compressive modulus close to the native muscular tissue stiffness (i.e. 5-20 kPa). Moreover, C2C12 cells were able to differentiate and align when seeded on top of Q-based micropatterned hydrogels. When encapsulated within the different hydrogel formulations, C2C12 cells were able to adhere, grow and align post-printing. Nevertheless, differentiation was only achieved when cells were bioprinted within cRGD-based hydrogels. Furthermore, innervated constructs were prepared by printing neuronal cells side-by-side to skeletal muscle cells, showing enhanced functionality as detected by calcium discharge and contractility. In the final work, we explored the potential of C2C12 laden in Q-biofunctionalized GG-based hydrogels to treat localized muscle injuries through direct in-situ injection. Post injection, C2C12-laden hydrogels promoted robust myogenesis, re-established a profound vascular network and neural filaments.Fundação para a Ciência e a Tecnologia (FCT

Injectable laminin-biofunctionalized gellan gum hydrogels loaded with myoblasts for skeletal muscle regeneration

Moderate muscular injuries that exceed muscular tissue's auto-healing capacity are still a topic of noteworthy concern. Tissue engineering appeared as a promising therapeutic strategy capable of overcoming this unmet clinical need. To attain such goal, herein we propose an in situ-crosslinking gellan gum (GG)-based hydrogel tethered with a skeletal muscle-inspired laminin-derived peptide RKRLQVQLSIRTC(Q) and encapsulated with skeletal muscle cells (SMCs). Pre-hydrogel solutions presented decreasing shear viscosity with increasing shear rate and shear stress, and required low forces for extrusion, validating their injectability. The GGDVS hydrogel was functionalized with Q-peptide with 30% of efficiency. C2C12 were able to adhere to the developed hydrogel, remained living and spreading 7 days post-encapsulation. Q-peptide release studies indicated that 25% of the unbound peptide can be released from the hydrogels up to 7 days, dependent on the hydrogel formulation. Treatment of a chemically-induced muscular lesion in mice with an injection of C2C12-laden hydrogels improved myogenesis, primarily promoted by the C2C12. In accordance, a high density of myoblasts (α-SA+ and MYH7+) were localized in tissues treated with the C2C12 (alone or encapsulated in the hydrogel). α-SA protein levels were significantly increased 8 weeks post-treatment with C2C12-laden hydrogels and MHC protein levels were increased in all experimental groups 4 weeks post-treatment, in relation to the SHAM. Neovascularization and neoinnervation was also detected in the defects. Altogether, this study indicates that C2C12-laden hydrogels hold great potential for skeletal muscle regeneration. Statement of significance We developed an injectable gellan gum-based hydrogel for delivering C2C12 into localized myopathic model. The gellan gum was biofunctinalized with laminin-derived peptide to mimic the native muscular ECM. In addition, hydrogel was physically tuned to mimic the mechanical properties of native tissue. To the best of our knowledge, this formula was used for the first time under the context of skeletal muscle tissue regeneration. The injectability of the developed hydrogel provided non-invasive administration method, combined with a reliable microenvironment that can host C2C12 with nominal inflammation, indicated by the survival and adhesion of encapsulated cells post-injection. The treatment of skeletal muscle defect with the cell-laden hydrogel approach significantly enhanced the regeneration of localized muscular trauma.The authors acknowledge the financial support from the project JUSThera (NORTE-01-0145-FEDER-000055), and from Portuguese Foundation for Science and Technology (FCT) for the PhD grant FCT PD/BD/128090/2016 under TERM program PD/59/2013 (OA) and when eligible, by COMPETE 2020 FEDER funds, under the Scientific Employment Stimulus - Individual Call (CEEC Individual) - 2020.01541.CEECIND/ CP1600/CT0024 (LdS). The authors would also like to acknowledge Dr. Reem Sweid for image segmentation using Matlab, Teresa Oliveira for her support in histology