Animals
Four 16-week-old New Zealand white male rabbits, each weighing 3.0 ~ 3.5 kg, were used. Animal selection, management, preparation, and the surgical protocol followed the protocol that was approved by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea.
Materials
Three-layer PLGA membrane
1. Manufacturing PLGA scaffold
0.18 g of poly (lactic-co-glycolic acid) (PLGA; 65:35; Sigma-Aldrich Chemical Co., USA) was stirred 1 ml of dioxane for one day in order to prepare the completely dissolved PLGA polymer solution, and the prepared polymer solution was injected into a cylindrical shaped polyethylene (PE) mold with 10.0 mm diameter. Ammonium bicarbonate (AB; Sigma Aldrich Chemical Co.) particles, 355 ~ 500 μm diameter particle size, was mixed in the polymer solution. The prepared solution was rapidly cooled down by using liquid nitrogen. During freeze-drying performed for four days, dioxane was removed from the PLGA solution, thus obtaining a rod type sample.After removal of dioxane, the PLGA rod was cut uniformly in a disk with a thickness of 1.0 mm, and residual dioxane was completely removed again through lyophilizing of the samples for 24 hrs. The entire PLGA disks were immersed in the 4 liter of distilled water at 45°C and a porous PLGA scaffold was obtained by removing the AB particles with gasification in distilled water, three times at intervals of one hour. After drying the obtained porous PLGA disks under reduced pressure for 24 hrs, the sample was collected (5% HA gel; Figure 1).
2) Induction of HA hydrogel in the pores of the porous PLGA disks
Both 0.01 g of hyaluronic acid-acrylate polymer powder (HA-Ac) and 0.01 g of hyaluronic acid derivative polymer powder with phosphate functional group (HA-TCEP) were separately dissolved in 0.2 ml of PBS solution to create each precursor solution. In brief, HA-Ac has been synthesized by grafting acrylic acid to the hydroxyl groups of HA via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. Hyaluronic acid- tri(2-carboxyethyl)phosphine (HA-TCEP) was synthesized through similar mechanism by grafting TCEP to those of HA by using EDC chemistry, which was reported by one of authors in this paper [17].
3) Manufacturing three-layer PLGA membraneBoth 0.005 g of HA-Ac powder and 0.005 g of HA-TCEP powder were separately dissolved in 0.1 ml of PBS buffer solution to create each precursor solution. The prepared HA derivative solutions were mixed and then loaded on the top side of the PLGA disk in a Teflon mold (1 mm x 1 mm). Gelation was proceeded in 4°C for 1 hr, thus obtaining a two-layered scaffold. This same process was performed on the other side of the two-layered scaffold, thus obtaining a three-layer PLGA membrane (Figure 1). 10% HA derivative solution was employed for manufacturing of 10% 3-layer PLGA membrane by following the same protocol as described above.
Collagen membrane
A non cross-linked collagen membrane with type I and III porcine collagen (Bio-Gide®, Geistlich-Pharma, Wolhusen, Switzerland) were employed as a control and used as supplied. It had a smooth, compact upper layer and a dense porous lower layer.
Monolayer PLGA membrane
A porous PLGA scaffold was manufactured in a membrane type with 2 mm thickness. The monolayer PLGA membrane group was employed as a control for comparison with the three-layer PGLA membrane group (2.0 mm thickness).
Study design
While both collagen and monolayer PLGA membrane groups were used as control groups, the three-layer PLGA membrane groups (5% and 10% HA gel) were employed for experimental groups. Bone graft material was not used to exclude possible influence of graft material on the study. Each group was sacrificed at 4 and 8 weeks after surgery. Two rabbits were assigned to each group, and total four rabbits were used for experiments.
Surgical Protocol
Hair removal and disinfection procedures were carried out with povidine iodine under general anesthesia with ketamine hydrochloride and xylazine. An incision was made in the sagittal plane across the cranium, and a full thickness flap was lifted to expose the calvarial bone under infiltration anesthesia with 2% lidocaine. Four bone defects were formed around sagittal plane with 8 mm diameter trephine bur under saline irrigation (Figure 2A). Collagen membrane, monolayer PLGA membrane and three-layer PLGA membrane (5% and 10% HA gel) were applied on prepared four bone defects (Figure 2B). After the barrier was positioned evenly, periosteum suture using resorbable suture material was performed to fix the membrane and skin closure with mono-filament nylon suture material was done.
After 4 and 8 weeks of surgery, the experimental animals were sacrificed with phenobarbital (100 mg/kg) by intravenous injection. Samples were fixed with 10% neutral formalin for 10 days and demineralized with 5% nitric acid for 5 days. Then the demineralized tissues were embedded in paraffin. Serial sections were performed with 7 μm thickness, stained with hematoxylin–eosin (H–E). All the animal experiments followed the animal guideline of Yonsei University Medical School (Project number: 2013–0042).
Evaluation methods
Clinical observations
During the procedures of dressing, stitch-out and 4 and 8 weeks of surgery, possible complications such as inflammation and abnormal reactions on the surgical sites were observed.
Histological observations
At the time points of 4 and 8 weeks after surgery, the appearance of new bone formation, membrane resorption, angiogenesis, cells of inflammation, and infiltration of external organisms were observed.