Porous pure PDLLA (D/L = 50/50 mol%; porosity calculated from apparent density, 70%; pore diameter, 40 - 400 μm) was provided by Takiron (Hyogo, Japan) (Figure 1) and was shaped as a tablet with a diameter of 5.0 mm and a height of 2.0 mm. The molecular weight (Mw) of the PDLLA was 130.9 kDa.

In these experiments, twenty-five 8-week-old male Wistar/ST rats were used. The animals were kept under standard housing conditions and allowed ad libitum access to water and a standard dry diet. The 25 rats were divided into 5 groups containing 5 rats each, based on the time points of 1, 2, 4, 12, and 24 weeks.

The rearing of animals and animal experiments were performed at the Institute of Laboratory Animals and were conducted in accordance with the guidelines for animal experimentation at Tokyo Medical and Dental University (approval no.0110231B). Anesthesia was induced using inhaled diethyl ether and then maintained by intramuscular administration of ketamine hydrochloride (1.64 ml/kg body weight) and celactal (0.36 ml/kg body weight). After skin incision, the panniculus between the epithelium and periosteum was exfoliated via blunt dissection. A PDLLA scaffold was placed onto the periosteum, and the 4 corners of the scaffold were fixed using 5/0 nylon sutures (SOFTRETCH, GC, Tokyo, Japan) (Figures 2 and 3). Two scaffolds were placed for 3 rats in each of the 4-week, 12-week, and 24-week groups, where one scaffold was fixed with nylon sutures and the other was not. All skin incisions were closed using 4/0 nylon sutures.

The animals were killed at 1, 2, 4, 12, and 24 weeks after surgery by an overdose of pentobarbital-natrium. The scaffolds were removed together with the cranial bone and surrounding tissues. The other scaffolds that were not fixed with nylon sutures were removed with the surrounding tissue. All samples were fixed with 10% formalin solution. Five samples from each group were embedded in paraffin for H & E staining. Other samples, which were not fixed with nylon sutures, were used for measurement of molecular weight.

The height of the scaffold was measured to evaluate

the preservation of the scaffolds. When the animals were sacrificed, the scaffolds were measured with digital calipers to determine their height. The height was provided for the line from the top position with more than twothird of the former material’s diameter to the lower base.

When the rats were sacrificed, the samples that were not fixed with nylon sutures were used for the measurement of molecular weight over time by using gel permeation chromatography (GPC; HLC-8320GPC; Tosoh Corporation, Japan).

The connective tissue area, sinusoidal capillaries, and necrotic tissue were measured to evaluate the replacement of the scaffold with connective tissue (Image J software) (Figure 4). The diameter of the connective tissue was measured in 5 slides at 24 weeks. Connective tissue that was narrow in the middle or that existed around the PDLLA was measured by dividing by the minimal width (Image J software 1.46 r, National Institutes of Health, USA).

The values have been expressed as mean ± standard deviation values. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey’s HSD test or the Games-Howell test, if necessary, by using the SPSS software for Windows (PASW Statistics 18.0; SPSS Inc., Japan).

The scaffold height was measured (Figure 5), and some of the values were at least 2.00 mm because the samples included tissues on the top of the scaffold. Significant differences were observed between the height at 24 weeks and those at 1, 2, and 4 weeks (p < 0.05).

The molecular weight was measured (Tables 1 and 2), and significant differences were observed over time (p < 0.05).

PDLLA was not observed on the slides because it was dissolved by organic solvents such as xylene that were used during paraffin embedding. However, the volume and shape of the tissue in the scaffold pores were main-