ABSTRACT
Implantation of ex vivo expanded and osteogenically differentiated mesenchymal stem cells (MSCs) for bone regeneration has drawbacks for clinical applications, such as poor survival of implanted cells and increased treatment expenses. As a new approach for bone regeneration that can circumvent these limitations, we propose dual delivery of substance P (SP) and bone morphogenetic protein-2 (BMP-2) to facilitate endogenous stem cell recruitment to bone defects by SP and subsequent in situ osteogenic differentiation of those cells by BMP-2. A heparin-conjugated fibrin (HCF) gel enabled dual delivery with fast release of SP and slow release of BMP-2, which would be ideal for prompt recruitment of endogenous stem cells in the first stage and time-consuming osteogenic differentiation of the recruited stem cells in the second stage. The HCF gels with SP and/or BMP-2 were implanted into mouse calvarial defects for 8 weeks. Local delivery of SP to the calvarial defects using HCF gel was more effective in recruiting MSCs to the calvarial defects than intraperitoneal or intravenous administration of SP. Many of the cells recruited by SP underwent osteogenic differentiation through local delivery of BMP-2. The efficacy of in vivo bone regeneration was significantly higher in the SP/BMP-2 dual delivery group. The dual delivery of SP and BMP-2 using the HCF gel therefore has potential as an effective bone regeneration strategy.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Bone Regeneration/drug effects , Mesenchymal Stem Cells/cytology , Substance P/pharmacology , Animals , Cattle , Cell Differentiation/drug effects , Cell Movement/drug effects , Fibrin/pharmacology , Flow Cytometry , Heparin/pharmacology , Humans , Inflammation/pathology , Mesenchymal Stem Cells/drug effects , Mice , Multipotent Stem Cells/cytology , Multipotent Stem Cells/drug effects , Osteogenesis/drug effects , Real-Time Polymerase Chain Reaction , Skull/drug effects , Skull/pathology , Tissue Scaffolds/chemistryABSTRACT
The culture surface can affect the in vitro differentiation of stem cells. In this study, we investigated whether modifying the culture surface with 3,4-dihydroxy-l-phenylalanine (DOPA), an element of mussel adhesion protein, could enhance the in vitro osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMMSCs). hBMMSCs cultured on DOPA-coated plates exhibited better cell adhesion and spreading compared with noncoated conventional tissue culture plates. The DOPA coated did not affect the apoptosis or viability of the cultured hBMMSCs. Also, hBMMSCs cultured on DOPA-coated plates exhibited a higher degree of osteogenic differentiation than did hBMMSCs cultured on noncoated plates, as evaluated with alkaline phosphate (ALP) activity, calcium content, and the mRNA expression of runt-related transcription factor 2, ALP, and osteocalcin. Further, hBMMSCs cultured on DOPA-coated plates demonstrated a higher capability of ectopic bone formation in vivo following implantation in the subcutaneous space of athymic mice compared with hBMMSCs cultured on noncoated plates, as evaluated with microcomputer topography analysis and histomorphometry. These results indicate that modifying the culture surface with DOPA can enhance the in vitro osteogenic differentiation of hBMMSCs.
