Gregory N. Bancroft

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces.

In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells. Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media of different viscosities while maintaining the fluid flow rate constant. This methodology allowed exposure of the cultured cells to increasing levels of mechanical stimulation, in the form of fluid shear stress, whereas chemotransport conditions for nutrient delivery and waste removal remained essentially constant. Under similar chemotransport for the cultured cells in the 3D porous scaffolds, increasing fluid shear forces led to increased mineral deposition, suggesting that the mechanical stimulation provided by fluid shear forces in 3D flow perfusion culture can indeed enhance the expression of the osteoblastic phenotype. Increased fluid shear forces also resulted in the generation of a better spatially distributed extracellular matrix inside the porosity of the 3D titanium fiber mesh scaffolds. The combined effect of fluid shear forces on the mineralized extracellular matrix production and distribution emphasizes the importance of mechanosensation on osteoblastic cell function in a 3D environment.

Design of a flow perfusion bioreactor system for bone tissue-engineering applications

Several different bioreactors have been investigated for tissue-engineering applications. Among these bioreactors are the spinner flask and the rotating wall vessel reactor. In addition, a new type of culture system has been developed and investigated, the flow perfusion culture bioreactor. Flow perfusion culture offers several advantages, notably the ability to mitigate both external and internal diffusional limitations as well as to apply mechanical stress to the cultured cells. For such investigation, a flow perfusion culture system was designed and built. This design is the outgrowth of important design requirements and incorporates features crucial to successful experimentation with such a system.

Flow Perfusion Enhances the Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds

In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 μm fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 × 105 cells in 300 μl, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.

Effect of Fibronectin- and Collagen I-Coated Titanium Fiber Mesh on Proliferation and Differentiation of Osteogenic Cells

The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells. Three main treatment groups were investigated in addition to uncoated titanium fiber meshes: meshes coated with fibronectin, meshes coated with collagen I, and meshes coated first with collagen I and then subsequently with fibronectin. Rat bone marrow cells were cultured for 1, 4, 8, and 16 days in plain and coated titanium fiber meshes. In addition, a portion of each of these coating treatment groups was cultured in the presence of antibodies against fibronectin and collagen I integrins. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, osteocalcin, and calcium content and alkaline phosphatase activity. There were no significant effects of the coatings on cellular proliferation as indicated by the DNA quantification analysis. When antibodies against fibronectin and collagen I integrins were used, a significant reduction (p < 0.05) in cell proliferation was observed for the uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. The different coatings also did not affect the alkaline phosphatase activity of the cells seeded on the coated meshes. However, the presence of antibodies against fibronectin or collagen I integrins resulted in significantly delayed expression of alkaline phosphatase activity for uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. Calcium measurements did not reveal a significant effect of fibronectin or collagen I coating on calcium deposition in the meshes. Also, no difference in calcium content was observed in the uncoated titanium meshes and meshes coated with fibronectin when antibodies against fibronectin or collagen I integrins were present. Meshes coated with both collagen I and fibronectin showed significantly higher calcium content when cultured in the presence of antibodies to collagen and fibronectin integrins. A similar phenomenon was also observed for collagen-coated meshes cultured in the presence of antibodies to fibronectin integrins. No significant differences in osteocalcin content were observed between the treatment groups. However, all groups exposed to antibodies against fibronectin integrins showed a significant decrease in osteocalcin content on day 16. These results show that a fibronectin or collagen I coating does not stimulate the differentiation of rat bone marrow cells seeded in a titanium fiber mesh.

Fluid flow increases mineralized matrix deposition in three-dimensional perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

The aim of the study is to investigate the effect of flow perfusion in three-dimensional titanium meshes seeded with rat marrow stromal cells (MSCs), on the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. Flow perfusion allows the continuous infusion of cell culture media throughout the porous network of the scaffold. It also permits the mechanical stimulation of the seeded cells by exposing them to shear forces.

Bone Tissue Engineering by Cell Transplantation

Bone defects that require skeletal reconstruction are a common problem facing physicians today. These defects often arise as a result of trauma, tumor resection, infection, or skeletal abnormalities. Traditional therapies to overcome these bone deficits include bone grafts, bone cement, and synthetic bone substitutes including plastics, ceramics, and metals. Each of these has limitations that preclude it from being an ideal bone replacement, indicating a need for bone tissue engineering. Tissue engineering seeks to regenerate this lost bone tissue by favorably utilizing the interactions between cells, growth factors, and scaffolding in novel bone tissue engineering constructs and therapies. Although different tissue engineering approaches may place emphasis on the cellular component, the involved growth factors, or implanted matrices, this review focuses on the use of cell transplantation to regenerate bone and enhance bone healing. By transplanting different cell populations including bone marrow cells, mesenchymal stem cells, osteoblasts, or genetically modified cells, researchers have shown the potential of these cell-based tissue engineering approaches to engineer new bone tissue. Because of this demonstrated ability, cell transplantation will undoubtedly play an important role in eventual tissue engineering therapies for treating bone defects.