Results/Discussion



Title: Role of Platelet-Rich Plasma in Acceleration of Bone Fracture Healing

Authors: Wylan Cornelius Peterson, M.D., Andrea Hoffmann, Ph.D., and

Richard Simman, M.D., CWS

Objectives: Platelet-rich plasma (PRP) is a common therapy for acceleration of healing in bone graft therapies. Benefits of PRP are attributed to platelet-dependent release of cytokines and growth factors that promote blood coagulation, tissue repair and bone mineralization. To date, no study has evaluated PRP in bone fracture healing.

Methods: Following open fracture of femurs from 16 inbred male Lewis rats, normal saline (control) or PRP (2.67±0.15 fold enriched platelets) was applied to the fracture sites before wound closure. At four weeks, radiographs were evaluated for callus to cortex ratio.  In addition, fracture sites were analyzed by a three-point loading test and immunohistochemistry.

Results: Radiographic analysis demonstrated an increasing trend in the callus to cortex ratio in the PRP group (1.6±0.09 PRP; 1.49±0.11 control), indicating PRP-enhanced bone fracture healing. Three-point load bearing capabilities were similar between the two groups (71.74±5.37 [Newton] PRP; 67.40±2.55 [Newton] control). Immunohistochemistry demonstrated enhanced formation of trabecular bone mass in association with advanced downregulation of TGF-( and bone morphogenetic protein 2 (BMP-2) in the PRP group compared to the control at 4 weeks suggesting earlier onset of bone formation.

Conclusion: PRP-released growth factors might act on acceleration of TGF(1 and BMP-2 production from bone cells within the fracture site, thereby triggering early down-regulation of TGF(1 and BMP-2 expression. Our findings suggest that PRP may have a positive effect on the healing process of a bone fracture. Further investigations are being conducted to validate our findings.

Discussion: To test the effect of PRP on bone fracture healing, four rats were exsanguinated via open chest and direct cardiac puncture followed by PRP purification according to the method described by Tozum et al and Marx et al. (2, 3). As quality control, the purified PRP (2.67±0.15 fold enrichment of platelets compared to normal blood) was tested for premature activation of growth factors by a soluble P-selectin ELISA kit (R&D Systems, Minneapolis, MN). Thus, soluble P-selectin was detected in PRP (4.65 ng/ml), the measured levels were approximately 5 to 50fold lower than soluble P-selectin baseline serum levels previously detected in rats and humans by other groups, indicating that the PRP contained intact thrombocytes (4, 5). The purified thrombin-activated PRP or normal saline (control) was applied to open femur fracture sites before wound closure. At four weeks, bone fractures were evaluated by three-point loading bearing to test the grade of bone reconstitution (rigidity), bone radiographs to assess the callus to cortex ratio as well as histomorphometric and immunohistochemical analysis for the determination of bone morphology and growth factor status.

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In addition, PRP-fractures showed higher manifestation of lamellar trabecular bone mass with low occurrence of woven (endochondral) bone. In comparison, saline-treated fractures showed less trabecular bone formation that mainly consisted of woven (endochondral) bone (Figure 3B). Additionally, the saline-control fractures contained multiple small cartilage islands with distribution at the edge and spreading throughout the fracture site. Expression of TGF(1 and BMP-2 was higher in the saline-treated fractures compared to the PRP-treated fracture sites (Figure 3 E,H and F,I). In agreement with previous publications, expression of TGF(1 and BMP-2 was found in the osteoids near the trabecular bones in both, saline control and PRP-treated fracture sites and in chondrocytes of the saline control (6-9). Thus, no expression of TGF(1 and BMP-2 could be found in the chondrocytes of PRP-treated animals. Compared to normal bone repair where TGF(1 and BMP-2 expression peaks within the first two weeks, followed by a gradual reduction after 4-6 weeks during onset of ossification, the observed data suggest that PRP might accelerate TGF(1 and BMP-2 production from chondrocytes and osteocytes within the fracture site (6-9). An acceleration in TGF(1 and BMP-2 production might trigger the faster accomplishment of the peak expression phase and, accordingly, early downregulation of TGF(1 and BMP-2 expression. In contrast, the higher cartilage content and the localization of TGF(1 and BMP-2 in osteoids and in cartilage tissue of the saline control suggest late onset of ossification in the control with TGF(1 and BMP-2 still being at their higher expression phase.

Summary: The histomorphometric results suggest that PRP may accelerate healing of rat femur fractures via early onset of TGF(1 and BMP-2 growth factor expression. The proposed future studies try to monitor the time-dependent aspects of PRP-facilitated bone healing and include larger group sizes for better statistical evaluation.

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References

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To test the stiffness or strength of the repaired bone mass, mechanical measurement of bending by three-point load bearing was used (1). Three-point load bearing analysis after four week fracture healing of PRP or saline (control) treated bone fractures demonstrated a similar (50% (p ................
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