Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices.
The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.
D000469:Alkaline Phosphatase / Q000378:metabolism; D001672:Biocompatible Materials; D001854:Bone Marrow Cells / Q000201:enzymology / Q000502:physiology*; D018786:Bone Substitutes; D002208:Caproates; D017886:Durapatite; D005109:Extracellular Matrix / Q000502:physiology*; D006801:Humans; D020100:Hydrogels; D007783:Lactones; D008648:Mesoderm / Q000166:cytology / Q000201:enzymology; D018613:Microscopy, Confocal; D015675:Osteocalcin / Q000378:metabolism; D011091:Polyesters; D013234:Stem Cells / Q000201:enzymology / Q000502:physiology*; D023822:Tissue Engineering
D001672:Biocompatible Materials; D018786:Bone Substitutes; D002208:Caproates; D020100:Hydrogels; D007783:Lactones; D011091:Polyesters; D015675:Osteocalcin; C016240:polycaprolactone; C121056:caprolactone; D017886:Durapatite; D000469:Alkaline Phosphatase