Introduction. In tissue engineering, the establishment of sufficient vascularization is essential for tissue viability and functionality. Inadequate vascularization disrupts nutrients and oxygen supply. Nonetheless, regenerating intricate vascular networks represents a significant challenge. Consequently, research efforts devoted to preserving and regenerating functional vascular networks in engineered tissues are of paramount importance. The present work aims to validate a decellularisation process with preservation of the vascular network and extracellular matrix (ECM) components in fasciocutaneous flaps. Method. Five vascularized fasciocutaneous flaps from cadaveric donors were carefully harvested from the anterolateral thigh (ALT), preserving the main perforator of the fascia lata. The entire ALT flap underwent decellularization by perfusion using a clinically validated chemical protocol. Fluoroscopy and computed tomography (CT) were used to analyze the persistence of the vascular network within the flap, pre- and post-decellularization. Histological analysis, including hematoxylin and eosin staining, and quantitative DNA assessment evaluated decellularization efficacy. Further qualitative (immunohistochemistry, IHC) and quantitative analyses were conducted to assess the preservation of ECM components, such as collagen, glycosaminoglycans, and elastin. Result. On average, the ALT flap maintains 82% of the perfusion area (p = 0.094) post-treatment. Histological analysis confirmed decellularization efficacy and revealed structural rearrangement. Paired analysis revealed a significant decrease in DNA levels (<14.8 ng/mg of dry weight, p****< 0.0001) and well-maintained ECM. IHC indicated the persistence of elastine, collagen IV and laminin. Quantitative analysis confirmed elastin (p = 0.44) and collagen persistence (+74%, p*** = 0.001, albeit with a decrease in matrix glycosaminoglycans (-41%, p*** = 0.01). Conclusion. Decellularization effectively removed cells, while preserving the ECM overall and maintaining some vascular network integrity. Yet, further study is needed to validate these findings, involving microCT examination of the vascular network and its ability to support cell colonization and viability

Introduction. Supraspinatus tears comprise most rotator cuff injuries, the leading cause of shoulder pain and an increasing problem with ageing populations. Surgical repair of considerable or persistent damages is customary, although not invariably successful. Tissue engineering presents a promising alternative to generate functional tissue constructs with improved healing capacities. This study explores tendon tissue constructs’ culture in a platform providing physiological mechanical stimulation and reports on the effect of different loading regimes on the viability of human tendon cells. Method. Porcine decellularized tendon scaffolds were fixed into flexible, self-contained bioreactor chambers, seeded with human tenocytes, allocated in triplicates to either static control, low (15±0.8Newtons [N]), medium (26±0.5N), or high (49±2.1N)-force-regime groups, connected to a perfusion system and cultured under standard conditions. A humanoid robotic arm provided 30-minute adduction/abduction stimulation to chambers daily over a week. A metabolic activity assay served to assess cell viability at four time points. Statistical significance = p<0.05. Result. One day after beginning mechanical stimulation, chambers in the medium and high-force regimes displayed a rise in metabolic activity by 3% and 5%, respectively. By the last experimental day, all mechanical stimulation regimes had induced an augment in cell viability (15%, 57% and 39% with low, medium, and high loads, respectively) matched against the static controls. Compared to all other conditions, the medium-force regime prompted an increased relative change in metabolic activity for every time point set against day one (p<0.05). Conclusion. Human tenocytes’ viability reflected by metabolic activity in a physiologically relevant bioreactor system is enhanced by loading forces around 25N when mechanically stimulating using adduction/abduction motions. Knowing the most favourable load regime to stimulate tenocyte growth has informed the ongoing exploration of the distinctive effect of different motions on tendon regeneration towards engineering tissue grafts. This work was supported by the Engineering and Physical Sciences Research Council EP/S003509/1

Introduction. The objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones by Medication-Related Osteonecrosis of the Jaw using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR (Elastin-Like Recombinamers) based hydrogels. Method. Chemical crosslinking facilitated the creation of multi-bioactive scaffolds using ELRs with reactive groups. Cell-loaded multi-bioactive scaffolds, prepared and incubated, underwent evaluation for adhesion, proliferation, angiogenic, and neurogenic potential. In vitro assessments utilized immunofluorescence staining and ELISA assays, while live-recorded monitoring and live-dead analysis ensured cytocompatibility. In rat and rabbit models, preformed scaffolds were subcutaneously implanted, and the regenerative process was evaluated over time. Rabbit models with MRONJ underwent traditional or percutaneous implantation, with histological evaluation following established bone histological techniques. Result. A 3D scaffold using ELR that combines various peptides with different degradation rates to guide both angiogenesis and neurogenesis has been developed. Notably, scaffolds with different degradation rates promoted distinct patterns of vascularization and innervation, facilitating integration with host tissue. This work demonstrates the potential for tailored tissue engineering, where the scaffold's bioactivities and degradation rates can control angiogenesis and neurogenesis. In an animal model of medication-related osteonecrosis of the jaw (MRONJ), the scaffold showed promising results in promoting bone regeneration in a necrotic environment, as confirmed by histological and imaging analyses. This study opens avenues for novel tissue-engineering strategies where precise control over vascularization and nerve growth is crucial. Conclusion. A groundbreaking dual approach, simultaneously targeting angiogenesis and innervation, addresses the necrotic bone in MRONJ syndrome. Vascularization and nerve formation play pivotal roles in driving reparative elements for bone regeneration. The scaffold achieves effective time/space control over necrotic bone regeneration. The authors are grateful for funding from the Spanish Government (PID2020-118669RA-I00)

Introduction. The healing of rotator cuff injuries poses significant challenges, primarily due to the complexity of recreating the native tendon-to-bone interface, characterized by highly organized structural and compositional gradients. Addressing this, our innovative approach leverages bioprinted living tissue constructs, incorporating layer-specific growth factors (GFs) to facilitate enthesis regeneration. This method aims to guide in situ zonal differentiation of stem cells, closely mirroring the natural enthesis tissue architecture. Method. Our strategy involves the utilization of advanced bioprinting technology to fabricate living tissue constructs. These constructs are meticulously designed with embedded microsphere-based delivery carriers, ensuring the sustained release of tenogenic, chondrogenic, and osteogenic growth factors. This layer-specific release mechanism is tailored to promote the precise differentiation of stem cells across different regions of the construct, aligning with the gradient nature of enthesis tissues. Result. In vitro studies demonstrated that our layer-specific tissue constructs significantly outperformed basal constructs without GFs, achieving region-specific differentiation of stem cells. More critically, in a rabbit model of rotator cuff tear, these bioprinted living tissue constructs expedited enthesis regeneration. Key outcomes included improved biomechanical properties, enhanced collagen deposition and alignment, and the formation of a gradient fibrocartilage interface with aligned collagen fibrils. After 12 weeks, the constructs achieved an ultimate load failure of 154.3 ± 9.5 N resembling that of native enthesis tissues, marking a notable achievement in tissue engineering. Conclusion. Our exploration introduces a viable and innovative strategy for engineering living tissue constructs that exhibit region-specific differentiation capabilities. This approach holds significant promise for the functional repair of gradient enthesis tissues, potentially revolutionizing the treatment of rotator cuff injuries by closely replicating the natural tendon-to-bone interface, thus offering a promising avenue for future clinical applications

Introduction. Ink engineering can advance 3D-printability for better therapeutics, with optimized proprieties. Herein, we describe a methodology for yielding 3D-printable nanocomposite inks (NC) using low-viscous matrices, via the interaction between the organic and inorganic phases by chemical coupling. Method. Natural photocurable matrices were synthesized: a protein – bovine serum albumin methacrylate (BSAMA), and a polysaccharide – hyaluronic acid methacrylate (HAMA). Bioglass nanoparticles (BGNP) were synthesized and functionalized via aminosilane chemistry. The functionalization of BSAMA, HAMA, and BGNP were quantified via NMR. To arise extrudable inks, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) chemistry was used to link innate carboxylic groups of BSAMA/HAMA and amine-functionalized BGNP. Different crosslinker and BGNP amounts were tested. Visible light photopolymerization is performed, using lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The NC's rheological, mechanical, and biological behavior was evaluated before 3D extrusion printability. Result. All composite formulations effectively immobilized and homogeneously dispersed the BGNP, turning low-viscous materials (< 1 Pa) into shear-thinning formulations with tunable increased elastic/viscous moduli (50-500 Pa). More pronounced increments were found with increasing EDC/NHS and BGNP concentrations. The resulting inks produce robust and stable scaffolds successfully retrieved after post-print photocrosslinking (1-5 kPa). Bioactivity in simulated body fluid and in vitro assays using adipose-derive stem cells revealed a similar calcium/phosphate ratio to that of hydroxyapatite, and increased viability and metabolic activity. BSAMA and HAMA demonstrated distinct natures not only in printability but also in overall cellular performance and mechanical properties, making these ideal for interfacial tissue engineering. Conclusion. This strategy demonstrated being effective and reproducible to advance nanocomposites for 3D printing using different types of biomaterials. Further, we envision using both inks to produce hierarchical constructs via extrusion printing, better mimicking bone-to-cartilage interfaces. Acknowledgements. FCT grants (DOI:10.54499/2022.04605.CEECIND/CP1720/CT0021), (BI/UI89/10303/2022), (PRT/BD/154735/2023); EU's Horizon 2020 research and innovation programs InterLynk (Nº953169) and SUPRALIFE (Nº101079482) projects; CICECO-Aveiro Institute of Materials projects (DOI:10.54499/UIDB/50011/2020), (DOI:10.54499/UIDP/50011/2020), and (DOI:10.54499/LA/P/0006/2020), financed by FCT/MCTES(PIDDAC)

Aims

To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration.

The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4). We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes. We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells. We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to hypertrophic cells at full transcriptomic breadth. Using this atlas as the ground truth for evaluating _in vitro_ cells, we found substantial variability in cell states produced by each protocol, with many showing little similarity to _in vivo_ cells, and all exhibiting off-target differentiation. Trajectory alignment between _in vivo_ and _in vitro_ single-cell data revealed key differences in gene expression dynamics between _in vitro_ and _in vivo cells,_ with several osteoblastic transcription factors erroneously unregulated _in vitro,_ including _FOXO1._. Using this information, we inhibited _FOXO1_ in culture to successfully increase chondrocyte yield _in vitro._. This study presents a new framework for evaluating tissue engineering protocols, using single-cell data to drive improvement and bring the prospect of true engineered cartilage closer to reality

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration

Our musculoskeletal system has a limited capacity for repair. This has led to increased interest in the development of tissue engineering and biofabrication strategies for the regeneration of musculoskeletal tissues such as bone, ligament, tendon, meniscus and articular cartilage. This talk will demonstrate how different musculoskeletal tissues, specifically cartilage, bone and osteochondral defects, can be repaired using emerging biofabrication and 3D bioprinting strategies. This will include examples from our lab where cells and/or growth factors are bioprinted into constructs that can be implanted directly into the body, to approaches where biomimetic tissues are first engineered in vitro before in vivo implantation. The efficacy of these different biofabrication strategies in different preclinical studies will be reviewed, and lessons from the relative successes and failures of these approaches to tissue regeneration will be discussed

Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario.

Cells typically respond to a variety of geometrical cues in their environment, ranging from nanoscale surface topography to mesoscale surface curvature. The ability to control cellular organisation and fate by engineering the shape of the extracellular milieu offers exciting opportunities within tissue engineering. Despite great progress, however, many questions regarding geometry-driven tissue growth remain unanswered. Here, we combine mathematical surface design, high-resolution microfabrication, in vitro cell culture, and image-based characterization to study spatiotemporal cell patterning and bone tissue formation in geometrically complex environments. Using concepts from differential geometry, we rationally designed a library of complex mesostructured substrates (10. 1. -10. 3. µm). These substrates were accurately fabricated using a combination of two-photon polymerisation and replica moulding, followed by surface functionalisation. Subsequently, different cell types (preosteoblasts, fibroblasts, mesenchymal stromal cells) were cultured on the substrates for varying times and under varying osteogenic conditions. Using imaging-based methods, such as fluorescent confocal microscopy and second harmonic generation imaging, as well as quantitative image processing, we were able to study early-stage spatiotemporal cell patterning and late-stage extracellular matrix organisation. Our results demonstrate clear geometry-dependent cell patterning, with cells generally avoiding convex regions in favour of concave domains. Moreover, the formation of multicellular bridges and collective curvature-dependent cell orientation could be observed. At longer time points, we found clear and robust geometry-driven orientation of the collagenous extracellular matrix, which became apparent with second harmonic generation imaging after ∼2 weeks of culture. Our results highlight a key role for geometry as a cue to guide spatiotemporal cell and tissue organisation, which is relevant for scaffold design in tissue engineering applications. Our ongoing work aims at understanding the underlying principles of geometry-driven tissue growth, with a focus on the interactions between substrate geometry and mechanical forces

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure

Biofabrication is a popular technique to produce personalized constructs for tissue engineering. In this study we combined laponite (Lap), gellan gum (GG) with platelet-rich plasma (PRP) aiming to enhance the endothelial regeneration through the synergistic effects of their individual properties. Laponite has the ability to form porous three-dimensional networks mimicking the extracellular matrix structure, and PRP delivery of growth factors stimulates the endothelial cell proliferation and migration, offering a composite bioink for cell growth and support. The sustained release of these growth factors from the GG-laponite-PRP composite material over time provides a continuous source of stimulation for the cells, leading to more effective tissue engineering strategies for endothelial tissue regeneration. Four blend compositions comprising 1% w/v GG and 0.5 or 1% w/v Lap and 25% v/v PRP were combined with Wharton jelly mesenchymal stem cells (WJ-MSCs) and bioprinted into vessel-like structures with an inner diameter of 3 mm and a wall thickness of 1 mm. Stress/strain analysis revealed the elastomeric properties of the hydrogels with Young modulus values of 10 MPa. Increasing the Lap concentration led to a non-significant decrease of swelling ratio from 93 to 91%. Live/dead assay revealed cell viability of at least 76%, with the 0.5%Lap-GG viability exceeding 99% on day 21. Gradual increase of glycosaminoglycans accumulation and collagen production indicate promotion of ECM formation. The expression and membranous localization of PECAM-1 from day 7 and the granular intracellular localization of vWF after 2 weeks demonstrate in vitro endothelial functionality. In vivo subcutaneous implantation indicated the absence of any adverse immunological reactions. The results reveal the expression of both vWF and PECAM-1 by WJ-MSCs entrapped in all four construct compositions with significantly higher expression of vWF in the presence of PRP

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration. Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors. Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering

A major obstacle in biofabrication is replicating the organization of the extracellular matrix and cellular patterns found in anisotropic tissues within bioengineered constructs. While magnetically-assisted 3D bioprinting techniques have the potential to create scaffolds that mimic natural biological structures, they currently lack the ability to accurately control the dispersion of magnetic substances within the bioinks without compromising the fidelity of the intended composite. To overcome this dichotomy, the concepts of magnetically- and matrix-assisted 3D bioprinting are combined here. This method preserves the resolution of printed structures by keeping low viscosity bioinks uncrosslinked during printing, which allows for the arrangement of magnetically-responsive microfibers without compromising the structural integrity of the design. Solidification is induced after the microfibers are arranged in the desired pattern. Furthermore, the precise design of these magnetic microfillers permits the utilization of low levels of inorganic materials and weak magnetic field strengths, which reduces the potential risks that may be associated with their use. The effectiveness of this approach is evaluated in the context of tendon tissue engineering, and the results demonstrate that combining the tendons like anisotropic fibrous microstructure with remote magneto-mechanical stimulation during in vitro maturation provides both biochemical and biophysical cues that effectively guide human adipose-derived stem cells towards a tenogenic phenotype In summary, the developed strategy allows the fabrication of anisotropic high-resolution magnetic composites with remote stimulation functionalities, opening new horizons for tissue engineering applications. Acknowledgments: ERC Grant CoG MagTendon nr 772817, BioChips PoC project nr 10106930, (PD/BD/129403/2017), (CEECIND/01375/2017), (2020.03410.CEECIND), (2022.05526.PTDC), (ED481B2019/025)

Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels. 2. to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis. Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies. Acknowledgements: The authors acknowledge the funding from the European Union's Horizon 2020 under grant No. 772817; from FCT/MCTES for scholarships PD/BD/143039/2018 & COVID/BD/153025/2022 (S.P.B.T.), and PD/BD/129403/2017 (S.M.B.), co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for contract 2020.03410.CEECIND (R.M.A.D.) and project 2022.05526.PTDC; and from Xunta de Galicia for grant ED481B2019/025 (A.P.)

Degenerative disc disease, associated to low back pain, afflicts more than 50% of humans, and represents a major healthcare problem, especially for the pathology initiation. Current treatments range from conservative strategies to more invasive surgical techniques, such as disc removal and vertebral fusion. In the Intervertebral Disease (IVD) the nucleus pulposus (NP) degeneration is a key factor for the pathology initiation. Several tissue engineering approaches aiming to restore the appropriate NP cell (NPCs) and matrix content, were attempted by using adult stromal cells either from bone marrow or adipose tissue, chondrocytes, notochordal cells and more recently also pluripotent stem cells. However, none was fully satisfactory since the NP acid and a-vascularized environment appeared averse to the implanted heterologous cells. Several studies demonstrated the efficacy of platelet derivatives such as platelet rich plasma (PRP) in promoting the regeneration of connective tissues. We investigated the efficacy of PRP on NPCs proliferation and differentiation with the goal to propose the direct stimulation of resident cells (stimulation of endogenous cells – less invasive surgical procedure) or the implantation of NPCs expanded in vitro in the presence of PRP as therapeutic agents in IVD degeneration. NPCs were isolated from small fragments of NP explants, cultivated in medium supplemented with PRP or FCS (standard condition control) and characterized by FACS analysis for the expression of the typical mesenchymal stem cells markers CD34, CD44, CD45, CD73, CD90 and CD105. NPCs cultured in PL showed a phenotypic profile like the cells cultured in FCS. However, compared to NPCs expanded in the presence of FCS, NPCs expanded in PRP showed a much better proliferation and differentiation capacity. NPCs differentiation was evaluated by the cell ability to produce an organized metachromatic cartilaginous matrix, confirmed by the positive immunohistochemical staining for chondrogenic markers

Skeletal muscle tissue engineering has made progress towards production of functional tissues in line with the development in materials science and fabrication techniques. In particular, combining the specificity of 3D printing with smart materials has introduced a new concept called the 4D printing. Inspired by the unique properties of smart/responsive materials, we designed a bioink made of gelatin, a polymer with well-known cell compatibility, to be 3D printed on a magnetically responsive substrate. Gelatin was made photocrosslinkable by the methacrylate reaction (GELMA), and its viscosity was finetuned by blending with alginate which was later removed by alginate lyase treatment, so that the printability of the bioink as well as the cell viability can be finetuned. C2C12 mouse myoblasts-laden bioink was then 3D printed on a magnetic substrate for 4D shape-shifting. The magnetic substrate was produced using silicon rubber (EcoFlex) and carbonyl iron powders. After 3D printing, the bioink was crosslinked on the substrate, and the substrate was rolled with the help of a permanent magnet. Unrolled (Open) samples were used as the control group. The stiffness of the bioink matrix was found to be in the range of 13–45 kPa, which is the appropriate value for the adhesion of C2C12 cells. In the cell viability analysis, it was observed that the cells survived and could proliferate within the 7-day duration of the experiment. As a result of the immunofluorescence test, compared to the Open Group, more cell nuclei were observed overlapping MyoD1 expression in the Rolled Group; this indicated that the cells in these samples had more cell-cell interactions and therefore tended to form more myotubes. Acknowledgements: This research was supported by the TÜBİTAK 2211-A and YÖK 100/2000 scholarship programs

Bottom-up tissue engineering (TE) strategies employing microscale living materials as building blocks provide a promising avenue for generating intricate 3D constructs resembling native tissues. These microtissue units exhibit high cell densities and a diverse extracellular matrix (ECM) composition, enhancing their biological relevance. By thoughtfully integrating different cell types, the establishment of vital cell-cell and cell-matrix interactions can be promoted, enabling the recreation of biomimetic micro-niches and the replication of complex morphogenetic processes. Notably, by co-assembling blood vessel-forming endothelial cells with supportive stromal cells, microtissues with stable capillary beds, referred to as vascular units (VUs), can be generated. Through a modular TE approach, these VUs can be further combined with other microtissues and biomaterials to construct large-scale vascularized tissues from the bottom up. Integration of VUs with technologies such as 3D bioprinting and microfluidics allows for the creation of structurally intricate and perfusable constructs. In this presentation, we will showcase examples of VUs and explore their applications in regenerative medicine and tissue modeling. Acknowledgements: This work was supported by project EndoSWITCH (PTDC/BTM-ORG/5154/2020) funded by FCT (Portuguese Foundation for Science and Technology)

The application of immune regenerative strategies to deal with unsolved pathologies, such as tendinopathies, is getting attention in the field of tissue engineering exploiting the innate immunomodulatory potential of stem cells [1]. In this context, Amniotic Epithelial Cells (AECs) represent an innovative immune regenerative strategy due to their teno-inductive and immunomodulatory properties [2], and because of their high paracrine activity, become a potential stem cell source for a cell-free treatment to overcome the limitations of traditional cell-based therapies. Nevertheless, these immunomodulatory mechanisms on AECs are still not fully known to date. In these studies, we explored standardized protocols [3] to better comprehend the different phenotypic behavior between epithelial AECs (eAECs) and mesenchymal AECs (mAECs), and to further produce an enhanced immunomodulatory AECs-derived secretome by exposing cells to different stimuli. Hence, in order to fulfill these aims, eAECs and mAECs at third passage were silenced for CIITA and Nrf2, respectively, to understand the role of these molecules in an inflammatory response. Furthermore, AECs at first passage were seeded under normal or GO-coated coverslips to study the effect of GO on AECs, and further exposed to LPS and/or IL17 priming to increase the anti-inflammatory paracrine activity. The obtained results demonstrated how CIITA and Nrf2 control the immune response of eAECs and mAECs, respectively, under standard or immune-activated conditions (LPS priming). Additionally, GO exposition led to a faster activation of the Epithelial-Mesenchymal transition (EMT) through the TGFβ/SMAD signaling pathway with a change in the anti-inflammatory properties. Finally, the combinatory inflammatory stimuli of LPS+IL17 enhanced the paracrine activity and immunomodulatory properties of AECs. Therefore, AECs-derived secretome has emerged as a potential treatment option for inflammatory disorders such as tendinopathies. Acknowledgement: This research is part of the P4FIT project ESR1, funded under the H2020-ITN-EJD-Marie-Skłodowska-Curie grant agreement 955685