Estudio experimental sobre la utilización de células madre humanas en la terapia de los defectos periodontales: resultados preliminares / Experimental study on the use of human stem cell therapy in periodontal defects: Preliminary results

La destrucción del ligamento periodontal y de la cresta alveolar causada por la enfermedad periodontal, constituye la principal causa de pérdida dentaria en pacientes adultos. Resulta difícil que la terapia en la enfermedad periodontal avanzada lleve a una restitutio ad integrum de la estructura que se ha perdido por la imposibilidad de regenerar la correcta anatomía del sistema perdido. Este estudio tiene como objetivo evaluar el potencial y la estabilidad de la utilización de células madre mesenquimales (MSC) en la regeneración periodontal de la estructura original. Su objetivo es determinar un método de regeneración de la estructura periodontal original que se ha perdido después de la enfermedad periodontal, cerca de los elementos dentales vitales sin patologías correlacionadas. Este estudio constituye una experimentación de fase 1 aprobada por el Instituto Superior de Salud sobre un número limitado de pacientes, cuyo objetivo principal será la evaluación de la biocompatibilidad y toxicidad in vivo de las células madre mesenquimales diferenciadas en sentido osteoblástico, sostenidas por andamios de colágeno biomiméticos en pacientes afectados por periodontopatía severa. El protocolo describe el transplante de células madre mesenquimales derivadas de muestras de un aspirado percutáneo de médula ósea. Las células de la médula ósea son manipuladas ex vivo para el aislamiento de la población de células madre mesenquimales. Las células madre adultas son diferenciadas en las líneas celulares que forman el sistema periodontal, sucesivamente inducidas y cultivadas en andamios adecuados definidos en base a características propias con el fin de obtener una distribución uniforme de las líneas celulares, e injertadas en el sitio receptor estimulado adecuadamente hasta la degradación de los andamios, en un tiempo suficiente para garantizar la estabilidad de las diferentes líneas celulares y la recíproca interacción, con el fin de recrear la correcta anatomía estructural del periodonto perdido. Después de una fase de expansión celular, estas células se introducen en una estructura biomimética (andamios en colágeno), y se inducen a la diferenciación en sentido osteogénico. El producto final que consta de andamios y células mesenquimales diferenciadas en sentido osteoblástico, se instala por último en el defecto alveolar periodontal. La experimentación se basa en la posibilidad de utilizar células madre mesenquimales autólogas a partir de la médula ósea humana. La finalidad es la de estimular la regeneración de PDL cerca de la pared radicular y la formación de hueso nuevo sobre el PDL, beneficiándose de las técnicas de regeneración-reconstrucción ósea, de manera que se recree la correcta anatomía periodontal. Los resultados presentados en este trabajo conciernen el primer paciente tratado con esta metodología y afectarán los estudios siguientes para la prueba con una cobertura más amplia (AU)

Destruction of periodontal ligament and alveolar ridge rerorption caused by periodontal disease is the leading cause of tooth loss in adults. Therapy of advanced periodontal disease leads for a total recovery of the structure lost by the inability to regenerate the pristine correct anatomy system. The aim of this study is to assess the potential use of mesenchymal stem cells (MSCs) in the regeneration of original periodontal structure. This study is an experimental phase 1 approved by the Italian Institute of Health on a limited number of patients; main objective will be to assess the biocompatibility and toxicity in vivo of differentiated mesenchymal stem cells into osteoblast sense, supported by biomimetic collagen scaffolds in patients with severe periodontal disease. The protocol describes transplantation of mesenchymal stem cells derived from samples of percutaneous bone marrow aspirate. The bone marrow cells are manipulated ex vivo to isolate mesenchymal stem cell population. After a phase of cell expansion, these cells are induced to differentiate into periodontal system cell lines in a biomimetic structure (collagen scaffold). Induction and culturation in an appropriate scaffold defined on specific characteristics lead to obtain an uniform distribution of cell lines. After 28 days Scs are grafted into the defect site of the patient. Scaffold degradation time will ensure stability of the different cell lines and reciprocal interaction, in order to recreate the correct structural anatomy of periodontium lost. The purpose is to stimulate the regeneration of PDL near the root wall and new bone formation on the PDL, benefiting from the techniques of bone regeneration, reconstruction, as to recreate the pristine periodontal anatomy. The results presented in this paper concern the first patient treated with this method and will affect following studies for test for a wider coverage (AU)

GMP-grade preparation of biomimetic scaffolds with osteo-differentiated autologous mesenchymal stromal cells for the treatment of alveolar bone resorption in periodontal disease.

BACKGROUND: Periodontal disease is a degenerative illness that leads to resorption of the alveolar bone. Mesenchymal stromal cells (MSC) represent a novel tool for the production of biologic constructs for the treatment of degenerative bone diseases. The preparation of MSC differentiated into osteogenic lineage for clinical use requires the fulfillment of strict good manufacturing practice (GMP) procedures. METHODS: MSC were isolated from BM samples and then cultured under GMP conditions. MSC were characterized phenotypically and for their differentiative potential. Cells were seeded onto collagen scaffolds (Gingistat) and induced to differentiate into osteogenic lineages using clinical grade drugs compared with standard osteogenic supplements. Alizarin Red S stain was used to test the deposition of the mineral matrix. Standard microbiologic analysis was performed to verify the product sterility. RESULTS: The resulting MSC were negative for CD33, CD34 and HLA-DR but showed high expression of CD90, CD105 and HLA-ABC (average expressions of 94.3%, 75.8% and 94.2%, respectively). Chondrogenic, osteogenic and adipogenic differentiation potential was demonstrated. The MSC retained their ability to differentiate into osteogenic lineage when seeded onto collagen scaffolds after exposure to a clinical grade medium. Cell numbers and cell viability were adequate for clinical use, and microbiologic assays demonstrated the absence of any contamination. DISCUSSION: In the specific context of a degenerative bone disease with limited involvement of skeletal tissue, the combined use of MSC, exposed to an osteogenic clinical grade medium, and biomimetic biodegradable scaffolds offers the possibility of producing adequate numbers of biologic tissue-engineered cell-based constructs for use in clinical trials.

Mesenchymal stem cells cultured on a collagen scaffold: In vitro osteogenic differentiation.

OBJECTIVE: Management of periodontal defects has always been a challenge in clinical periodontics. Recently mesenchymal stem cells (MSC) have been proposed for tissue regeneration in periodontal disease and repair of large bone defects. Bone regeneration has to be supported by a scaffold which has to be biocompatible, biodegradable, and able to support cell growth and differentiation. The aim of this study was to evaluate osteogenic differentiation of MSC seeded on a collagen scaffold. DESIGN: MSC were obtained from adult rat bone marrow, expanded and cultured in plastic dishes or seeded in a collagen scaffold (Gingistat). MSC were induced towards osteogenic differentiation using osteogenic supplements. Cell differentiation and calcium deposits were evaluated by immunoblotting, immunohistochemistry, histochemical techniques, enzymatic activity assay, and SEM-EDX analysis. Biomaterial in vitro degradation was evaluated by measuring mass reduction after incubation in culture medium. RESULTS: Rat MSC osteogenic differentiation was demonstrated by osteopontin and osteocalcin expression and an increase in alkaline phosphatase activity. MSC were distributed homogeneously in the collagen scaffold. Nodular aggregates and alizarin red stained calcium deposits were observed in MSC induced towards osteogenic differentiation cultured in dishes or seeded in the collagen scaffold. SEM-EDX analysis demonstrated that calcium co-localized with phosphorous. The biomaterial in vitro degraded in 4-5 weeks. CONCLUSIONS: MSC from bone marrow differentiate towards osteogenic lineage, representing a suitable cell source for bone formation in periodontal regeneration. Gingistat collagen scaffold supports MSC distribution and differentiation, but its short degradation time may be a limitation for a future application in bone tissue regeneration.