Yonsei Med J.  2009 Dec;50(6):825-831. 10.3349/ymj.2009.50.6.825.

Effects of Polycaprolactone-Tricalcium Phosphate, Recombinant Human Bone Morphogenetic Protein-2 and Dog Mesenchymal Stem Cells on Bone Formation: Pilot Study in Dogs

• Kim SJ
• Kim MR
• Oh JS
• Han I
• Shin SW

• KMID: 1777098
• DOI: http://doi.org/10.3349/ymj.2009.50.6.825

Abstract

PURPOSE
The aim of this study was to evaluate the survival, proliferation, and bone formation of dog mesenchymal stem cells (dMSCs) in the graft material by using Polycaprolactone-tricalcium phosphate (PCL-TCP), auto-fibrin glue (AFG), recombinant human bone morphogenetic protein-2 (rhBMP-2), and dMSCs after a transplantation to the scapula of adult beagle dogs.
MATERIALS AND METHODS
The subjects were two beagle dogs. Total dose of rhBMP-2 on each block was 10 microg with 50 microg/mg concentration. The cortical bone of the scapula of the dog was removed which was the same size of PCL-TCP block (Osteopore International Pte, Singapore; 5.0x5.0x8.0 mm in size), and the following graft material then was fixed with orthodontic mini-implant, Dual-top(R) (Titanium alloy, Jeil Co. Seoul, Korea). Four experimental groups were prepared for this study, Group 1: PCL-TCP + aFG; Group 2: PCL-TCP + aFG + dMSCs; Group 3: PCL-TCP + aFG + dMSCs + rhBMP-2; Group 4: PCL-TCP + aFG + dMSCs + rhBMP-2 + PCL membrane. The survival or proliferation of dMSCs cells was identified with an extracted tissue through a fluorescence microscope, H-E staining and Von-Kossa staining in two weeks and four weeks after the transplantation.
RESULTS
The survival and proliferation of dMSCs were identified through a fluorescence microscope from both Group 1 and Group 2 in two weeks and four weeks after the transplantation. Histological observation also found that the injected cells were proliferating well in the G2, G3, and G4 scaffolds.
CONCLUSION
This study concluded that bone ingrowth occurred in PCL-TCP scaffold which was transplanted with rhBMP-2, and MSCs did not affect bone growth. More sufficient healing time would be needed to recognize effects of dMSCs on bone formation.

Keyword

PCL-TCP scaffold; dog MSCs; recombinant human bone morphogenic protein-2; auto-fibrin glue; bone formation

MeSH Terms

Animals
Bone Morphogenetic Proteins/*pharmacology
Calcium Phosphates/*pharmacology
Cell Proliferation/drug effects
Cell Survival/drug effects
Cells, Cultured
Dogs
Fibrin Tissue Adhesive/pharmacology
Humans
Mesenchymal Stem Cell Transplantation
Mesenchymal Stem Cells/*cytology/*drug effects/physiology
Microscopy, Fluorescence
Osteogenesis/*drug effects
Polyesters/*pharmacology
Recombinant Proteins/*pharmacology
Transforming Growth Factor beta/*pharmacology

Figure

• 

  Fig. 1 Position of graft materials in both scapular bones. PCL-TCP scaffold in size 5.0×5.0×8.0 mm was used, and the grafted material was fixed by orthodontic mini-implant. PCL-TCP, Polycaprolactone-tricalcium phosphate.

• 

  Fig. 2 Specimens taken 2 weeks after grafting. Grafts G1 and G2 (the non-BMP group) were easily detached from the recipient beds, whereas grafts G3 and G4 (the BMP group) were difficult to detach due to the bone-like tissue at the junction of the scaffolds and the scapular bone. BMP, bone morphogenetic protein.

• 

  Fig. 3 Light and fluorescence micrographs of the specimens taken 2 weeks after grafting. (A) Light micrograph shows that calcium is deposited in the bone matrix of grafts G3 and G4 (Von-Kossa stain, original magnification ×100). (B) Fluorescence micrograph reveals that dMSCs which are stained with PKH26 survive (fluorescence stain, original magnification ×100). dMSC, dog mesenchymal stem cell.

• 

  Fig. 4 Fluorescence micrographs of the specimens taken 4 weeks after grafting. Cellular proliferation was more active in the grafts without rhBMP-2 (G2) than in those with it (G3). rhBMP-2, recombinant human bone morphogenetic protein-2.

• 

  Fig. 5 Light micrographs of the specimens taken 2 weeks after grafting. Active cell proliferation was observed in the scaffolds of all groups. Bone formation from the area adjacent to the scapular bone was noted in grafts G3 and G4 (the BMP group) (H-E stain, original magnification ×40). BMP, bone morphogenetic protein.

• 

  Fig. 6 Light micrographs of the specimens taken 4 weeks after grafting. Bone formation was not observed in grafts G1 and G2. However, in grafts G3 and G4, bone formation was more active in the grafts taken 4 weeks after grafting than those taken 2 weeks after grafting (H-E stain, original magnification ×40).

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