J Periodontal Implant Sci.  2012 Jun;42(3):95-104. 10.5051/jpis.2012.42.3.95.

Heat or radiofrequency plasma glow discharge treatment of a titanium alloy stimulates osteoblast gene expression in the MC3T3 osteoprogenitor cell line

• Rapuano BE
• Hackshaw K
• MacDonald

• KMID: 2094733
• DOI: http://doi.org/10.5051/jpis.2012.42.3.95

Abstract

PURPOSE
The purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat (600degrees C) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line.
METHODS
Quantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (alpha1), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix.
RESULTS
Osteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectin's bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression.
CONCLUSIONS
The results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectin's bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms.

Keyword

Cell differentiation; Dental implants; Fibronectins; Integrin alpha5beta1; Osteoblasts

MeSH Terms

Alkaline Phosphatase
Alloys
Cell Differentiation
Cell Line
Collagen Type I
Dental Implants
Fees and Charges
Fibronectins
Gene Expression
Hot Temperature
Integrin alpha5beta1
Integrin-Binding Sialoprotein
Osteoblasts
Osteocalcin
Osteopontin
Parathyroid Hormone-Related Protein
Plasma
Proteins
Real-Time Polymerase Chain Reaction
RNA, Messenger
Titanium
Alkaline Phosphatase
Alloys
Collagen Type I
Dental Implants
Fibronectins
Integrin alpha5beta1
Integrin-Binding Sialoprotein
Osteocalcin
Osteopontin
Parathyroid Hormone-Related Protein
Proteins
RNA, Messenger
Titanium

Figure

• 

  Figure 1 Effects of heat and radiofrequency plasma glow discharge (RFGD) pretreatments on (A) Runx2 and (B) osterix mRNA expression in MC3T3 osteoprogenitor cells cultured on Ti6Al4V disks. Both untreated and pretreated disks were either uncoated (control) or precoated (fibronectin, FN) with 1 nM FN overnight and plated with MC3T3 cells. The levels of Runx2 and osterix mRNA expression measured by quantitative real time polymerase chain reaction for treated disks at the indicated points in time are presented as a percentage of the corresponding levels of mRNA expression measured for untreated disks. The data represent means±standard error for 3 to 7 independent cultures at each point in time. a)Significantly greater (P<0.05) than untreated disks. b)Significantly greater (P<0.01) than untreated disks. c)Significantly greater (P<0.05) than heat-pretreated disks based on analysis of variance.

• 

  Figure 2 Effects of heat and radiofrequency plasma glow discharge (RFGD) pretreatments on (A) type I collagen (α1), (B) parathyroid hormon-related peptide (PTH-rP), and (C) alkaline phosphatase mRNA expression in MC3T3 osteoprogenitor cells cultured on Ti6Al4V disks. The levels of type I collagen (α1), PTH-rP, and alkaline phosphatase mRNA expression measured by quantitative real time polymerase chain reaction for treated disks at the indicated points in time are presented as a percentage of the levels of mRNA expression measured for untreated disks. Data represent means±standard error for 3 to 8 independent cultures at each point in time. FN: fibronectin. a-c)Significantly greater (P<0.05, 0.01, 0.001) than untreated disks. d-f)Significantly greater (P<0.05, 0.01, 0.001) than RFGD-pretreated disks based on analysis of variance.

• 

  Figure 3 Effects of heat and radiofrequency plasma glow discharge (RFGD) pretreatments on (A) osteopontin, (B) bone sialoprotein, and (C) osteocalcin mRNA expression in MC3T3 osteoprogenitor cells cultured on Ti6Al4V disks. The levels of osteopontin, bone sialoprotein, and osteocalcin mRNA expression measured by quantitative real time polymerase chain reaction for treated disks at the indicated points in time are presented as a percentage of the levels of mRNA expression measured for untreated disks. Data represent means±standard error for 3 to 9 independent cultures at each point in time. FN: fibronectin. a)Significantly greater (P<0.05) than untreated disks. b)Significantly greater (P<0.05) than heat-pretreated disks based on analysis of variance.

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