Assessment of stability of surface anchors for antibacterial coatings and immobilized growth factors on titanium.

Titanium (Ti) has been functionalized with biomolecules for biomedical purposes. However, there is very limited information on the stability of such functionalities. Ti surface functionalized with carboxymethyl chitosan (CMCS) and bone morphogenetic protein 2 (BMP-2) has been reported to inhibit bacterial colonization while at the same time enhances osteoblast functions. In this work, three types of anchoring molecules, (3-aminopropyl) triethoxysilane (Silane), dopamine (DA), and polydopamine (PDA), were used for immobilizing the CMCS on Ti. The CMCS-modified surfaces were subjected to 70% ethanol treatment, autoclaving, and prolonged immersion in phosphate buffered saline (PBS). After the treatment procedures, the ability of the CMCS-modified substrates to inhibit colonization by Staphylococcus epidermidis (S. epidermidis) was assessed to evaluate the stability of the immobilized CMCS. The bacterial adhesion assays showed that the CMCS-DA- and CMCS-PDA-modified Ti remained stable after 70% ethanol treatment, autoclaving, and prolonged immersion in PBS, whereas the CMCS-Silane-modified Ti was less stable after autoclaving and prolonged immersion in PBS. The CMCS-DA- and CMCS-PDA-modified Ti substrates were functionalized with BMP-2 and used to support osteoblast growth. Evaluation of alkaline phosphatase (ALP) activity and calcium deposition from osteoblasts cultured on these substrates, which have been treated with 70% ethanol, or subjected to autoclaving, and prolonged immersion in PBS indicated that the immobilized BMP-2 on these surfaces retained its bioactivity.

An in vitro assessment of fibroblast and osteoblast response to alendronate-modified titanium and the potential for decreasing fibrous encapsulation.

Fibrous encapsulation can impair implant osseointegration and cause implant failure but currently there are limited strategies to address this problem. Since bisphosphonates (BPs), a class of drugs widely used to treat bone diseases, was recently found to induce fibroblast apoptosis, we hypothesize that by loading BPs on titanium (Ti) implant surface, fibrous encapsulation may be inhibited with simultaneous enhancement of implant osseointegration. This strategy of local administration can also be expected to minimize the adverse side effects of BPs, which are associated with intravenous injections. To verify this hypothesis, alendronate was loaded on Ti surface via a hydroxyapatite (CaP) coating, and the effects of the loaded alendronate on fibroblast proliferation and apoptosis, and osteoblast proliferation, alkaline phosphatase (ALP) activity, and apoptosis were investigated in vitro. With a surface density of loaded alendronate 0.046 mg/cm(2) or higher, fibroblast proliferation was suppressed due to increased apoptosis, while osteoblast proliferation and ALP activity increased with minimal apoptosis. In a coculture of fibroblasts and osteoblasts in a 1:1 ratio, ~60% of the cells on these alendronate-loaded substrates were osteoblasts 1 day after cell seeding. The percentage of osteoblasts increased to about 75% 4 days after cell seeding. These results suggest that fibroblasts and osteoblasts respond differently toward the alendronate-modified substrates, and this phenomenon can potentially be capitalized to reduce fibrous encapsulation.

An in vitro assessment of titanium functionalized with polysaccharides conjugated with vascular endothelial growth factor for enhanced osseointegration and inhibition of bacterial adhesion.

The long-term success of orthopedic implants may be compromised by defective osseointegration and bacterial infection. An effective approach to minimize implant failure would be to modify the surface of the implant to make it habitable for bone-forming cells and anti-infective at the same time. In this in vitro study, the surfaces of titanium (Ti) substrates were functionalized by first covalently grafting either dopamine followed by carboxymethyl chitosan (CMCS) or hyaluronic acid-catechol (HAC). Vascular endothelial growth factor (VEGF) was then conjugated to the polysaccharide-grafted surface. Antibacterial assay with Staphylococcus aureus (S. aureus) showed that the polysaccharide-modified substrates significantly decrease bacterial adhesion. The CMCS-functionalized Ti demonstrated better antibacterial property than the HAC-functionalized Ti since CMCS is bactericidal while HA only inhibits the adhesion of bacteria without killing them. Osteoblast attachment, as well as alkaline phosphatase (ALP) activity and calcium deposition were enhanced by the immobilized VEGF on the polysaccharide-grafted Ti. Thus, Ti substrates modified with polysaccharides conjugated with VEGF can promote osteoblast functions and concurrently reduce bacterial adhesion. Since VEGF is also known to enhance angiogenesis, the VEGF-polysaccharide functionalized substrates will have promising applications in the orthopedic field.