Development of a contaminated ischemic porcine wound model and the evaluation of bromelain based enzymatic debridement.

OBJECTIVES: There are no well accepted animal models of chronic wounds, limiting advances in understanding and treatment of chronic ulcers. We developed a porcine wound model which combines multiple factors involved in chronic wounds to create a contaminated necrotic eschar and evaluated the debriding efficacy of a novel bromelain based enzymatic debriding agent (EscharEx). METHODS: Contaminated ischemic wounds were created on the flanks of domestic pigs by 'sandwiching' the skin between 2 'O' rings (1 placed on the surface of the skin and the other underneath the skin) for 24h prior to dermatomal excision of the necrotic eschar and its contamination with Staphylococcus aureus and Candida albicans. After confirming the development of infected eschars, additional animals were used to compare the effects of daily application of topical EscharEx or its hydrating vehicle on eschar debridement as a control. RESULTS: In all cases, application of the 'O' rings resulted in full thickness necrotic ecshars with invasive infections, which did not reepithelialize and sloughed off spontaneously within 14-21 days. All wounds reepithelialized within 28-42 days forming contracted scars. All EscharEx treated eschars were completely debrided within 7-9 days, while no debridement was evident in eschars treated with the control gel. CONCLUSIONS: Our model simulates the initial phase of chronic wounds characterized by a contaminated necrotic eschar allowing evaluation of wound debriding agents, and that a bromelain-based debriding agent completely debrides the contaminated necrotic eschars within one week in this model.

Effects of surface microtopography on the assembly of the osteoclast resorption apparatus.

Bone degradation by osteoclasts depends on the formation of a sealing zone, composed of an interlinked network of podosomes, which delimits the degradation lacuna into which osteoclasts secrete acid and proteolytic enzymes. For resorption to occur, the sealing zone must be coherent and stable for extended periods of time. Using titanium roughness gradients ranging from 1 to 4.5 µm R(a) as substrates for osteoclast adhesion, we show that microtopographic obstacles of a length scale well beyond the range of the 'footprint' of an individual podosome can slow down sealing-zone expansion. A clear inverse correlation was found between ring stability, structural integrity and sealing-zone translocation rate. Direct live-cell microscopy indicated that the expansion of the sealing zone is locally arrested by steep, three-dimensional 'ridge-like barriers', running parallel to its perimeter. It was, however, also evident that the sealing zone can bypass such obstacles, if pulled by neighbouring regions, extending through flanking, obstacle-free areas. We propose that sealing-zone dynamics, while being locally regulated by surface roughness, are globally integrated via the associated actin cytoskeleton. The effect of substrate roughness on osteoclast behaviour is significant in relation to osteoclast function under physiological and pathological conditions, and may constitute an important consideration in the design of advanced bone replacements.

Substrate adhesion regulates sealing zone architecture and dynamics in cultured osteoclasts.

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data.

Nano-topography sensing by osteoclasts.

Bone resorption by osteoclasts depends on the assembly of a specialized, actin-rich adhesive 'sealing zone' that delimits the area designed for degradation. In this study, we show that the level of roughness of the underlying adhesive surface has a profound effect on the formation and stability of the sealing zone and the associated F-actin. As our primary model substrate, we use 'smooth' and 'rough' calcite crystals with average topography values of 12 nm and 530 nm, respectively. We show that the smooth surfaces induce the formation of small and unstable actin rings with a typical lifespan of approximately 8 minutes, whereas the sealing zones formed on the rough calcite surfaces are considerably larger, and remain stable for more than 6 hours. It was further observed that steps or sub-micrometer cracks on the smooth surface stimulate local ring formation, raising the possibility that similar imperfections on bone surfaces may stimulate local osteoclast resorptive activity. The mechanisms whereby the physical properties of the substrate influence osteoclast behavior and their involvement in osteoclast function are discussed.

Surface-induced regulation of podosome organization and dynamics in cultured osteoclasts.

Bone is continuously repaired and remodeled through the well-coordinated activity of osteoblasts, which form new bone, and osteoclasts, which resorb it. How osteoclasts sense the properties of the bone surface remains unclear. By combining light and electron microscopy, we compared osteoclast behavior on three distinct surfaces: glass, calcite single crystals, and bone. Podosomes, the basic units of the adhesion structure, and their organization into superstructures were found to be common to cells that were attached to all three substrates, whereas the structure of the resorption organelle, the so-called "ruffled border," markedly differed. Moreover, the integrity, stability, and dynamic behavior of the adhesion superstructures also fundamentally differed, depending on the substrate. We conclude that osteoclasts sense the local properties of the underlying substrate and respond to these signals, both locally and globally.

The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly.

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions.