David A. Grant

Assessment of the biocompatibility and stability of a gold nanoparticle collagen bioscaffold.

Collagen has been utilized as a scaffold for tissue engineering applications due to its many advantageous properties. However, collagen in its purified state is mechanically weak and prone to rapid degradation. To mitigate these effects, collagen can be crosslinked. Although enhanced mechanical properties and stability can be achieved by crosslinking, collagen can be rendered less biocompatible either due to changes in the overall microstructure or due to the cytotoxicity of the crosslinkers. We have investigated crosslinking collagen using gold nanoparticles (AuNPs) to enhance mechanical properties and resistance to degradation while also maintaining its natural microstructure and biocompatibility. Rat tail type I collagen was crosslinked with AuNPs using a zero-length crosslinker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Several characterization studies were performed including electron microscopy, collagenase assays, ROS assays, and biocompatibility assays. The results demonstrated that AuNP-collagen scaffolds had increased resistance to degradation as compared to non-AuNP-collagen while still maintaining an open microstructure. Although the biocompatibility assays showed that the collagen and AuNP-collagen scaffolds are biocompatible, the AuNP-collagen demonstrated enhanced cellularity and glycoaminoglycans (GAG) production over the collagen scaffolds. Additionally, the Reactive Oxygen Species (ROS) assays indicated the ability of the AuNP-collagen to reduce oxidation. Overall, the AuNP-collagen scaffolds demonstrated enhanced biocompatibility and stability over non-AuNP scaffolds.

Materials characterization of explanted polypropylene, polyethylene terephthalate, and expanded polytetrafluoroethylene composites: Spectral and thermal analysis

This study utilized spectral and thermal analysis of explanted hernia mesh materials to determine material inertness and elucidate reasons for hernia mesh explantation. Composite mesh materials, comprised of polypropylene (PP) and expanded polytetrafluoroethylene (ePTFE) mesh surrounded by a polyethylene terephthalate (PET) ring, were explanted from humans. Scanning electron microscopy (SEM) was conducted to visually observe material defects while attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to find chemical signs of surface degradation. Modulated differential scanning calorimetry (MDSC) and thermogravimetric analysis (TGA) gave thermal stability profiles that showed changes in heat of fusion and rate of percent weight loss, respectively. ATR-FTIR scans showed higher carbonyl peak areas as compared to pristine for 91% and 55% of ePTFE and PP explants, respectively. Ninety-one percent of ePTFE explants also exhibited higher C--H stretch peak areas. Seventy-three percent of ePTFE explants had higher heats of fusion while 64% of PP explants had lower heats of fusion with respect to their corresponding pristines. Only 9% of PET explants exhibited a lower heat of fusion than pristine. Seventy-three percent of ePTFE explants, 73% of PP explants, and only 18% of PET explants showed a decreased rate of percent weight loss as compared to pristine. The majority of the PP and ePTFE mesh explants demonstrated oxidation and crosslinking, respectively, while the PET ring exhibited breakdown at the sites of high stress. The results showed that all three materials exhibited varied degrees of chemical degradation suggesting that a lack of inertness in vivo contributes to hernia mesh failure.

Materials characterization and histological analysis of explanted polypropylene, PTFE, and PET hernia meshes from an individual patient

During its tenure in vivo, synthetic mesh materials are exposed to foreign body responses, which can alter physicochemical properties of the material. Three different synthetic meshes comprised of polypropylene, expanded polytetrafluoroethylene (ePTFE), and polyethylene terephthalate (PET) materials were explanted from a single patient providing an opportunity to compare physicochemical changes between three different mesh materials in the same host. Results from infrared spectroscopy demonstrated significant oxidation in polypropylene mesh while ePTFE and PET showed slight chemical changes that may be caused by adherent scar tissue. Differential scanning calorimetry results showed a significant decrease in the heat of enthalpy and melt temperature in the polypropylene mesh while the ePTFE and PET showed little change. The presence of giant cells and plasma cells surrounding the ePTFE and PET were indicative of an active foreign body response. Scanning electron micrographs and photo micrographs displayed tissue entrapment and distortion of all three mesh materials.

In vitro electromagnetic stimulation to enhance cell proliferation in extracellular matrix constructs with and without metallic nanoparticles.

Extremely low frequency electromagnetic fields (ELF-EMFs) can induce beneficial effects including enhanced protein synthesis and cell proliferation on healing bone and skin wounds. This study investigated the effects of ELF-EMFs on acellular tissue constructs with and without gold nanoparticles (AuNPs) to determine if cell proliferation could be increase and thus provide an enhanced mechanism for in vitro cell seeding on tissue engineered constructs. Different sized AuNPs, 20 and 100 nm, were conjugated to acellular porcine tissue, seeded with L929 murine fibroblasts and exposed to a continuous 12 gauss, 60 Hz electromagnetic field for 2 hours each day up to 10 days. Scanning electron microscopy and cell culture assays were performed to ascertain cell proliferation and viability before and after exposure. Results indicate the ELF-EMF stimulation significantly increased cell proliferation. The presence of AuNPs did not boost the stimulatory effects, but they did demonstrated higher rates of proliferation from day 3 to day 10. In addition, unstimulated 100 nm AuNPs constructs resulted in significant increases in proliferation as compared to unstimulated crosslinked constructs. In conclusion, ELF-EMF stimulation enhanced cellular proliferation and while the presence of AuNPs did not significantly enhance this effect, AuNPs resulted in increased proliferation rates from day 3 to day 10.

A comparative study of the remodeling and integration of a novel AuNP-tissue scaffold and commercial tissue scaffolds in a porcine model.

The extracellular matrices of a variety of human and animal tissues have been utilized as scaffold materials for soft tissue applications including hernia repair, dermal grafts, and tendon, ligament, and cartilage reconstruction. While these biological scaffolds are expected to demonstrate superior tissue integration, there is very little evidence documenting the properties and behavior of these materials in vivo. This in vivo study investigated four biological scaffolds: two commercially available (a moderately crosslinked scaffold and a noncrosslinked scaffold) and two novel porcine diaphragm biological scaffolds (one with and one without the incorporation of gold nanoparticles). The scaffolds were implanted in a porcine model and evaluated over 1, 3, and 6 months. The moderately crosslinked scaffolds demonstrated the least cellular infiltration and evidence of fibrosis. The noncrosslinked scaffolds demonstrated the greatest cellular infiltration, but these scaffolds were delaminated and exhibited a rapid loss of integrity. The porcine diaphragm scaffolds with and without nanoparticles showed evidence of tissue remodeling and cellular infiltration, with no evidence of encapsulation. While there were no significant differences in the performance of the two novel scaffolds, the gold nanoparticle scaffold typically exhibited higher cellular infiltration. This study demonstrated the potential biocompatibility of a gold nanoparticle-tissue scaffold.

In vivo bone tunnel evaluation of nanoparticle-grafts using an ACL reconstruction rabbit model.

Acellular human gracilis tendons conjugated with gold nanoparticles (AuNP) and hydroxyapatite nanoparticles (nano-HAp) were used as a graft in an anterior cruciate ligament (ACL) reconstruction rabbit model. The ACLs of 11 New Zealand rabbits were reconstructed using grafts conjugated without nanoparticles, with AuNP only, and with both AuNP and nano-HAp. Semi-quantitative histological scoring of bone tunnel portion of grafts was performed after 14 weeks. Bone tunnels were scored for graft degeneration, graft remodeling, percentage of new host fibrous connective, collateral connection, head-to-head connection, graft collagen fiber organization, new host fibrous connective tissue organization, and graft and interface vascularity. All grafts were intact at 14 weeks. Results of bone tunnel scoring indicate remodeling in all graft types with new organized host fibrous connective tissue, head-to-head connection to bone and mild inflammation associated with remodeling. Components of the 20 nm AuNP grafts have significantly more graft degeneration, more new host fibrous connective tissue, and more vascularity compared to crosslinked grafts. Comparison between femoral and tibial tunnel scores indicate more degeneration in femoral tunnels compared to tibial tunnels. Overall results indicated potentially enhanced remodeling from the use of 20 nm AuNP grafts.

Materials characterization of explanted polypropylene hernia mesh: Patient factor correlation:

This study quantitatively assessed polypropylene (PP) hernia mesh degradation and its correlation with patient factors including body mass index, tobacco use, and diabetes status with the goal of improving hernia repair outcomes through patient-matched mesh. Thirty PP hernia mesh explants were subjected to a tissue removal process followed by assessment of their in vivo degradation using Fourier transform infrared, differential scanning calorimetry, and thermogravimetric analysis analyses. Results were then analyzed with respect to patient factors (body mass index, tobacco use, and diabetes status) to determine their influence on in vivo hernia mesh oxidation and degradation. Twenty of the explants show significant surface oxidation. Tobacco use exhibits a positive correlation with modulated differential scanning calorimetry melt temperature and exhibits significantly lower TGA decomposition temperatures than non-/past users. Chemical and thermal characterization of the explanted meshes indicate measurable degradation while in vivo regardless of the patient population; however, tobacco use is correlated with less oxidation and degradation of the polymeric mesh possibly due to a reduced inflammatory response.

Controlled Ion Release from Novel Polyester/Ceramic Composites Enhances Osteoinductivity

Due to the growing number of patients suffering from musculoskeletal defects and the limited supply of and sub-optimal outcomes associated with biological graft materials, novel biomaterials must be created that can function as graft substitutes. For bone regeneration, composite materials that mimic the organic and inorganic phases of natural bone can provide cues which expedite and enhance endogenous repair. Specifically, recent research has shown that calcium and phosphate ions are inherently osteoinductive, so controllably delivering their release holds significant promise for this field. In this study, unique aliphatic polyesters were synthesized and complexed with a rapidly decomposing ceramic (monobasic calcium phosphate, MCP) yielding novel polymer/ceramic composite biomaterials. It was discovered that the fast dissolution and rapid burst release of ions from MCP could be modulated depending on polymer length and chemistry. Also, controlled ion release was found to moderate solution pH associated with polyester degradation. When composite biomaterials were incubated with mesenchymal stems cells (MSCs) they were found to better facilitate osteogenic differentiation than the individual components as evidenced by increased alkaline phosphate expression and more rapid mineralization. These results indicate that controlling calcium and phosphate ion release via a polyester matrix is a promising approach for bone regenerative engineering.

Development and Characterization of a Rapid Polymerizing Collagen for Soft Tissue Augmentation

Abstract A liquid collagen has been developed that fibrilizes upon injection. Rapid polymerizing collagen (RPC) is a type I porcine collagen that undergoes fibrillization upon interaction with ionic solutions, such as physiological solutions. The ability to inject liquid collagen would be beneficial for many soft tissue augmentation applications. In this study, RPC was synthesized and characterized as a possible dermal filler. Transmission electron microscopy, ion induced RPC fibrillogenesis tests, collagenase resistance assay, and injection force studies were performed to assess RPCs physicochemical properties. An in vivo study was performed which consisted of a 1‐, 3‐, and 6‐month study where RPC was injected into the ears of miniature swine. The results demonstrated that the liquid RPC requires low injection force (<7 N); fibrillogenesis and banding of collagen occurs when RPC is injected into ionic solutions, and RPC has enhanced resistance to collagenase breakdown. The in vivo study demonstrated long‐term biocompatibility with low irritation scores. In conclusion RPC possesses many of the desirable properties of a soft tissue augmentation material.

Fluorescence imaging preparation methods for tissue scaffolds implanted into a green fluorescent protein porcine model

Green fluorescent protein (GFP) animal models have become increasingly popular due to their potential to enhance in vivo imaging and their application to many fields of study. We have developed a technique to observe host tissue integration into scaffolds using GFP expressing swine and fluorescence imaging. Current fluorescence imaging preparation methods cannot be translated to a full GFP animal model due to several challenges and limitations that are investigated here. We have implanted tissue scaffolds into GFP expressing swine and have prepared explanted scaffolds for fluorescence imaging using four different methods including formalin fixation and paraffin embedding, vapor fixation, freshly prepared paraformaldehyde fixation, and fresh frozen tissue. Explanted scaffolds and tissue were imaged using confocal microscopy with spectral separation to evaluate the GFP animal model for visualization of host tissue integration into explanted scaffolds. All methods except fresh frozen tissue induced autofluorescence of the scaffold, preventing visualization of detail between host tissue and scaffold fibers. Fresh frozen tissue preparation allowed for the most reliable visualization of fluorescent host tissue integration into non-fluorescent scaffolds. It was concluded that fresh frozen tissue preparation is the best method for fluorescence imaging preparation when using scaffolds implanted into GFP whole animal models.