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, 14, 3831-3843
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Effect of Mussel Adhesive Protein Coating on Osteogenesis in Vitro and Osteointegration in Vivo to Alkali-Treated Titanium With Nanonetwork Structures

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Effect of Mussel Adhesive Protein Coating on Osteogenesis in Vitro and Osteointegration in Vivo to Alkali-Treated Titanium With Nanonetwork Structures

Derong Yin et al. Int J Nanomedicine.

Abstract

Purpose: On the basis of reasonable superposition of various surface treatment methods, alkali-treated titanium with nanonetwork structures (TNS) was coated with mussel adhesive protein (MAP) and named TNS-MAP. The aims were to optimize the biological properties of TNS, endue it with new properties, and enhance its utility in clinical dental applications. Methods: TNS disks were coated with MAP and the product surface was characterized. Its osteogenic properties were determined by evaluating its effects on cell adhesion, cell proliferation, the expression of osteogenesis-related genes, and in vivo experiments. Results: The treated materials showed excellent hydrophilicity, good surface roughness, and advantages of both TNS and MAP. TNS-MAP significantly promoted initial cell attachment especially after 15 mins and 30 mins. At every time point, cell adhesion and proliferation, the detection rate of osteogenesis-related markers in the extracellular matrix, and the expression of osteogenesis-related genes were markedly superior on TNS-MAP than the control. The in vivo experiments revealed that TNS-MAP promoted new bone growth around the implants and the bone-implant interface. Conclusion: We verified through in vitro and in vivo experiments that we successfully created an effective TNS-MAP composite implant with excellent biocompatibility and advantages of both its TNS and MAP parent materials. Therefore, the new biocomposite implant material TNS-MAP may potentially serve in practical dentistry and orthopedics.

Keywords: biocomposite; bone marrow mesenchymal stem cell; extracellular matrix; nanopore; polydopamine.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 8
Figure 8
In vivo experimental operation. Notes: (A) Incision and exposure of surgical area, (B) implantation model formation, (C) implant embedment, and (D) suture of surgical area; bar =1 cm.
Figure 1
Figure 1
SEM micrographs and AFM images of TNS and TNS-MAP.Notes: (AC) TNS surface, and (DF) TNS-MAP surface. Abbreviations: SEM, scanning electron microscopy; AFM, atomic force microscopy; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 2
Figure 2
Comparison of contact angle measurements for pure Ti, TNS, and TNS-MAP.Notes: Contact angles of Ti, TNS, and TNS-MAP were measured with a VSA2500 XE contact angle measurement system after application of 2 µL ddH2O to the sample surface at room temperature (***P<0.001; *P<0.05). Abbreviations: Ti, titanium; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 3
Figure 3
Surface chemical compositions of specimens examined by XPS. Abbreviations: XPS, X-ray photoelectron spectroscopy; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 4
Figure 4
FTIR analysis of TNS and TNS-MAP.Notes:(A) cm−1 from 1,000 to 4,000, and (B) cm−1 from 1,350 to 1,750. Abbreviations: FTIR, Fourier transform infrared spectroscopy; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 5
Figure 5
Cell adhesion, proliferation, and morphological analysis of rBMMSCs on sample disks.Notes: (A and B) TNS and TNS-MAP disks were incubated with rBMMSCs and cell adhesion and proliferation were evaluated after 15 mins, 30 mins, 1 day, 3 days and 7days, respectively, with a CellTiter-Blue® Cell Viability Assay (Promega, Madison, WI, USA), (C and D) TNS and TNS-MAP disks were incubated with rBMMSCs for 6 hrs, stained with phalloidin (F-actin) and DAPI (nuclei), and visualized by fluorescence microscopy; bar =100 μm. (***P<0.001; **P<0.01). Abbreviations: rBMMSCs, rat bone marrow mesenchymal stem cells; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 6
Figure 6
ALP activity and calcium deposition in cells grown on sample disks.Notes: rBMMSCs were cultivated on TNS and TNS-MAP disks for up to 28 days and the levels of (A) ALP activity (7 days and 14 days) and (B) calcium deposition (21 days and 28 days) were evaluated as described in the “Materials and Methods” (***P<0.001; **P<0.01; *P<0.05). Abbreviations: ALP, alkaline phosphatase; rBMMSCs, rat bone marrow mesenchymal stem cells; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 7
Figure 7
Expression of osteogenesis-related genes in cells grown on sample disks.Notes: (A–D): Expression levels of genes encoding Runx2, ALP, Bglap, and BMP-2 were evaluated in rBMMSCs cultivated on TNS and TNS-MAP disks at days 3 and 7 and days 14 and 21, respectively, by real-time RT-PCR (***P<0.001; **P<0.01; *P<0.05). Abbreviations: Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; Bglap, bone γ-carboxyglutamate (gla) protein; BMP-2, bone morphogenetic protein 2; rBMMSCs, rat bone marrow mesenchymal stem cells; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 9
Figure 9
Transverse micro-CT reconstructed images of proximal tibiae showing ROI status.Notes: Implant (red), newly formed bone with relatively low density (kelly green), and cortical bone with high density (blue). (A) 8-week TNS group, and (B) 8-week TNS-MAP group; bar =2 mm. Abbreviations: micro-CT, micro-computed tomography; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein; ROI, region of interest.
Figure 10
Figure 10
Micro-CT quantitative evaluation within the ROI.Notes: (A) Bone volume fraction of the two groups, (B) Tb.N of the two groups, (C) Tb.Th of the two groups, and (D) Tb.Sp of the two groups (***P<0.001; **P<0.01). Abbreviations: micro-CT, micro-computed tomography; ROI, region of interest; BV/TV, bone volume/total volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 11
Figure 11
Histological sections with Villanueva staining showing bone tissue morphology around the implant (black).Notes: (A) TNS surface, and (B) TNS-MAP surface; bar =200 μm (***P<0.001; **P<0.01). Abbreviations: TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.
Figure 12
Figure 12
Quantitative histomorphometric analysis within the region of measurement (BA and BIC).Notes: (A) Percentage of new bone formation (BA) and (B) percentage of direct BIC (***P<0.001; *P<0.05). Abbreviations: BA, bone area ratio; BIC, bone–implant contact; TNS, titanium with nanonetwork structures; TNS-MAP, titanium with nanonetwork structures coated with mussel adhesive protein.

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