Repair of surgically created diaphragmatic defect in rat with use of a crosslinked porous collagen scaffold.

Large defects in congenital diaphragmatic hernia are closed by patch repair, which is associated with a high complication risk and reherniation rate. New treatment modalities are warranted. We evaluated the feasibility of using an acellular biodegradable collagen bioscaffold for a regenerative medicine approach to close a surgically created diaphragmatic defect in a rat model. Scaffold degradation, cellular ingrowth and regeneration of the diaphragm were studied. In 25 rats, a subcostal incision was made and one third of the right hemidiaphragm was resected. Crosslinked porous type I collagen scaffolds (Ø ~ 14 mm) were sutured into the lesion. Rats were sacrificed at 2, 4, 8, 12 or 24 weeks after scaffold implantation. Implants were evaluated macroscopically and (immuno)histologically. Survival after surgery was 88% with no evidence of reherniation. Histological examination showed that the collagen scaffold degraded slowly and new collagen, elastin and mesothelium were deposited. Blood vessels were observed primarily at the outer borders of the scaffold; their number gradually increased in time. Muscle fibres were found on the scaffold covering up to 10% of the defect. Macroscopically, adhesion of the scaffold to the liver was observed. Use of a collagen scaffold to close a surgically created diaphragmatic defect is feasible, with evidence of new tissue formation. The use of crosslinked collagen scaffolds allows targeted modification; e.g. addition of growth factors to further stimulate growth of muscle cells.

Lyophilisomes as a new generation of drug delivery capsules.

Nanoparticulate drug delivery systems are currently explored to overcome critical challenges associated with classical administration forms. In this study, we present a drug delivery system based on a novel class of proteinaceous biodegradable nano/micro capsules, lyophilisomes. Lyophilisomes can be prepared from biomolecules without the need for amphiphilicity. Albumin-based lyophilisomes were prepared by freezing, annealing and lyophilizing, resulting in capsules ranging from 100 to 3000 nm. Lyophilisomes were loaded with the anti-tumor drugs doxorubicin and curcumin using different concentrations and time/temperature regimes. Incubation in 0.1 mg/ml doxorubicin or 1.0 mg/ml curcumin resulted in an entrapment efficiency of 95±1% and 4±1%, respectively. This corresponds to a drug loading of 0.24 mg doxorubicin per milligram albumin and 0.10 mg curcumin per milligram albumin. Drug release profiles from doxorubicin and curcumin-loaded lyophilisomes were studied in culture medium and showed slow release for doxorubicin (2.7% after 72 h), and rapid release for curcumin (55% after 72 h). When applied to cells, non-loaded lyophilisomes did not influence cell viability, even at high concentrations (1 mg/ml). Lyophilisomes were internalized by cells. When loaded with doxorubicin and curcumin, lyophilisomes strongly reduced cell proliferation and viability of SKOV-3 and HeLa cells, respectively, to a level similar or better compared to an equal amount of free drugs. In conclusion, albumin lyophilisomes show potential as (nano)carriers of drugs for tumor cell elimination.

Preparation and characterization of injectable fibrillar type I collagen and evaluation for pseudoaneurysm treatment in a pig model.

OBJECTIVE: Despite the efficacy of collagen in femoral artery pseudoaneurysm treatment, as reported in one patient study, its use has not yet gained wide acceptance in clinical practice. In this particular study, the collagen was not described in detail. To further investigate the potential of collagen preparations, we prepared and characterized highly purified injectable fibrillar type I collagen and evaluated its use for femoral artery pseudoaneurysm (PSA) treatment in vivo using a pig model. METHODS: Purified fibrillar type I collagen was characterized using electron microscopy. The effect of three different sterilization procedures, ie, hydrogen peroxide gas plasma (H2O2), ethylene oxide gas (EtO), and gamma irradiation, was studied on both SDS-PAGE and platelet aggregation. Different collagen injectables were prepared (3%, 4%, and 5%) and tested using an injection force test applying a 21-gauge needle. To evaluate the network characteristics of the injectable collagen, the collagen was suspended in phosphate buffered saline (PBS) at 37°C and studied both macroscopically and electron microscopically. To determine whether the collagen induced hemostasis in vivo, a pig PSA model was used applying a 4% EtO sterilized collagen injectable, and evaluation by angiography and routine histology. RESULTS: Electron microscopy of the purified type I collagen revealed intact fibrils with a distinct striated pattern and a length<300 µm. Both SDS-PAGE and platelet aggregation analysis of the sterilized collagen indicated no major differences between EtO and H2O2 sterilization, although gamma-irradiated collagen showed degradation products. Both 3% and 4% (w/v) collagen suspensions were acceptable with respect to the force used (<50 N). The 4% suspension was selected as the preferred injectable collagen, which formed a dense network under physiologic conditions. Testing the collagen in vivo (n=5), the angiograms revealed that the PSA partly or completely coagulated. Histology confirmed the network formation, which was surrounded by thrombus. CONCLUSIONS: Collagen injectables were prepared and EtO sterilized without major loss of structural integrity and platelet activity. In vivo, the injectable collagen formed a dense network and triggered (partial) local hemostasis. Although optimization is needed, an injectable collagen may be used as a therapeutic agent for femoral PSA treatment.