Non-communicable diseases (NCDs) are a worldwide health issue because of their prevalence, negative impacts on human welfare, and economic costs. Protease enzymes play important roles in viral and NCD diseases. Slowing disease progression by inhibiting proteases using small-molecule inhibitors or endogenous inhibitory peptides appears to be crucial.

Osseointegration is vital to success in orthopedic and dental reconstructions with implanted materials. The bone matrix or cells—particularly osteoblasts—are required to achieve functional contact on the implant surface. Osteoblast induction is therefore essential for osteogenesis to occur. Enhancement of osteoblast adhesion, proliferation, and differentiation, particularly by implant surface modifications, have been found challenging to develop. Secretory Leukocyte Protease Inhibitor (SLPI), a cation ionic protein with anti-inflammatory and anti-bacterial activities, showed activation in osteoblast proliferation and differentiation. However, the effects of coating recombinant human (rh) SLPI on a titanium alloy surface on human osteoblast adhesion, proliferation, and differentiation has never been investigated. In this study, titanium alloys (Ti–6Al–4V) were coated with rhSLPI, while human osteoblast adhesion, proliferation, differentiation, actin cytoskeletal organization, and gene expressions involved in cell adhesion and differentiation were investigated. The results indicate that coating titanium with 10–100 µg/ml rhSLPI enhanced the physical properties of the Ti surface and enhanced human osteoblast (hFOB 1.19) cell adhesion, activated actin dynamic, enhanced adhesive forces, upregulated integrins α1, α2, and α5, enhanced cell proliferation, mineralization, alkaline phosphatase activity, and upregulated ALP, OCN, and Runx2. This is the first study to demonstrate that coating SLPI on titanium surfaces enhances osseointegration and could be a candidate molecule for surface modification in medical implants.

Titanium and its alloys are used in dental and orthopedic implants. However, long-term stability remains a clinical challenge. To overcome this limitation, surface modification has been investigated to improve surface properties. Our previous study demonstrated that the immobilization of secretory leukocyte protease inhibitor (SLPI) on the titanium surface promotes osteoblast adhesion, proliferation, and differentiation in vitro. The current study demonstrated the first in vivo evaluation of SLPI as a bioactive coating for medical implants. Grade 5 titanium screws were coated with 10 µg/mL of recombinant human SLPI (rhSLPI) for 24 h via simple physical adsorption, and the results were preliminarily validated via FE-SEM and ELISA. These SLPI-coated titanium screws (TiSs) were then placed in the tibia of Sprague–Dawley rats for 4 and 8 weeks. The hematological and biochemical parameters (BUN, Creatinine, AST, and Troponin I) demonstrated no acute systemic alterations within the 8-week period across all groups. Moreover, micro-computed tomography (micro-CT) and histological analysis revealed significantly higher bone volume fraction (%BV/TV) at 4 weeks compared to uncoated controls (20.64% ± 2.452% vs. 11.73% ± 0.524%). Finally, the biomechanical stability of implants, assessed using the removal torque test, showed that TiSs showed higher strength compared to Ti at both 4 and 8 weeks. In conclusion, this study represents a novel approach to transitioning rhSLPI-coated titanium evaluation from in vitro models to an in vivo rat model. rhSLPI surface functionalization enhances early-stage osseointegration and improves implant mechanical stability without acute hematological and biochemical alterations. These proof-of-concept findings suggest the potential of SLPI as a bioactive coating strategy.

Titanium (Ti) implants are the gold standard for orthopedic and dental applications, but their bioinert surfaces lack the osteoinductive properties. Combining alkaline etching for biomimetic surface topography and biofunctionalization with secretory leukocyte protease inhibitor (SLPI) has potential to promote osteogenic activities. Thus, this study is the aim to develops and characterizes a novel strategy to immobilize recombinant human SLPI (rhSLPI) onto alkaline-etched Ti (TiOH) surfaces via plasma polymerization. The grade 5 Ti and TiOH were polymerized utilizing a plasma-polymerized interlayer, followed by UV-graft polymerization of polyacrylic acid (PAAc) and EDC/NHS-mediated covalent coupling to immobilize 10 µg/mL rhSLPI (TiS and TiOHS, respectively) to immobilize 10 µg/mL rhSLPI (TiS and TiOHS, respectively). The ELISA and XPS confirmed the successful rhSLPI immobilization. Moreover, the SEM revealed the pillar-like morphology on the TIOH and TiOHS groups, whereas the Ti and TiS showed smooth surfaces, which is consistent with the profilometer. Additionally, the TiOH and rhSLPI-immobilized groups showed significantly higher hydrophilicity. Moreover, all sample types showed biocompatible to hFOB 1.19 cell. The TiOHS demonstrated highest cell adhesion, while TiS promoted highest cell proliferation. Alizarin red S (ARS) staining demonstrated TiOHS promoted the highest mineral deposition. In conclusion, the rhSLPI immobilization on alkaline-etched Ti via plasma polymerization, PAAc grafting and EDC/NHS covalent coupling. The combined platform (TiOHS) demonstrated enhanced physicochemical and biofunctional properties, resulting in promoted in vitro human osteoblast cell adhesion and differentiation.

Ischemic Heart Disease (IHD) is the main global cause of death. Previous studies indicated that recombinant human secretory leukocyte protease inhibitor (rhSLPI) exhibits a cardioprotective effect against myocardial ischaemia/reperfusion (I/R) injury. However, SLPI has a short half-life in vivo due to digestion by protease enzymes in circulation. The application of nanoparticle encapsulation could be beneficial for SLPI delivery. Several types of nanoparticles have been developed to encapsulate SLPI and applied in some disease models. However, silica nanoparticles for rhSLPI delivery, particularly on myocardial I/R injury, have never been studied. In this study, we aimed to fabricate gelatin-covered silica nanoparticles (GSNPs) to encapsulate rhSLPI and cardioprotective effect of GSNP-SLPI against an in vitro simulated ischaemia/reperfusion (sI/R). Silica dioxide nanoparticles (SNPs) were fabricated followed by incubation with 0.33 mg/mL of rhSLPI. Then, SNPs containing rhSLPI were coated with gelatin (GSNPs). The GSNPs and rhSLPI-GSNPs were characterized by particle size, zeta potential, and morphology scanning electron microscope (SEM). The concentration of rhSLPI in rhSLPI-GSNPs and drug release was determined by ELISA. Then, cytotoxicity and cardioprotective effect were determined by incubation of GSNPs or rhSLPI-GSNPs with rat cardiac myoblast cell line (H9c2) subjected to simulated ischaemia/reperfusion (sI/R). The results showed the particle size of SNPs, GSNPs, and rhSLPI-GSNPs was 273, 300, and 301 nm, with a zeta potential of −57.21, −22.40, and −24.50 mV, respectively. One milligram of rhSLPI-GSNPs contains 235 ng of rhSLPI. The rhSLPI-GSNPs showed no cytotoxicity on cardiac cells. Treatment with 10 μg/ml of rhSLPI-GSNPs could significantly reduce sI/R induced cardiac cell injury and death. In conclusion, this is the first study to show successful of fabricating novel rhSLPI-encapsulating gelatin-covered silica nanoparticles (rhSLPI-GSNPs) and the cardioprotective effects of rhSLPI-GSNPs against cardiac cell injury and death from myocardial ischaemia/reperfusion.