Award Date

5-15-2018

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Dental Medicine

First Committee Member

Brian Chrzan

Second Committee Member

Tanya Al-Talib

Third Committee Member

Karl Kingsley

Fourth Committee Member

Bryan Spangelo

Number of Pages

93

Abstract

Periostin is a secreted, extracellular matrix (ECM) protein widely expressed within collagen-rich fibrous connective tissues of the body including the periodontal ligament (PDL), bone, skin, heart, and cornea. Periostin has been shown to serve many important regulatory functions including cell adhesion, cell motility, wound healing and of particular importance to the dental field, differentiation of osteoblasts. The deletion of periostin compromises osteoblast attachment to bone matrix and induces a reduction in mineralization and expression of bone markers, including type I collagen, osteocalcin, osteopontin and alkaline phosphatase. Periostin has also been shown to play a significant role in collagen fibrillogenesis by enhancing the proteolytic activation of lysyl oxidase, which is required for collagen cross-linking. Immunohistochemistry studies have revealed high levels of periostin expression in the PDL with the periostin expressing cells identified as the fibroblastic cells in the PDL and osteoblastic cells on the alveolar bone surfaces. The significance of periostin to bone and PDL development was further demonstrated in a study on periostin knockout mice. These mice displayed a unique phenotype with significant changes to the periodontium: gingival tissue atrophy, PDL damage and loss of bone around the molars, underscoring the importance of periostin as a key ECM protein within the PDL. In controlling osteoblast differentiation and collagen fibrillogenesis, periostin has a critical role in maintaining bony architecture and density, which suggests that it may be able to act as another layer of therapeutic control on bone homeostasis.

Cytokines of the transforming growth factor-beta (TGF-β) superfamily of proteins including TGF-β1 have been implicated in the regulation of periostin expression in bone. In particular, studies have shown that periostin expression is enhanced with increasing levels of TGF-β1 until bone mineralization begins. At that point, periostin expression is negatively regulated as mineralization proceeds, suggesting that TGF-β1 may play a role during the initiation of bone mineralization. Another member of the TGF-β superfamily, bone morphogenetic protein 2 (BMP2), has also been demonstrated to enhance periostin expression in osteoblasts. Recently, there has been increasing interest in bone morphogenetic proteins (BMPs) due to their therapeutic potential in orthopedics, oral surgery and other disciplines. BMPs are members of the TGF-β superfamily of proteins and act as regulators during embryogenesis and bone and cartilage formation and repair. Research in the area of BMP action has revealed great complexity with far reaching effects among the many members of the various BMPs. Although periostin expression in pre-osteoblastic cells, specifically MC3T3-E1 mouse pre-osteoblasts, has been studied in response to TGF-β1 and BMP2, other BMP members have not been considered. Given that different BMP family members are differentially expressed in tissues of the body with various physiological functions, it is reasonable to assume that they may have different effects on periostin expression as well. For example, BMP2, BMP4 and BMP7 all play key roles in bone and cartilage development whereas BMP3 has been characterized as an antagonist to the osteogenic effects of the other BMPs.

The objective of this study was to demonstrate the in vitro expression of periostin in MC3T3-E1 mouse pre-osteoblast cells in response to different BMPs. Previous studies describing the regulation of periostin expression by TGF-β1 suggests that periostin has the potential to be a downstream effector of the TGF-β superfamily of proteins. In this study, the expression of periostin was hypothesized to increase with BMP2, BMP4 and BMP7 treatment, supporting the notion of these BMPs as enhancers or agonists of periostin expression. In contrast, BMP3 was hypothesized to suppress periostin expression due to its innate inhibitory potential. However, BMP3 does possess modulator activity and could also enhance periostin expression under some conditions as well. Unlike other studies, this research is unique in attempting to determine not only the effects of BMP2, but also BMP4 and BMP7 on periostin expression, which, to our knowledge, have never been considered. In addition, no previous studies have considered the antagonistic effects of BMP3. As a way to further analyze this relationship, the effect of concentration was to be considered as well. Ultimately, understanding the effects of BMPs on periostin expression will contribute to our overall understanding of the complex mechanisms involved in maintaining osteoblasts in an undifferentiated state as well as their therapeutic applications in the clinical setting. In the future, this knowledge may have important clinical implications in the modulation of osteoblast activity, which may be applicable to the dental field in the regulation of tooth movement, regeneration of the periodontium and de novo bone formation.

MC3T3-E1 pre-osteoblast cells were prepared and treated in duplicate with BMP2, BMP3, BMP4 and BMP7 with two concentrations: 10 ng/mL and 25 ng/mL. After 24 hours of incubation under controlled conditions, cells were lysed and total RNA was purified, extracted and stored at -80°C. Reverse transcription polymerase chain reaction (RT-PCR) was performed on all samples using periostin primers and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. These particular primers were selected to amplify a region of periostin cDNA as well as the typical housekeeping protein (GAPDH) as a normalization factor for cell number. Amplified products were run on a 2% agarose gel for two hours followed by visualization and image capture under a UV light. Expected products were identified against the known base pair values of periostin and GAPDH. Pixel density was quantified for each band and periostin bands were normalized against their corresponding GAPDH bands. Results indicated that periostin expression was significantly increased in both BMP2 and BMP3 treatments. The following increases in periostin expression from baseline were observed: BMP2 (10 ng/mL): +29%, BMP2 (25 ng/mL): +26%, BMP3 (10 ng/mL): +24% and BMP3 (25 ng/mL): +17%. Periostin expression was also increased under BMP4 (+9% and +11% for 10 ng/mL and 25 ng/mL concentrations respectively) and BMP7 (+5% and +11% for 10 ng/mL and 25 ng/mL concentrations respectively) conditions, although this was not statistically significant. These findings confirm the observation from other studies that BMP2 enhanced periostin expression. However, BMP3 showed contrasting results and actually increased periostin expression, suggesting a modulator role for BMP3 on periostin expression. BMP4 and BMP7 did not elicit significant changes on periostin expression, which may be due to a number of factors. Concentration-dependence was not observed for any of the BMPs. Future studies are needed to further evaluate the relationship between periostin expression and BMPs.

Keywords

BMPs; BMP treatment; Bone mineralization; Bone morphogenetic proteins; Periostin; Periostin gene expression

Disciplines

Biochemistry | Biology | Dentistry

Language

English


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