**1. Introduction**

Dental implants have been generally used as credible and secure treatments for the restoration of function and aesthetics of edentulous patients [1]. However, patients who have insufficient bone quality and quantity, or poor healing and regenerative capacities have been reported to experience unfavorable results after implant treatment [2]. To improve the success rate of these patients, it is important to increase the initial fixation of implant fixtures and to shorten the time required for the upper prosthesis to connect [3]. Recently, developments have focused on biomimetic treatment techniques based on applying biomolecules, such as bone morphogenetic protein (BMP) or platelet-derived growth factor (PDGF), to implant surfaces to address these problems [4–6].

BMP is a well-known growth factor that enhances bone regeneration by inducing the differentiation of mesenchymal stem cells to osteoblasts and promotes biosynthesis of bone matrix by regulating factors that are required for osteoinduction [7,8]. BMP-2, which is one of the 16 members of the BMP family, has been proven to be used in a variety of medical treatments by animal and clinical studies [4]. In particular, in one study an anodized titanium implant coated with recombinant human BMP-2 (rhBMP-2) produced by genetic recombination was found to be an effective carrier of rhBMP-2 [5]. However, several studies have reported that rhBMP-2 has no significant effect on bone formation [9,10]. These negative results were suggested to be due to large initial release of rhBMP-2, lack of standardization of the optimal rhBMP-2 concentration, and the use of only one type of growth factor, as natural regeneration process in man involves multiple growth factors [11–14].

Platelet-derived growth factor (PDGF), which is well-characterized tissue growth factor has been used in numerous in vivo and clinical studies [15–20], and has been shown to effectively promote bone, ligament, and cement regeneration in the periodontology field. [21,22]. PDGF is present in bone matrix and is secreted from platelets locally at fracture sites during initial fracture repair [23,24]. PDGF-BB is one of the five PDGF isoforms and is biologically the most potent and binds with greatest affinity to osteoblasts [6,25]. PDGF-BB has both mitogenic and chemotactic effects on osteoblasts and stimulates collagen I synthesis by osteoblasts [23]. It is also important for embryologic skeletal development, and when used topically, it can accelerate fracture healing in animals [26]. PDGF-BB has also been efficaciously used to treat osteoporosis in rodents, in which it improved trabecular bone strength and density [27].

Heparin is a natural linear polysaccharide and a highly sulfated glycosaminoglycan that binds strongly with various growth factors [28]. Biomaterial systems containing heparin exhibit controlled growth factor release [29,30]. When heparin was covalently grafted on anchored free amino positive groups on titanium surfaces, the primary amine groups of growth factors, such as BMP-2 or PDGF-BB, were found to bind to the carboxyl groups of bound heparin [31]. In a previous study, we suggested PDGF-BB/Hepa-Ti system exhibited promising potentials for the enhancements the functions of osteoblast [32]. Also in another previous study on PDGF-BB and BMP-2 co-delivery system on Hep-Ti substrates, the co-delivery system positively promoted functions of osteoblasts [6]. Many experiments have been performed on heparin and growth factor combinations in attempts to induce proper growth factor release, but these experiments were performed at the cellular level or under conditions too far removed from clinical situations.

Therefore, the purpose of this study was to confirm the effects of rhPDGF-BB and rhBMP-2 co-delivery in large animals using clinically reproducible conditions. rhPDGF-BB or/and rhBMP-2 were immobilized onto the surfaces of heparinized-Ti implants and inserted into open defects in beagle dog models. Histomorphometric analysis was conducted to evaluate the effect of rhPDGF-BB, rhBMP-2, and rhPDGF-BB/rhBMP-2 implants on osseointegration and bone regeneration.
