Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival soft tissue and alveolar bone following tooth extraction. For target identification and validation, hard and soft tissue were extracted from mini-pigs at the indicated times after tooth extraction. From triplicate experiments, 56 proteins in soft tissue and 27 proteins in alveolar bone were found to be differentially expressed before and after tooth extraction. The expression of 21 of those proteins was altered in both soft tissue and bone. Comparison of the activated networks in soft tissue and alveolar bone highlighted their distinct responsibilities in bone and tissue healing. Moreover, we found that there is crosstalk between identified proteins in soft tissue and alveolar bone with respect to cellular assembly, organization, and communication. Among these proteins, we examined in detail the expression patterns and associated networks of ATP5B and fibronectin 1. ATP5B is involved in nucleic acid metabolism, small molecule biochemistry, and neurological disease, and fibronectin 1 is involved in cellular assembly, organization, and maintenance. Collectively, our findings indicate that bone regeneration is accompanied by a profound interaction among networks regulating cellular resources, and they provide novel insight into the molecular mechanisms involved in the healing of periodontal tissue after tooth extraction.Healthy dental gingival tissue and the alveolar bone that surrounds the teeth are essential for the proper function of teeth, as well as for a good appearance and good general health. Socket healing after tooth extraction is a useful experimental model for investigating the communication between gingival tissue and alveolar bone after tooth extraction. Preservation of the alveolar socket after tooth extraction requires the formation of a biological connection between the living and osseous tissue, which has to be created during the healing process. The success of such dental remodeling is dependent on the establishment of a soft tissue barrier that is able to shelter the underlying osseous structures and the osseo-integration of the soft tissue surrounding the alveolar bone. Understanding the processes governing soft and hard tissue healing and maintenance around the alveolar socket is paramount for oral health.Several studies have reported significant structural changes and bone reabsorption in fresh sockets following tooth extraction, with important dimensional changes in the surrounding alveolar bone (1–3). A reduction of alveolar bone may present problems after tooth extraction, especially in aged individuals in whom bone volume is important for both physiological and medical reasons. Although it has been shown that reduction defects in alveolar bone can be completely repaired using surgical techniques such as guided bone regeneration (4, 5), bone autograft, bone allograft, and xenograft (6, 7), these techniques are not broadly applicable (8). However, the introduction of biomimetic agents such as enamel matrix derivatives (9), platelet-rich plasma (10), platelet-derived growth factor (11, 12), and bone morphogenic proteins (BMPs)¹ (9) promises potentially better outcomes with bone regeneration treatments, although their efficacy remains controversial.The proteins present in bone are essential for all of the life processes ongoing in bone, and they are the most important final products of the homeostatic signaling pathways. Profiling those proteins is vital for a thorough understanding of bone biology. To date, proteome research on bone has been focused mainly on in vitro analysis of bone-forming cells (osteoblasts and osteoclasts) to determine which proteins are expressed under a given set of experimental conditions (13–16). Although important, such studies cannot identify the actual protein profile in oral alveolar bone. Recently, the extraction of proteins directly from skull bone for proteome analysis was reported (17, 18). The extracted proteins were first separated using two-dimensional gel electrophoresis, after which spots of interest were excised and the proteins were identified via mass spectrometry (MS). However, using two-dimensional gel electrophoresis to analyze extreme proteins (e.g. extremely basic or acidic, extremely small or large, extremely hydrophobic) is challenging. Shotgun proteomics, which is a method of high-throughput proteome analysis (19–21), avoids the intrinsic limitations of two-dimensional gel electrophoresis. Despite an interesting need for large-scale characterization of the bone proteome, one study has been reported to apply shotgun proteomics for proteome analysis of rat femur bone (22). However, they identified only 133 proteins, because they analyzed bone proteins using a one-step method without a demineralization stage. The other report showed only that bone proteins extracted from the skull bone of an adult beagle are carried using a demineralization step (23). There are no reports regarding the interaction between alveolar bone and soft tissue yet.The efficient extraction of bone proteins is a critical issue for proteome analysis (24). Because bone is largely mineralized, and therefore nearly solid, classical protein extraction methods used for soft tissues and cells may not be appropriate for bone. It is therefore necessary to develop methods to efficiently extract protein from bone. In earlier bone proteome analyses (17, 18, 22), the bones were first ground to powder, after which the proteins were extracted by means of incubating the powder in lysis buffer. However, mechanically breaking bones down into powder is laborious, especially for large animal bones. More important, large amounts of collagen and proteoglycans also are extracted, and this can impair the detection of low-abundance proteins and strongly affect isoelectric focusing (25). For the present study, we adopted an alternative method of demineralizing bone tissue and then investigated the efficiency of protein extraction from the demineralized bone tissue. This method was based on a recently reported sequential protein extraction protocol that was used to extract proteins from skull for comprehensive analysis of its proteome. Two-dimensional high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) was then applied to analyze the protein extracts, enabling the identification of 2479 proteins (23). We employed a similar method to extract and identify proteins in tooth alveolar bone.Given that a large number of proteins are likely involved in the healing of bone, as well as of soft tissues, another goal of the present study was to examine protein expression and putative signaling during bone healing after tooth extraction. Here, we used nano-UPLC-MS^E-based label-free quantitative proteomics to analyze alveolar bone and the adjacent soft tissue. The environment surrounding healing bone would be expected to affect the specific signaling networks involved in bone regeneration. We suggest that determining the protein networks in alveolar bone and gingival tissue will enable improvement of the soft tissue interface, aspects of the hard tissue, and dental appearance during and after therapy.