Adipose-derived multipotent stromal cells and natural latex protein fraction (Hevea brasiliensis) associated with polycaprolactone and graphene scaffolds in experimental osteogenesis

Author(s)

Guilherme Ferreira Caetano

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2017-03-20

Examining board members

Marco Andrey Cipriani Frade; Paulo Jorge da Silva Bártolo; Celso Herminio Ferraz Picado; Maria Carolina de Oliveira Rodrigues; Adalberto Luiz Rosa

• Advisor: Marco Andrey Cipriani Frade

Abstract

The increment of life expectancy and frequency of chronic diseases in the population has led to an increasing incidence of chronic bone defects. Conventional treatment is based on autologous transplantation, which depends on extremely invasive approaches. A promising proposal is to obtain healthy tissues in laboratories using porous three-dimensional matriz (scaffolds), which enable cellular growth and differentiation of mesenchymal stem cells (MSCs). Scaffolds can be optimized to provide adequate vascularization, a critical event to bone regeneration. MSCs have potential for differentiation, immunoregulation and angiogenesis. F1 natural latex protein from Hevea brasiliensis rubber tree presents important angiogenic and healing activity. The objective of this work was to investigate the influence of pre-colonized polycaprolactone (PCL) scaffolds on experimental in vitro and in vivo osteogenesis (xenotransplantation) and also the safety and influence of F1 protein on MSCs seeded on PCL and PCL reinforced with different concentrations of graphene scaffolds (PCL/graphene) in cell proliferation and osteogenic differentiation. MSCs were isolated from human adipose tissue (ADSCs), characterized by positive and negative markers and also in vitro differentiation. PCL Scaffolds, produced by an additive manufacturing technique, were evaluated for cell adhesion/viability potential (MTT assay), osteogenic differentiation (alizarin red) and in vivo xenogenic grafting potential for osteointegration and osteoinduction evaluated by histology and immunohistochemistry. F1 latex protein, prepared in different concentrations, was evaluated in contact with ADSCs and 3T3 fibroblasts culture in vitro regarding to cytotoxicity (MTT), proliferative potential (tritiated thymidine), migratory (scratch assay) and osteogenic induction (alkaline phosphatase). PCL scaffolds were reinforced with graphene (PCL/graphene scaffolds), coated with F1 protein (adsorption) and evaluated for cell viability/proliferation assay (Alamar blue) and osteogenic differentiation (alkaline phosphatase and alizarin red). ADSCs showed low percentage for negative markers, high percentage for positive markers and differentiation properties in vitro, providing enough information on the successful isolation and maintenance of human ADSCs. PCL scaffolds showed non-toxicity activity and osteogenic differentiation induced by culture medium. PCL scaffolds, pre-colonized and non-colonized with ADSCs, were implanted in a critical calvarial defect in rats. The group of rats which received scaffolds with ADSCs to treat the bone defect had improved bone formation with direct and indirect participation of ADSCs to the bone repair process. To in vitro assays with F1 (2D culture model), proliferative stimulus was observed to F1 0.00001% and 0.0001% samples, in addition to a higher percentage of cell migration to 0.001% and 0.0001%, different from control. PCL/graphene scaffolds demonstrated proliferative stimulation when colonized by ADSCs and this stimulus was even higher to F1 coated PCL/graphene scaffolds, mainly to 0.75% graphene. Although F1 have not enhanced osteogenic differentiation on 2D cell culture model, the stimulus was observed to F1 coated scaffolds with higher alkaline phosphatase activity. This work demonstrated success in the use of ADSCs and scaffolds for bone regeneration and presented two promising products to be applied in tissue engineering field, such as, scaffolds with graphene reinforcement at low concentration and F1 latex protein to improve cell proliferation and differentiation. (AU)

• FAPESP's process: 13/09055-2 - Osteo-differentiation of human mesenchymal stem cells attached to natural latex proteins from the rubber tree Hevea brasiliensis on scaffold of polycaprolactone and polylactic acid (PCL/PLA) to bone xenografting
• Grantee: Guilherme Ferreira Caetano
• Support Opportunities: Scholarships in Brazil - Doctorate