Four adult male New Zealand rabbits (Oryctolagus cuniculus) with a mean weight of 4.0 kg (range, 3.6–4.3 kg) were used in the experiment. Randomization was performed using the online resource random.org. Each rabbit was housed individually in a separate cage at a controlled temperature of 18‒20 ^oC and fed a standard pellet diet (Oxbow Garden Select pellets; Oxbow Animal Health). All animal procedures and care were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC No. #KMAP22-03) and performed in accordance with all Association for Assessment and Accreditation of Laboratory Animal Care International and IACUC standards.

Preoperatively, 10 mL of blood was collected from each rabbit’s ear into a vacuum flask containing 2 mL of the anticoagulant citrate dextrose solution formula A. The collected blood was then centrifuged at 180×g for 10 minutes at room temperature. The supernatant, including the buffy coat, was subsequently separated from the lower layer of red blood cells. Then a second centrifugation step was performed at 500×g for 10 minutes to remove the plasma floating in the upper layer. The remaining concentrated platelet solution contained an average platelet count of 7.5×10⁵ cells.

All surgical procedures were performed under sterile conditions. General anesthesia was induced by intramuscular injection of ketamine (0.25 mL/kg) and xylazine (0.05 mL/kg), while anesthesia was maintained through inhalation of isoflurane via a face mask. Skin incisions were made on the back fascia over the trapezius and latissimus muscles of each experimental rabbit, creating a total of eight pocket spaces per rabbit. Intact ADM (Strattice; Allegheny Technologies), 1.5–2.0 mm thick, was precisely cut into rectangular shapes measuring 2×1.2 cm, and the top and bottom of the porous cross sections were carefully wrapped with silicone to prevent vertical-axis neovascularization. Prior to implantation, the ADM rectangles designated for PRP treatment were immersed in 200 μL of PRP, which was concentrated at 7.5×10⁵ platelets per μL in plasma for 5 minutes to ensure thorough saturation [14]. These silicone-wrapped ADMs were then inserted into the subfascial spaces (Figs. 1, 2). Four pocket spaces were implanted with unsoaked ADM, while the remaining four sites were implanted with ADM soaked in PRP (Fig. 1). All implants were fixed with a 5-0 round needle PROLENE suture. One pure ADM and one PRP-soaked ADM were harvested from each rabbit at weeks 1, 2, 3, and 4 (four pairs per week). The surgical sites were monitored for infections and other complications throughout the recovery period.

To ensure accurate measurements, the ADM abutting the fascia was removed prior to histological analysis, and only the portion surrounded by silicone was used for examination. The prepared specimens intended for histologic analysis were rapidly excised and fixed in 10% formalin for paraffin sectioning and then sectioned into 4-μm-thick slices for hematoxylin and eosin (H&E) staining. Light microscopy was used to identify vascular endothelial cells formed within the dermal matrix and to analyze the extent of transverse-axis neovascularization. ImageJ software version 1.53t (National Institutes of Health) was used to calculate these measurements. All histological measurements were performed by two independent observers who were blinded to the experimental conditions.

Descriptive statistical analysis was conducted in the control group to determine whether transverse-axis neovascularization occurs in the absence of PRP. Given the non-normal distribution of the experimental values, a Mann-Whitney U test was used to compare the control and experimental groups. Additionally, a repeated measures analysis of variance (ANOVA test) was performed to assess potential differences between the groups at each weekly time point. All statistical analyses were performed using IBM SPSS software version 28.0 (IBM Corporation). Statistical significance was set at P<0.05.

All animals in the experiment survived and remained in good health. None of the rabbits showed any adverse reactions to ADM or silicone. All stitches were removed on postoperative day 7 following implantation or sampling.

The results of H&E staining indicated that in both the control and ADM+PRP groups, the implanted ADM retained its original structure, with the presence of surrounding inflammatory cells. Neovascularization was observed in both groups (Fig. 3). Histological analysis of the two groups revealed no microscopic differences in inflammatory cell activity or the number of fibroblasts.

The extent of transverse-axis neovascularization within the dermal matrix was quantitatively assessed by measuring the distance from the margin to the continuously generated vascular endothelial cells (Fig. 4). Throughout the 4-week experimental observation, transverse-axis neovascularization was observed even in the untreated ADM group (Table 1). However, attributing clinical significance to these findings is challenging when considering the weekly variation and the specific measurements. Moreover, the extent of neovascularization, although present, did not demonstrate a consistent trend over time, suggesting limited clinical relevance in the untreated ADM scenario.

A Mann-Whitney U test was conducted to evaluate the statistical significance of the differences in neovascularization between the control and PRP groups, revealing no significant differences (P=0.158) (Table 2). This result suggests that the application of PRP might not significantly enhance the extent of transverse-axis neovascularization in ADMs.

In the analysis using repeated measures ANOVA, the impact of PRP treatment on the extent of neovascularization was assessed across all measured time points (Fig. 3). The results indicated that the application of PRP did not significantly influence the degree of neovascularization at any time point throughout the study period (P=0.693). Furthermore, post-hoc analysis using Tukey’s honest significant difference test revealed no significant pairwise differences between the groups at any time point (P>0.05). These results suggest that the extent of neovascularization did not vary significantly between the control and PRP groups throughout the study and did not exhibit significant changes over time, irrespective of the treatment.