Evangelia Bellas

Functionalized Silk Biomaterials for Wound Healing

Silk protein-biomaterial wound dressings with epidermal growth factor (EGF) and silver sulfadiazine were studied with a cutaneous excisional mouse wound model. Three different material designs and two different drug incorporation techniques were studied to compare wound healing responses. Material formats included silk films, lamellar porous silk films and electrospun silk nanofibers, each studied with the silk matrix alone and with drug loading or drug coatings on the silk matrices. Changes in wound size and histological assessments of wound tissues showed that the functionalized silk biomaterial wound dressings increased wound healing rate, including reepithelialization, dermis proliferation, collagen synthesis and reduced scar formation, when compared to air-permeable Tegaderm tape (3M) (- control) and a commercial wound dressing, Tegaderm Hydrocolloid dressing (3M) (+ control). All silk biomaterials were effective for wound healing, while the lamellar porous films and electrospun nanofibers and the incorporation of EGF/silver sulfadiazine, via drug loading or coating, provided the most rapid wound healing responses. This systematic approach to evaluating functionalized silk biomaterial wound dressings demonstrates a useful strategy to select formulations for further study towards new treatment options for chronic wounds.

In Situ Cross-linkable Hyaluronan Hydrogels Containing Polymeric Nanoparticles for Preventing Postsurgical Adhesions

Objective:To develop a combined barrier method and drug delivery system (“hybrid system”) for preventing postoperative peritoneal adhesions, which could combine the biocompatibility and ease of application of in situ cross-linkable hydrogels with the controlled release features of polymeric nanoparticles. Methods:Poly(lactic-co-glycolic acid) nanoparticles were dispersed in aldehyde- and hydrazide-modified hyaluronic acids (HA), then combined via a double-barreled syringe. The material was subjected to mechanical testing and was assayed for in vitro cytotoxicity to murine mesothelial cells. Subsequently, it was tested for biocompatibility by intraperitoneal injection in mice. The hybrids effectiveness in preventing postsurgical adhesions was assessed using a rabbit sidewall defect-cecum abrasion model, where it was applied to both injured surfaces. Results:The in situ hybrid gel system formed a flexible and durable hydrogel in less than 10 seconds. It had low in vitro cytotoxicity. In the mouse, the cross-linked HA maintained the polymeric nanoparticles in the peritoneum for 1 week, which we had previously shown would have cleared in less than 2 days, and no animals developed adhesions. Notably, the hybrid gel, even in the absence of encapsulated drug, was highly effective in preventing peritoneal adhesions in the rabbit model employed. Animals treated with the hybrid (n = 8) had no adhesions in 62.5% of cases, and none had adhesions that could only be separated by sharp dissection. In contrast, only 4.2% of untreated animals (n = 24) had no adhesions, and 58.3% developed adhesions requiring sharp dissection. Conclusions:The hybrid cross-linked HA-nanoparticle system described here appears to be a biocompatible and highly effective adhesion barrier, which could also deliver antiadhesion drugs.

Local Myotoxicity from Sustained Release of Bupivacaine from Microparticles

Background:Sustained release of local anesthetics is frequently associated with myotoxicity. The authors investigated the role of particulate delivery systems and of the pattern of drug release in causing myotoxicity. Methods:Rats were given sciatic nerve blocks with bupivacaine solutions, two types of bupivacaine-containing microparticles (polymeric microspheres and lipid–protein–sugar particles), or blank particles with or without bupivacaine in the carrier fluid. Myotoxicity was scored in histologic sections of the injection sites. Bupivacaine release kinetics from the particles were measured. Myotoxicity of a range of bupivacaine concentrations from exposures up to 3 weeks was assessed in C2C12 myotubes, with or without microparticles. Results:Both types of bupivacaine-loaded microparticles, but not blank particles, were associated with myotoxicity. Whereas 0.5% bupivacaine solution caused little myotoxicity, a concentration of bupivacaine that mimicked the amount of bupivacaine released initially from particles caused myotoxicity. Local anesthetics showed both concentration and time-dependent myotoxicity in C2C12s. Importantly, even very low concentrations that were nontoxic over brief exposures became highly toxic after days or weeks of exposure. The presence of particles did not increase bupivacaine myotoxicity in vitro but did in vivo. Findings applied to both particle types. Conclusions:Whereas the release vehicles themselves were not myotoxic, both burst and extended release of bupivacaine were. A possible implication of the latter finding is that myotoxicity is an inevitable concomitant of sustained release of local anesthetics. Particles, and perhaps other vehicles, may enhance local toxicity through indirect mechanisms.

In vitro 3D Full-Thickness Skin-Equivalent Tissue Model Using Silk and Collagen Biomaterials

Current approaches to skin equivalents often only include the epidermis and dermis. Here, a full-thickness skin equivalent is described including epidermis, dermis, and hypodermis, that could serve as an in vitro model for studying skin biology or as a platform for consumer product testing. The construct is easy to handle and is maintained for >14 d while expressing physiological morphologies of the epidermis and dermis, seen by keratin 10, collagens I and IV expression. The skin equivalent produces glycerol and leptin, markers of adipose metabolism. This work serves as a foundation for understanding a few necessary factors needed to develop a stable, functional model of full-thickness skin.

Tetrodotoxin for prolonged local anesthesia with minimal myotoxicity

Conventional local anesthetics such as bupivacaine cause considerable myotoxicity and neurotoxicity, whereas tetrodotoxin (TTX) does not. Tetrodotoxin combined with bupivacaine or vasoconstrictors produces long‐duration nerve blockade. To assess whether these prolonged blocks can be produced without increased myotoxicity, Sprague‐Dawley rats were injected with bupivacaine, TTX, and both, or TTX plus epinephrine. Median durations of thermal nociceptive blockade were, respectively, 188, 401, 882, and 972 min. On dissection 4 days later, all tissues appeared macroscopically pristine. Muscle injury was at most mild to moderate in all animals, and the muscle injury scores for the combination formulations were not higher than for bupivacaine alone. Similarly, in differentiated cells from a myoblast cell line (C2C12), TTX caused either no or minimal worsening of cell viability from bupivacaine at 2 or 7 days. Epinephrine did not worsen TTXs relatively minimal cytotoxicity. Tetrodotoxin may thus be useful in producing prolonged nerve block with minimal myotoxicity and perhaps neurotoxicity. Muscle Nerve, 2006

Characterization of metabolic changes associated with the functional development of 3D engineered tissues by non-invasive, dynamic measurement of individual cell redox ratios

Non-invasive approaches to assess tissue function could improve significantly current methods to diagnose diseases and optimize engineered tissues. In this study, we describe a two-photon excited fluorescence microscopy approach that relies entirely on endogenous fluorophores to dynamically quantify functional metabolic readouts from individual cells within three-dimensional engineered tissues undergoing adipogenic differentiation over six months. Specifically, we employ an automated approach to analyze 3D image volumes and extract a redox ratio of metabolic cofactors. We identify a decrease in redox ratio over the first two months of culture that is associated with stem cell differentiation and lipogenesis. In addition, we demonstrate that the presence of endothelial cells facilitate greater cell numbers deeper within the engineered tissues. Since traditional assessments of engineered tissue structure and function are destructive and logistically intensive, this non-destructive, label-free approach offers a potentially powerful high-content characterization tool for optimizing tissue engineering protocols and assessing engineered tissue implants.

Sustained volume retention in vivo with adipocyte and lipoaspirate seeded silk scaffolds

Current approaches to soft tissue regeneration include the use of fat grafts, natural or synthetic biomaterials as filler materials. Fat grafts and natural biomaterials resorb too quickly to maintain tissue regeneration, while synthetic materials do not degrade or regenerate tissue. Here, we present a simple approach to volume stable filling of soft tissue defects. In this study, we combined lipoaspirate with a silk protein matrix in a subcutaneous rat model. Silk biomaterials can be tailored to fit a variety of needs, and here were processed silk biomaterials into a porous sponge format to allow for tissue ingrowth while remaining mechanically robust. Over an 18 month period, the lipoaspirate seeded silk protein matrix regenerated subcutaneous adipose tissue while maintaining the original implanted volume. A silk protein matrix alone was not sufficient to regenerate adipose tissue, but yielded a fibrous tissue, although implanted volume was maintained. This work presents a significant improvement to the standard approaches to filling soft tissue defects by matching biomaterial degradation and tissue regeneration profiles.

Effect of chemical permeation enhancers on nerve blockade.

Chemical permeation enhancers (CPEs) have the potential to improve access of local anesthetics to the nerve, thereby improving nerve block performance. We assessed the effects of six CPEs on nerve blockade from tetrodotoxin (TTX) and from bupivacaine. Each of the six surfactants, representing three CPE subgroups (anionic, cationic, and nonionic surfactants) was coinjected with TTX or bupivacaine at the sciatic nerve of Sprague-Dawley rats. Myotoxicity of CPEs, alone and with TTX, was assessed in vitro in C2C12 myotubes and in vivo via histological analysis. All enhancers produced marked concentration-dependent improvements in the frequency and duration of block with TTX but not bupivacaine. An in vitro toxicity assay showed a wide range of CPE myotoxicity, but in vivo histological assessment showed no signs of muscle or nerve damage at concentrations of CPEs that produced a half-maximal increase in the duration of block of TTX (except in the case of the cationic surfactant DDAB). This study demonstrates that CPEs can provide marked prolongation of nerve blockade from TTX but not bupivacaine, without apparent local tissue toxicity. These results may enhance the clinical applicability of TTX for prolonged-duration local anesthesia.

Injectable Silk Foams for Soft Tissue Regeneration

Soft tissue fillers are needed for restoration of a defect or augmentation of existing tissues. Autografts and lipotransfer have been under study for soft tissue reconstruction but yield inconsistent results, often with considerable resorption of the grafted tissue. A minimally invasive procedure would reduce scarring and recovery time as well as allow the implant and/or grafted tissue to be placed closer to existing vasculature. Here, the feasibility of an injectable silk foam for soft tissue regeneration is demonstrated. Adipose-derived stem cells survive and migrate through the foam over a 10-d period in vitro. The silk foams are also successfully injected into the subcutaneous space in a rat and over a 3-month period integrating with the surrounding native tissue. The injected foams are palpable and soft to the touch through the skin and returning to their original dimensions after pressure is applied and then released. The foams readily absorb lipoaspirate making the foams useful as a scaffold or template for existing soft tissue filler technologies, useful either as a biomaterial alone or in combination with the lipoaspirate.

Noninvasive Metabolic Imaging of Engineered 3D Human Adipose Tissue in a Perfusion Bioreactor

The efficacy and economy of most in vitro human models used in research is limited by the lack of a physiologically-relevant three-dimensional perfused environment and the inability to noninvasively quantify the structural and biochemical characteristics of the tissue. The goal of this project was to develop a perfusion bioreactor system compatible with two-photon imaging to noninvasively assess tissue engineered human adipose tissue structure and function in vitro. Three-dimensional (3D) vascularized human adipose tissues were engineered in vitro, before being introduced to a perfusion environment and tracked over time by automated quantification of endogenous markers of metabolism using two-photon excited fluorescence (TPEF). Depth-resolved image stacks were analyzed for redox ratio metabolic profiling and compared to prior analyses performed on 3D engineered adipose tissue in static culture. Traditional assessments with H&E staining were used to qualitatively measure extracellular matrix generation and cell density with respect to location within the tissue. The distribution of cells within the tissue and average cellular redox ratios were different between static and perfusion cultures, while the trends of decreased redox ratio and increased cellular proliferation with time in both static and perfusion cultures were similar. These results establish a basis for noninvasive optical tracking of tissue structure and function in vitro, which can be applied to future studies to assess tissue development or drug toxicity screening and disease progression.