Yonsei Med J.  2016 Jul;57(4):1006-1015. 10.3349/ymj.2016.57.4.1006.

Preclinical Study of Cell Therapy for Osteonecrosis of the Femoral Head with Allogenic Peripheral Blood-Derived Mesenchymal Stem Cells

• Fu Q
• Tang NN
• Zhang Q
• Liu Y
• Peng JC
• Fang N
• Yu LM
• Liu JW
• Zhang T

• KMID: 2374136
• DOI: http://doi.org/10.3349/ymj.2016.57.4.1006

Abstract

PURPOSE
To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH).
MATERIALS AND METHODS
rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR.
RESULTS
After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually.
CONCLUSION
We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.

Keyword

Osteonecrosis of the femoral head (ONFH); peripheral blood; mesenchymal stem cells (MSCs); cell transplantation; disease model; rabbit

MeSH Terms

Animals
Blood Cells/*cytology
Bone Morphogenetic Protein 2/genetics
*Cell- and Tissue-Based Therapy
Femur Head Necrosis/metabolism/*pathology/*therapy
Gene Expression Regulation
*Mesenchymal Stem Cell Transplantation
Mesenchymal Stromal Cells/*cytology
Osteonecrosis/*pathology/*therapy
PPAR gamma/genetics
Rabbits
Transplantation, Homologous
Bone Morphogenetic Protein 2
PPAR gamma

Figure

• 

  Fig. 1 Operation process of the establishment of rabbit model by liquid nitrogen freezing. (A) Blunt separation of the gluteus maximus muscle, gluteus medius muscle, and gluteus minimus muscle. (B) Dislocation of femoral head and covering it with a rubber funnel. (C) Freezing the femoral head with liquid nitrogen by medical cotton stickers. (D) Drilling a 5 mm-depth hole at an angle of 45° from the posteromedial femoral neck according to the direction of the arrow until the bit arrived at the underside of articular cartilage. (E) Injection of PBMSCs of passage 2 or passage 3 after drilling. (F) Suture of the wound layer by layer. (G) Direction of the drilling. PBMSCs, peripheral blood-derived mesenchymal stem cells.

• 

  Fig. 2 Morphology of cultured rPBMSCs. (A) Primary colony growth of rPBMSCs for 3 d ×40. (B) Primary confluent growth of rPBMSCs for 10 d ×100. (C) The second passage of rPBMSCs ×200. (D) The second passage of rPBMSCs ×200. rPBMSCs, peripheral blood-derived mesenchymal stem cells from rabbits.

• 

  Fig. 3 Phenotypic analysis of mobilized rPBMSCs. (A) Demonstration of the positive expression of CD29 and the negative expression of CD14 by FCM. (B) Positive expression of CD44, CD105, and CD90, and negative expression of CD45 and CD34 by immunocytochemical staining ×200. rPBMSCs, peripheral blood-derived mesenchymal stem cells from rabbits; FCM, flow cytometer; PBS, phosphate buffer saline.

• 

  Fig. 4 CFE and differentiation capacities of cultured rPBMSCs. (A) Colonyforming ability of CFU-F isolated from mobilized rabbit peripheral blood. (B) One CFU-F of mobilized peripheral blood under a light microscope. (C) Colony-forming ability of CFU-F isolated from non-mobilized rabbit peripheral blood. (D) CFU-F of non-mobilized peripheral blood under a light microscope. (E) Comparison of CEF between mobilized peripheral blood and non-mobilized one. (F) Osteogenesis-committed differentiation of PBMSCs by alizarin red ×400. (G) Adipogenesis-committed differentiation of PBMSCs stained by oil red O ×400. (H) Chondrogenesis-committed differentiation of PBMSCs stained by alcian blue ×400. **p<0.01. CFE, colony forming efficiency; rPBMSCs, peripheral blood-derived mesenchymal stem cells from rabbits; CFU, colony forming units; PBMNCs, peripheral blood mononuclear cells.

• 

  Fig. 5 Labelling in vitro and survival in vivo of mobilized rPBMSCs after implantation. (A) View of PKH26-labelled PBMSCs in vitro under phase contrast microscope ×100. (B) View of PKH26-labelled PBMSCs in vitro under a fluorescent microscope ×100. (C) rPBMSCs marked with PKH26 and DAPI could be found in the lesion site at 2 weeks, less conspicuously at 6 weeks, and faintly at 8 weeks as the arrow pointed ×100. rPBMSCs, peripheral blood-derived mesenchymal stem cells from rabbits.

• 

  Fig. 6 Radiographs, histopathologic changes, and expression of BMP-2 and PPAR-γ of FHs in all rabbit groups. (A) There were obvious collapse, lessened shape, serious defect, greater joint space, and even a changed para position in the control group. (B) The central zone density was lower, while the edge higher and the sclerosis lines appeared in the CD group. (C) High-density image and bone trabeculae appeared in the PBMSCT group at 8 w. (D) Post-operation HE staining of the model group, (E) CD group, and (F) PBMSCT group at 8 w ×100. (G) Percentage of area of bone trabeculae. (H) Expression of BMP-2 at 2 weeks. (I) Expression of PPAR-γ at 2 weeks. (J) Expression of BMP-2 at 8 weeks. (K) Expression of PPAR-γ at 8 weeks. *p<0.05, **p<0.01. FH, femoral head; CD, core decompression; HE, hematoxylin and eosin; PBMSCT, peripheral blood-derived mesenchymal stem cells transplantation.

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