J Periodontal Implant Sci.  2011 Dec;41(6):293-301. 10.5051/jpis.2011.41.6.293.

Comparative study on the cellular activities of osteoblast-like cells and new bone formation of anorganic bone mineral coated with tetra-cell adhesion molecules and synthetic cell binding peptide

• Yu HS
• Noh WC
• Park JW
• Lee JM
• Yang DJ
• Park KB
• Suh JY

• KMID: 2094726
• DOI: http://doi.org/10.5051/jpis.2011.41.6.293

Abstract

PURPOSE
We have previously reported that tetra-cell adhesion molecule (T-CAM) markedly enhanced the differentiation of osteoblast-like cells grown on anorganic bone mineral (ABM). T-CAM comprises recombinant peptides containing the Arg-Gly-Asp (RGD) sequence in the tenth type III domain, Pro-His-Ser-Arg-Asn (PHSRN) sequence in the ninth type III domain of fibronectin (FN), and the Glu-Pro-Asp-Ilu-Met (EPDIM) and Tyr-His (YH) sequence in the fourth fas-1 domain of betaig-h3. Therefore, the purpose of this study was to evaluate the cellular activity of osteoblast-like cells and the new bone formation on ABM coated with T-CAM, while comparing the results with those of synthetic cell binding peptide (PepGen P-15).
METHODS
To analyze the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, andto analyze gene expression, northernblot was performed. Mineral nodule formations were evaluated using alizarin red stain. The new bone formations of each group were evaluated using histologic observation and histomorphometrc analysis.
RESULTS
Expression of alkaline phosphatase mRNA was similar in all groups on days 10 and 20. The highest expression of osteopontin mRNA was observed in the group cultured with ABM/P-15, followed by those with ABM/T-CAM and ABM on days 20 and 30. Little difference was seen in the level of expression of collagen type I mRNA on the ABM, ABM/T-CAM, and ABM/P-15 cultured on day 20. There were similar growth and proliferation patterns for the ABM/T-CAM and ABM/P-15. The halo of red stain consistent with Ca2+ deposition was wider and denser around ABM/T-CAM and ABM/P-15 particles than around the ABM particles. The ABM/T-CAM group seemed to have bone forming bioactivity similar to that of ABM/P-15. A complete bony bridge was seen in two thirds of the defects in the ABM/T-CAM and ABM/P-15 groups.
CONCLUSIONS
ABM/T-CAM, which seemed to have bone forming bioactivity similar to ABM/P-15, was considered to serve as effective tissue-engineered bone graft material.

Keyword

Bone substitutes; Cell adhesion molecules; Cell survival

MeSH Terms

Alkaline Phosphatase
Anthraquinones
Artificial Cells
Bone Substitutes
Cell Adhesion Molecules
Cell Survival
Collagen Type I
Fibronectins
Gene Expression
Oligopeptides
Osteogenesis
Osteopontin
Peptide Fragments
Peptides
RNA, Messenger
Tetrazolium Salts
Thiazoles
Transplants
Alkaline Phosphatase
Anthraquinones
Bone Substitutes
Cell Adhesion Molecules
Collagen Type I
Fibronectins
Oligopeptides
Osteopontin
Peptide Fragments
Peptides
RNA, Messenger
Tetrazolium Salts
Thiazoles

Figure

• 

  Figure 1 Optical density measured after culture for 1, 4, and 7 days at a wavelength of 579 nm by an enzyme-linked immunosorbent assay reader (n=3 in each groups). The optical density of anorganic bone mineral/tetra-cell adhesion molecule (ABM/T-CAM) and ABM/P-15 was significantly higher than ABM alone on days 4 and 7. a)A statistically significant difference as compared with ABM (P<0.01). ABM: OsteoGraf/N-300 particles in polystyrene petri dishes, ABM/T-CAM: OsteoGraf/N-300 particles absorbed T-CAM in polystyrene petri dishes, ABM/P-15: OsteoGraf/N-300 particles absorbed P-15 (PepGen P-15) in polystyrene petri dishes.

• 

  Figure 2 Cellular viability of anorganic bone mineral (ABM), ABM/tetra-cell adhesion molecule (ABM/T-CAM), and ABM/P-15 at 4 day culture stained by H&E. (A) ABM (×40), (B) ABM (×100), (C) ABM/T-CAM (×40), (D) ABM/T-CAM (×100), (E) ABM/P-15 (×40), (F) ABM/P-15 (×100). MC3T3-E1 cells were attached in remarkably great numbers on ABM/T-CAM and ABM/P-15 than on ABM.

• 

  Figure 3 Gene expression of alkaline phosphatase (ALP) of MC3T3-E1 cells cultured on anorganic bone mineral (ABM), ABM/tetra-cell adhesion molecule (ABM/T-CAM), and ABM/P-15 for 10 and 20 days. The ALP activity of MC3T3-E1 cells on ABM/T-CAM and ABM/P-15 was similar to that of ABM. ABM: OsteoGraf/N-300 particles in polystyrene petri dishes, ABM/T-CAM: OsteoGraf/N-300 particles absorbed on T-CAM in polystyrene petri dishes, ABM/P-15: OsteoGraf/N-300 particles absorbed on P-15 (PepGen P-15) in polystyrene petri dishes.

• 

  Figure 4 Gene expression of osteopontin (OPN) and α1 (I) collagen (Col I) of MC3T3-E1 cells cultured on anorganic bone mineral (ABM), ABM/tetra-cell adhesion molecule (ABM/T-CAM), ABM/P-15 for 10, 20, and 30 days. The expression of OPN mRNA on ABM/T-CAM and ABM/P-15 was similar to that of ABM at 10 days culture, but was remarkably higher on ABM/T-CAM and ABM/P-15 than ABM at 20 days culture. The expression of Col 1 mRNA on ABM/T-CAM and ABM/P-15 was similar to that of ABM at 20 days culture. ABM: OsteoGraf/N-300 particles in polystyrene petri dishes, ABM/T-CAM: OsteoGraf/N-300 particles absorbed T-CAM in polystyrene petri dishes, ABM/P-15: OsteoGraf/N-300 particles absorbed P-15 (PepGen P-15) in polystyrene petri dishes.

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  Figure 5 Staining with alizarin red S on anorganic bone mineral (ABM), ABM/tetra-cell adhesion molecule (ABM/T-CAM), and ABM/P-15 at 30 day culture. (A) ABM (×40), (B) ABM (×100), (C) ABM/T-CAM (one particle ×100), (D) ABM/T-CAM (particles ×100), (E) ABM/P-15 (one particle ×100), (F) ABM/P-15 (particles ×100). There were similar growth and proliferation patterns for ABM/T-CAM and ABM/P-15. In these groups, the cells formed multicellular layers around the graft material and interparticular bridges between them. Multicellular layers were also observed in the cultures with ABM, but fewer. The halo of red stain consistent with Ca2+ deposition is wider and denser around the ABM/T-CAM and ABM/P-15 particles than around the ABM particles.

• 

  Figure 6 Histologic findings of defects filled with anorganic bone mineral/tetra-cell adhesion molecule (ABM/T-CAM) (A) and ABM/P-15 (B) at 4 weeks (Masson's Trichorome stain). (a) Whole findings (×25), (b) High magnification (×100), (c) High magnification (×100). (▼: margin defect, G: graft material, NB: new bone). A complete bony bridge was seen in two thirds of the defects in the ABM/T-CAM and ABM/P-15 groups (A-a and b). Newly formed bone was seen in direct apposition to the graft material (A-b). The new mineralized tissue consisted of woven bone characterized by a high number of randomly distributed large osteocytes and by irregularly arranged fiber bundles within the new bone matrix (A-c). At the margin of the defect, the new bone that had emerged from the old bone was mature (B-b). Among the graft materials, the direct union of new bone was favorably observed (A and B-c).

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