ABSTRACT
The osteoinductivity of human growth-and-differentiation factor-5 (GDF-5) is well established, but a reduced amount of ectopic bone is formed compared to other members of the bone morphogenetic protein (BMP) family like BMP-2. We hypothesized that swap of two BMP-receptor-interacting residues of GDF-5 to amino acids present in BMP-2 (methionine to valine at the sites 453 and 456) may improve the bone formation capacity of the mutant GDF-5. Heterotopic bone formation of a mutant GDF-5 coated beta-TCP carrier was compared to carriers coated with similar amounts (10 microg) of GDF-5 and BMP-2 in SCID mice. Four week explants revealed 6-fold higher ALP activity in the mutant GDF-5 versus the wild type GDF-5 group (p < 0.0001) and 1.4-fold higher levels compared to BMP-2 (p < 0.006). Bone area in histology was significantly higher in mutant GDF-5 versus all other groups at 4 weeks; however, at 8 weeks BMP-2 reached a similar neo-bone formation like mutant GDF-5. Micro-CT evaluation confirmed higher values in the mutant GDF-5 and BMP-2 groups compared to wild type GDF-5. In conclusion, the mutant GDF-5 showed superior bone formation capacity than GDF-5, and a faster induction at similar final outcome as BMP-2. Mutant GDF-5 thus represents a promising new GDF-5 variant for bone regeneration possibly acting via an increased binding affinity to the BMP-type I receptor.
Subject(s)
Calcium Phosphates/chemistry , Choristoma/pathology , Growth Differentiation Factor 5/genetics , Growth Differentiation Factor 5/pharmacology , Osteogenesis/drug effects , Point Mutation/genetics , Tissue Scaffolds/chemistry , Alkaline Phosphatase/metabolism , Animals , Bone and Bones/diagnostic imaging , Bone and Bones/drug effects , Bone and Bones/enzymology , Bone and Bones/pathology , Enzyme Activation/drug effects , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/metabolism , Humans , Mice , Mice, SCID , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , X-Ray MicrotomographyABSTRACT
The in vitro effect of platelet-rich plasma (PRP) on cell loading, proliferation, and osteogenic differentiation of human mesenchymal stem cells (MSC) is assessed on distinct resorbable and synthetic calcium phosphate scaffolds. A high specific surface area scaffold composed of calcium-deficient hydroxyapatite (CDHA; 48m2/g) is compared with one made out of beta-tricalcium phosphate (beta-TCP; surface area <0.5 m2/g). Fivefold concentrated fresh PRP is applied to scaffolds loaded with 2 x 10(5) MSC (n = 5). These constructs are kept in a medium with osteogenic supplements for 3 weeks. The addition of PRP leads to a higher cell loading efficiency of MSC on CDHA (p = 0.0001), that reaches the values of beta-TCP. Proliferation over 21 days is improved by PRP both on CDHA (p = 0.0001) and beta-TCP (p = 0.014) compared to MSC/calcium phosphate composites. Without the addition of PRP, CDHA has a lower cell loading efficiency (p= 0.0001) and proliferation (p= 0.001) than beta-TCP. The ALP activity is higher in the MSC/ceramics groups than in the monolayer controls (p<0.05). The addition of PRP does not significantly affect ALP activity. However, ALP activity varies considerably within the cell donors and different PRP-pools (p = 0.001), while the cell numbers do not vary within these two parameters. PRP generates a positive effect on the loading efficiency of MSC on the high specific surface scaffold CDHA that thereby reaches the loading efficiency of beta-TCP. PRP improved proliferation, but its osteogenic properties on both calcium phosphate scaffolds are weak.
Subject(s)
Biocompatible Materials/chemistry , Calcium Phosphates/chemistry , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Osteoblasts/cytology , Osteogenesis/physiology , Platelet-Rich Plasma/metabolism , Tissue Engineering/methods , Bone Substitutes/chemistry , Cell Culture Techniques/methods , Cell Differentiation , Cell Proliferation , Cells, Cultured , Humans , Materials Testing , Osteoblasts/physiologyABSTRACT
The interaction of stem cells and ceramics in bone regeneration is still poorly understood. The aim of this study was to examine the influence of the porosity (25%, 65% and 75%) of beta-tricalcium phosphate (TCP) ceramics on osteogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. For the in vitro portion of the study, TCP scaffolds loaded with MSC were kept in osteogenic induction medium for 21 days. For the in vivo portion of the study, scaffolds loaded with undifferentiated MSC were implanted subcutaneously into SCID mice for 8 weeks and compared with similarly implanted controls that were not loaded with MSC. Measurements of total protein as well as specific alkaline phosphatase (ALP) activity were taken as indicators of growth/matrix production and osteogenic differentiation. An increase in the total protein concentration was noted from day 1 to day 21 on the in vitro TCP 65% and TCP 75% scaffolds (p<0.05) with no such increase noted in the TCP 25% specimens. However, the specific alkaline phosphatase activity increased from day 1 to day 21 in all three in vitro specimens (p<0.02) and reached similar levels in each specimen by day 21. In vivo, ALP activity of cell-loaded TCP 65% ceramics was higher when compared with both the TCP 25% and TCP 75% specimens (p<0.046), and higher in the TCP 75% than TCP 25% specimens (p=0.008). Histology revealed mineralization by human cells in the pores of the TCP ceramic scaffolds with a trend toward greater calcification in TCP 65% and 75%. In summary, a higher porosity of TCP scaffolds does not necessarily mean a higher ALP activity in vivo. The distribution and size of the pores, as well as the surface structure, might play an important role for osteogenic differentiation in vivo.
