Preparation of RYR extract
RYR was purchased from a local Chinese medicine store and was identified morphologically and histologically according to standard Chinese herbal identification procedures [9]. A voucher specimen including identification and classification of the plant was preserved in the Hard Tissue Laboratory, University of Hong Kong. RYR extract was prepared according to the protocol for injection preparation of traditional Chinese medicine [10]. For every 4 g of RYR powder, 40 ml of HPLC grade water was added and the mixture was boiled with stirring on a hot plate for 4 hours. HPLC grade water was added occasionally to prevent the mixture from drying. After boiling, the final volume of the mixture was made up to 4 ml by adding HPLC grade water. The mixture was cooled to room temperature and then centrifuged (4648 × g). The supernatant was collected and filtered with a 0.22 μm sterile syringe filter (25JP020AS, MPS, USA) into a sterile glass bottle. Each ml of the supernatant contained 1 g of RYR extract.
In vivo qualitative study
The method and animal model used in the present study have been previously described [4]. Four 10 × 5 mm2 full-thickness bone defects were created in the parietal bones of two inbred New Zealand white rabbits. The rabbits were five months old (adult stage) and weighed between 3.5 kg to 4.0 kg. One of them was used as control while the other was used as test animal. The animal handling and experimental protocol was approved by the Committee on the Use of Live Animals in Teaching and Research, University of Hong Kong. Two defects of the test animal were grafted with collagen matrix with RYR extract; two defects of the control animal were grafted with collagen matrix alone. The animals were pre-medicated one hour before surgery with oxytetracycline hydrochloride (200 mg/ml, 30 mg/kg body weight, Tetroxyla, Bimeda, Ireland) and buprenorphine hydrochloride (0.3 ml/kg body weight, Hypnorm, Janssen Pharmaceutical, Belgium), supplemented with diazepam (5 mg/ml, 1 mg/kg body weight, Valium 10, Roche). In order to maintain the level of neuroleptanalgesia, increments of Hypnorm (0.1 ml/kg) were given at 30-minute intervals during the operation.
The surgical procedure consisted of the creation of two 10 × 5 mm2 full-thickness (approximately 2 mm) cranial defects, devoid of periosteum, using templates, in the parietal bones. The defects were produced with round stainless steel burs (1 mm in diameter) on a low speed dental drill. Outlines of the defects were made initially by making holes of full thickness in the parietal bone with a stainless steel wire template bent to the required size of the defect. The holes were joined to complete the process. During the cutting of the bones, copious amounts of sterile saline were used for irrigation and to minimize thermal damage to the tissues. In the test animal, the defects were filled with 0.02 g of purified absorbable fibrillar collagen matrix (Collagen Matrix, USA) with 0.2 ml of RYR extract (1 g/ml). The grafts were prepared 15 minutes before grafting. In the control animal, the defects were grafted with 0.02 g of the same collagen matrix with 0.2 ml water for injection.
All wounds were closed with interrupted 3/0 black silk sutures. No attempt was made to approximate the periosteum to prevent the barrier effect. Postoperatively, the rabbits were given oxytetracycline hydrochloride daily for ten days and buprenorphine hydrochloride daily for two weeks.
Two weeks after surgery, the animals were sacrificed with sodium pentobarbitone. Immediately after death, defects and surrounding tissues were removed for histological preparation and examination.
Tissues were fixed in 10% buffered formal saline solution, demineralized with K's Decal Fluid (sodium formate/formic acid) and double embedded in celloidin-paraffin wax. Serial, 5-μm-thick sections of the whole defect were cut perpendicular to the long axis. The slides were stained with Periodic acid-Schiff stain for identification of new bone formation.
In vitro quantitative study
Cell culture
UMR 106, a rat osteoblastic cell line derived from osteosarcoma, was used as a model system for in vitro studies of the effect of parathyroid hormone on osteoblasts [11]. UMR 106 cells (ATCC, CRL-1661) were cultured in Dulbecco's modified Eagle's medium (Gibco, UK) which contained fetal bovine serum (10% vol, Gibco, UK) and antibiotics penicillin G sodium (100 units/ml, Gibco, UK) and streptomycin (100 μl/ml, Gibco, UK), and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air [12].
In the control group, cells were cultured without intervention for three durations (24 hours, 48 hours and 72 hours). In the RYR group, cells were cultured with RYR extract of various concentrations (0.001 g/ml, 0.005 g/ml and 0.01 g/ml) for three durations (24 hours, 48 hours and 72 hours) in 96-well tissue culture plates (4–5 × 104 cells/well, Corning, USA). Each experiment had eight samples and was repeated once.
BCA assay for total protein
The total protein is an indicator for the biosynthetic capacity of bone cell cultures. The bicinchoninic acid (BCA) assay is a biochemical assay for determining the total level of protein in a solution using colorimetric techniques [13]. This method combines the reduction of the Cu2+ to Cu+ by proteins in an alkaline medium with the highly sensitive and selective colorimetric detection of the Cu+ using a BCA containing reagent [14]. The purple-colored reaction product of this assay is a chelation of two BCA molecules with one Cu+. This water-soluble compound exhibits a strong absorbance at 562 nm and is linearly proportional to the protein concentrations over a broad range (20–2000 μg/ml) [15]. Cells were lysed and the cellular material was transferred to 250 μl of buffer (10 mM Tris HCl, pH 7.5, 0.5 mM MgCl2 and 0.1% Triton-X100). The cellular material was homogenized by two freeze-and-thaw cycles [15]. The cellular protein concentration was determined by a BCA protein assay kit (Pierce, USA).
MTT assay for cell viability and mitochondrial activity
The cell viability and mitochondrial activity of the bone cells after exposure to various concentrations of RYR extract were determined by the colorimetric MTT assay which detects the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, USA) to a purple formazan product. MTT is a pale yellow tetrazolium salt that produces a dark blue formazan product when incubated with living cells. MTT ring is cleaved in active mitochondria in living cells [13]. The MTT assay was used to measure the bone cell viability and mitochondrial activities. Cells were incubated with 0.5 mg/ml of MTT in the final four hours of each interval. The medium was then decanted; the formazan salt was dissolved in 150 μl of acid-isopropanol; and the optical density was determined at 570 nm against a reference wavelength of 690 nm with an ELISA reader [13].
ALP assay for osteogenic activity
Alkaline phosphatase (ALP) assay is a standard method to measure osteogenic activity of bone cells in vitro. The ALP assay protocol of Declercq et al. [12] was followed. Mono-layers of cultured cells were rinsed with Ringer solution. Cells were lysed and the cellular material was transferred to 250 μl of buffer (10 mM Tris HCl, pH 7.5, 0.5 mM MgCl2 and 0.1% Triton X-100). The cellular material was homogenized by two freeze-and-thaw cycles. The ALP assay was performed using p-nitrophenylphosphate as substrate. Each sample (50 μl) was added to 50 μl of p-nitrophenylphosphate (4.34 mM) in buffer (100 mM glycine, pH 10.3, 1 mM MgCl2). The mixture was incubated at 37°C for 30 minutes on a bench shaker. The enzymatic reaction was stopped by adding 50 μl of 1 M NaOH. The reaction product was quantified by absorbance measurement at 405 nm against p-nitrophenol (PNP) standards. The ALP activity is expressed as mM PNP/μg protein.
Statistical analysis
Data were analyzed with software SPSS 15.0 for Windows (SPSS, USA). Normality test (Kolmogorov-Smirnov test, P > 0.05) was performed. The unpaired t-tests with Bonferroni correction (P = 0.05) were used to compare the control with various concentrations of RYR extract (0.001 mm, 0.005 mm and 0.01 mm). The results with P < 0.05 were considered to be statistically significant.
