Local gentamicin application does not interfere with bone healing in a rat model.

For the prophylaxis and treatment of bony infections antibiotics are locally used. Since several decades antibiotics mixed with bone cement (methylmethacrylate) are successfully used in prosthetic surgery and a gentamicin coated tibial nail is approved in Europe for fracture stabilization. The goal of the present study was to investigate if gentamicin, locally applied from a polymeric coating of intramedullary nails, might interfere with the bone healing process. Female Sprague Dawley rats (n = 72) were used and the tibiae were intramedullary stabilized with Kirschner-wires (k-wires) after osteotomy. This model was established earlier and shows a delayed healing with a prolonged inflammatory reaction. The open approach is clinically more relevant compared to a closed one because it mimics the clinically critical case of an open fracture, which has a higher risk of infection. The k-wire was either coated with the polymer poly(d,l-lactide) (control group) or with 10% gentamicin incorporated into the polymer (gentamicin group). In vivo µCT analyses were performed at days 10, 28, 42, and 84 after osteotomy. Mechanical torsional testing and histological evaluation were done at the days of sacrifice: 28, 42, and 84. The µCT analyses revealed an increase in tissue mineral density (TMD) over the healing period in both groups. In the control group, the torsional stiffness and maximum load did not reach the values of the intact contralateral side at any time point. At day 84 the gentamicin treated tibiae, however, showed significantly better maximum load compared to the control group. The histology showed no bony bridging in the control, whereas in 2 of 5 calluses of the gentamicin group mineralized bridging occurred. Significantly more mineralized tissue was measured in the gentamicin group. This study shows that the local gentamicin application does not negatively interfere with the long term healing process. Local infection prophylaxis is effective without negative effects on bone healing.

Effect of ß-tricalcium phosphate coated with zoledronic acid on human osteoblasts and human osteoclasts in vitro.

The combination of a bone graft material with bisphosphonates (BPs) might be advantageous for an optimal balance of material resorption and stimulation of bone formation. This study investigated the effect of ß-tricalcium phosphate (ß-TCP) bone grafts coated with zoledronic acid (ZOL) on osteoblast-like cells and osteoclast-like cells (OLC). As a drug carrier, the polymer poly(D,L-lactide) was used and three different concentrations of ZOL were tested. ß-TCP coated with ZOL stimulated the production of osteocalcin (OC), osteoprotegerin, and sRANKL in osteoblast-like cells. The polymer coating alone caused a significant increase in collagen type 1 and OC production. OLC viability was inhibited and the tartrate-resistant acidic phosphatase isoform-5b was significantly decreased after cultivation on polymer-coated ß-TCP for 12 days. The three different concentrations of ZOL decreased cell viability and no TRAPiso-5b was detectable, indicating a strong reduction of the TRAPiso-5b after 12 days in culture. After 21 days in culture, only the higher ZOL concentrations significantly reduced cell viability and TRAPiso-5b. The results of this study show that coating of ß-TCP with ZOL has stimulating effects on osteoblast-like cells. Additionally, an inhibition of osteoclasts was seen. The combination of this bone grafting material with BPs might, therefore, be effective in the treatment of large bone defects.

Cocultures of osteoblasts and osteoclasts are influenced by local application of zoledronic acid incorporated in a poly(D,L-lactide) implant coating.

The antiresorptive activity of bisphosphonates such as zoledronic acid (ZOL) has been shown in vitro to be because of their effect on osteoclasts and osteoblasts. However, whether the effect of ZOL on monocultures might be reproducible on cocultures and whether cell interactions might influence this effect has not been described. The aim of the study was to investigate the effect of ZOL on cocultures of osteoblasts and osteoclasts in vitro. ZOL was incorporated in an implant coating based on poly(D,L-lactide) in different concentrations (10-50 microM). Cell number was measured, and procollagen I synthesis, osteoprotegerin (OPG) secretion and soluble receptor activator of nuclear factor-kappaB ligand (sRANKL) were analyzed. Moreover, TRAP-positive cells and resorption lacunas on dentin chips were counted. Results showed that cell viability was not affected when treated with doses equivalent up to 50-microM ZOL-coated implants (ZOL-CI). Procollagen I and OPG synthesis was highest when treated with 10 microM ZOL-CI, whereas sRANKL showed no significant decrease when treated with the investigated concentrations of ZOL-CI. TRAP-positive cells were decreased when treated with ZOL-CI in a dose-dependent manner. Resorption activity of osteoclasts was not significantly decreased when treated with investigated concentrations of ZOL-CI. Exposure to specific concentrations of ZOL-CI showed a beneficial effect on osteoblast differentiation and protein synthesis. Formation of osteoclast was decreased, whereas a significant decrease in sRANKL secretion and resorption activity of osteoclasts could not be shown. The investigated effect on cocultures might be clinically useful to support fracture healing and to reduce orthopedic implant loosening.

Effect of mechanical stimulation on osteoblast- and osteoclast-like cells in vitro.

Bone-forming osteoblasts and bone-resorbing osteoclasts play an important role during maintenance, adaptation and healing of bone, and both cell types are influenced by physical activity. The aim of the present study was to investigate the effect of a narrow mechanical stimulation window on osteoblast- and osteoclast-like cells. Primary human cells were cultured on a bone-like structure (dentine) and three-point bending with approximately 1,100 microstrain was applied to the dentine at varying frequencies (0.1 and 0.3 Hz) and duration (1, 3 and 5 min daily over 5 days) resulting in different patterns of mechanical stimulation of osteoblast- and osteoclast-like cells. The longest stimulation (5 min at 0.1 Hz) induced a significant increase in osteoblast alkaline phosphatase activity and a significant decrease in osteoprotegerin (OPG) production, and resulted in a significant increase in the soluble receptor activator of NF-kappaB ligand (sRANKL)/OPG ratio towards sRANKL in comparison to the unstimulated osteoblast-like cells. All stimulations caused a significant decrease in collagen type 1 synthesis. Stimulation for 1 min at 0.3 Hz decreased the fusion and resorption activity of the osteoclast-like cells. These results demonstrate a direct effect of mechanical stimuli on osteoblast-like cells as well as on osteoclast formation and activity in vitro. The change in the sRANKL/OPG ratio towards the stimulation of osteoclastogenesis stresses the necessity to investigate the effect of the same stimulation parameter on the co-culture of both cell types.

The effect of zoledronic acid incorporated in a poly(D,L-lactide) implant coating on osteoblasts in vitro.

Bisphosphonates such as zoledronic acid (ZOL) are used in diseases associated with osteoclast-mediated bone loss. However, their antiresorptive activity is partly due to their effect on osteoblasts. Local application might increase the therapeutical fence and their local efficiency and reduce systemic side effects. Aim of the study was to investigate the effect of ZOL on human osteoblasts like cells in vitro with special focus on the synthesis of factors mediating osteoclast differentiation (RANKL, OPG). ZOL was incorporated in an implant coating based on poly(D,L-lactide) (PDLLA) in different concentrations (10-150 microM). Control groups were treated with uncoated implants, PDLLA-coated implants, and ZOL pure substance in corresponding concentrations. After an experimental period of 144 h, primary human osteoblasts were stained with alamar blue and cell viability was measured. Procollagen I synthesis, osteoprotegerin (OPG) secretion, and soluble receptor activator of nuclear factor-kappaB ligand (sRANKL) were analyzed. Results showed that cell viability was not affected when treated with doses equivalent up to 100 microM ZOL-coated implants (ZOL-CI). Procollagen I synthesis was highest when treated with 50 microM ZOL-CI. OPG increased significantly in the 10 microM ZOL-CI group, whereas sRANKL decreased significantly with different concentrations of ZOL-CI. Higher concentrations or exposure to the pure substance showed a decrease in cell viability, collagen I, OPG, and sRANKL synthesis. In conclusion, exposure to specific concentrations of ZOL-CI showed a beneficial effect on osteoblast differentiation and protein synthesis without influencing their proliferation. Changes in sRANKL and OPG production may contribute to the inhibition of osteoclastic bone resorption. This local antiresorptive effect might be clinically useful in osseous implant integration and fracture healing.

Quantification of various growth factors in different demineralized bone matrix preparations.

Besides autografts, allografts, and synthetic materials, demineralized bone matrix (DBM) is used for bone defect filling and treatment of non-unions. Different DBM formulations are introduced in clinic since years. However, little is known about the presents and quantities of growth factors in DBM. Aim of the present study was the quantification of eight growth factors important for bone healing in three different "off the shelf" DBM formulations, which are already in human use: DBX putty, Grafton DBM putty, and AlloMatrix putty. All three DBM formulations are produced from human donor tissue but they differ in the substitutes added. From each of the three products 10 different lots were analyzed. Protein was extracted from the samples with Guanidine HCL/EDTA method and human ELISA kits were used for growth factor quantification. Differences between the three different products were seen in total protein contend and the absolute growth factor values but also a large variability between the different lots was found. The order of the growth factors, however, is almost comparable between the materials. In the three investigated materials FGF basic and BMP-4 were not detectable in any analyzed sample. BMP-2 revealed the highest concentration extractable from the samples with approximately 3.6 microg/g tissue without a significant difference between the three DBM formulations. In DBX putty significantly more TGF-beta1 and FGFa were measurable compared to the two other DBMs. IGF-I revealed the significantly highest value in the AlloMatrix and PDGF in Grafton. No differences were accessed for VEGF. Due to the differences in the growth factor concentration between the individual samples, independently from the product formulation, further analyzes are required to optimize the clinical outcome of the used demineralized bone matrix.

Quantification of growth factors in allogenic bone grafts extracted with three different methods.

BACKGROUND: Bony allografts are used for defect filling. A reliable sterilization method is the peracetic acid-ethanol sterilization procedure (PES). Several studies showed the antimicrobiological efficacy of this method. Aim of this study was the quantification of growth factors necessary for bone formation in PES sterilized allografts (n = 9). METHODS: To extract the growth factors from the tissue three different methods were used: (a) use of collagenase 1 for extraction, (b) incubation of the material in a proteinase inhibitor cocktail (Complete), and (c) extraction with guanidine HCl. The supernatants from the different methods were analyzed for the total protein concentration and different growth factors. RESULTS: The extraction with guanidine HCl resulted in the highest amount of protein measurable in the supernatants of the samples. For comparison of the individual growth factor values the results were normalized to the protein content. The highest growth factor amount/protein was detectable for BMP-2 using the GndHCL method followed by FGFa, IGF-I, TGF-beta1, VEGF, and PDGF. Comparing the three extraction methods, significant differences were measured for the individual growth factor content. CONCLUSION: PES sterilized bony allografts contain several growth factors. Depending on the extraction method, the quantity of the analyzed growth factors varies.

Differences in the fusion and resorption activity of human osteoclasts after stimulation with different growth factors released from a polylactide carrier.

Previous in vivo studies were able to demonstrate the efficacy of locally released growth factors IGF-I, TGF-beta1, and BMP-2 from a poly(D,L-lactide) (PDLLA) implant coating on fracture healing. In vitro studies using human osteoblast-like cells showed an enhanced collagen-1 production due to growth factor application without an effect of the PDLLA on the investigated parameter. Both bone-forming osteoblasts and bone-resorbing osteoclasts are important during bone formation and fracture healing. The aim of this study was to investigate the influence of different growth factors and the polylactide coating into which they were incorporated on isolated osteoclasts. In vitro studies using human osteoclast-like cells derived from peripheral blood mononuclear cells (PBMNCs) were performed. Titanium K-wires coated with the lactide loaded with IGF-I and TGF-beta1 (alone and in combination) or BMP-2 were added to the culture in a non-contact manner and the fusion, resorption activity (pit formation assay), and TRAP 5b synthesis of the cells were analyzed. Differences in the effect of the growth factors were seen depending on the differentiation state of the cells. The fusion of the monocytes to multinuclear osteoclasts was significantly enhanced by the application of TGF-beta1 both alone and in combination with IGF-I. No effect was seen after application of IGF-I alone or BMP-2. The resorption activity of the osteoclasts analyzed on dentine chips was significantly enhanced after application of TGF-beta1 or BMP-2. These results indicate a differentiation-dependent effect of growth factors on osteoclasts. TGF-beta1 affects both the osteoclastogenesis and the activity of osteoclasts, whereas BMP-2 had an effect only on the activity of mature osteoclasts but not on the fusion of the PBMNCs.