A New Form of Artificial Skin to Promote Permanent Wound Coverage: A Preliminary Report.

BACKGROUND: Although tendon-exposed or bone-exposed wounds can be resurfaced with flaps, such surgeries may not be feasible in patients with poor general or local conditions. Biosynthetic artificial skin is an alternative for critical wound coverage. We designed a new artificial skin bilayer to close difficult wounds permanently. AIM AND OBJECTIVES: This study compares incorporation and wound contraction between silicone acellular porcine dermis (SAPD) and the Integra graft (Integra Life Sciences Corp., Billerica, Mass) in a rat model. MATERIALS AND METHODS: The SAPD was manufactured according to our previously described standard procedures. Integra grafts were obtained commercially. We included 24 male adult Sprague-Dawley rats and divided them into 2 groups. After creating a 3 × 4-cm full-thickness wound on the back, we transplanted the same-sized SAPD and Integra grafts onto the rat wounds. Autologous full-thickness skin (FTS) was grafted onto the acellular porcine dermal matrix (APDM) of the SAPD and the Integra dermal matrix (IDM) 2 weeks later. We measured the wound size and contraction rate of recipient wounds, studied the incorporation of FTS on the dermal matrix, and did pathological examination. Generalized estimating equations were used to assess the data from repeated wound and scar contraction measurements using SAS v9.2. RESULTS: The sizes of wounds of both groups decreased over time. No difference in wound contraction was observed between the SAPD and Integra groups at weeks 2, 4, or 6 after grafting. However, the contraction rates in both groups increased significantly. The pathological examination showed that the FTS was well incorporated in the APDM and IDM. The recipient wounds showed new vessels and cell infiltration in the new matrix, but no severe inflammation. Skin appendages were regenerating in the FTS. There was no rejection sign. CONCLUSIONS: Both SAPD and Integra are double-layered artificial skin products. Our results demonstrate that APDM and IDM are good templates and show excellent incorporation with autologous FTS graft. The results also demonstrated gradual wound contraction over time, but the contraction rate was not different between SAPD and Integra 6 weeks after grafting in a rat model.

Characterization of chitosan-gelatin scaffolds for dermal tissue engineering.

Porous scaffolds for dermal tissue engineering were fabricated by freeze-drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well-distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin-based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue-engineered skin.

Development of a new HPLC method for the simultaneous determination of ticarcillin and clavulanic acid in pharmaceutical formulations.

A new HPLC method has been developed and validated for the simultaneous determination of ticarcillin (TIC) and clavulanic acid (CA) in pharmaceutical formulations. The HPLC separation was achieved on a beta-cyclodextrin column (Cyclobond I, 250 x 4.6 mm, 5 microm) with methanol-16 mM pH 6.0 ammonium acetate buffer (50 + 50, v/v) mobile phase at a flow rate of 0.8 mL/min. Detection was at 220 nm. Validation of the method was performed by evaluating specificity, robustness, accuracy, and precision. The calibration curves were linear in the range of 1-100 microg/mL for CA and 2-200 microg/mL for TIC. The LOQs based on the standard regression lines were 0.42 and 1.42 microg/mL for CA and TIC, respectively, and the LOD were 0.14 and 0.47 microg/mL, respectively. Total recoveries of synthetic mixtures (CA:TIC = 1:10, 1:15, and 1:30) were 99.25-100.99% for CA and 99.54-100.82% for TIC. Compared with the U.S. Pharmacopeia method, the proposed method has the advantage of a relatively low flow rate and short analysis time. The proposed method was successfully applied for the simultaneous determination of these two drugs in sterilized H20 and 5% dextrose injection solutions.

Simultaneous determination of ampicillin, cefoperazone, and sulbactam in pharmaceutical formulations by HPLC with beta-cyclodextrin stationary phase.

An accurate and reproducible method for the simultaneous determination of ampicillin (AMP), sulbactam (SUL), and cefoperazone (CFP) in pharmaceutical formulations by using HPLC with beta-CD stationary phase was developed. It involved the use of the added tetraethylammonium acetate (TEAA) reagent, pH, and methanol as the significant parameters to find the optimum separation condition. A high resolution and selectivity of analytes was obtained by running the mobile phase in methanol-5 mM TEAA buffer = 35:65 (v/v, pH 4.5) at 280 nm. The mean recoveries ranged from 96.6 to 103.3% for AMP in the synthetic mixture, 97.6 to 103.0% for SUL, and 97.0 to 104.0% for CFP. The low LOD (<1.8 microg/mL) and low CV (<0.9%) assured that this method was sensitive and reproducible. The assay of analytes in commercial products exhibited that it was convenient and reproducible for routine analyses of these components in sterilized H(2)O, saline, or 5% dextrose injection solutions.

The application of new biosynthetic artificial skin for long-term temporary wound coverage.

Temporary dressings protect wounds from desiccation and infection. In our previous study, we used meshed acellular porcine dermis (APD) to enhance wound healing and decrease wound contraction; however, the wounds showed meshed scar. In this study, we produced an artificial skin composed of a cross-linked silicon sheet on the surface of APD which we have called silicone acellular porcine dermis (SAPD). This new artificial skin can protect the wound long enough to promote wound healing either by second intention or covered long enough until cultured epithelium autograft (CEA) or autologous skin graft can be harvested for permanent coverage. We delivered 4 cm x 5 cm full-thickness wound on the back of 350 g Sprague-Dawley rats. Thirty-six rats were divided into two groups. Eighteen rats had SAPD and the other 18 were covered with Biobrane. The wounds were first examined 2 weeks after grafting and followed weekly for an additional 4 weeks to evaluate the wound and study pathological changes by using H.E. and Masson's stains. Wound size was calculated by ruler and analyzed by Student's t-test. At the 2-week inspection, both SAPD and Biobrane showed tight adherence to the wound with no change of wound size. Both the SAPD and Biobrane dermal templates were pink. In the Biobrane-covered group, the wounds contracted soon after the tie-over dressing was removed. Its dermal layer is a layer of thin porcine dermal substance, which was promptly digested by tissue hyaluronidase and provides no real dermal template. In the SAPD-covered group however, the wound size was maintained significantly from third to sixth week after grafting (p<0.001). SAPD was designed with thick epidermal silicone and a well-organized porcine dermis so that it incorporates into the recipient wound. Clinically the silicone layer of SAPD dislodged from APD about 6-7 weeks after grafting and was followed by dermal matrix exposure and infection. In pathological examination, much like a human skin graft, new vessels were found in APD about 1 week after grafting with minimal inflammatory cells infiltrated in the graft and wound. Six weeks after grafting, the collagen of APD incorporated into the wound, showing palisade arrangement and no sign of rejection. In the Biobrane group however, the wounds showed severe inflammation, the porcine dermal matrix was digested and disappeared 3 weeks after coverage. In conclusion, SAPD is a thick biosynthetic artificial skin, which protects the rat wound significantly longer than Biobrane and prevents contraction. We expect that using of SAPD for temporary wound coverage will provide enough time to grow autologous-cultured epithelium or to reharvest skin grafts.

Poly(2-hydroxyethyl methacrylate) wound dressing containing ciprofloxacin and its drug release studies.

An improved wound dressing with a long-term drug diffusion-efficacy has been developed by UV-radiation technique. It involves incorporation of ciprofloxacin (CIP), at the concentration of 0.5-2.0% (w/v), into a water mixture of 2-hydroxymethacrylate (HEMA) monomer, benzoin isobutyl ether (BIE) initiator and different content of ethylene glycol dimethacrylate (EGDMA) cross-linker. Increasing the concentration of EGDMA would reduce the releasing ratio of CIP from pHEMA. T1/2 is increased from 2.64 to 45.67 h when the EGDMA is added from 1 to 8%. In the ranges of 0< or = F < or = 0.6, the n value of 1%CIP-pHEMA membranes is increased from 0.48 to 0.81. It indicates that the mechanism of drug release falls between the Fickian and Case II diffusion model. The antibacterial activity of the drug impregnated into the membrane was evaluated by in vitro drug kinetic agar plate method. Higher concentration of EGDMA, up to 8% of the cross-linker, extends the drug release. Comparison with the drug-soaked membranes, the newly synthesized 1% CIP-pHEMA membrane (cross-linked with 4% EGDMA) sustains the release of the entrapped drug and maintains the antibacterial activity up to 12 days.

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide.

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.

In vitro and in vivo antipseudomonal activity, acute toxicity, and mode of action of a newly synthesized fluoroquinolonyl ampicillin derivative.

Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the alpha-amino-group of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity. Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both. The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa. Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule. The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration. Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA. However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity.