ABM/P-15 modulates proliferation and mRNA synthesis of growth factors of periodontal ligament cells

P. Emecen; A.C. Akman; S.S. Hakki; E.E. Hakki; B Demiralp; T.F. Tozum; R.M. Nohutcu. “ABM/P-15 modulates proliferation and mRNA synthesis of growth factors of periodontal ligament cells.” Acta Odontologica Scandinavica, 2009, 67(2): 65-73.

Introduction
Over the past ten years, ABM/P-15 has been studied as a potential matrix for bone repair by promoting the concentration-dependent binding of dermal fibroblasts and osteoblasts on ABM. The ABM (anorganic bovine-derived bone mineral) particles are coated with a synthetic cell-binding 15-residue peptide, P-15, which mimics the cell-binding region of the α1 chain in Type 1 collagen. These particles have been shown in the literature to enhance cell-attachment in periodontal ligament fibroblasts, dermal fibroblasts, and human osteosarcoma cells and thus suggest that ABM/P-15 could be a viable matrix and alternative to bone-grafts and less biocompatible methods for bone repair. Few studies are available that evaluate the mechanisms of how ABM/P-15 affects the proliferation and mineralization of cells once they have achieved attachment to ABM. In order to expand this library of knowledge, the authors study the effect of ABM/P-15 on the proliferation and mineralization of periodontal ligament cells and elucidate how ABM/P-15 regulates growth factors necessary (i.e. biomarkers) for periodontal regeneration in vitro.

Summary – of methods, of results
The authors in this study isolated periodontal ligament (PDL) cells from the root surfaces of healthy premolar teeth by harvesting PDL tissue from the root and culturing pieces in Petri dishes at 37C for five passages total in the appropriate media. Cells from the fifth and third passage were used in subsequent experiments. The 5x104 PDL cells and 1x104 PDL cells were cultured in either 5% FBS or 5% FBS+ABM/P-15 (50mg/ml) on 24 well tissue-culture plates; all experiments were done in triplicate. Cell morphology was investigated on Day 7 using DMEM and a phase contrast microscope. The cells for the control and particles (Fig. 1A, 1B) were spindle-shaped and polarized; the particles did not seem to affect the morphology expected of these cells and the cells are seen to closely associate with the particles in an orthogonal manner (Fig. 1B). A proliferation assay was performed by staining the cells with trypan blue to check for cell viability and viable cells were counted with a hemacytometer on Days 1, 6, 8, and 10. The ABM/P-15 treated cells showed consistently higher proliferation that the control group.

























The Von Kossa staining method was used to analyze the mineralization of the cells every week for four weeks; no mineralization was observed for either the control or cells treated with particles. The mRNA expression of growth factors TGF-beta, BMP-2, IGF-I, b-FGF, PDGF, COL-1, and VEGF were studied using RT-PCR and the appropriate cDNA primers on the 3rd and 7th day. The authors claim to observe an increase in expression for BMP-2 TGF-beta as well as a decrease in b-FGF and IGF-I for the ABM/P-15 treated cells versus the control group (Fig 3). No difference was observed in COL-1 or VEGF expressions, while the PDGF seemed to first be greater in the particle-treated cells on Day 3, they decrease to a signal equivalent to the control group by Day 7.













Critique
This paper served its intended purpose in expanding our knowledge of ABM/P-15 on different cell types by investigating their effect on periodontal ligament (PDL) cells. They use the cells in the third and fifth passages to conduct their studies, without commenting on the fourth. It is not clear if they mixed the cells from both passages before seeding the cells into wells for each assay; if they did not, there may be some discrepancy in the health of the cells between each passage and this would definitely influence the results. Different concentrations of PDL cells were seeded for the proliferation and mineralization assays. The reason for this was not explained, and the fact that no mineralization was observed in the control or particle-treated group may be attributed to the lower cell concentration. The authors also claim that the cells were spindle-shaped, densely packed, and well-oriented with the ABM/P-15 particles; however, the images of the control and particle-treated group show no real difference. In fact, the particle-treated cells align themselves perpendicular to the particles; it would seem that if the cell integrins were actually binding to the particles, they would be oriented parallel instead to increase interactions. There is a statistically significant increase in cell number in the particle-treated group, though, so this suggests that cell binding is increased. A cell attachment assay should be performed to confirm how much the cells are interacting with P-15 on the particle. The mRNA results show that COL-1 did not change between Day 3 and Day 7 and it is not clear whether or not the particles are faster and just reached a plateau sooner than the control, or if there is no real difference.. They should consider performing these assays at earlier time points to verify. Also, to help with their mineralization assay, the authors should consider evaluating the expression of alkaline phosphatase (ALP), an early differentiation marker for the osteoblastic phenotype (high levels of ALP are necessary for mineralization potential).