Functional Hyaluronic Acid-Polylactic Acid/Silver Nanoparticles Core-Sheath Nanofiber Membranes for Prevention of Post-Operative Tendon Adhesion

In this study, we prepared core-sheath nanofiber membranes (CSNFMs) with silver nanoparticles (Ag NPs) embedding in the polylactic acid (PLA) nanofiber sheath and hyaluronic acid (HA) in the nanofiber core. The PLA/Ag NPs sheath provides mechanical support as well as anti-bacterial and anti-inflammatory properties. The controlled release of HA from the core could exert anti-adhesion effects to promote tendon sliding while reducing fibroblast attachment. From the microfibrous structural nature of CSNFMs, they function as barrier membranes to reduce fibroblast penetration without hampering nutrient transports to prevent post-operative peritendinous adhesion. As the anti-adhesion efficacy will depend on release rate of HA from the core as well as Ag NP from the sheath, we fabricated CSNFMs of comparable fiber diameter, but with thick (Tk) or thin (Tn) sheath. Similar CSNFMs with thick (Tk+) and thin (Tn+) sheath but with embedded Ag NPs in the sheath were also prepared. The physico-chemical properties of the barrier membranes were characterized in details, together with their biological response including cell penetration, cell attachment and proliferation, and cytotoxicity. Peritendinous anti-adhesion models in rabbits were used to test the efficacy of CSNFMs as anti-adhesion barriers, from gross observation, histology, and biomechanical tests. Overall, the CSNFM with thin-sheath and Ag NPs (Tn+) shows antibacterial activity with low cytotoxicity, prevents fibroblast penetration, and exerts the highest efficacy in reducing fibroblast attachment in vitro. From in vivo studies, the Tn+ membrane also shows significant improvement in preventing peritendinous adhesions as well as anti-inflammatory efficacy, compared with Tk and Tn CSNFMs and a commercial adhesion barrier film (SurgiWrap®) made from PLA.

Bacteriostatic Behavior of PLA-BaTiO3 Composite Fibers Synthesized by Centrifugal Spinning and Subjected to Aging Test

The present work investigated the effect of Polylactic acid (PLA) fibers produced by centrifugal spinning with incorporated BaTiO3 particles to improve their bacteriostatic behavior. The PLA matrix and three composites, presenting three different amounts of fillers, were subjected to UV/O3 treatment monitoring the possible modifications that occurred over time. The morphological and physical properties of the surfaces were characterized by different microscopic techniques, contact angle, and surface potential measurements. Subsequently, the samples were tested in vitro with human dermal fibroblasts (HDF) to verify the cytotoxicity of the substrates. No significant differences between the PLA matrix and composites emerged; the high hydrophobicity of the fibers, derived by the polymer structure, represented an obstacle limiting the fibroblast attachment. Samples underwent bacterial exposure (Staphylococcus epidermidis) for 12 and 24 h. Increasing the concentration of BT, the number of living bacteria and their distribution decreased in comparison with the PLA matrix suggesting an effect of the inorganic filler, which generates a neutralization effect leading to reactive oxygen species (ROS) generation and subsequently to bacterial damages. These results suggest that the barium titanate (BT) fillers clearly improve the antibacterial properties of PLA fibers after aging tests made before bacterial exposure, representing a potential candidate in the creation of composites for medical applications.

Collagen Film Activation with Nanoscale IKVAV-Capped Dendrimers for Selective Neural Cell Response

Biocompatible neural guidance conduits are alternatives to less abundant autologous tissue grafts for small nerve gap injuries. To address larger peripheral nerve injuries, it is necessary to design cell selective biomaterials that attract neuronal and/or glial cells to an injury site while preventing the intrusion of fibroblasts that cause inhibitory scarring. Here, we investigate a potential method for obtaining this selective cellular response by analysing the responses of rat Schwann cells and human dermal fibroblasts to isoleucine-lysine-valine-alanine-valine (IKVAV)-capped dendrimer-activated collagen films. A high quantity of nanoscale IKVAV-capped dendrimers incorporated onto pre-crosslinked collagen films promoted rat Schwann cell attachment and proliferation, and inhibited human dermal fibroblast proliferation. In addition, while pre-crosslinked dendrimer-activated films inhibited fibroblast proliferation, non-crosslinked dendrimer-activated films and films that were crosslinked after dendrimer-activation (post-crosslinked films) did not. The different cellular responses to pre-crosslinked and post-crosslinked films highlight the importance of having fully exposed, non-covalently bound biochemical motifs (pre-crosslinked films) directing certain cellular responses. These results also suggest that high concentrations of nanoscale IKVAV motifs can inhibit fibroblast attachment to biological substrates, such as collagen, which inherently attract fibroblasts. Therefore, this work points toward the potential of IKVAV-capped dendrimer-activated collagen biomaterials in limiting neuropathy caused by fibrotic scarring at peripheral nerve injury sites.

Hierarchical Composite Meshes of Electrospun PS Microfibers with PA6 Nanofibers for Regenerative Medicine

One of the most frequently applied polymers in regenerative medicine is polystyrene (PS), which is commonly used as a flat surface and requires surface modifications for cell culture study. Here, hierarchical composite meshes were fabricated via electrospinning PS with nylon 6 (PA6) to obtain enhanced cell proliferation, development, and integration with nondegradable polymer fibers. The biomimetic approach of designed meshes was verified with a scanning electron microscope (SEM) and MTS assay up to 7 days of cell culture. In particular, adding PA6 nanofibers changes the fibroblast attachment to meshes and their development, which can be observed by cell flattening, filopodia formation, and spreading. The proposed single-step manufacturing of meshes controlled the surface properties and roughness of produced composites, allowing governing cell behavior. Within this study, we show the alternative engineering of nondegradable meshes without post-treatment steps, which can be used in various applications in regenerative medicine.

Genipin Attachment of Conjugated Gold Nanoparticles to a Decellularized Tissue Scaffold

Decellularized allograft tissue is used for a wide array of tissue injuries and repair with tenons and ligament repair being among the most common. However, despite their frequent use there is concern over the lengthy inflammatory period and slow healing associated with allografts. One promising solution has been the use of nanoparticles. There is currently no easy, fast method to achieve consistent conjugation of nanoparticles to tissue. The available conjugation methods can be time-consuming and/or may create numerous cytotoxic byproducts. Genipin, a naturally derived crosslinking agent isolated from the fruits of Gardenia jasminoides was investigated as a conjugation agent to achieve fast, consistent crosslinking without cytotoxic byproducts. The rational of utilizing genipin is that is reacts spontaneously with amino-group-containing compounds such as proteins, collagens, and gelatins, and does not require extensive washing after conjugation. Porcine diaphragm tendons were decellularized and then immersed in cysteamine functionalized gold nanoparticles and genipin for various time points. Tissue scaffolds were tested for the degree of crosslinking, gold nanoparticle concentrations, and fibroblast attachment and biocompatibility. Results demonstrated that genipin can successfully and reproducibly attach gold nanoparticles to tissue in as little as 15 min. The genipin had no cytotoxic effects and improved fibroblast attachment and proliferation. Genipin can be used to attach gold nanoparticles to tissue in a fast, cell safe manner.

Nonthermal plasma treatment of polymers modulates biological fouling but can cause material embrittlement

Plasma-based treatment is a prevalent strategy to alter biological response and enhance biomaterial coating quality at the surfaces of biomedical devices and implants, especially polymeric materials. Plasma, an ionized gas, is often thought to have negligible effects on the bulk properties of prosthetic substrates given that it alters the surface chemistry on only the outermost few nanometers of material. However, no studies to date have systematically explored the effects of plasma exposure on both the surface and bulk properties of a biomaterial. This work examines the time-dependent effects of a nonthermal plasma on the surface and bulk properties of polymeric implants, specifically polypropylene surgical meshes and sutures. Findings suggest that plasma exposure improved resistance to fibrinogen adsorption and Escherichia coli attachment, and promoted mammalian fibroblast attachment, although increased duration of exposure resulted in a state of diminishing returns. At the same time, it was observed that plasma exposure can be detrimental to the material properties of individual filaments (i.e. sutures), as well as the structural characteristics of knitted meshes, with longer exposures resulting in further embrittlement and larger changes in anisotropic qualities. Though there are few guidelines regarding appropriate mechanical properties of surgical textiles, the results from this investigation imply that there are ultimate exposure limits for plasma-based treatments of polymeric implant materials when structural properties must be preserved, and that the effects of a plasma on a given biomaterial should be examined carefully before translation to a clinical scenario.

Ibuprofen-Loaded Hyaluronic Acid Nanofibrous Membranes for Prevention of Postoperative Tendon Adhesion through Reduction of Inflammation

A desirable multi-functional nanofibrous membrane (NFM) for prevention of postoperative tendon adhesion should be endowed with abilities to prevent fibroblast attachment and penetration and exert anti-inflammation effects. To meet this need, hyaluronic acid (HA)/ibuprofen (IBU) (HAI) NFMs were prepared by electrospinning, followed by dual ionic crosslinking with FeCl3 (HAIF NFMs) and covalent crosslinking with 1,4-butanediol diglycidyl ether (BDDE) to produce HAIFB NFMs. It is expected that the multi-functional NFMs will act as a physical barrier to prevent fibroblast penetration, HA will reduce fibroblast attachment and impart a lubrication effect for tendon gliding, while IBU will function as an anti-inflammation drug. For this purpose, we successfully fabricated HAIFB NFMs containing 20% (HAI20FB), 30% (HAI30FB), and 40% (HAI40FB) IBU and characterized their physico-chemical properties by scanning electron microscopy, Fourier transformed infrared spectroscopy, thermal gravimetric analysis, and mechanical testing. In vitro cell culture studies revealed that all NFMs except HAI40FB possessed excellent effects in preventing fibroblast attachment and penetration while preserving high biocompatibility without influencing cell proliferation. Although showing significant improvement in mechanical properties over other NFMs, the HAI40FB NFM exhibited cytotoxicity towards fibroblasts due to the higher percentage and concentration of IBU released form the membrane. In vivo studies in a rabbit flexor tendon rupture model demonstrated the efficacy of IBU-loaded NFMs (HAI30FB) over Seprafilm® and NFMs without IBU (HAFB) in reducing local inflammation and preventing tendon adhesion based on gross observation, histological analyses, and biomechanical functional assays. We concluded that an HAI30FB NFM will act as a multi-functional barrier membrane to prevent peritendinous adhesion after tendon surgery.