Effects of titanium surface roughness on mesenchymal stem cell commitment and differentiation signaling.
OBJECTIVE
Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces.


METHODS
hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium.


RESULTS
The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins.


CONCLUSIONS
These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface.
Balloni
Stefania
S
Department of Experimental Medicine and Biochemical Science, University of Perugia, Italy.
Calvi
Edoardo Maria
EM
Damiani
Francesco
F
Bistoni
Giovanni
G
Calvitti
Mario
M
Locci
Paola
P
Becchetti
Ennio
E
Marinucci
Lorella
L
D000134:Acid Etching, Dental / Q000379:methods; D000328:Adult; D000469:Alkaline Phosphatase / Q000032:analysis; D001854:Bone Marrow Cells / Q000502:physiology; D055396:Bone Morphogenetic Protein 2 / Q000032:analysis; D002448:Cell Adhesion; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D024042:Collagen Type I / Q000032:analysis; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D004247:DNA / Q000096:biosynthesis; D003764:Dental Materials / Q000737:chemistry*; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D039902:Multipotent Stem Cells / Q000502:physiology; D010006:Osteoblasts / Q000166:cytology; D015675:Osteocalcin / Q000032:analysis; D053495:Osteopontin / Q000032:analysis; D011392:Proline / Q000378:metabolism; D012795:Sialoglycoproteins / Q000032:analysis; D000076182:Sp7 Transcription Factor; D013499:Surface Properties; D013936:Thymidine / Q000378:metabolism; D014025:Titanium / Q000737:chemistry*; D014157:Transcription Factors / Q000032:analysis
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D003764:Dental Materials; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; C493709:RUNX2 protein, human; D012795:Sialoglycoproteins; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; D015675:Osteocalcin; D053495:Osteopontin; D003094:Collagen; D004247:DNA; D011392:Proline; D014025:Titanium; D000469:Alkaline Phosphatase; D013936:Thymidine