COMPARACIÓN DE LA DIFERENCIACIÓN OSTEOGÉNICA DE CÉLULAS MADRE DE PULPA DENTAL EN ANDAMIOS IMPRESOS EN 3D Y BIOIMPRESOS CON UNA BIOTINTA DE ALGINATO, GELATINA, NANOHIDROXIAPATITA Y L-PRF.
Bone loss in the oral cavity a}ects individuals' quality of life and can result from various systemic and local conditions. Although synthetic biomaterials and demineralized bone matrices are used in clinical treatments to replace lost tissue, their e}iciency, cost, and osteogenic potential lim...
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| Tipo de recurso: | tesis doctoral |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/413609 |
| Acceso en línea: | http://hdl.handle.net/10261/413609 |
| Access Level: | acceso abierto |
| Palabra clave: | Células madre de pulpa dental Fibrina rica en plaquetas y leucocitos alginato nanohidroxiapatita bioimpresión biotinta regeneración ósea http://metadata.un.org/sdg/3 Ensure healthy lives and promote well-being for all at all ages |
| Sumario: | Bone loss in the oral cavity a}ects individuals' quality of life and can result from various systemic and local conditions. Although synthetic biomaterials and demineralized bone matrices are used in clinical treatments to replace lost tissue, their e}iciency, cost, and osteogenic potential limitations have driven the development of tissue engineering as a promising alternative. This discipline employs three-dimensional biocompatible sca}olds combined with cells and growth factors to promote the formation of functional tissues. Various techniques have been explored to design an ideal sca}old to address tissue regeneration in the oral cavity. 3D printing and bioprinting have emerged as innovative technologies that allow the creation of bioinks for fabricating printed constructs using hydroxyapatite, chitosan, gelatin, and alginate, providing an environment conducive to cell adhesion and growth. These cells can be either injected or integrated into the material during printing. Among the growth factors studied to enhance cell growth and proliferation, leukocyte- and platelet-rich fibrin (LPRF) stands out. Similarly, human dental pulp stem cells (DPSCs) have shown great promise due to their di}erentiation potential, particularly towards the osteoblastic lineage for bone tissue formation. However, there is a lack of studies that integrate a biocompatible matrix, growth factors, and dental-derived stem cells with osteogenic properties for applications in oral surgery. Thus, the main objective of this study was to compare the in vitro osteogenic di}erentiation of DPSCs on sca}olds fabricated using 3D printing and bioprinting, employing alginate, gelatin, nanohydroxyapatite, and LPRF. The project was developed in three stages. In the first stage, various bioinks were synthesized and characterized through rheological analysis, printability assessments, and cell viability tests. 3D printing and bioprinting techniques were performed using the CellInk Inkredible printer, integrating cells with the material. The second stage involved the physicochemical and biological characterization of the printed sca}olds, utilizing scanning electron microscopy (SEM) to analyze morphology, Fourier transform infrared spectroscopy (FT-IR) to identify functional groups, biodegradability tests, and biocompatibility evaluations. Finally, in the third stage, osteogenic di}erentiation was assessed through gene expression analysis via RT-qPCR, growth factor release using LUMINEX, and cell proliferation using Ki-67 immunofluorescence. The results showed that bioinks composed of alginate-gelatin-nanohydroxyapatite-L-PRF exhibited pseudoplastic behavior with an 80% recovery rate, making them suitable for printing. The storage modulus predominated over the loss modulus, and, in combination with calcium chloride (CaCl₂), they formed a robust material conducive to cell growth. FT-IR analyses identified key functional groups (-PO₄, -COO, -OH), confirming cross-linking with CaCl₂. SEM revealed a 90% geometric fidelity. The sca}olds exhibited controlled degradation, retaining over 50% of their structure at 30 days, and showed 130% swelling within 24 hours, favoring cell di}erentiation at 14 days. Cell viability assays using MTS confirmed the absence of cytotoxicity, and LIVE/DEAD assays demonstrated high viability (>80%) in both 3D-printed and bioprinted sca}olds. Additionally, growth factors (PDGF-BB, FGF-B, EGF, VEGF-C, and BMP-2) were released up to 21 days, and their biological activity was confirmed via immunohistochemistry using Ki-67,evidencing their role in cell proliferation. Gene expression analysis by RT-PCR indicated higher osteogenic gene expression in bioprinted sca}olds compared to 3D-printed sca}olds and undi}erentiated pulp cells, including RUNX2, OSX, OPG, OPN, VEGF-β, TGF-β, BMP-2, COL1A1, and ALP, suggesting superior osteogenic capacity in bioprinted sca}olds. In conclusion, this study developed alginate-gelatin-nanohydroxyapatite-LPRF sca}olds using 3D printing and bioprinting, demonstrating their biocompatibility, controlled degradation, and ability to promote DPSC osteogenesis. The bioprinted sca}olds stood out for their higher cell viability, highlighting the key role of L-PRF in bone regeneration, positioning them as a promising solution for clinical applications in regenerative dentistry. |
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