Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics.
BACKGROUND
Mesenchymal stem cells (MSC) applied to bone substitution materials can improve bone healing. Bone formation in biocomposites is highly dependent on the kind of biomaterial, its pre-treatment and the applied cells. Potentially immunogenic or infectious supplements such as fetal calf serum (FCS) should be avoided in cell expansion media. Therefore, we developed an expansion protocol free of xenogenic supplements. Cells expanded with two different media were tested on distinct biomaterials for their bone formation capacity after ectopic implantation in vivo, as well as for their growth rate and differentiation capacity in vitro.


METHODS
MSC of six donors were expanded with cell expansion medium containing FCS (2%) or platelet-rich plasma (PRP, 3%). Their growth rate and osteogenic, adipogenic and chondrogenic differentiation capacity were compared in vitro. For the in vivo bone formation assay, expanded cells (2 x 105 or 2 x 106) were seeded on calcium-deficient hydroxyapatite (CDHA; n = 12) and on beta-tricalcium phosphate (beta-TCP; n = 12) blocks, which had been coated with either fibronectin or human serum. They were then implanted subcutaneously in severe combined immunodeficient mice (SCID), harvested after 8 weeks and analysed by histology. Bone formation was assessed by a semi-quantitative bone score, after toluidine blue and alizarin red staining. Human cells were detected by an in situ hybridisation for human-specific alu sequences.


RESULTS
PRP-supplemented expansion medium yielded two-fold higher cell numbers compared to medium with FCS (P = 0.046) after 3 weeks (four passages) and retained a similar capacity to differentiate towards the osteogenic, chondrogenic and adipogenic lineage. In vivo bone formation was equal for cells expanded with PRP and FCS and depended on the specific surface area of the carrier. CDHA (specific surface area (SSA) 48 m2/g) showed a significantly better bone formation in deep layers (P = 0.005) than beta-TCP (SSA 0.5 m2/g). Fibronectin-coating of the ceramics was slightly superior to coating with human serum (P = 0.045).


CONCLUSIONS
The replacement of FCS by PRP eliminated risks connected with the use of xenogeneic supplements. It improved expansion of MSC and retained their differentiation and in vivo bone formation capacity in a setting adaptable to autogenous use.
Vogel
Julia P
JP
Department of Orthopaedic Surgery, University of Heidelberg, Schlierbacher Landstr. 200a, 69118 Heidelberg, Germany.
Szalay
Krisztian
K
Geiger
Florian
F
Kramer
Martin
M
Richter
Wiltrud
W
Kasten
Philip
P
D000818:Animals; D001672:Biocompatible Materials; D001792:Blood Platelets / Q000502:physiology*; D002130:Calcium Phosphates; D002450:Cell Communication / Q000502:physiology; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D002516:Ceramics / Q000737:chemistry / Q000627:therapeutic use*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003470:Culture Media / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D006801:Humans; D008422:Materials Testing; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D010012:Osteogenesis; D023822:Tissue Engineering / Q000379:methods*; D014183:Transplantation, Heterologous
D001672:Biocompatible Materials; D002130:Calcium Phosphates; D020099:Coated Materials, Biocompatible; D003470:Culture Media; D017077:Culture Media, Conditioned; C020243:calcium phosphate