RESUMO
Due to the increasing demand for a bone marrow study model, we developed a natural scaffold from decellularized bovine bone marrow (DeBM). The obtained bioscaffold was analyzed after the decellularization process; histological staining, scanning and transmission electron microscopy confirmed the preservation of its native 3D-architecture; including blood vessels and cell niches as well as the integrity of important components of the extracellular matrix; Collagen III, IV and fibronectin. In addition to biochemical composition, physical properties of the bone marrow were also conserved. We evaluated the suitability of this bio-scaffold as a tridimensional culture platform. Seeding experiments with umbilical cord-derived hematopoietic stem cells and human bone marrow stromal cell line HS5 demonstrated that this scaffold is capable of supporting hematopoietic and stromal cell adhesion and proliferation without the need of exogenous factors. DeBM provided an inductive environment for the repopulation of the bone marrow inducing the expression of SDF-1, HGF and SCF by seeded stromal cells. The presence of these potent hematopoietic chemoattractants would be crucial for ex vivo long-term culture of HSCs, and for recreating the natural microenvironment of the bone marrow for bioengineering applications. We conclude that the decellularization process succeeded in preserving the 3D structure and mechanical properties of the bone marrow. The resulting scaffold is suitable for cell culture, representing an advantageous bone marrow experimental model, and potentially an effective platform for CD34+ HSC expansion and differentiation for clinical applications.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas/citologia , Animais , Bovinos , Células Cultivadas , Técnicas de Cocultura , Humanos , Células-Tronco Mesenquimais/citologia , Tamanho da PartículaRESUMO
The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-ß1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.