Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

Abstract

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and β-tricalcium phosphate/hydroxyapatite (β-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29(+), CD44(+) and CD166(+) after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β-TCP/HA matrix comparable to the application of 60 μg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with β-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.

© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Figures

Fig 1

Sheep MSC were characterized using FACS and RT-PCR analysis. (A) With RT-PCR analysis CD29, CD44 and CD166 expression of MSC could be proofed on mRNA level. As indicated by increased CD45 expression, ratio of hematopoietic cells was higher in directly auto-transplanted MSC as compared to expanded MSC. (B–D) FACS analysis revealed sheep MSC to express CD29, CD44 and CD166. Expanded MSC (B) were negative for the hematopoietic markers CD31 and CD45. Directly auto-transplanted cells (C) had a different expression pattern than expanded MSC. The directly auto-transplanted MSC had a weaker CD29 and CD166 but a stronger CD45 expression. Mean fluorescent indices are shown in (D).

Fig 1

Sheep MSC were characterized using FACS and RT-PCR analysis. (A) With RT-PCR analysis CD29, CD44 and CD166 expression of MSC could be proofed on mRNA level. As indicated by increased CD45 expression, ratio of hematopoietic cells was higher in directly auto-transplanted MSC as compared to expanded MSC. (B–D) FACS analysis revealed sheep MSC to express CD29, CD44 and CD166. Expanded MSC (B) were negative for the hematopoietic markers CD31 and CD45. Directly auto-transplanted cells (C) had a different expression pattern than expanded MSC. The directly auto-transplanted MSC had a weaker CD29 and CD166 but a stronger CD45 expression. Mean fluorescent indices are shown in (D).

Fig 2

DiI-labelled MSC at passage 5. MSC DiI labelling was effective and was stable over several passages in cell culture. Nuclei are counterstained with DAPI (blue).

Fig 3

MSC in a fibrinogen–thrombin matrix were implanted subcutaneously on the sheep’s back (groups 1–3). Explants were harvested after 2 weeks (shown in A and B) and 4 weeks (shown in C and D) to investigate apoptosis and sufficient DiI labelling of the implanted MSC. Constructs with expanded MSC are shown in (A) and (C) versus constructs with directly auto-transplanted MSC shown in (B) and (D). DiI+ cells (red) were identified at all time-points in the subcutaneous implants. The staining intensity of expanded MSC was stronger in comparison to directly auto-transplanted MSC. Apoptotic cells (TUNEL assay) are shown in green. Both expanded and directly auto-transplanted cells had a decreasing apoptosis during the implantation period.

Fig 4

MSC in a fibrinogen–thrombin matrix were implanted subcutaneously on the sheep’s back (groups 1–3). Explants were harvested after 2 weeks (shown in A and B) and 4 weeks (shown in C and D) to investigate proliferation of the implanted MSC. Constructs with expanded MSC are shown in (A) and (C) versus constructs with directly auto-transplanted MSC shown in (B) and (D). DiI-labelled MSC are shown in red. Proliferating cells (Ki67) are shown in green. Both expanded and directly auto-transplanted cells had a constant proliferation during the implantation period.

Fig 5

β-TCP/HA granules were implanted subcutaneously in sheep with different BMP-2 concentrations (groups 5–7). Haematoxylin and eosin (A–C) and collagen I (D–F) staining were performed. A/D 2.5 μg/ml BMP-2, B/E 12.5 μg/ml BMP-2, C/F 60 μg/ml BMP-2. Only initial signs of bone formation could be observed while using 2.5 or 12.5 μg/ml BMP-2 nearby the β-TCP/HA granules. Trabecular, osteon-like bone formation could be detected in combination using 60 μg/ml BMP-2.

Fig 6

β-TCP/HA granules were implanted subcutaneously in sheep with different BMP-2 concentrations (groups 4–7) (A and B). The expression of genes specific for bone can be detected in the 60 μg/ml constructs. Expression levels in bone serve as controls. (C) Osteocalcin, osteonectin, osteopontin and collagen I are up-regulated in 60 μg/ml constructs compared to the control group using β-TCP/HA granules with fibrinogen–thrombin matrix without growth factors or cells.

Fig 7

For determination of the cell type which is qualified best for bone tissue engineering purposes, different groups (expanded versus directly auto-transplanted MSC, groups 8–10) were investigated. In both groups cells were DiI labelled prior to implantation and implanted subcutaneously with or without BMP-2. Bone formation is shown in haematoxylin and eosin (A–C) and collagen I staining (D–F). Directly auto-transplanted (B, E) or expanded MCS (A, D) and BMP-2 in combination with directly auto-transplanted MSC (C, F) contribute to bone formation in subcutaneous sheep implants. Bone formation was observed in all groups. (G) There is no difference between the groups with expanded or directly auto-transplanted MSC. Even the combination with BMP-2 in this setting did not improve the bone mass.

Fig 8

For determination of the cell type which is qualified best for bone tissue engineering purposes, different groups (expanded versus directly auto-transplanted MSC, groups 8–10) were investigated. In both groups cells were implanted subcutaneously with or without BMP-2. (A–C) The expression of bone-specific genes is proofed by RT-PCR analysis. (D) Osteocalcin and osteopontin are up-regulated in all three groups in comparison to the control group using β-TCP/HA granules with fibrinogen–thrombin matrix without growth factors or cells.

Fig 9

For determination of the cell type which is qualified best for bone tissue engineering purposes, different groups (expanded versus directly auto-transplanted MSC, groups 8–10) were investigated. In both groups cells were DiI labelled prior to implantation and implanted subcutaneously with or without BMP-2. (A–C) Expanded MSC (A), directly auto-transplanted MSC (B), BMP-2 in combination with directly auto-transplanted MSC (C). DiI-labelled MSC (red) could be found close to β-TCP/HA granules contributing to the newly formed bone parts. In the explants with directly auto-transplanted MSC a higher section of the DiI-labelled cells were found in the connective tissue parts of the constructs compared to the explants with expanded MSC or directly auto-transplanted MSC with BMP-2. (D–F) Sections of constructs of the groups with expanded MSC (D), directly auto-transplanted MSC (E), BMP-2 in combination with directly auto-transplanted MSC (F) were evaluated for vascularization. The constructs in all three groups are well vascularized as shown by CD31 immunohistochemistry (green). Nuclei are counterstained with DAPI (blue).