Objectives

Oxidative stress plays a major role in the onset and progression of involutional osteoporosis. However, classical antioxidants fail to restore osteoblast function. Interestingly, the bone anabolism of parathyroid hormone (PTH) has been shown to be associated with its ability to counteract oxidative stress in osteoblasts. The PTH counterpart in bone, which is the PTH-related protein (PTHrP), displays osteogenic actions through both its N-terminal PTH-like region and the C-terminal domain.

Objectives

Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied.

Summary Statement

Description of an original in vitro protocol for assessing combined bacteria and cell competitive adherence on the surface of biomaterials of medical interest

Summary Statement

Parathytorid hormone-related protein (107–111) loaded onto biopolymer-coated nanocrystalline hydroxyapatite (HA_Glu) improves the bone repair in a cavitary defect in rat tibiae.

Type 1 diabetes mellitus (DM) is associated with a decreased bone formation. Osteoblastic expression of parathyroid hormone-related protein(PTHrP) -an important modulator of osteoblast differentiation- decreases in age-related osteopenia. We here examined the putative role of PTHrP on the decreased osteoblastic function in DM.

We performed marrow ablation in the tibiae of diabetic mice after streptozotocin injection (glycemia > 300mg/dl). Some mice were treated with PTHrP(1–36) (100 ng/g/every other day, s.c.) or vehicle for 2 weeks. Both tibiae were then removed for histological evaluation or total RNA isolation. In vitro, MC3T3-E1 cells were grown in differentiation medium (a-MEM), with or without high glucose(HG) (25 mM) (or mannitol, as osmotic control), supplemented (or not) with PTHrP(1–36) (100 nM). In some experiments, anti-PTHrP N-terminal antibody C13 (1:100) or PTHrP(7–34) (1 μM) were added to normal-glucose medium. RANKL secretion was measured in the cell-conditioned medium by ELISA. Gene expression was analyzed by real-time PCR.

DM induced a 10–15% weight loss and a decrease (20–40%;p< 0.05) in the gene expression of the following osteoblastic factors in the regenerating tibia for 6 days: PTHrP, the PTH/PTHrP type1 receptor (PTH1R), osteocalcin, VEGF and its receptors 1 and 2; and in the OPG/RANKL ratio, related to an increased PPAR-γ mRNA expression. Compared to control mice, the regenerating tibia of DM mice showed a 5-fold increase in adipocyte number, and a decreased osteoblast number and osteoid surface. In MC3T3-E1 cells, HG decreased (20–40%) the OPG/RANKL ratio and the gene expression of both PTHrP/PTH1R and VEGF systems. PTHrP(1–36) reversed these HG-related effects in vivo and in vitro. Similar inhibitory effects were induced by a neutralizing PTHrP antibody or the antagonist PTHrP(7–34) in these cells in normal glucose.

In conclusion, a deficit in PTHrP production by osteoblasts seems to be at least in part responsible for the DM-related decreased bone formation in mice.

SBA-15 is a siliceous mesoporous ordered material with hexagonal arrangement of 9-nm tubular pores connected by micropores, high pore volume and abundance of silanol groups. This functionalised material could thus tailor the release kinetics of specific biomolecules to the clinical needs. Non-functionalized SBA-15 and its C8- or C3-alkyl-derivatives were coated with parathyroid hormone–related protein (PTHrP)(107–111) to assess their relative effects on osteoblastic cell growth and function.

SBA-15 was functionalized with either octyl or propyl trimethoxysilane (C8 or C3 precursor, respectively) in ACN for 24h and then were coated (or not) by dipping in 10 nM PTHrP (107–111) solution for 24 h at 4°C. After air drying, biomaterials were transferred to culture dishes. MC3T3-E1 cells were cultured in differentiation medium with SBA-15, C3-SBA-15 and C8-SBA-15, loaded or not with the peptide. Cell viability and proliferation were evaluated by trypan blue exclusion and a proliferation kit (Promega), respectively. Alkaline phosphatase (ALP) activity and collagen secretion were determined by colorimetric methods. Gene expression was analyzed by real-time PCR. Mineralization was assessed by alizarin red staining.

PTHrP(107–111)-coated SBA-15 increased cell proliferation (50%), cell viability (20%), and ALP activity (15%) over control values within 2–4 days. At day 2, collagen secretion increased (20%), and also the gene expression of ALP, PTHrP, and VEGF, which normalized at day 8, in these cells. An increase (by 30–40%) in all of these parameters was induced by peptide-coated C3-SBA-15 at day 4. Similar stimulatory effects were also observed with PTHrP(107–111)-coated C8-SBA-15 but only at day 8. At day 10, collagen secretion slightly increased (10–15%), and also mineralization (30–40%) with both functionalized materials coated with the PTHrP peptide.

In conclusion, PTHrP(107–111)-coated SBA-15 stimulates osteoblastic function in vitro; an effect delayed by C3- or C8-functionalization. These data further support the clinical impact of this bioceramic as functionalized implants in vivo.