Abstract
Summary Statement
Parathytorid hormone-related protein (107–111) loaded onto biopolymer-coated nanocrystalline hydroxyapatite (HAGlu) improves the bone repair in a cavitary defect in rat tibiae.
Introduction
Biopolymer-coated nanocrystalline hydroxyapatite (HAGlu) made as macroporous foams are promising candidates as scaffolds for bone tissue engineering applications. They exhibit optimal features, promoting internalization, proliferation and differentiation of osteoprogenitors, with an adequate cell colonization over the entire scaffold surface. Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Its 107–111 epitope (osteostatin) exhibits osteogenic properties at least in part by directly acting on osteoblasts. The main aim of this study was to evaluate whether osteostatin loading into HAGlu scaffolds might improve their bone regeneration capacity.
Materials and Methods
HAGlu scaffolds were prepared as previously described (Sánchez-Salcedo S et al. J. Mater. Chem. 2010; 20:6956-61). Osteostatin was adsorbed onto HAGlu material by dipping into a solution containing this peptide at 100 nM (in phosphate-buffered saline, pH 7.4), following a standard protocol. We performed a cavitary defect (2 mm in diameter and 3 mm in depth) in both distal tibial metaphysis using a drill under general anesthesia in male Wistar rats (n=8) of 6 months of age. Unloaded HAGlu material (7 mg) was implanted into left tibial defects, whereas rigth tibial defects received the osteostatin-loaded material. Animals were sacrificed after 4 weeks for histological, μ-computerised tomography and gene expression analysis of the callus. Our protocol was approved by the Institutional Animal Care and Use Committee at the IIS-FJD. Mouse osteoblastic MC3T3-E1 cells were grown in differentiation medium (α-MEM with 10% fetal bovine serum, 50 µg/ml ascorbic acid, and 10 mM β-glycerolphosphate), in the presence or absence of HAGlu material with or without osteostatin. Cell viability (assessed by trypan blue staining), alkaline phosphatase (ALP) activity and mineralization (alizarin red) were analyzed at different culture times.
Results
The mean uptake of osteostatin by HAGlu scaffolds was about 60 % (representing 0.7 μg/implanted scaffold) after 24 h of loading, and they released a mean of 80 % of loaded peptide to the surrounding medium within 1–24 h. At 4 weeks, this osteostatin-containing HAGlu material significantly increased the bone volumen fraction and trabecular thickness of regenerating bone in the tibial methaphysis, compared to those observed with unloaded HAGlu scaffolds. In addition, osteostatin-coated HAGlu scaffolds increased (2-fold) the gene expression of osteocalcin and vascular cell adhesion molecule 1, but decreased (2-fold) that of the Wnt inhibitors, SOST and Dickkopf homolog 1 (DKK-1) in the fracture callus. In MC3T3-E1 cell cultures, osteostatin-loaded HAGlu material increased cell viability and ALP activity (each by 30%), and matrix mineralization (by 50%) at days 4 and 10, respectively.
Conclusions
These results indicate that osteostatin loading improves the bone regeneration capacity of HAGlu scaffolds. Our findings suggest that these scaffolds might be promising implants in orthopaedic applications. This work has been supported by a grant from Comunidad Autónoma de Madrid (S-2009/MAT/1472).
