Peptide-Enriched Silk Fibroin Sponge and Trabecular Titanium Composites to Enhance Bone Ingrowth of Prosthetic Implants in an Ovine Model of Bone Gaps

Abstract

Osteoarthritis frequently requires arthroplasty. Cementless implants are widely used in clinics to replace damaged cartilage or missing bone tissue. In cementless arthroplasty, the risk of aseptic loosening strictly depends on implant stability and bone-implant interface, which are fundamental to guarantee the long-term success of the implant. Ameliorating the features of prosthetic materials, including their porosity and/or geometry, and identifying osteoconductive and/or osteoinductive coatings of implant surfaces are the main strategies to enhance the bone-implant contact surface area. Herein, the development of a novel composite consisting in the association of macro-porous trabecular titanium with silk fibroin (SF) sponges enriched with anionic fibroin-derived polypeptides is described. This composite is applied to improve early bone ingrowth into the implant mesh in a sheep model of bone defects. The composite enables to nucleate carbonated hydroxyapatite and accelerates the osteoblastic differentiation of resident cells, inducing an outward bone growth, a feature that can be particularly relevant when applying these implants in the case of poor osseointegration. Moreover, the osteoconductive properties of peptide-enriched SF sponges support an inward bone deposition from the native bone towards the implants. This technology can be exploited to improve the biological functionality of various prosthetic materials in terms of early bone fixation and prevention of aseptic loosening in prosthetic surgery.

Keywords: bone; osseointegration; prosthetic implant; silk fibroin; titanium.

FIGURE 1

Implant design. (A) trabecular titanium (TT) implant dimensions with 3 and 1 mm concentric gap models. (B) TT implant incorporated into the peptide-enriched silk fibroin sponge [TT+ silk fibroin (SF)/Cs].

FIGURE 2

Physical properties of silk fibroin (SF)/Cs sponges. (A) attenuated total reflection (ATR)-fourier transform infrared (FTIR) spectra, and (B) differential scanning calorimetry (DSC) thermograms of SF sponges without Cs peptides and with different amounts of peptides (5, 10, and 25% w/w).

FIGURE 3

Cross-section morphology of silk fibroin (SF) sponges by scanning electron microscopy (SEM) analysis. (A) SF sponge without peptides. Magnification 120× (scale bar 500 μm) (B–D). SF sponges enriched with 5, 10, and 25% w/w Cs peptides, respectively. Magnification 100× (scale bar 500 μm).

FIGURE 4

Viability assays. (A) MTT assay on NIH-3T3 fibroblasts indirectly cultured with silk fibroin (SF) sponges without peptides (0%), with different concentrations of peptides (Cs 2.5, 5, 7.5, and 10% w/w) or cultured in fresh medium as a negative control (NC). The positive control (PC) represents cells treated with Triton-X100. Significance: *p < 0.05; **p < 0.01; a, ***p < 0.001. (B) Live&Dead assay of NIH-3T3 fibroblasts directly cultured onto SF sponges without peptides (0%), with different concentrations of peptides (Cs 7.5 and 10%).

FIGURE 5

Trabecular titanium (TT) and TT+silk fibroin (SF)/Cs implants. (A) scanning electron microscopy (SEM) image of the trabecular structure of the TT device before impregnation with the SF/Cs sponge. Magnification 50× (scale bar 1 mm). (B–D) Optical Microscopy images at increasing magnification 4×, 10×, 40×, respectively (scale bars: 5, 2.5, and 1 mm, respectively) of the TT+SF/Cs implant device where the surface spongy material was removed. The trabecular section of the device is fully infiltrated by the spongy material. No macroscopic voids are visible.

FIGURE 6

Surgical procedure and fluoroscopic imaging. (A) Creation of the bone defect (11 mm Ø × 20 mm h); trabecular titanium (TT) and TT+ silk fibroin (SF)/Cs implants fixation within the defects by press fit. (B) Representative lateral and oblique fluoroscopic projections of implanted tibia and femur.

FIGURE 7

Histological and histomorphometric analyses of trabecular titanium (TT) and TT+ silk fibroin (SF)/Cs implants in femoral and tibial sites. (A) Overview of one representative section per group. Two months after surgery, new bone matrix in variable stages of mineralization (stained in blue) have surrounded and enclosed the TT+SF/Cs implant, mostly in femoral sites within both gaps. Toluidine Blue and Pyronin Yellow staining. (B) Histograms reporting the tissue implant contact (TIC) as the percentage of newly formed tissue in contact with the implant surface. *p < 0.05. Black colored circles refer to TT samples, Black colored square shapes refer to TT+SF/Cs.

FIGURE 8

Qualitative and quantitative histology. (A) Representative histological sections reporting the stages of the newly bone matrix organization: the low mineralized tissue (osteoid and woven bone) is stained in blue, the more mineralized tissue (mature bone) is stained in purple/brown. Osteoid matrix presents as a three-dimensional network of collagen bundles, while the woven bone is characterized by a compact structure of collagen fibers resembling primitive trabeculae not yet completely mineralized. The lamellar bone is formed by islands of mineralized interconnected by bridges of not mineralized bone matrix. Toluidine Blue and Pyronin Yellow staining. Total magnification 100×. (B) Histograms report the semi-quantitative histomorphometric analysis of the three different stage of bone matrix organization: osteoid matrix, woven bone, lamellar bone. *p < 0.05. Black colored circles refer to TT samples, Black colored square shapes refer to TT+SF/Cs.

FIGURE 9

Backscattered electron microscopy (BSE)-scanning electron microscopy (SEM) analyses of trabecular titanium (TT) and TT+ silk fibroin (SF)/Cs implants in femoral and tibial sites. (A) Overview of one representative section per group. Two months after surgery, calcified bone/hydroxyapatite crystals in gray are present in the area that surrounded the TT+SF/Cs implant trabeculae (in white), mostly in femoral sites within both gaps. (B) Histograms reporting the bone implant contact (BIC) as the percentage of newly calcified bone in contact with the implant surface. *p < 0.05. Black colored circles refer to TT samples, Black colored square shapes refer to TT+SF/Cs.

FIGURE 10

Inflammatory reaction in trabecular titanium (TT)+silk fibroin (SF)/Cs sites, and histograms. (A) Localization of the inflammatory infiltrate within the core of the samples (red box). (B) Subacute reaction with various inflammatory cells (granulocytes, plasma cells, and lymphocytes) around blood vessels. (C) Osteoblast- and osteoclast-like cells lining along new bone trabeculae (red boxes). (D) Multinucleated giant cells (red arrow) in the proximity of the remodeling site. Toluidine Blue and Pyronin Yellow, magnification 40× (A), 400× (B,C), 600× (D). (E) Semi-quantitative analysis of the inflammatory infiltrates, *p < 0.05. Black colored circles refer to TT samples, Black colored square shapes refer to TT+SF/Cs.