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Evaluation of a bilayer artificial skin capable of sustained release of an antibiotic

Kazuya Matsuda, Shigehiko Suzuki, Nobuhiko Isshiki, Kazuo Yoshioka*. Ryoichi Wada*, S.-H. Hyon* and Yoshito Ikada* Department of Plastic Surgery, Faculty of Medicine and *Research Center for Biomedical Engineering, Kyoto University, Kyoto, 606 Japan

A bilayer artificial skin, composed of an upper silicone sheet and a lower collagen sponge, has been developed by modifying a technique proposed by Yannas and Burke. We have applied it clinically with success, but infection sometimes occurred in the area where the artificial skin was placed. To use it safely in an infected wound, we developed a new type of artificial skin capable of sustained release of antibiotic. Microspheres of poly-L-lactic acid containing an antibiotic, were installed in the upper silicone sheet. The usefulness of the new type of artificial skin was suggested by in vitro studies.

Keywords: Wound dressings, antibiotics, drug delivery

Received 23 August 1990; revised 20 November 1990; accepted 20 December 1990

Yannas, Burke and Dagalakis et al.‘-’ developed a stage 1 membrane comprising an upper silicone sheet and a lower collagen sponge sheet containing chondroitin 6-sulphate, a kind of glycosaminoglycans (GAGS). Modifying their technique, we developed a bilayer artificial skin and used it in experimental animals and clinically, both with success”’ I*. In a previous paper, we studied the effect of GAGS in the collagen sponge by measuring the scanning electron microscopic (SEM) structure, mechanical properties and the proliferation of fibroblasts into collagen sponge with GAGS. The results of these studies indicated no significant effect from mixing to GAGS the collagen sponge sheet”, and no GAGS was therefore added to the collagen sheet of our artificial skin.

The most frequent complication of the artificial skin was infection, which sometimes occurred about 1 wk after applying it to patients. The infection was not serious because it subsided by partially removing the silicone sheet over the infected area and using a conservative wound dressing. But to use the artificial skin safely even on infected wounds, it was necessary to develop one resistant to infection.

For this purpose, an artificial skin capable of sustained release of an antibiotic was developed. Microspheres containing an antibiotic were incorporated underneath the silicone sheet of the artificial skin. This paper describes the effect of the new type of artificial skin evaluated by in vitro studies.

Correspondence to Dr K. Matsuda.

MATERIALS AND METHODS

Materials

A matrix material of the microsphere is poly-L-lactic acid (PLA), a synthetic biodegradable polymer, with a mol wt of 4700. The diameter of the microsphere is 50-100 pm. Tobramycin (TOB) was first selected as an antibiotic to be incorporated in the microsphere because it has effectiveness on many kinds of microbes. The ratio of TOB in the microsphere was settled about 25 wt/wt%. An original technique for the well-controlled release of hydrophilic drug from a microsphere composed of PLA was developed by Wada et a1.13.

Methods

PLA weighing 0.75 g was dissolved in 10 ml of acetonitrile mixed with water by 10 ~01%. Both hydrophobic PLA and hydrophilic TOB are soluble in this mixed solvent. TOB weighing 0.25 g was added to this solution, followed by a dropwise addition of water to effect complete dissolution of TOB. This solution with PLA and TOB was added in a dropwise manner under agitation to cottonseed oil containing an emulsifier. The temperature was raised to 4645°C to evaporate the solvent from this oil-in-oil type emulsion. The microsphere was hardened by this process, and then filtered by sieves to obtain a fraction of microsphere containing TOB (M-TOB) ranging from 56 to 100pm in diameter. After that, excessive emulsifier and cottonseed oil on the surface of M-TOB were

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120 Artificial skin resistant to infection: K. Matsuda et al.

removed by washing with petroleum ether. M-TOB was dried up under reduced pressure and sterilized with EOG gas.

M-TOB weighing 630 mg was put in 30 g of solution containing 0.3% of atelocollagen @H 3, type 1 collagen, donated by Nitta-Gelatin Co., Japan) and the mixture was stirred immediately. The stirred collagen solution was poured into a mould [the size 10 X 7 cm’) frozen rapidly at -20°C and stored overnight. After this, the frozen contents were directly freeze-dried for 46 h to yield a collagen layer. The collagen layer contains M-TOB at the density of 9 mg/cm’ (TOB 2.25 mg/cm’). The collagen layer serves as a reservoir of M-TOB, but the collagen sponge sheet does not contain M-TOB (Figure 3). The collagen layer with M-TOB was attached to a silicone layer of 25 pm thickness to fabricate the combined sheet (Figure I). The silicone dope was cross-linked at room temperature overnight. The combined sheet, which permits sustained release of TOB, constitutes the upper layer of the new bilayer artificial skin (Figure I). Figure 2 shows the SEM structure of the combined sheet.

Release of TOB from free M-TOB and the combined sheet The 18 mg of M-TOB and the combined sheet with the same amount of M-TOB were placed in test tubes containing 4 ml saline. The test tubes continued to be shaken in a 37% water bath. At days 1, 3, 5, 7,9, 11 and 13, the test tubes were centrifuged and the supernate was sampled for quantifying the released amount of TOB by bioassay (Figure 3).

contaikng TOE (M-TOE)

> silicone layer

I

collagen layer with M-TOE

@ microsphere containing TOE

swinging - . c silicone layer

collagen layer with miaosDhere containing (18mg)

saline 4ml w&r bath

Figure 3 Procedures of release experiment.

Inhibition of proliferation of a microbe using an agar method The combined sheet with M-TOB, 6 mm in diameter, was placed on the centre of the simple agar in a dish, 90 mm in diameter. After 24 h, the combined sheet was peeled off the simple agar. Subsequently, Mueller Hinton agar with 1.0 X lO’/ml of Pseudomonas Aeruginosa NCYC 10490 was poured on the simple agar in the dish. The dish was incubated at 37’C in 5% CO, for 16-20 h. After culturing, the inhibitory zone on the dish was measured. The combined sheet, once peeled off, was placed again on the simple agar in another dish and the same procedures were repeated every l-3 d till day 16 (Figure 4). Using five combined sheets, the inhibitory zones were measured at every sampling time. As a control, five paper discs, 0.5 mm in thickness, were used, which were the same in diameter and contained the same amount of TOB as the combined sheet.

Figure 1 Structure of a new type of artificial skin with sustained release of Tobramycin (TOW.

Figure2 Microscopic structure of the combined sheet. (*) Collagen sponge sheet (figure 7), (arrowhead) microsphere containing Tobramycin (M-TOB).

TO6

GneO

RESULTS

Release of TOB from free M-TOB and the combined sheet In Figure 5 the burst release is observed in the beginning for both the M-TOB and the combined sheet. The amount of TOB released from the combined sheet on the first day was controlled, compared with that from M-TOB. However, sustained release of TOB to saline from both the M-TOB and the combined sheet continued at least for 13 d from day 3.

measurement of

Combined Collagen Sheet Mueller Hinton agar with Pseudomonas

inhibitory zone

with M-TOE (66mm) Aeruginosa

I 1.0x106/ml (4ml)

I simole aear I I I

---zTz m

24hr. 37°C incubation for 16-20hr.

Figure4 Procedures of the experiment for inhibition of proliferation.

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Artificial skin resistant to infection: K. Matsuda et al. 121

released amount/contained amount (%) the inhibitory zone of the combined sheet was twice as large as that of the control and almost twice as thick as the minimum inhibitory concentration (MIC) against P. aeruginosa. The difference between the combined sheet and the control, as arrow in the graph, was found statistically significant by a Student’s t test.

40

30

20

0

M microsphere

x-x combined sheet

===: 1 I I I I I I

1 3 5 7 9 11 13 (days)

Figure 5 Release of Tobramycin (TOB) from the microspheres containing TOB and the combined sheet to day 13. (.- 0) Microsphere, (X -X) combined sheet.

Inhibition of proliferation of a microbe using an agar method Figure 6 compares the inhibitory zone between the combined sheet and the paper disc. The inhibitory zone of the paper disc was larger than that of the combined sheet till day 4. After that, the inhibitory zone of the combined sheet outgrew that of the control. On day 14,

lnhibltory Zone

(mm)

60

x-x paper disk (OSmm thickness)($6mm)

- combwed sheet ($6mm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 (days)

Figure 6 Change of inhibitory zones. Significant difference was noted, as arrows, between them (P < 0.01) by Students t test. (X ----X) Paper disc (0.5 mm thickness, 6 mm diameter), (0 -0) combined sheet (6 mm diameter).

DISCUSSION

Silicone, known as quite bioinert, is the biomedical polymeric material most frequently used. It has high permeability for hydrophobic drugs, making their release from silicone easier. Hydrophilic drugs, including most of the antibiotics are, however, much less permeable through silicone. It is reported’4-‘6 that an addition of substances having both hydrophobic and hydrophilic

groups, such as polyethylene glycol and glycerin, accelerated the release of hydrophilic drugs from silicone. Various materials may be utilized as the carrier of an antibiotic which should be released from silicone in a sustained fashion. In our previous study17, the sustained release of TOB was compared for three different materials: PLA, silicone to which poly vinyl alcohol (PVA) is added, and hydrophilic polyurethane. The results revealed: (1) sustained release of TOB from the microsphere composed of PLA, (2) no release of TOB from the silicone sheet-added PVA, and (3) release of almost all TOB on the first day from the hydrophilic polyurethane. These results suggest that no materials other than PLA is suitable as the carrier of TOB. PLA is a harmless synthetic polymer which is degradated to H,O and CO, by hydrolysis in the body. TOB may be released mostly through diffusion from the PLA microsphere during degradation on the wound.

Our release study shows that TOB was released to saline from the combined sheet continuously for at least 13 d after day 3, similar to M-TOB. The experiment on agar cuture reveals that the combined sheet with M-TOB inhibited proliferation of the microbe for a significant period of time. The paper disc which simulated such dressing material as simply containing TOB proved less effective than the combined sheet after 4 d. These in vitro studies indicate that the artificial skin with the sustained release system of TOB may be useful for infected wounds.

Before such artificial skin capable of antibiotic release was developed, infection occurred clinically in five out of the thirteen regions where the artificial skin without the sustained release system of TOB was placed, but no infection has ever occurred in any of the six areas where the new type of artificial skin was used’*. Three of six with the new artificial skin were regions suffered from burns, where infection usually occurs. Usefulness of the new type artificial skin was confirmed clinically.

REFERENCES

1 Burke, J.F., Yannas, I.V., Quinby, W.C., Bondoc, CC. and Jung, W.K., Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury, Ann. Surg. 1981, 194, 413-427

2 Burke, J.F., Observation on the development of artificial skin: Presidential address 1982 American Burn Associa- tion Meeting, J. nauma 1983, 23, 543-551

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