CorNeat KPro: Ocular Implantation Study in Rabbits.

PURPOSE: The purpose of this study was to evaluate surgical feasibility and long-term integration of the CorNeat Keratoprosthesis (KPro), a novel synthetic cornea, in rabbits. METHODS: The CorNeat KPro is a synthetic corneal implant designed to treat corneal blindness by using a polymeric scaffold for biointegration, consequently assimilating synthetic optics within ocular tissues. Eight New Zealand White rabbits were implanted unilaterally with the CorNeat KPro and observed for 6 months. Animals were regularly monitored by a certified ophthalmologist using slit-lamp biomicroscopy. One animal developed postoperative endophthalmitis and was removed from the study 7 weeks postsurgery. At termination, eyes were enucleated and evaluated histologically to assess local tissue integration and inflammatory response. RESULTS: The surgical procedure was found feasible. The CorNeat KPro integrated into all operated eyes, resulting in a retention rate of 87.5% at the conclusion of the 6-month follow-up period. We observed minimal-to-mild conjunctival and iridial congestion and did not find additional inflammatory indicators, such as anterior chamber fibrin, flare, or cells. The optical element of the device remained clear with zero incidence of retroprosthetic membrane formation. Histopathological evaluation revealed comparable tissue and cellular reaction in all eyes, consisting of the presence of fibroblasts and associated collagen fibrils within the device's skirt component. Some eyes showed a mild foreign body reaction surrounding the skirt. CONCLUSIONS: Clinical and histological findings indicate the integration of the implanted device into the surrounding tissue, evident by the retention rate and the diffuse infiltration of fibroblasts with collagen deposition among the device's fibrils. These data hold promise for clinical application in humans.

Treatment of contaminated radial fracture in Sprague-Dawley rats by application of a degradable polymer releasing gentamicin.

Fracture-related infections remain a leading cause of morbidity and mortality. We aimed to establish a simple contaminated radial osteotomy model to assess the efficacy of a biodegradable polymer poly(sebacic-co-ricinoleic acid) [p(SA-RA)] containing 20% w/w gentamicin. A unilateral transverse osteotomy was induced in Sprague-Dawley (SD) rats, followed by application of Staphylococcus aureus suspension over the fracture. After successfully establishing the contaminated open fracture model, we treated the rats either systemically (intraperitoneal cefuroxime), locally with p(SA-RA) containing gentamicin, or both. Control groups included non-contaminated group and contaminated groups that were either untreated or treated with the polymer alone. After 4 weeks, the bones were subjected to micro-CT scanning and microbiological and histopathology evaluations. Micro-CT analysis revealed similar changes in the group subjected to both local and systemic treatment as in the non-contaminated control group. Lack of detectable bacterial growth was noted in most animals of the group subjected to both local and systemic treatment, and all samples were negative for S. aureus. Histopathological evaluation revealed that all treatment modalities containing antibiotics were highly effective in reducing infection and promoting callus repair, resulting in early bone healing. While p(SA-RA) containing gentamicin treatment showed better results than cefuroxime, the combination of local and systemic treatment displayed the highest therapeutic potential in this model.

Local Tolerability and Performance Evaluation in Domestic Pigs of a Fractional Radiofrequency Device for Dermatologic Treatment.

Information on the safety of energy-based dermatological surgical devices in domestic pigs, and fractional radiofrequency (RF) devices in particular, is very limited. The aim of this study was to evaluate in a GLP-compliant study in domestic pigs the local reaction and performance of a novel fractional RF device. Five female domestic pigs were subjected to fractional RF pulses, using different energy and pulse durations and depth of penetration of the pulses. The animals were evaluated clinically and histologically at different time points (days 0, 1, 3, 7, and 14) postenergy exposure. There were no microscopic or macroscopic local adverse effects in any tested power settings, and there was time-related progressive healing, reaching complete macroscopic and microscopic healing by 7 days postapplication. As expected, there was power-related progressive increase in the incidence of ablation (destruction of skin tissue by vaporization) and coagulative necrosis of the dermis from low to high power setting. This comprehensive study, using multiple power settings (both ablative and coagulative) and several time points, will be of benefit for future studies evaluating new fractional RF devices.

Biodegradability and Safety Study of LifeMesh™, a Novel Self-adhesive Mesh, in Sprague-Dawley Rats.

Self-adhesive meshes are being developed to avoid complications due to traumatic fixation methods. LifeMesh™ is a novel self-adhesive mesh with a biodegradable gelatin adhesive layer developed for hernia repair. The aim of this study was to assess the safety and biodegradability of LifeMesh in Sprague-Dawley (SD) rats for 6 weeks, in comparison to a bare polypropylene (BPP) mesh fixed with sutures. LifeMesh was tolerated well and its implantation did not result in any adverse local reaction, and its adhesive layer was substantially degraded after 4 weeks. Histopathological examination revealed that the presence of the adhesive contributed to a uniform thickness of the granulation tissue surrounding the mesh, in contrast to a nonuniform granulation tissue with BPP. Nonuniform granulation tissue suggests that there will be poorer integration of the mesh to the abdominal wall. The use of LifeMesh also resulted in less adhesions of internal organs with a smaller surface area of involvement. These findings lend support to the potential benefit of LifeMesh for hernia repair in humans and expand the available information on the typical histopathological findings expected with biodegradable implants in the peritoneal cavity of SD rats.

Long-term Local and Systemic Safety of Poly(L-lactide-co-epsilon-caprolactone) after Subcutaneous and Intra-articular Implantation in Rats.

The use of biodegradable materials is gaining popularity in medicine, especially in orthopedic applications. However, preclinical evaluation of biodegradable materials can be challenging, since they are located in close contact with host tissues and might be implanted for a long period of time. Evaluation of these compounds requires biodegradability and biocompatibility studies and meticulous pathology examination. We describe 2 preclinical studies performed on Sprague-Dawley rats for 52 weeks, to evaluate clinical pathology, biocompatibility, biodegradability, and systemic toxicity after implantation of 2-layered films or saline-inflated balloon-shaped implants of downsized InSpace™ devices (termed "test device"). The test devices are made from a copolymer of poly-L-lactide-co-∊-caprolactone in a 70:30 ratio, identical to the device used in humans, intended for the treatment of rotator cuff tears. Intra-articular film implantation and subcutaneous implantation of the downsized device showed favorable local and systemic tolerability. Although the implanted materials have no inherent toxic or tumorigenic properties, one animal developed a fibrosarcoma at the implantation site, an event that is associated with a rodent-predilection response where solid materials cause mesenchymal neoplasms. This effect is discussed in the context of biodegradable materials along with a detailed description of expected pathology for biodegradable materials in long-term rodent studies.

Self-activation of Caspase-6 in vitro and in vivo: Caspase-6 activation does not induce cell death in HEK293T cells.

Caspase-6 (Casp6) is a short pro-domain caspase that is activated early in Alzheimer disease, yet, little is known on the mechanism of activation of this caspase. In this study, critical proteolytic processing events required for Casp6 activation in vitro and in vivo were evaluated by site directed mutagenesis of the D23 pro-domain, and D179 and D193 linker processing sites. We found that (1) Casp6 was self-processed and activated in vitro and in vivo, (2) uncleavable Casp6 possessed low activity in vitro but not in vivo, (3) the pro-domain of Casp6 entirely prevented self-processing and activation in vivo but not in vitro, (4) removal of the pro-domain promoted Casp6 activation, (5) cleavage at either D179 or D193 was sufficient to generate activity in vitro and in vivo, and (6) Casp6 activity did not induce cell death in HEK293T cells. We conclude that the Casp6 is activated through proteolytic cleavage, as are the effector Caspase-3 and -7. However, unlike other effector caspases, Casp6 can be entirely self-activated and its activation does not necessarily induce cell death.

Targets of caspase-6 activity in human neurons and Alzheimer disease.

Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals. The active Caspase-6 is localized in neuritic plaques, in neuropil threads, and in neurofibrillary tangles containing neurons that are not morphologically apoptotic in nature. To investigate the potential consequences of the activation of Caspase-6 in neurons, we conducted a proteomics analysis of Caspase-6-mediated cleavage of human neuronal proteins. Proteins from the cytosolic and membrane subcellular compartments were treated with recombinant active Caspase-6 and compared with undigested proteins by two-dimensional gel electrophoresis. LC/MS/MS analyses of the proteins that were cleaved identified 24 different potential protein substrates. Of these, 40% were cytoskeleton or cytoskeleton-associated proteins. We focused on the cytoskeleton proteins because these are critical for neuronal structure and function. Caspase-6 cleavage of alpha-Tubulin, alpha-Actinin-4, Spinophilin, and Drebrin was confirmed. At least one Caspase-6 cleavage site was identified for Drebrin, Spinophilin, and alpha-Tubulin. A neoepitope antiserum to alpha-Tubulin cleaved by Caspase-6 immunostained neurons, neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer disease and co-localized with active Caspase-6. These results imply that the early and neuritic activation of Caspase-6 in Alzheimer disease could disrupt the cytoskeleton network of neurons, resulting in impaired neuronal structure and function in the absence of cell death. This study provides novel insights into the pathophysiology of Alzheimer disease.

Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors.

We describe novel peptide-based caspase inhibitors. Potent and comparatively selective compounds containing a dipeptide scaffold and a substituted oxymethyl ketone as a warhead were developed. The newly synthesized compounds were tested for inhibition in in vitro enzymatic assays of caspases-1, -3, -6, -8, and -9. The benzyloxycarbonyl-phenylglycyl-aspartyl benzoyloxymethyl ketone (Z-Phg-Asp-CH2OCO-Ph, coded as HU44) was the most potent inhibitor of caspase-1 and caspase-3. Of several analogs of HU44 that were made, the beta-Asp methyl ester (2) is an effective inhibitor against caspase-3 and caspase-8, and less effective against caspase-1. These compounds did not inhibit caspase-6 and caspase-9 significantly.

Proteasomal degradation of caspase-6 in 17beta-estradiol-treated neurons.

In primary cultures of human neurons, 17beta-estradiol (17beta-E2) prevents caspase-6-mediated cell death and induces a caspase inhibitory factor (CIF) inhibiting active caspase-6 (Csp-6) in vitro. Here, we show that treatment of neurons with 17beta-E2 results in a proteasomal-dependent but ubiquitin-independent degradation of endogenous and exogenous active Csp-6 in live neurons and in cell free assays, respectively. We further show that the proteasomal-dependent degradation of Csp-6 is not required for its inhibition. Using several protease inhibitors, we find that leupeptin, E-64, and ALLN prevent inhibition of recombinant active Csp-6 (R-Csp-6) in 17beta-E2-treated neuronal protein extracts. Because all three protease inhibitors have the ability to inhibit cysteine proteases, we believe that a cysteinyl protease activity may be required for 17beta-E2-mediated inhibition of active Csp-6. However, we exclude caspases, calpains, and cathepsins as potential cysteinyl proteases involved in the 17beta-E2-mediated Csp-6 inhibition. The results suggest that a proteolytic activity inhibited by leupeptin, E-64, and ALLN is needed to inhibit Csp-6 and that the inhibited Csp-6 is subsequently degraded by the proteasome. The mechanism of 17beta-E2-mediated inhibition of Csp-6 is different from the ubiquitin-dependent proteasomal degradation of Csp-3 and Csp-7 by XIAP and cIAP2 but consistent with the mechanism of Baculovirus p35 inhibition of caspases.