Ultrastructural and immunohistochemical evaluation of hyperplastic soft tissues surrounding dental implants in fibular jaws

Abstract

In reconstructive surgery, complications post-fibula free flap (FFF) reconstruction, notably peri-implant hyperplasia, are significant yet understudied. This study analyzed peri-implant hyperplastic tissue surrounding FFF, alongside peri-implantitis and foreign body granulation (FBG) tissues from patients treated at the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital. Using light microscopy, pseudoepitheliomatous hyperplasia, anucleate and pyknotic prickle cells, and excessive collagen deposition were observed in FFF hyperplastic tissue. Ultrastructural analyses revealed abnormal structures, including hemidesmosome dilation, bacterial invasion, and endoplasmic reticulum (ER) swelling. In immunohistochemical analysis, unfolded protein-response markers ATF6, PERK, XBP1, inflammatory marker NFκB, necroptosis marker MLKL, apoptosis marker GADD153, autophagy marker LC3, epithelial-mesenchymal transition, and angiogenesis markers were expressed variably in hyperplastic tissue surrounding FFF implants, peri-implantitis, and FBG tissues. NFκB expression was higher in peri-implantitis and FBG tissues compared to hyperplastic tissue surrounding FFF implants. PERK expression exceeded XBP1 significantly in FFF hyperplastic tissue, while expression levels of PERK, XBP1, and ATF6 were not significantly different in peri-implantitis and FBG tissues. These findings provide valuable insights into the interconnected roles of ER stress, necroptosis, apoptosis, and angiogenesis in the pathogenesis of oral pathologies, offering a foundation for innovative strategies in dental implant rehabilitation management and prevention.

Keywords: Endoplasmic reticulum stress; Fibula free flap; Foreign body granulation; Hyperplastic tissue; Implant; Unfolded protein response.

Figure 1

The flow of the current study: specimen collection and selection and preparation for histopathological, ultrastructural, and immunohistochemistry (IHC) analyses. Abbreviations: GVT, gingivectomy; H&E, hematoxylin–eosin; SEM, scanning electron microscope; TEM, transmission electron microscope. Antibodies abbreviations: *αSMA, α-smooth muscle actin; ATF6, activating transcription factor 6; E-cadherin, epithelial cadherin; GADD153, growth arrest-and DNA damage-inducible gene 153; HIF-1, hypoxia-inducible factor 1; IL-8, interleukin 8; LC3, microtubule-associated protein 1 light chain 3; MLKL. mixed lineage kinase domain-like protein; NFκB, nuclear factor kappa-light-chain-enhance of activated B cells; PERK, protein kinase RNA activated (PKR-like ER kinase); TGFβ, transforming growth factor beta; XBP1, xbox-binding protein 1.

Figure 2

Histology findings from peri-implant hyperplastic tissue. The stratum spinosum in specimen A-2 was filled by pyknotic and anucleated prickle cells (A). Keratin granules were observed in the B-1 specimen (B; arrows). Pseudo-epitheliomatous hyperplasia of the epidermis on the Ce-1 slide (C). Abundant inflammatory cells on slide D-3 (D). Vellus hair without a functional structure was observed on slide F-1 (E). Collagen fibers and abundant blood vessels on slide H-4 (F).

Figure 3

SEM findings from the D-1 specimen revealed stratum spinosum consisting of mixed anucleated and nucleated prickle cells (a1). Mast cells, eosinophils, and lymphocytes were observed in D-1 (a2). Abundant neutrophils were observed in the D-2 specimen (b1), with bacterial invasion in the cell (b2). The D-3 specimen showed metaplasia of prickle cells into cuboidal cells (c1), and proto-myofibroblasts were observed in the reticular dermis (c2). A multinucleated giant cell was observed in D-2, and the energy dispersive x-ray revealed components of titanium, aluminum, zirconium, and zinc (d).

Figure 4

Fibroblast cells with early signs of ERS. The continuation of the ER can still be seen, although swelling and fragmentation of the ER are beginning to be visible (a1-2). Swelling, fragmentation, and vacuolization of the ER were observed in fibroblasts (b1-2; representative images of group A, d1-2; representative images of group D, e3–5; representative images of group E, f1-2; representative images of group F, g3-4; representative images of group G) and prickle cells (c1-2; representative images of group C, e1-2; representative images of group E, g1-2; representative images of group G) from each group A, C, D, E, F, G. In the TEM examination, severely dilated ER cisternae were observed (d5, d6).

Figure 5

The IHC for UPR.