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. 2022 Aug 2:9:rbac054.
doi: 10.1093/rb/rbac054. eCollection 2022.

Laminin 332-functionalized coating to regulate the behavior of keratinocytes and gingival mesenchymal stem cells to enhance implant soft tissue sealing

Lipeng Liu  1 , Jing Wang  1 , Ying Li  2 , Bing Liu  2 , Wei Zhang  1 , Weikang An  1 , Qing Wang  1 , Boya Xu  1 , Lingzhou Zhao  2 , Chufan Ma  1   2
Affiliations

Laminin 332-functionalized coating to regulate the behavior of keratinocytes and gingival mesenchymal stem cells to enhance implant soft tissue sealing

Lipeng Liu et al. Regen Biomater. .

Abstract

Peri-implant epithelial sealing is the first line of defense against external pathogens or stimuli; hence, an essential process to prevent peri-implantitis. Laminin 332 (LN332) is the main component of the internal basal lamina and participates in peri-implant epithelial sealing by forming hemidesmosomes (HDs) with integrin α6β4. In this work, poly (D, L-lactide) (PDLLA)-LN332 composite coating was successfully constructed by a method similar to layer-by-layer assembly, displaying staged LN332 release for as long as 28 days. The PDLLA-LN332 composite coating can activate the intracellular PI3K-Akt pathway via binding to cellular integrin α6β4, which can promote adhesion, migration and proliferation of HaCaT cells and further enhance the expression of keratinocyte HD-related molecules, including integrin α6β4, LN332 and plectin. Furthermore, the PDLLA-LN332 composite coating can promote the adhesion, spreading and proliferation of gingival mesenchymal stem cells and accelerate their epithelial differentiation. Therefore, the PDLLA-LN332 composite coating can enhance implant soft tissue sealing, warranting further in vivo study.

Keywords: PDLLA; gingival mesenchymal stem cells; keratinocytes; laminin 332; soft tissue sealing.

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Figures

None
Graphical abstract
Figure 1.
Figure 1.
Implant surface characterizations of different Ti plates. (A) FE-SEM observation of Ti, TiP and TiPLN. (B) Phase contrast and 3D of surface height fluctuations of AFM scanning images (5 μm × 5 μm) of Ti, TiP and TiPLN. (C) XPS survey spectra of Ti, TiP and TiPLN samples. (D) The peak fitting of high-resolution C1s for TiP and TiPLN surfaces. (E) High-resolution T2p for Ti, TiP and TiPLN substrates. (F) High-resolution N1s for Ti, TiP and TiPLN substrates. (G) FTIR spectra of Ti, TiP and TiPLN substrates. (H) Water contact angle measurement of Ti, TiP and TiPLN. (I) The cumulative release rate of preloaded LN332 from TiPLN in PBS (pH 7.4) at 37°C for 28 days (***P < 0.001).
Figure 2.
Figure 2.
The morphology, adhesion, migration and proliferation of HaCaT cells. (A) SEM morphology of HaCaT cells on different substrates for 6, 12 and 24 h; (B) CLSM images of the stained cytoskeleton of HaCaT cells on different substrates for 2, 6 and 24 h. (C) Representative nuclei staining (D) and quantitative counting of adherent cells on different substrates for 6 and 24 h incubation. (E) CLSM images of the stained cytoskeleton (F) and relative migration rate of HaCaT cells on different substrates in the scratch-wound healing assay. (G) Cell growth curves of CCK8 assay for HaCaT cells on different substrates (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 3.
Figure 3.
Immunofluorescence staining and semi-quantification. Immunofluorescence staining for (A) integrin α6, (B) LN332, (C) integrin β4 and (D) plectin in HaCaT cells cultured for 48 h on different substrates. (E) Relative fluorescence intensity per cell of integrin α6, LN332, integrin β4 and plectin of HaCaT cells (***P < 0.001).
Figure 4.
Figure 4.
Western blot analysis and semi-quantification. (A) Western blot analysis and semi-quantification of integrin α6, laminin332, integrin β4 and plectin of HaCaT cells on different substrates at 3 days. (B) Western blot analysis and semi-quantification of integrin α6, laminin332, integrin β4 and plectin of HaCaT cells on different substrates at 7 days. (C) Western blot analysis and semi-quantification of pPI3K, PI3K, pAkt and Akt of HaCaT cells of different substrates (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 5.
Figure 5.
Inhibition of the PI3K pathway (PI3K inhibitor LY294002, 50 μM). (A) Western blot analysis and semi-quantification of pPI3K, pAkt and integrin β4 of HaCaT cells on TiPLN and TiPLN+LY. (B) CLSM images of the stained cytoskeleton of HaCaT cells on TiPLN and TiPLN+LY at 24 h. (C) Representative nuclei staining and quantitative counting of adherent cells on TiPLN and TiPLN+LY at 24 h. (D) CLSM images of the stained cytoskeleton and relative migration rate of HaCaT cells on TiPLN and TiPLN+LY in the scratch-wound healing assay at 24 h. (E) Cell growth curves of CCK8 assays on TiPLN and TiPLN+LY (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 6.
Figure 6.
The effects of PDLLA-LN332 composite coating on the behavior of HaCaT cells in terms of the PI3K-Akt signal pathway.
Figure 7.
Figure 7.
GMSCs characterization. (A) Expression of CD14, CD34, CD45, CD44, CD90 and CD105 was assessed by flow cytometry. (B) The osteogenesis potential was examined using ALP staining. (C) The osteogenesis potential was examined using alizarin red staining. (D) The adipogenesis was assessed with oil red O staining (Bar =100 μm).
Figure 8.
Figure 8.
The morphology, adhesion, migration and proliferation of GMSCs. (A) SEM morphology of GMSCs cells on different substrates for 6, 12 and 24 h. (B) CLSM images of the stained cytoskeleton of GMSCs on different samples for 2, 6 and 24 h. (C) Representative staining of nuclei and (D) quantitative counting of adherent cells on different substrates after 6 and 24 h of incubation. (E) Cell growth curves by CCK8 assays for GMSCs on different substrates (*P < 0.05; **P < 0.01).
Figure 9.
Figure 9.
Western blot analysis and semi-quantification of GMSCs. (A) Western blot analysis and semi-quantification of CK14, CK18 and CK19 of GMSCs on Ti, TiP and TiPLN substrates at 3 days. (B) Western blot analysis and semi-quantification of CK14, CK18 and CK19 of GMSCs on Ti, TiP and TiPLN substrates at 7 days (**P < 0.01; ***P < 0.001).
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