MPC

J Med Dent Sci 2005; 52: 115–121

Original Article Evaluation of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated dressing on surgical wounds

Osamu Katakura1,3, Nobuyuki Morimoto2,3, Yasuhiko Iwasaki2,3, Kazunari Akiyoshi2,3 and Shohei Kasugai1,3 1) Oral Implantology and Regenerative Dental Medicine, Graduate school, Tokyo Medical and Dental University 2) Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University 3) Center of Excellence Program, Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University

The ideal dressing material is bio-inert and keeps the wound site moist. It is equally important that no regenerative tissue is peeled off on the removal of the dressing. 2-Methacryloyloxyethyl phosphorylcholine (MPC) has a phospholipid polar group that mimics a biomembrane. We prepared poly [MPC-co-n-dodecyl methacrylate (DMA)] (PMD), using conventional radical polymerization with 2,2’-azobisisobutyronitrile as an initiator, and coated it on polyurethane (PU; TecoflexÑ 60 Thermedics Inc.) membrane. Fullthickness surgical wounds were made on the dorsal skin of rats and wound healing was compared under the following three conditions: air-exposed control (no dressing), PU dressing, and PMD dressing. At 3, 4 and 7 days after the operation, the wound sizes of the PMD dressings were smaller than the non-dressed wound, and at 6 and 7 days after the operation, the wound sizes of PU dressing were smaller than that of the air-exposed group. But there were no significant difference between the PMD dressing group and PU dressing group. Histologically, scab formation was not observed on Corresponding Author: Osamu Katakura Oral Implantology and Regenerative Dental Medicine, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan Tel: +81-3-5803-4664 Fax: +81-3-5803-5934 E-mail: [email protected] Received December 6, 2004; Accepted March 18, 2005

the PU or PMD-dressed wounds. However, in the air-exposed control, a scab was formed and reepithelialization of the wound site was prevented. Additionally, no damage was observed in the histological section of PMD dressed wound after the wound was cured. These results indicate that PMD dressing (PMD-coated PU membrane) has the potential to provide an inert environment for wound healing as well as PU. Key words:

dressing, wound healing, bio-inert, moist, MPC

Introduction Several materials are currently used for wound dressing. Although myths still remain concerning wound healing, the factors, which affect healing, have been identified. Humidity, temperature and oxygen tension of the wound site affect healing1-5. Although remedies for wounds depend on their type, degree and site, theoretically, if a wound dressing protects the wound site and maintains ideal conditions for healing, wound healing will be promoted. Infection of the wound should obviously be avoided, and it is now accepted that dryness at the wound site inhibits healing. Indeed, in a wet environment, wounds heal smoothly, and the newly formed epidermis is of good quality1-5. Thus, dressing material is required not only to

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be bio-inert but also to seal the wound and prevent desiccation. It is also apparent that various cells and endogenous growth factors at the wound site play an important role in the healing process4,5. The dressing material should be bio-inert and protect the wound site and should ideally possess the ability to maintain optimal conditions at the wound site, consequently accelerating the healing process. On the other hand, 2-methacryloyloxyethyl phosphorylcholine (MPC) is a synthetic molecule, which has phosphorylcholine in its structure that mimics the cellular surface6-24. Poly [MPC-co-n-dodecyl methacrylate (DMA)] (PMD) is an MPC polymer with the following unique characteristics. Protein absorption onto the PMD surface is negligible, and although fibroblasts are not able to attach to the PMD sheet, fibroblasts maintain their proliferative capacity while in contact with it7-11. For example, fibroblasts do not attach to MPC polymer and they do not spread on its surface; however, after leaving the MPC polymer surface, they start to proliferate on a more appropriate substrate. Importantly, PMD has high blood compatibility and does not activate platelets12-23. Thrombus formation is inhibited if an artificial blood vessel made of a synthetic polymer, such as polyurethane, is coated with MPC polymer14-23. Thus, no blood coagulation cascade is elicited when blood comes into contact with the PMD surface. In addition, PMD has moisture-retaining ability6,7,8. Based on these unique characteristics of PMD, we speculated that a PMD dressing might provide an ideal set of conditions for wound healing. The purpose of this study was to evaluate whether PMD could be applied as a dressing material.

J Med Dent Sci

pared using a solvent evaporation technique from a 1 wt% chloroform solution on a TeflonÑ plate. The plate was left in a sealed chloroform environment overnight and then dried under vacuum for 1 day. These membranes were attached to the polyurethane membrane using ether as a solvent and sterilized under ultraviolet light. The surfaces of the PMD membranes were smooth and clean (Fig. 2). Thirty minutes before we applied the PMD dressings to the surgical wounds, the dressings were soaked in sterilized PBS solution to equilibrate their surfaces. This procedure made the phosphate residues protrude from the outer surface. Retainer We used a retainer to hold the dressing membrane on the wound site. The retainer was made from a plastic plate 0.2 mm thick. A hole 10 x 10 mm in size was made at the center of the retainer. The retainer holding the dressing membrane covered the wound site and was attached to the dorsal skin with a suture (Fig. 3). Surgical procedure Thirty male Wistar rats, 15 weeks old, weighing approximately 230 g, were used. They were divided into three groups. A full-thickness square surgical wound,

Figure 1. Chemical structure of PMD

Materials and Methods Polyurethane membrane Polyurethane (TecoflexÑ 60 Thermedics Inc.) is commercially available, and 2.5 % polyurethane in chloroform was prepared. The solution was poured into a casting dish and left in a sealed chloroform environment overnight. To evaporate the chloroform completely, the disk-shaped polyurethane membranes were dried under vacuum overnight and sterilized under ultraviolet light. Attaching the PMD membrane to the polyurethane surface PMD was synthesized from MPC and dodecylmethacrylate (Fig. 1). PMD membranes were pre-

Figure 2. SEM image of PMD membrane surface. (x 5000)

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Figure 3. Cross-sectional images of dressings and retainer. The upper and the lower drawing demonstrated the layers in the dressings used for the PU group and the PMD group, respectively.

10 x 10 mm in size, was made on the dorsal skin of each rat under Nembutal anesthesia. In the airexposed control group, only the retainer was applied to the wound site. In the polyurethane group, the wound was covered with the polyurethane membrane that we had prepared, then fixed with another polyurethane TM membrane (Tegaderm , 3M); the retainer was then sutured to the dorsal skin. In the PMD group, the wounds were first covered with the PMD membranes. The PMD membrane was then held in place with TegadermTM (3M) and the retainer (Fig. 3). Wound size measurement The cephalocaudal and horizontal axis of the wound were measured every day. The wound area and wound area ratio were calculated using the following formulas. Wound area (mm2) = size of cephalocaudal axis (mm) x size of horizontal axis (mm) Wound area ratio (%) = [wound area on measurement day / wound area on day 0] x 100 For statistical analysis, repeated measure ANOVA and Tukey test were used. Statcel 2 (OMS-Publishing) was used for all statistical analysis in this study. Histological examination Four days, 7 days and 1 month after the operation, the tissue of the wound site was harvested and fixed in 10% formalin. The specimens were embedded in paraffin, cut into 4-Òm frontal sections, and stained with hematoxilin and eosin. Masson trichrome staining was carried out to observe the collagen fiber alignment.

Results In the present study, we used polyurethane (PU) membrane because PU itself is currently used as a

Figure 4. Wound area ratio after the operation. A Value of p ＜ 0.05 is accepted as statistically significant. *: p ＜ 0.05; **: p ＜ 0.01.

dressing. This group was positive control of this experiment. And as the experimental group, PMD, one type of MPC polymer, was attached to the polyurethane (PU) membrane because PMD polymer membrane alone has low mechanical strength. Changes in wound area ratio are shown in Figure 4. The wound area ratio of the PMD group was smaller than that of the air-exposed control group after at 3, 4 and 7 days after the operation. The difference between air-exposed control group and PU group was observed at 6 and 7 after the operation. Furthermore, the speed of reduction of the wound area ratio of the PMD group has a tendency to be high before the fifth operative day. Therefore there was no significant difference between the PMD and PU groups all through the experimental period. One week after the operation, the wound area ratio of the air-exposed control group was approximately 60%, whereas the value of both PMD and PU groups was approximately 30%. In the air-exposed group, a scab was observed over the wounds 4 days and 7 days after the operation. In this group, the new epithelium migrated under the scab. In PU and PMD group, no scab was formed, and the epithelial tissue had migrated smoothly beneath the membranes at 4 days and 7 days after the operation (Figs. 5,6). At Days 4 and 7, uniform infiltrations of inflammatory cells were observed in all three groups. However, one month after the operation, the epidermis in the PMD groups was thicker than in the air-exposed control and PU dressing group. In all groups no appendages were regenerated after the wounds were completely healed (Fig. 7). In the dermis, more blood vessels were regenerated in PMD group, compared with the other groups. And no anomaly was observed in

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J Med Dent Sci

Figure 5-1. Low-magnification histological images on Day 4. (A) Open group wound, (B) PU group wound, (C) PMD group wound. The arrows indicate the edges of the epithelial tissue that has migrated from the peripheral tissue of the wound. Hematoxylin and eosin staining (x 20).

Figure 5-2. High-magnification histological images on Day 4. (A) Open group wound, (B) PU group wound, (C) PMD group wound. Hematoxylin and eosin staining (x 100).

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Figure 6. High-magnification histological images on Day 7. (A) Open group wound, (B) PU group wound, (C) PMD group wound. Hematoxylin and eosin staining (x 200).

Figure 7. High-magnification histologial images of 1 month after the operation. (A) Open group wound, (B) PU group wound, (C) PMD group wound. Hematoxylin and eosin staining (x 100).

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Figure 8. Histological images after 1 month after the opration. (A) Open group wound, (B) PU group wound, (C) PMD group wound. Masson trichrome staining (x 200).

the collagen bundles of the dermis of the PMD group (Fig. 8).

Discussion The present results demonstrated that PMD and PU dressing enhanced wound closure than air-exposed group, although there was no difference in wound closure rate between the PMD and PU dressing all through the experimental period. In the air-exposed control, a scab was formed and wound closure was extremely retarded. Histological observation revealed that in both the PMD and PU group, the re-epithelialized areas at the wound site were smooth. Since the number of animals is limited, quantitative data in histological images was not obtained unfortunately. However, the following histological differences were observed. The epidermis of PMD groups was thicker than in the air-exposed control and the PU group at 1 month after the operation. The alignment of fibrous bundles in the dermis of the PMD group looked mature than the PU group. In addition to that, large number of regenerated blood vessels was observed in the PMD group compared to the PU and air-exposed groups. Although these finding indicate high potentiality of PMD as a dressing material, further

additional experiments including quantitative histological analyses are required to confirm this. PMD is unique bio-compatible material and we had expected PMD dressing would be superior as wound dressing. However, contrary to our expectation, significant difference between PU group and PMD group was not detected. Present results demonstrated at least that PMD dressing is not inferior to the ordinary PU dressing i.e. that PMD dressing is comparable to PU dressing. Mechanical property of PMD is easily manipulated by changing the ratio of MPC and DMA. Utilizing PMD, it is also possible to prepare a dressing, which retains moisture and/or which releases a drug or growth factor to enhance wound healing. It is likely that newlydesigned dressing of PMD would be superior to the ordinary dressing. Conclusively, this is the first report of the PMD application to wound and although we could not prove PMD dressing is superior to PU dressing, the potentiality of PMD as a dressing material was suggested in the present study.

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