Biochemical and Biophysical Analyses of Tissue-Engineered Bone Obtained from Three-Dimensional Culture of a Subset of Bone Marrow Mesenchymal Stem Cells

Grafts of tissue-engineered bone represent a promising alternative in the treatment of large and small bone defects. Current approaches are often badly tolerated by patients because of invasiveness, ethical problems, culture, and possibility of infection. Autologous grafts have been indicated as a solution to such problems. Because of tissue availability, many have proposed the use of cultured cells derived from bone marrow expanded in culture and induced to differentiate in bone tissue. Data reported in the literature show that it is possible to produce tissue substitutes in vitro indeed, but results are not always concordant regarding the in vitro produced bone quality. In the present work, we investigated bone formation in aggregates of human bone marrow-derived mesenchymal stem cells induced to differentiate in bone. After osteoinduction we characterized the mineral matrix produced using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray powder diffraction. Cells were obtained from bone marrow, subjected to immunodepletion for CD3, CD11b, CD14, CD16, CD19, CD56, CD66b, and glycophorin A using RosetteSep and cultured in a new formulation of medium for four passages and then were allowed to form spontaneous aggregates. At the end of proliferation before aggregation, cells were analyzed by fluorescent activated cell sorting (FACS) for markers routinely used to characterize expanded mesenchymal stem cells and were found to be remarkably homogeneous for CD29 (99% +/- 1%), CD73 (99% +/- 1%), CD90 (95% +/- 4%), CD105 (96% +/- 4%), and CD133 (0% +/- 1%) expression. Our results show that not only aggregated cells express the major markers of osteogenic differentiation, such as osteocalcin, osteonectin, osteopontin, and bone sialoprotein, but also the inorganic matrix is made of an apatite structurally and morphologically similar to native bone even without a scaffold.