Study design and ethics statement
All procedures of platelet-rich fibrin and bone marrow stem cells isolation and surgery on experimental animals were carried out according to the guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee (IACUC approval: NTU-102-EL-82; NTU-102-EL-91).
Harvest and cultivation of bone marrow stem cells
Bone marrow aspirates were obtained aseptically from mouse femurs. Bone marrow specimen was collected from the disposed aspirates using a 10 mL syringe. The aspirates were immediately mixed with 0.5 mL of sodium-heparin (10000 U/mL) and diluted in equal volume of phosphate-buffered saline (PBS). The cell suspension was then fractionated on a Lymphoprep (Fisher Scientific, Goteborg, Sweden) and centrifuged at 400 g for 30 minutes. The interface fraction enriched with bone marrow stem cells (BMSC) was collected and plated onto a 10 cm dish containing 10 mL of α Modified Eagles Medium (αMEM) containing 10% of fetal bovine serum (FBS) (Gibco, Paisley, UK), and 1X P/S/A (penicillin/ streptomycin/ fungizone). After washing out non-adherent hematopoietic cells, the adherent BMSCs were cultured in 5% CO2 at 37°C with medium changed every 3~4 days. When the cells reached 80% confluence, they were trypsinized and passaged into new 10-cm dishes at a cell density of 5 x 105 cells/ dish. The cells were sub-cultured till passage 2 (P2). P2 cells were then seeded at a cell density of 6.5 x 10 3 cells/ cm2 for further in vitro tests.
Flow cytometry analysis
BMSCs were fixed with ethanol overnight at –20 °C. Aliquots of 5 x 105 cells were incubated with each of the fluorochrome-conjugated antibodies against a panel of cell surface markers, including CD 34 (BD 553731, Biosciences, USA), CD 45 (AB 10558, Abcam, USA), CD 44 (AB 119335, Abcam, USA) and CD 90 (BD 554895, BD Biosciences, USA) at 4 C°. Cells were resuspended in Con’s tube (BD) containing 200 uL of PBS/ 1% bovine serum albumin and analyzed by flow cytometry using the FACScan system (Becton Dickinson, USA).
Preparation of platelet-rich fibrin releasates
A laboratory mouse has a circulating blood volume of about 1.5-2.5 mL (6-8% of the body weight) , thus it would be difficult to obtain sufficient amount of mouse blood for experimental use. Since rabbits are of the order lagomorphs similar to rodents, thus large lot sizes available from pooled rabbit donors may be an alternative blood source . In this study, blood samples were collected from the New Zealand White rabbits (mean weight 3~3.5 kg) under inhalational anesthesia with isoflurane. The PRF gel was prepared using the technique described by Choukroun et al . After adequate skin cleaning, disinfection and sterilization, 8 mL of venous blood was harvested from rabbits in sterile tubes without anticoagulant supplement, and then centrifuged at 400 g for 10 minutes in a DSC-200A-2 table top centrifuge (Digisystem, Laboratory Instruments Inc., New Taipei City, Taiwan). The acquired PRF gel was located between the red blood cells and the acellular plasma. The PRF gel was then transferred into 15 mL sterile centrifuge tube and was allowed to stand quiescence for 5 hours to allow PRF releasates (PRFr) formation. According to Su et al. study, the PRFr collected at 5 hours contains notably higher concentration of growth factors which would be beneficial for bone formation . Later, the PRF gel was centrifuged at 3000 g for 10 minutes and the resultant PRFr located in supernatant fraction was carefully aspirated and stored at -20 °C until use.
Cytokine assay of Platelet-rich fibrin releasate
The PRFr derived from blood samples of five different rabbits were collected for quantitation of cytokine content. The concentrations of cytokine including platelet-derived growth factor (PDGF)-AA and BB, transforming-growth factor-β (TGF-β), and bone morphogenetic protein-2 (BMP-2) were measured using commercially available bead-based sandwich immune-assay kits ((PDGF AA & BB: Bioassay Technology Laboratory, Shanghai, China; TGF-β: Abbkine, CA, USA; BMP-2: Cloud-clone, TA, USA). Standards and samples were assayed in five replicates and mean values were recorded. The detection limit was 5, 2, 15 and 15.6 pg/mL for PDGF-AA, PDGF-BB, TGF-β and BMP-2 respectively.
Osteogenic differentiation assay
The osteogenic potential of BMSCs was assessed by osteogenic differentiation assay. Osteogenic induction medium was prepared using αMEM medium supplemented with 15 % FBS, 50 mg/mL L-ascorbate-2-phosphate, 10-7 M dexamethasone and 10 mM β-glyceralphosphate. The P2 BMSCs were suspended in αMEM medium at a density of 1 × 104 cells/mL per well and loaded on 24-well plate. After 24 hours, the medium was removed and 1 mL of αMEM medium or osteogenic medium were added, respectively. The medium was changed every 3 days, for a total culture period of 14 days. To detect calcium deposition, the differentiated BMSCs were fixed with 4% formaldehyde at room temperature for 30 minutes and rinsed rapidly with distilled water. Then, 1 mL of pH 4.2 Alizarin Red S solution (Sigma, St. Louis, USA) was added to cover cell surface for 5 minutes, followed by washing thoroughly with distilled water. The calcium deposits exhibited as orange red sediments on the cell surface and were recorded microscopically. For quantitative analysis, the intensity of calcium deposition in each group was counted with image analysis software (Image J, Version 1.52a, National Health Institute) in triplicate.
Establishment of mouse osteoporotic model via bilateral ovariectomy
Postmenopausal osteoporosis mouse model was established through bilateral ovariectomy (OVX). The female ICR mice (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) were maintained in polycarbonate cages at 21 ± 2 °C with a 12 hours’ light/dark cycle and given access to food and water ad libitum for 4 weeks as an adaptation period. Before surgery, the mice were placed under general anesthesia consisting of an intramuscular injection (0.25 mL/kg) with 1:1 mixture of Zoletil®50 (Virbac Laboratories, Carros, France) and xylazine (Balanzine®, Health-Tech Pharmaceutical Co., Taiwan). The animals were infiltrated with 1% lidocaine to the incision site to reduce the operative pain. A 1-cm longitudinal incision was made at the intersection which was 0.5 cm from the central spine and 1 cm below the lower rib edge. In control group (non-OVX), the mice had received sham surgery without ovary removal. For OVX-mice, the ovarian fat pad was lifted, and the bilateral ovaries were exteriorized and carefully removed followed by suture ligations to minimize bleeding. The abdominal muscles were then closed using an absorbable suture (Vicryl 3.0, ETHICON Inc., Somerville, NJ, USA). The OVX mice were then observed twice a day for a total period of 14 days.
Micro-computed tomography (Micro-CT)
Eight weeks postoperatively, micro-CT was used to evaluate the changes in bone mineral density (BMD) and trabecular bone morphology between non-OVX and OVX mice. The protocol of micro-CT used in this study was described as follow. The left tibia of animal was harvested and was placed in a custom jig with water and scanned with a SkyScan 1176 Micro CT scanner (SkyScan, Kontich, Belgium) operating at 40 kV, 600 μA, 0.3 μ of rotation step, 0.5 mm A1 filter and 9 mm/pixel of scan resolution. The image slices were reconstructed using the NRecon (v. 1.4.4, Skyscan) software system. The three dimensional (3D) parameters of bone microarchitecture were calculated using CTAn (v.17.0.0, Skyscan) software. For trabecular bone, the proximal tibia was selected for analysis within a conforming volume of interest commencing at the growth plate and extending a further longitudinal distance of 0.5-1.5 mm in the proximal direction to assess site-specific responses to ovariectomy (Figure 2A). Microstructural measurements included the percentage of bone volume versus total tissue volume (BV/TV, %), bone mineral density (BMD, g/cm3), trabecular number (Tb.N, /mm), and trabecular separation (Tb.Sp, mm).
In Vivo intravenous administration of BMSCs and PRFr
Forty-two OVX mice were used for experiment and were allocated into 7 groups. The OVX mice which received no treatment served as control. To evaluate the accumulative effects of PRFr and BMSCs on bone mass, two different treatment groups were compared, known as the single-injection group or quadruple-injection group. PRFr and cell suspension were administrated through tail vein injection using a 26-gauge needle. Basically, in single-injection group, mice received single dose of either 0.6 mL of PRFr, 3 x 105 BMSCs or 0.6 mL of PRFr containing 3 x 105 of BMSCs. In quadruple-injection group, mice received injection of either 0.6 mL of PRFr, 3 x 105 of BMSCs or 0.6 mL of PRFr containing 3 x 105 of BMSCs once a week, for four consecutive weeks. Eight weeks after treatment, the mice were euthanized by carbon dioxide inhalation with flow rate 3 litre/minute. For all animals, the carbon dioxide inhalation was continued until one minute after breathing stops. The euthanized animals were then subjected for micro-CT analysis followed the protocol as previously described.
For histological evaluation, the tibia specimens were decalcified and fixed in 10% buffered formalin for at least 48 hours before routine processing. The samples were then embedded in paraffin and the blocks were cut into serial 5-μm thick sections and stained with hematoxylin and eosin (H&E). The specimen sections were examined under light microscopy (Leica, Japan) for evaluation of trabecular changes.
For the quantitative assay, each data point was derived from three independent experiments or an experiment of quadruplicate assay and was presented as mean with standard deviation. All analyses were performed using GraphPad Prism 8.0 analytic software. Statistical significance was set at a p value of <0.05. The data were analyzed using student t-test, and one-way ANOVA followed by post hoc scheffe test and multiple comparisons Dunnett test. The statistical significances among the experimental groups were indicated with asterisks. Groups labeled with asterisk superscript letters, indicate that the statistic difference between the two groups had the p value less than 0.05, and was considered significantly different.