J Adv Prosthodont.  2010 Mar;2(1):7-13. 10.4047/jap.2010.2.1.7.

Angiogenic factor-enriched platelet-rich plasma enhances in vivo bone formation around alloplastic graft material

• Kim ES
• Kim JJ
• Park EJ

• KMID: 1975171
• DOI: http://doi.org/10.4047/jap.2010.2.1.7

Abstract

PURPOSE
Although most researchers agree that platelet-rich plasma (PRP) is a good source of autogenous growth factors, its effect on bone regeneration is still controversial. The purpose of this study was to evaluate whether increasing angiogenic factors in the human PRP to enhance new bone formation through rapid angiogenesis. MATERIAL AND METHODS: In vitro, the human platelets were activated with application of shear stress, 20 microgram/ml collagen, 2 mM CaCl2 and 10U thrombin/1 x 109 platelets. Level of vascular endothelial growth factor (VEGF) and platelet microparticle (PMP) in the activated platelets were checked. In the animal study, human angiogenic factors-enriched PRP was tested in 28 athymic rat's cranial critical bone defects with beta-TCP. Angiogenesis and osteogenesis were evaluated by laser Doppler perfusion imaging, histology, dual energy X-ray densinometry, and micro-computed tomography.
RESULTS
In vitro, this human angiogenic factors-enriched PRP resulted in better cellular proliferation and osteogenic differentiation. In vivo, increasing angiogenic potential of the PRP showed significantly higher blood perfusion around the defect and enhanced new bone formation around acellular bone graft material.
CONCLUSION
Angiogenic factor-enriched PRP leads to faster and more extensive new bone formation in the critical size bone defect. The results implicate that rapid angiogenesis in the initial healing period by PRP could be supposed as a way to overcome short term effect of the rapid angiogenesis.

Keyword

Platelet-rich plasma; Angiogenesis; Human; Athymic rat; Cranial defect

MeSH Terms

Angiogenesis Inducing Agents
Animals
Blood Platelets
Bone Regeneration
Calcium Phosphates
Cell Proliferation
Collagen
Durapatite
Humans
Intercellular Signaling Peptides and Proteins
Osteogenesis
Perfusion
Perfusion Imaging
Platelet-Rich Plasma
Rats, Nude
Transplants
Vascular Endothelial Growth Factor A
Angiogenesis Inducing Agents
Calcium Phosphates
Collagen
Durapatite
Intercellular Signaling Peptides and Proteins
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 A: Release of growth factors measured with ELISA. Platelet-derived growth factor (upper left), transforming growth factor-beta (upper right), basic fibroblast-growth factor (lower left), vascular endothelial growth factor (lower right). B: Production of platelet microparticles (PMPs) detected with Western blot. Standard; molecular weight standard of GPIIb α(125kD). Group A; activation with 10% CaCl2 and platelet-poor plasma (PPP), Group B; lysis with 0.1% Triton-X, Group C; activation with 142.8 U/mL thrombin and 4.3 mg/ml CaCl2, Group D; activation with shear force, 20 mg collagen, 10 U/ml thrombin and 2 mM CaCl2. Values represent mean and standard deviation.

• 

  Fig. 2 A: Proliferation of cord blood-derived outgrowth endothelial-like cells (left) and human bone marrow stromal cells (hBMSCs) (right) as indicated by absorbance of formazan at 490 nm after 48 hrs. B: Osteogenic differentiation of hBMSCs modulated by human cord blood-derived outgrowth endothelial-like cells (hCBOECs). Activity of alkaline phosphatase (left, unit: intensity of fluorescence). Production of osteocalcin (right, ng/mL). hBMSCs (1 × 104/cm2) and hCBOECs (1 × 104/cm2) were co-cultured with IMDM media, 1% FBS, 10-7 M dexamethasone and each PRP (100 µl/ml). Group A; activation with 10% CaCl2 and plateletpoor plasma (PPP), Group B; lysis with 0.1% Triton-X, Group C; activation with 142.8 U/ml thrombin and 4.3 mg/ml CaCl2, Group D; activation with shear force, 20 mg/ml collagen, 10 U/ml thrombin and 2 mM CaCl2. Values represent mean and standard deviation.

• 

  Fig. 3 A-C: Laser Doppler perfusion images of periosteal region of the cranial defect at 2 weeks after graft procedures. A. β-TCP only, B. β-TCP and conventional PRP activated with 142.8 U/ml thrombin and 4.3 mg/ml CaCl2, C. β-TCP and angiogenic factor-enriched PRP (PRP activated with shear force, 20 mg/ml collagen, 10 U/ml thrombin and 2 mM CaCl2, and containing peripheral blood mononuclear cells), D. Mean perfusion of each group (P < .05). The mean blood flows are normalized to the perfusion measured in the tail of the same animal (unit: voltage). White dotted circles manifest the original defects. Increased blood flow was indicated as color change from dark red to navy blue.

• 

  Fig. 4 Staining for vWF in critical size (8 mm) rat craniotomy defects following implantation of β-TCP only (A) and β-TCP + angiogenic factorsenriched PRP (A-PRP, B) at 2 weeks. Blood vessels are identified as circular structure with dark brown color: Quantitative analysis of the number of blood vessels in both groups (C). Values represent the mean and standard deviation.

• 

  Fig. 5 Photomicrographs of histological sections from critical size 8 mm rat craniotomy defects following implantation of β-TCP (Synthograft®) and several types of PRP at 7 weeks. A: β-TCP only, B: β-TCP and 0.1% triton-X treated PRP, C: β-TCP and PRP activated with 142.8 U/ml thrombin and 4.3 mg/ml CaCl2, D: β-TCP and angiogenic factorsenriched PRP (activated with shear force, 20 mg/ml collagen, 10 U/ml thrombin and 2 mM CaCl2 and containing peripheral blood mononuclear cells).

• 

  Fig. 6 Bone mineral density (A) and bone mineral content (B) of critical size (8 mm) rat craniotomy defects following implantation of β-TCP (Synthograft®) and several types of human PRP in athymic rats at 7 weeks. Group A. β-TCP only, Group B. β-TCP and 0.1% triton-X treated PRP, Group C. β-TCP and PRP activated with 142.8 U/ml thrombin and 4.3 mg/ml CaCl2, Group D. β-TCP and angiogenic factors-enriched PRP (activated with shear deformation, 20 mg/ml collagen, 10 U/ml thrombin and 2 mM CaCl2 and containing peripheral blood mononuclear cells). Values represent mean and standard deviation.

• 

  Fig. 7 MicroCT image reconstruction of critical size (8 mm) rat craniotomy defects at 7 weeks. A: β-TCP only, B: β-TCP and human angiogenic factors-enriched PRP in athymic rat. It shows positive effect of PRP in our experimental group.

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