Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro.
BACKGROUND
Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery.


METHODS
This in vitro study investigates cell seeding efficiency, metabolism, gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX), synthetic alpha-TCP (Biobase), synthetic beta-TCP (Vitoss), synthetic beta-TCP (Chronos), processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds, respectively. On days 2, 6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed.


RESULTS
The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; p<0.05) followed by Chronos (47.5+/-19.5, p<0.05), Actifuse ABX (19.1+/-4.4), Biobase (15.7+/-9.9), Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters, depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds, beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC.


CONCLUSIONS
Our results indicate that various bone-graft substitutes influence cell seeding efficiency, metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro, which may be important for bony integration in the clinical setting.
Seebach
Caroline
C
Department of Trauma Surgery, Johann-Wolfgang-Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany. ccseebach@aol.com
Schultheiss
Judith
J
Wilhelm
Kerstin
K
Frank
Johannes
J
Henrich
Dirk
D
D001672:Biocompatible Materials / Q000494:pharmacology; D001861:Bone Regeneration / Q000502:physiology*; D018786:Bone Substitutes / Q000494:pharmacology*; D016025:Bone Transplantation / Q000379:methods*; D002130:Calcium Phosphates / Q000494:pharmacology*; D002516:Ceramics / Q000494:pharmacology; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods
D001672:Biocompatible Materials; D018786:Bone Substitutes; D002130:Calcium Phosphates