Speaker
Description
Introduction: Implantable materials, e.g. barriers hindering the formation of peritoneal adhesions, are often produced from natural biopolymers. It is generally assumed that using biocompatible polymers such as hyaluronan (HA) will ensure the safety of the resulting product. But even products based on HA can elicit foreign body response (FBR) leading to their encapsulation (and thus to lower efficiency of treatment). The first reaction to the implanted material is protein adsorption. The protein layer can then facilitate cell adhesion. In this work, we studied the ability of different HA-based implantable films to attract proteins and cells both in vitro and in vivo in a mouse model. We correlated the results with the level of FBR induced by these films after implantation in the mouse peritoneum.
Methodology: Two commercial HA-based films (Seprafilm and Hyalosafe) and several in-house prepared films from either hydrophobized HA or cross-linkable HA derivatives were tested. The films were characterized by water contact angle and swelling degree measurements. Their solubilization and HA release rates were determined by LC-MS. These materials were implanted in the peritoneum of healthy mice and collected after 15 min or incubated for 15 min in vitro with different protein solutions including FBS and partially heparinized blood. These materials were then used for proteomic analysis of adsorbed proteins and in vitro cell adhesion analysis.
Results: Standard model biological fluids, such as FBS or blood, yielded different protein spectrum adsorbed onto materials than the real peritoneal fluid in vivo. We consequently improved the in vitro protocol to better reflect the in vivo conditions: films were first treated with a solution of fibrinogen and thrombin. This increased their cell adhesiveness to the level observed in samples incubated in the peritoneum. However, the film's ability to adsorb proteins was found to be of secondary importance. Instead, film stability in biological media measured by the release of HA and evidenced by material gelation or fragmentation proved to be decisive for cell adhesion. Film stability correlated with the observed frequency of FBR.
Conclusions: The often-used biological media such as FBS, blood, or BSA solution are not good models for peritoneal fluid and possibly other body fluids due to the limited spectrum of protein they contain. Hyalosafe is made of hydrophobized HA (with 100% degree of substitution), but surprisingly its surface is hydrophilic. It is also the most stable biomaterial tested and is known to elicit FBR. In-house materials both from partially hydrophobized HA and hydrophilic crosslinked HA were able to support the adhesion and growth of cells only in the more stable variants. Those solubilized after 1-3 day in culture media was generally non-adhesive for cells after implantation similar to Seprafilm. Thus, the biocompatibility of the film material itself or its properties such as hydrophilicity were not sufficient to prevent the FBR.
Supported by the European Regional Development Fund - Project INBIO (No. CZ.02.1.01/0.0/0.0/16_026/0008451)
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