Effect of microgrooves and fibronectin conjugation on the osteoblast marker gene expression and differentiation

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Abstract

PURPOSE

To determine the effect of fibronectin (FN)-conjugated, microgrooved titanium (Ti) on osteoblast differentiation and gene expression in human bone marrow-derived mesenchymal stem cells (MSCs).

MATERIALS AND METHODS

Photolithography was used to fabricate the microgrooved Ti, and amine functionalization (silanization) was used to immobilize fibronectin on the titanium surfaces. Osteoblast differentiation and osteoblast marker gene expression were analyzed by means of alkaline phosphatase activity assay, extracellular calcium deposition assay, and quantitative real-time PCR.

RESULTS

The conjugation of fibronectin on Ti significantly increased osteoblast differentiation in MSCs compared with non-conjugated Ti substrates. On the extracellular calcium deposition assays of MSCs at 21 days, an approximately two-fold increase in calcium concentration was observed on the etched 60-µm-wide/10-µm-deep microgrooved surface with fibronectin (E60/10FN) compared with the same surface without fibronectin (E60/10), and a more than four-fold increase in calcium concentration was observed on E60/10FN compared with the non-etched control (NE0) and etched control (E0) surfaces. Through a series of analyses to determine the expression of osteoblast marker genes, a significant increase in all the marker genes except type I collagen α1 mRNA was seen with E60/10FN more than with any of the other groups, as compared with NE0.

CONCLUSION

The FN-conjugated, microgrooved Ti substrate can provide an effective surface to promote osteoblast differentiation and osteoblast marker gene expression in MSCs.

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Most cited references 27

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Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.

Neelam Jaiswal, Stephen E. Haynesworth, Arnold I. Caplan … (1997)

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.

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Regulation of bone development and extracellular matrix protein genes by RUNX2.

Toshihisa Komori (2010)

RUNX2 is a multifunctional transcription factor that controls skeletal development by regulating the differentiation of chondrocytes and osteoblasts and the expression of many extracellular matrix protein genes during chondrocyte and osteoblast differentiation. This transcription factor plays a major role at the late stage of chondrocyte differentiation: it is required for chondrocyte maturation and regulates Col10a1 expression in hypertrophic chondrocytes and the expression of Spp1, Ibsp, and Mmp13 in terminal hypertrophic chondrocytes. It is essential for the commitment of pluripotent mesenchymal cells to the osteoblast lineage. During osteoblast differentiation, RUNX2 upregulates the expression of bone matrix protein genes including Col1a1, Spp1, Ibsp, Bglap, and Fn1 in vitro and activates many promoters including those of Col1a1, Col1a2, Spp1, Bglap, and Mmp13. However, overexpression of Runx2 inhibits osteoblast maturation and reduces Col1a1 and Bglap expression. The inhibition of RUNX2 in mature osteoblasts does not reduce the expression of Col1a1 and Bglap in mice. Thus, RUNX2 directs pluripotent mesenchymal cells to the osteoblast lineage, triggers the expression of major bone matrix protein genes, and keeps the osteoblasts in an immature stage, but does not play a major role in the maintenance of the expression of Col1a1 or Bglap in mature osteoblasts. During bone development, RUNX2 induces osteoblast differentiation and increases the number of immature osteoblasts, which form immature bone, whereas Runx2 expression has to be downregulated for differentiation into mature osteoblasts, which form mature bone. During dentinogenesis, Runx2 expression is downregulated, and RUNX2 inhibits the terminal differentiation of odontoblasts.

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A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

P Ducy, Jian Shen, G Pinero … (1999)

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

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Author and article information

Journal

Journal ID (nlm-ta): J Adv Prosthodont

Journal ID (iso-abbrev): J Adv Prosthodont

Journal ID (publisher-id): JAP

Title: The Journal of Advanced Prosthodontics

Publisher: The Korean Academy of Prosthodontics

ISSN (Print): 2005-7806

ISSN (Electronic): 2005-7814

Publication date (Print): December 2015

Publication date (Electronic): 30 December 2015

Volume: 7

Issue: 6

Pages: 496-505

Affiliations

[1 ]Department of Biomaterials & Prosthodontics, Kyung Hee University Hospital at Gangdong, Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea.

[2 ]Department of Dentistry, Graduate School of Dentistry, Kyung Hee University, Seoul, Republic of Korea.

[3 ]ED Dental Clinic, Seoul, Republic of Korea.

[4 ]Department of Oral and Maxillofacial Surgery, Kyung Hee University Hospital at Gangdong, Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea.

[5 ]Department of Prosthodontics, National Health Insurance Medical Center Ilsan Hospital, Goyang, Gyeonggi, Republic of Korea.

[6 ]Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea.

Author notes

Corresponding author: Sang Cheon Lee. Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Rep. of Korea. Tel. 82 2 961 2317, schlee@ 123456khu.ac.kr

Corresponding author: Suk Won Lee. Department of Biomaterials & Prosthodontics, Kyung Hee University Hospital at Gangdong, Institute of Oral Biology, School of Dentistry, Kyung Hee University, 892 Dongnam-ro, Gangdong-gu, Seoul 05278, Republic of Korea. Tel. 82 2 440 7518, ysprosth@ 123456hanmail.net

Author information

Su-Jung Park http://orcid.org/0000-0002-4111-2231

Richard Leesungbok http://orcid.org/0000-0002-8381-723X

Su-Jin Ahn http://orcid.org/0000-0003-2128-1561

Byung-Jin Im http://orcid.org/0000-0001-5661-2258

Do Yun Lee http://orcid.org/0000-0002-8638-7423

Yu-Jin Jee http://orcid.org/0000-0003-2526-4005

Joon-Ho Yoon http://orcid.org/0000-0002-4571-7342

Taixing Cui http://orcid.org/0000-0002-9528-7299

Sang Cheon Lee http://orcid.org/0000-0002-8560-0173

Suk Won Lee http://orcid.org/0000-0003-2726-3567

Article

DOI: 10.4047/jap.2015.7.6.496

PMC ID: 4722154

PubMed ID: 26816580

SO-VID: 558c6576-bd76-4044-a7a7-7d2f15995fe7

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 25 November 2015

Date revision received : 18 December 2015

Date accepted : 22 December 2015

Funding

Funded by: National Research Foundation of Korea, CrossRef http://dx.doi.org/10.13039/501100003725;

Award ID: 2010-0021268

Award ID: 2013R1A2A2A01009239

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Effect of microgrooves and fibronectin conjugation on the osteoblast marker gene expression and differentiation

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Abstract

PURPOSE

MATERIALS AND METHODS

RESULTS

CONCLUSION

Related collections

Arab Journal of Forensic Sciences & Forensic Medicine (AJFSFM)

Most cited references 27

Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.

Regulation of bone development and extracellular matrix protein genes by RUNX2.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

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Cited by 2

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