Nitric Oxide Releasing Bionanomatrix Coating for Percutaneous Osseointegrated Prostheses

Introduction: : 1.6 million people in the US live with an amputation. Amputations occur for many different reasons in the population including peripheral artery disease, injury, tumors, or infection. Percutaneous osseointegrated prostheses (POP) are a relatively recent and promising advancement in prosthetic technology, allowing direct skeletal attachment of the prosthesis. However, several challenges remain, particularly in achieving successful osseointegration, minimizing inflammation, and promoting vascularization at the implant site. To date, no prosthetic system has effectively enhanced both osseointegration and vascularization without increasing inflammation. Our approach seeks to address this challenge through the use of a nitric oxide (NO)-releasing nanomatrix. NO is known for its osteogenic, angiogenic, and anti-inflammatory properties. Our study has demonstrated that NO can promote angiogenesis while simultaneously reducing inflammation. We hypothesize that incorporating NO into the nanomatrix will enhance both osseointegration and vascularization while mitigating the inflammatory response commonly associated with POP implants.

Materials and

Methods: : The nanomatrix is made of NO releasing self-assembled peptide amphiphiles (PA). These PAs have the effects of highly controlled NO release, enzyme mediated degradation, high biocompatibility, and self-assembled multilayered nanofibrillar coating with stable and uniform structure without needing to be in organic solvents. The NO releasing nanomatrix will be made of two constituent parts: PA with cell adhesive ligands (RGDS) that will help promote osseointegration by recruiting human mesenchymal stem cells (hMSCs) and a second PA with the NO releasing donor group (KKKKK) that will help with vascularization and anti-inflammatory responses. The nanomatrix has been tested in vitro and in vivo. The in vitro study involved assessing the proliferation of hMSCs and U937s when cultured on top of nanomatrix coated titanium disks. The in vivo study focused on evaluating the efficacy of the nanomatrix through implantation of a POP into a rat femur. Histological analysis was performed on the rat femurs at either 2- or 4-weeks post operation when the rats were euthanized. The main samples that were collected were the right femur with the implant and the surrounding soft tissue. The extracted tissues were then fixed in formalin and decalcified in daily EDTA for 3 to 4 weeks. After the tissues were fixed and decalcified, they were cut into 5mm thick pieces and embedded in paraffin. Histological analysis consisting of hematoxylin-eosin staining and immunohistochemical staining was then performed.

Results, Conclusions, and Discussions::

Results: Our in vitro studies showed the proliferation and growth of hMSCs showed increased proliferation on the NO releasing nanomatrix coated titanium disk compared to the uncoated control. Our in vivo model showed osteocalcin and osteoprotogerin (OPG) have increased expression at the 2-week time point in rat femurs exposed to NO. Osteocalcin and OPG are proteins that have important roles in bone remodeling, metabolism, and formation. Figure 1 demonstrated elevated levels of CD31, a marker of endothelial cell adhesion and integrity. These increased levels suggest that nitric oxide (NO) contributed to enhanced endothelial cell expression and vascular stability. CD68, a marker expressed primarily in macrophages and monocytes, decreased in the femur exposed to NO when compared to the control. Additionally, the in vivo model demonstrated reduced inflammation and immune response within the bone marrow and surrounding soft tissues. Figure 2 presents a hematoxylin and eosin-stained cross-section of a rat femur after four weeks. The femur exposed to NO exhibited significantly less inflammation and improved bone healing compared to the control.

Discussion/

Conclusion: Our in vitro studies have demonstrated that hMSCs exhibit increased proliferation when cultured on NO-releasing nanomatrix-coated titanium discs compared to uncoated controls, indicating that the NO nanomatrix supports stem cell growth. Further in vitro studies are currently underway. In vivo results show that the NO nanomatrix coating enhances osseointegration, as evidenced by elevated levels of osteocalcin and osteoprotegerin. Additionally, increased CD31 expression suggests enhanced angiogenesis at the implant site and a decreased expression of CD68 suggests a reduction in immune response at the implant site. Together, these findings indicate that the PA-NO nanomatrix coating has the potential to significantly improve the performance and integration of percutaneous osseointegrated prostheses (POP).

Acknowledgements and/or References (Optional)::