EUROPEAN TISSUE REPAIR SOCIETY

STEM CELLS II

INTERACTION BETWEEN MESENCHYMAL STEM CELLS AND OSTEOGENIC CYTOKINES IN VITRO AND IN VIVO

Acompromised, larger and deteriorated bone defect requires new therapeutic modalities other than the conventional bone grafting, vascularized bone flaps, which often suffer from severe donor-site modalities.¹ The self-renewal and potential for subsequent differentiation into osteogenic lineage of the adult mesenchymal stem cell in the bone marrow may be induced by osteogenic signalling cues such as cytokines.²

Background

Cytokines such as bone morphogenetic protein-2 (BMP- 2) greatly induce osteoblastic differentiation, while angiogenic cytokines such as basic fibroblast growth factor (bFGF) can increase the DNA content in vitro. Osteogenic cytokine treatment to the human mesenchymal stem cells may demonstrate the osteogenic property and transplantable source.

Present status of the treatment

Human mesenchymal stem cells with BMP-2 and bFGF in vitro were tested in osteogenic medium containing dexamethasone, ascorbic acid and beta-glycerophosphate for alkaline-phosphatase (ALP)-positive cell numbers and for cell proliferation in serum-free medium.

A nude rat (F344/N Jcl-rnu) model, which is deficient in T cell immunity and more tolerant to the human-derived cells, was used for orthotopic bone healing and ectopic bone formation.^3,4 The human mesenchymal stem cells (hMSC), derived from a single donor, were purchased from BioWhittaker, Inc., Walkersville, MD, USA (Cat # PT- 2501) and the cryopreserved cells were immediately thawed at 37 °C. Recombinant human BMP-2 was kindly provided from Yamanouchi Pharmaceutical Co., Ltd., Tokyo and Kaken Pharmaceutical Co., Ltd., and recom-binant human FGF (Trafermin®) was purchased commercially.

ALP-positive cell numbers significantly increased by day 9 for hMSCs treated with combined BMP-2 (50 ng ml^-1) and bFGF (2.5 ng ml^-1) compared to the untreated hMSCs controls (p > 0.01, 29.0 ± 1.76 versus 11.2 ± 1.55, respectively). The cell number was significantly greater (p > 0.01) by day 3 in either bFGF alone or in BMP-2 and bFGF combined (6.4 ± 0.72 x 10³, 8.2 ± 0.81 x 10³) respectively, compared with controls cells (2.6 ± 0.27 x 103).

A nude rat (F344/N Jcl-rnu) calvarial defect showed significantly enhanced bone mineral density 4 weeks after treatment with hMSC in combination with either bFGF (10 micrograms), BMP-2 (10 micrograms) or with both cytokines combined, compared to those treated with hMSC alone or phosphate-buffered saline (PBS) only. Histologically, apparent basophilic mineralization was observed in hMSC with bFGF alone and partial lamellarity was formed in hMSC with BMP-2 alone, while the most remarkable bone ridge formation and osteoblast lining was detected when hMSC treated with a combination of two cytokines were used. By four weeks, hMSC alone demonstrated basophilic mineralization and partial lamellarity with a background stromal cell pattern. Bone marrow space formation and well-structured lamellarity were observed in hMSC with BMP-2 treatment, while the apparent osteocytes surrounded by an osteoblast expression pattern were demonstrated by hMSC treated with a combination of two cytokines. By nine weeks, all treatments demonstrated clear, organized and mature bone ridge formation.

In a 4 x 4 cm axial-pattern modified epigastric nude rat flap model, the hMSC and/or two cytokines in 0.1 ml of PBS were injected via a 30-gauge needle into the proximal site of the femoral artery after distal sites to the bifurcation of the femoral artery to the epigastric artery and vein were clamped.⁴ All clamps were removed after 10- minute incubation of the injected agents. The flap was tightly wrapped around a gelatin sponge and sutured back under the superficial skin. The specimen was investigated for sequential bone mineral density, histology and osteocalcin expression.

Bone mineral density significantly increased by day 7 post-transplantation in hMSC treated with bFGF or a combination of the two cytokines (Figure 1). By two weeks, faint but clear bone formation was demonstrated in hMSC treated with a combination of the two cytokines. By four weeks, the bone ridge demonstrated greater density in hMSC treated with both cytokines (Figure 2).

BMP-2 and bFGF can synergistically mediate mesenchymal stem cell differentiation towards bone formation and can promote proliferation in vitro and in two in-vivo models. Interaction between mesenchymal stem cells and osteogenic cytokines was clarified.

Figure 1: Bone Mineral Density of the ectopic bone formation model
Cell+ group (hMSCs with BMP-2 and bFGF treatment), cellgroups (BMP-2 and bFGF with no cells), soak (hMSCs and cytokines are soaked in the gelatin sponge and wrapped with the flap), rat calvarial defect (positive control of the 14 weeks of the bone healing). At day 7, cell+ group demonstrated significant bone density compared to cell- group (p<0.01, 48.4 ± 1.87 mg/ cm², 34.0 ± 1.11 mg/cm², respectively).

Figure 2: Histology of hMSCs and BMP-2 and bFGF treatment (H & E staining, _ 200)
Presumable bone ridge formation of the basophilic mineral structure spread in a network fashion. There are fibroblastlike undifferentiated cells in the interstitial space. These cells line along the mineral structure. The endothelial cells are well developed, suggesting there is sufficent blood flow.

References

1. Hartman EH, Spauwen PH and Jansen JA. Donorsite complications in vascularized bone flap surgery. J Invest Surg 2002; 15: 185–197.
2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143–147.
3. Dazai S, Akita S, Hirano A, et al. Leukaemia inhibitory factor enhances bone formation in calvarial bone defect. J Craniofac Surg 2000; 11: 513–520.
4. Akita S, Rashid MA, Ishihara H, Daian T, Dazai S, et al. Cytokine-Dependent gp130 Receptor Subunit Regulates Rat Modified Axial-Pattern Epigastric Flap. J Invest Surg 2002; 15: 137–151.

Sadanori Akita, MD, PhD, Masashi Fukui, MD and Tohru Fujii, MD.
Division of Plastic and Reconstructive Surgery
Department of Developmental and
Reconstructive Medicine
Graduate School of Biomedical Sciences
Nagasaki University, Japan
Kozo Akino, MD, PhD
Division of Anatomy and Neurobiology
Department of Developmental and
Reconstructive Medicine
Graduate School of Biomedical Sciences
Nagasaki University, Japan