Abstract
This study evaluated the effect of platelet-rich plasma (PRP) on the bone formation of marrow stromal cells (MSCs) in porous coral. MSCs in 50 μl of PRP were seeded into natural coral disks (diameter 8.0 mm; thickness 2.0 mm). The composites were clotted and cultured in vitro or implanted subcutaneously into nude mice. Coral scaffolds loading MSCs or PRP alone acted as control. Alkaline phosphatase (ALP) activity of the specimens cultured in vitro for 7 and 14 days was measured, and the level of ectopic bone formation was investigated 4 and 8 weeks after operation. The samples from the coral/PRP/MSC group exhibited significantly higher ALP activity, compared with that from the coral/MSC group or the coral/PRP group ( p < 0.05). New bone and/or cartilage formation could be observed in specimens from both coral/PRP/MSC and coral/MSC groups in ectopic sites, and osteogenesis followed the pattern of endochondral bone formation. Histomorphometric analyses showed enhanced cartilage and/or bone formation in the coral/PRP/MSC group, 4 and 8 weeks after implantation. No bone or cartilage formation could be observed in the coral/PRP group. The authors concluded that PRP could improve the ALP activity of MSCs on coral and increase ectopic bone formation.
Platelet-rich plasma (PRP) is an autologous source of various growth factors that is obtained by sequestering and concentrating freshly drawn venous blood. PRP was found to promote tissue healing and has been widely used in oral and maxillofacial surgery . Previous studies showed that, when activated with thrombin and calcium chloride, the platelets in the PRP delivered a high concentration of growth factors into the recipient bed, including platelet-derived growth factor, transforming growth factors-β (TGF-β), insulin-like growth factor-I, vascular endothelial growth factor, basic fibroblast growth factor, and platelet activating factor-4, all of which are involved in reparative processes such as bone healing . In vitro , a dose-dependent mitogenic effect of PRP has been described for marrow stromal cells (MSCs) and osteoblast-like cells .
MSCs have a high proliferative capacity and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes . MSCs can be loaded onto biomaterials to create a bioactive composite to improve bone formation . The influence of PRP on expanded MSCs for osteogenesis and ectopic bone formation remains to be elucidated .
In conventional tissue engineering, a cell/scaffold composite is obtained by seeding high-density cell suspension into scaffolds and transplanting them into the body with or without a short period of in vitro incubation to allow cell attachment. In this strategy, the efficiency of tissue regeneration is decreased due to the liquidity of cell suspension. To improve the efficacy of cell seeding, collagen gel or fibrin glue is commonly used to trap cells in the scaffold . In the current experiment, the authors used an autologous PRP to trap cells. Compared with PRP, collagen gel and fibrin glue are foreign materials and lack growth factors.
The hypothesis of this study was that the combination of expanded MSCs with PRP in natural coral would promote osteogenesis and enhance ectopic bone formation. Bone formation and osteogenic differentiation were evaluated by measuring of alkaline phosphatase (ALP) activity in vitro and histological observation after ectopic implantation.
Materials and methods
Natural coral (San’ya, Hai’nan, China) was carefully moulded into a disk with diameter of 8.0 mm and a thickness of 2.0 mm ( Fig. 1 A ). The material was immersed in 5% sodium hypochlorite for 8 days to remove alien protein and to enlarge the pores. The material was washed with distilled water and sterilized before use . The prepared coral scaffold had three dimensional and inter-connective pores ( Fig. 1 B).
New Zealand White rabbits (2 months old, 2–2.5 kg) were obtained from Jinling Hospital Animal Center (Nanjing, China). All procedures involving the use of rabbits were approved by the Animal Research Committee of Jinling Hospital. After general anaesthesia the iliacs were aseptically excised and cleaned of soft tissue. The bone grafts were split in half, and the bone marrow was flushed out with Dulbecco modified Eagle medium. MSCs were harvested and expanded as previously described .
1 h before cell seeding, 8 ml of blood was drawn from the central ear artery of the MSCs donor rabbit and transferred into a tube containing 2 ml citrate phosphate dextrose. Approximately 1 ml PRP was obtained by the process described by W u et al. .
A standard hemocytometer was used to measure platelet counts in whole blood and PRP. Concentration of TGF-β1 in whole blood and PRP (before clotting) was quantified with enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions. Briefly, both the samples and TGF-β standards were added to a microplate precoated with antibody against TGF-β. After unbound substances were rinsed away, an enzyme-linked polyclonal antibody for TGF-β was added to the wells. After a second wash, substrate solution was added, and colour developed in proportion to the amount of TGF-β bound. The colour development was stopped, and the intensity of the colour was then measured.
About 5 × 10 6 MSCs were suspended in 50 μl of PRP. The mixture was seeded into a coral disc. Ten microliter of activating solution (10% CaCl 2 and 1000 units/ml bovine thrombin) was added on the coral disc to obtain coral/PRP/MSC composites. A suspension of 5 × 10 6 MSCs in 50 μl medium or 50 μl PRP was seeded in to coral discs to obtain coral/MSC and coral/PRP composites, respectively.
Composites were cultured for 7 and 14 days, and osteogenic medium was changed every other day. The composites were processed for ALP content. The specimen was incubated with 1 ml 0.01% Triton X-100 (Sigma) and homogenized. Total protein concentration in the cell lysate was assessed by measuring the optical density of a colour reaction at 570 nm. ALP activity was assessed by measuring the conversion to P-nitrophenol after 30 min as described .
Composites were coated with gold after fixation with 5% glutaraldehyde and serial dehydration with ethanol. Samples were examined with a scanning electron microscope (Hitachi S-3000N, Japan) at 10 Kv. Composites were transferred into phosphate buffered saline containing 2 μg/ml carboxyfluorescein diacetate (CFDA) and incubate for 10 min at 37 °C . The composites were then taken out and processed for fluorescent microscope observation.
Four experimental groups were tested: coral/MSC/PRP; coral/MSC; coral/PRP; and coral. After the mice (BALB/c-nu, 6-week-old, female, Vital River Laboratories Co. Ltd., Beijing, China) were anesthetized, subcutaneous pockets were bluntly created through a 1 cm incision on the back. One composite from each group was inserted into each subcutaneous pocket (four samples in each group). The wound was closed with single interrupted sutures.
4 and 8 weeks after implantation, the specimens were harvested and processed for haematoxylin–eosin staining and histological observation. Three sequential sections at a defined depth of 0.75 mm were examined with a microscope for histomorphometry per sample, and the percentage of bone and/or cartilage occupying space within the constructs was measured using an image analysis system (KS400, Zeiss, Munich, Germany). The bone and/or cartilage area present within the pores of the scaffold in the available pore space (bone area/pore area × 100%) was expressed as the percentage of bone area on 8 samples per donor per group per time point.
Statistical analysis
The mean value and SEM were calculated. The effects of the independent variables were examined by analysis of variance (ANOVA). Differences between the groups were checked in post hoc tests by least significant difference (LSD) tests. Alpha error was consequently adjusted, p values < 0.05 were considered significant. All data were analysed using Graphpad Prism version 4.0 for Windows, GraphPad Software (San Diego, CA, USA).
Results
Platelet concentration in PRP is 47.2 ± 6.1 × 10 10 /l, which is 5.2 times the platelet concentration (9.1 ± 1.3 × 10 10 /l) in the whole blood. Mean values for TGF-β1 were 145.2 ng/ml in PRP, and 38.6 ng/ml in the whole blood.
The specific ALP activity was significantly higher in the samples from the coral/PRP/MSC group compared with those from the coral/MSC and coral/PRP groups ( p < 0.05) at 7 and 14 days in vitro ( Fig. 2 ).
SEM examination of the coral/PRP specimen showed netlike fibronectin in the pores of the coral scaffold ( Fig. 3 A ). In the coral/MSC group, seeded cells adhered and spread well on the surface of the coral scaffold ( Fig. 3 B). SEM examination of coral/PRP/MSC indicated that seeded cell adhered on the surface coral scaffold and trapped fibronectin in the pores of the coral scaffold ( Fig. 3 C). Using fluorescent microscopy, cells labelled green with CFDA were observed to line up against the surface of the porous scaffold in coral/MSC specimen ( Fig. 3 D). In the coral/PRP/MSC group, cells were on the surface and in the pores of the coral scaffold ( Fig. 3 E).
In vivo implantation
All the animals survived the experimental procedure, and no visible inflammatory reactions, infections, or extrusions were observed. Specimens from the coral/PRP/MSC and coral/MSC groups showed red, smooth surfaces ( Fig. 4 B and C ).
