title: Effect of the harvest procedure and tissue site on the osteogenic function of and gene expression in human mesenchymal stem cells.
abstract: Evidence has indicated that mesenchymal stem cells (MSCs) harvested with the Reamer/Irrigator/Aspirator (RIA) procedure exhibited an improved osteogenic differentiation capability compared with MSCs obtained by bone marrow aspiration from the iliac crest. In the present study, we hypothesized that the harvest procedure indeed influences the osteogenic activity of human MSCs more than the tissue site itself. Concentration [by colony forming unit-fibroblast (CFU-F) assay], calcification (by von Kossa staining), collagen deposition, gene expression and the gene methylation of the bone morphogenetic protein (BMP)-2 pathway [BMP2, SMAD5 and runt-related transcription factor 2 (RUNX2)], the Wnt pathway [WNT3, dickkopf-1 (DKK1), low-density lipoprotein receptor‑related protein 5 (LRP5) and β-catenin] and osteogenic genes [alkaline phosphatase (ALP), collagen, type I, alpha 1 (COL1A) and osteocalcin] were analyzed in the MSCs isolated intraoperatively from the iliac crest with a spoon (n=14), from the femur with a spoon (n=7), from the femur with the RIA procedure (n=13) and from the iliac crest by fine-needle aspiration (n=8, controls). A Bonferroni-Holm corrected p-value <0.05 indicated a statistically significant difference. The concentration of CFU-F in the MSCs was increased in the RIA debris in comparison with that in the iliac crest aspirates (trend) and the femur (spoon, significant). Calcium deposition was highest in the femur-derived MSCs (by RIA) and was significantly increased in comparison with that in the iliac crest-derived MSCs (spoon, aspirate). The gene expression of BMP2, SMAD5, RUNX2, osteocalcin, and COL1A was significantly increased in the femur-derived MSCs (spoon) and the iliac crest aspirate derived-MSCs in comparison with that in the femur-derived MSCs (by RIA). There was no significant diversity between the samples obtained using a spoon (from the femur or iliac crest). Calcium deposition and osteogenic gene expression decreased significantly with the increasing passage number in all the samples. The methylation of genes did not correlate with their respective gene expression and inconsistent differences were observed between the groups. Herein, we provide evidence that the harvest procedure is a critical factor in the osteogenesis of MSCs in vitro. The MSCs isolated from the femur and iliac crest using a spoon exhibit no significant differences. The altered gene expression and function of the femur-derived MSCs (by RIA) may be due to the harsh isolation procedure. The variable differentiation ability of the MSCs, which depends on the harvest site and the harvest technique, as well as the rapid loss of the osteogenic differentiation capacity with the increasing culture duration should be taken into consideration when using MSCs as a potential therapeutic application for bone tissue engineering.
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Henrich
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Nau
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Saskia Bo
authors.initials: SB
authors.lastname: Kraft
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Zollfrank
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Kontradowitz
authors.affiliation: Department of Visceral and Abdominal Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Elsie
authors.initials: E
authors.lastname: Oppermann
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Schultheiss
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Meier
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Frank
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Marzi
authors.affiliation: Department of Trauma, Hand and Reconstructive Surgery, Goethe University Hospital, 60590 Frankfurt am Main, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Seebach
mesh_terms: D000328:Adult; D000368:Aged; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D019175:DNA Methylation; D005260:Female; D005269:Femur / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D007085:Ilium / Q000166:cytology; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D008875:Middle Aged; D010012:Osteogenesis; D013234:Stem Cells; D020858:Tissue and Organ Harvesting / Q000379:methods*; D060449:Wnt Signaling Pathway
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2