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

Abstract

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.

Acellular Bone Colonization and Aggregate Culture Conditions Diversely Influence Murine Periosteum Mesenchymal Stem Cell Differentiation Potential in Long-TermIn VitroOsteoinductive Conditions

“…Routinely after osteogenic differentiation, the onset of mineral deposition was assessed with von Kossa assay, although this staining technique alone is not sufficient to confirm that in vitro mineralization represents bone formation. 17 Therefore, aggregates and colonized woven bone were subjected to X-ray powder diffraction analysis to evaluate the presence and the structure of the bone principal constituent, the carbonate HA. 32,33 In Figure 4b the X-ray diffraction (XRD) patterns obtained from aggregates after 3 months (ii) and 1 year (iii) induction time clearly show a reflection at 25.8°and a triplet of reflections around 32°of 2y, plus other weak reflections.…”

“…26,30 Real-time PCR confirmed the differences between culture conditions, evidencing in general the higher expression of osteoblastic and chondroblastic markers in aggregates after 3 months as well as after 1 year differentiation, compared with the acellular bone colonization experiments. 4,13,17 On the other hand, cells in colonization samples maintained a higher expression of NFATc1 and cathepsin K after 3 months as well as after 1 year, compared with aggregates.…”

“…Tissue engineering represents the opportunity to promote repair through tissue regeneration, and our previously published study 17 underlined the importance of the matrix characterization when using tissue-engineered bone in therapeutic applications. The purpose of the present study was to establish and characterize the clonal Pe-MSC osteoblastic potential, their ability to elaborate, mineralize bone matrix, and investigate the scaffold's influence after long-term in vitro osteoblastic induction.…”

Adipose Tissue-Derived Stem Cell in Vitro Differentiation in a Three-Dimensional Dental Bud Structure

“…ASCs were maintained semiconfluent to prevent cell differentiation, and approximately 80% of the medium was replaced every 3 days. Primary human bone marrow mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) were obtained as described by Ferro et al 12 Human bone MSCs were obtained by flushing the femur head content through a 26-gauge needle. Flushed cells were diluted in Hanks' balanced salt solution (Sigma), layered on top of Ficoll (Amersham), and centrifuged.…”

“…Whole cell extracts for positive and negative controls and proliferating and dental-induced ASCs were prepared by using lysis buffer [25 mmol/L HEPES (pH 7.6) 0.3 mol/L NaCl, 1.5 mmol/L MgCl 2 , 0.2 mmol/L EDTA, 0.5% Nonidet P40, 0.5 mmol/L dithiothreitol, 1ϫ protease inhibitor, 1 mmol/L NaF, and 1 mmol/L Na ortovanadate, all from Sigma] as described by Ferro et al 12 Samples protein content was quantified by BCA protein assay reagent kit (Pierce) and the program Labsystems genesis V2. 16.…”