RESEARCH ARTICLE
The protective effect of platelet released growth factors and bone augmentation (Bio-Oss®) on ethanol impaired osteoblasts

https://doi.org/10.1016/j.aanat.2017.07.002Get rights and content

Abstract

Background

Chronic alcohol consumption is a known limiting factor for bone healing. One promising strategy to improve bone augmentation techniques with Bio-Oss® in oral and maxillofacial surgery might be the supportive application of platelet-concentrated biomaterials as platelet-released growth factor (PRGF). To address this matter, we performed an in vitro study investigating the protective effects of PRGF and Bio-Oss® in ethanol (EtOH) treated osteoblasts.

Methods

The SAOS-2 osteosarcoma cell line, with and without EtOH pretreatment was used. The cell viability, proliferation and alkali phosphatase activity (ALP) after application of 0%, 5% and 10% PRGF and Bio-Oss® were assessed.

Results

The application of PRGF and Bio-Oss® in EtOH impaired osteoblasts showed a significant beneficial influence increasing the viability of the osteoblasts in cell culture. The synergistic effect of Bio-Oss® and 5% PRGF on the proliferation of osteoblasts was also demonstrated. Bio-Oss® only in combination with PRGF increases the alkaline phosphatase (ALP) activity in EtOH pretreated cells.

Conclusions

These results indicate that the simultaneous application of PRGF and Bio-Oss® inhibits EtOH induced bone healing impairment. Furthermore, in the cells, PRGF induced a protective mechanism which might promote bone regeneration.

Introduction

Natural bone deficiency is the most limiting factor to comprehensive functional and aesthetic rehabilitation with dental implants. Nowadays, augmentation of the alveolar bone or sinus floor with autogenous bone, xenografts or synthetic biomaterials using diverse surgical grafting procedures are the commonly used solutions. On the other hand, the use of additional regenerative autologous supplements such as platelet rich plasma (PRP), plasma rich growth factors or platelet-released growth factors (PRGF) during bone augmentation in the area of the bone augmentation has been widely practiced (Anitua, 2001, Anitua et al., 2009).

Next to smoking, chronic consumption of alcohol is a high risk factor associated with bone loss due to a reduced osteoblast number, osteoid synthesis and osteoid mineralization rate (Chakkalakal, 2005, Chen et al., 2010, Chen et al., 2011, Chen et al., 2008, Gonzalez-Calvin et al., 1999). A linear relationship was found between alcohol consumption, fracture risk, and femoral neck bone mineral density. Chronic heavy alcohol consumption above ≥70 g/setting or 100–200 g/day is considered a high-risk factor for the loss of bone mineral density and impairment of bone remodeling (Gaddini et al., 2016, Maurel et al., 2012). Bone repair by fracture healing is impaired by decreased quality and quantity of new bone while an abnormally high apoptosis of osteoblasts can be encountered (Alund et al., 2017, Elmali et al., 2002). EtOH treatment in a dose-dependent manner accelerates the aging process in bone marrow-derived mesenchymal stem cells (Chen et al., 2017). Furthermore, Ethanol (EtOH) intake leads to the production of reactive oxygen species (ROS), which results in increased oxidative stress in the cells (Chen et al., 2011, Chen et al., 2008). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a potent regulator of oxidative defense inducing genes like Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H:quinine oxidoreductase-1 (NQO-1) and other intracellular antioxidants by detoxifying ROS and various other toxic substances (Al-Sawaf et al., 2015, Ishii et al., 2000, Kensler et al., 2007). Nrf2 is a transcription factor that is known to maintain cellular defense against the toxicity of electrophiles and reactive oxygen species (ROS). Nrf2 binds to the cis-acting enhancer elements, called antioxidant response element (ARE) in the promoters of cytoprotective antioxidant genes (Kensler et al., 2007). In our previous work (Lippross et al., 2014), it has been shown that the Nrf2-deficient mice showed impaired bone healing.

PRP and PRGF are a rich source of growth factors. They have been found effective in accelerating significant tissue repair and regeneration, as well as in releasing massive quantities of growth factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), insulin-like growth factor (IGF) and transforming growth factor (TGF), but also antimicrobial peptides (Anitua, 2001, Anitua et al., 2017, Sonmez et al., 2013, Tohidnezhad et al., 2011a, Tohidnezhad et al., 2012). Recently, the use of PRP gel in wound healing has become popular in nearly all fields of surgery, in particular in oral and maxillofacial surgery (Anitua, 2001, Anitua et al., 2009, Sonmez et al., 2013). Plasma free PRGF, as our concept is a cell debris free fraction of PRP (Tohidnezhad et al., 2011b). In vitro studies have shown that PRGF plays an important role in the detoxification of reactive oxygen species in cells via activation of Nrf2-ARE pathway (Tohidnezhad et al., 2011b, Tohidnezhad et al., 2012, Tohidnezhad et al., 2014).

The quality and the quantity of the alveolar bone around the implant affect successful osseointegration. There are various therapeutic challenges due to the alveolar ridge atrophy or sinus pneumatization. Autologous bone graft is considered the gold standard, but it includes many drawbacks, in particular donor site morbidity, the need for two surgical sites, and postoperative discomfort (Pasquali et al., 2015). Due to these reasons, bone augmentation techniques using biocompatible xenografts like Bio-Oss® are often considered an alternative in clinical practice and have been widely used during dental surgical procedures. The high osteoconductive properties and the slow resorption of their particles maintain the stability and the required volume for the new bone structure. It can be used to augment the missing bone or it can be inserted into the tooth extraction sockets to prevent the loss of alveolar bone in all three dimensions.

This source of growth factors may be able to reduce the detrimental effects of alcohol consumption during bone remodeling. To address this issue, we investigated a) the effect of PRGF, Bio-Oss® and a combination of both on osteoblasts and b) the osteoblast activity under the influence of ethanol in Bio-Oss® xenograft with and without PRGF in vitro.

We hypothesize that i) PRGF increases the osteoblasts cell viability and proliferation, ii) the combination of PRGF and Bio-Oss® improves these effects and increases ALP activity and iii) PRGF and the combination of PRGF and Bio-Oss ® increase the cell viability and ALP activity of ethanol impaired osteoblasts.

Section snippets

Preparation of human PRGF

The preparation of PRGF was first described in our previous study (Tohidnezhad et al., 2011b). PRGF was produced from liquid-preserved human platelet concentrates obtained by platelet apheresis that were not older than one day, in accordance with the current German ethics laws (EK116/10 local ethical board RWTH Aachen University). The 2.2 ml platelet concentrate of 9 × 109 per ml which contained less than 5 × 104 leukocytes was centrifuged at 2000 × g for 10 min. The pellet was washed twice using

Effects of PRGF and Bio-Oss® on osteoblasts cell viability

To measure the cell viability of osteoblasts, the Cell Titer-Blue® cell viability assay was run after 24 h treatment of cells with 0%, 5%, 10% PRGF, Bio-Oss® (Geistlich, Pharma AG, Wolhusen, Switzerland) and a combination of them. The results demonstrated that neither PRGF at different concentrations nor Bio-Oss® treatment alone had any cytotoxic effects compared to the untreated control cells (Fig. 1). In contrast, the treatment with PRGF, Bio-Oss® led to increased cell viability in SAOS-2

Discussion

Additional use of bio-supplements is becoming increasingly attractive in modern surgical procedures including cell protective effects. Platelet released growth factors (PRGF) is a refined supplement from our subject studies, which has been shown as an effective stimulant for cell protection involving the main cellular antioxidant Nrf2-ARE pathway (Tohidnezhad et al., 2011b, Tohidnezhad et al., 2012, Tohidnezhad et al., 2014). Another major advantage of PRGF is that it only involves growth

Conclusion

This study underscores the biological effectiveness of PRGF as a pure source of growth factors in difficult clinical cases like impaired bone healing in the presence of chronic alcohol consumption. These results suggest that the simultaneous use of Bio-Oss® and PRGF may be a valuable therapeutic option worth further investigation for adjunctive use in surgery in the oral and maxillofacial region.

Funding

No.

Declarations

Ethics approval and consent to participate: blood preparations were obtained from healthy anonymous human donors, in accordance with the current German ethics laws (EK116/10 local ethical board RWTH Aachen University).

Consent for publication

Not applicable.

Acknowledgements

The Bio-Oss® used in this study was sponsored by Geistlich Biomaterials (Baden-Baden). The study was supported by MSD SHARP & DOHME GmbH; by a grant from the Interdisciplinary Centre for Clinical Research (IZKF, T9-5 and T11-3); ERS, BOOST OPBF071 within the Faculty of Medicine at the RWTH University of Aachen.

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      The PRGF activation solution was prepared by adding thrombin (T4648-1KU: 100 U/ml resolved in 0.1% bovine serum albumin stored at −20 °C) and 50 mM calcium chloride (CaCl2) in a ratio of 1:9. Subsequently, the pellet was re-suspended in a total of 1.1 ml PRGF activation solution and culture medium in a ratio of 1:9 to prevent gelifying by the removal of fibrinogen and to achieve 8- to 10-fold platelet concentration (Sönmez et al., 2017; Tohidnezhad et al., 2017). It was shaken until there were no clots and then soaked in a warm bath (37 °C) for 30 min to improve thrombin activity.

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