Comparison of the effects of different bone allografts with and without growth factors on proliferation, osteogenic differentiation and mineralization of MG-63 osteoblast-like cells

Abstract:

Background and aim: Predictable regeneration of alveolar bone defects has always been an important therapeutic challenge in implant dentistry. Allografts including FDBA and DFDBA are some substitutes being widely used and reported as having osteoinductive activities with some degrees of controversy. The aim of this study is to determine the effect of growth factors on osteoinductive activities of different bone materials.

Materials and Methods: MG-63 cells were exposed to 60 mg amounts of four different commercially available freeze-dried bone allografts with or without 5 ng/mL of two growth factors (singular or in combination). After 24 and 72 hours of incubation, the effect of water- soluble allograft released materials and soluble growth factors on cell viability and proliferation was assessed using methyl thiazol tetrazolium (MTT) assay. Cell differentiation and mineralization was respectively assessed by real-time quantitative reverse transcription PCR (qRT-PCR) and alizarin red staining after 72 hours of exposure.

Results: The effect of different GFs on cell/allograft containing plates was affected by the allograft type. Early proliferative and late osseoinductive effects of GFs were more consistent in TGF-β rather than PDGF. PDGF only showed limited osseoinductivity in terms of accelerating BSP and OC genes.
Conclusion: based on the results of this study, TGF-β can have additional osseoinductive effect on allografts/cells combination and its application may be beneficial in in vitro and clinical regenerative studies.
Key words: Bone allograft, Growth factor, Osteoblast differentiation, Mineralization


Introduction:

Alveolar bone loss is a common consequence of periodontal disease progression which can finally lead to tooth mobility and then, an inevitable tooth loss. Additionally, it can complicate further implant therapy due to some residual bone defects (1). Since the prevalence of generalized periodontitis has been reported as high as 5-15%, the predictable and complete regeneration of alveolar defects has been an important therapeutic challenge in implant dentistry (2). Using various procedures like guided tissue regeneration (GTR), guided bone regeneration (GBR), and using enamel-matrix proteins are some examples to overcome this challenge (3). In 1923, bone grafts were first used by Hedegus for the regeneration of defects caused by periodontal lesions (4), and later in 1965 by O'Leary and Nabers (5).
Among all four types of bone graft materials, allografts seem to overcome some complications related to using autografts, like patient's more pain and discomfort (6), more cost and the limitation of intraoral donor sites (7). Demineralized Freeze-Dried Bone Allograft (DFDBA) has been successfully used in the treatment of periodontal, peri-implant, and furcation defects for three decades (8). The exposure of osteoinductive factors like bone

morphogenic protein (BMP) by in vitro HCL acid demineralization in DFDBA, has improved its osteoinductive potential via accelerating osteoblastic/chonroblastic differentiation from precursor cells (9-11). It has been said that the osteoinductivity of these graft materials can largely vary depending on the manufacturers' preparation protocol, donor's properties, and the particle size; therefore, it appears that many of current commercial product may have a limited osteoinductive activity, if not totally lack it (12-15).
In our previous in vitro study (16) on the osteoinductivity of different DFDBAs (Osseo+, Alloss, Cenobone), we concluded that all three were osteoinductive and had the ability to promote osteogenic differentiation of SaOs-2 cells. On the other hand, it has also been shown that the quantity of BMPs in almost all different commercial DFDBAs is less than which can be really osteoinductive and so it is necessary to add complementary BMP to them (17). Platelet-derived growth factor-BB (PDGF-BB), a potent chemotactic and mitotic factor on mesenchymal cells, can evidently promote healing. Also, it has shown a potent stimulatory effect on extracellular matrix (ECM) synthesis (18). The effect of rhPDGF-BB on granulation tissue development in diabetic ulcers has also been well confirmed (19). Transforming growth factor-β (TGF-β) stimulates chemotaxis and survival of osteoblasts (20), ectopic bone formation, and when delivered by Matrigel matrix and implanted in class II and III furcation defects of mandibular molars, induced periodontal tissue regeneration in a primate model (21). So, it is likely that we can benefit from these two growth factors (PDGF-BB and TGF- β) to enhance bone regeneration in defect sites selected for further implant placement.
Considering the lack of enough knowledge and consistency about the real benefits of adding growth factors (GFs) to current bone allografts, we designed this experimental in vitro study primarily aimed at evaluating osseoinductivity of two commercial FDBAs (CenoNoneTM, NonDeminTM) and two DFDBAs (CenoBoneTM and OsteoDeminTM) with or without growth factors (PDGF-BB and TGF-β) via assessing cell viability/proliferation (quantitative MTT assay), cell differentiation (osteogenic gene expression via quantitative real-time PCR) and cell mineralization (qualitative Alizarin red staining) in human osteoblast cell line (MG-63).

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