Anke Kadow-Romacker

Sequential release kinetics of two (gentamicin and BMP-2) or three (gentamicin, IGF-I and BMP-2) substances from a one-component polymeric coating on implants.

The local application of antibiotics in combination with timely controlled growth factor delivery might be beneficial for the prevention of infections and to stimulate bone healing. Therefore, in this study a variable sequential drug delivery system with three distinctly different release profiles was developed: i) a burst release of gentamicin, ii) a burst release of IGF-I followed by a sustained release, and iii) a slow sustained release of BMP-2 out of an implant coating. Only one polymer [poly(D,L-lactide)], incorporating gentamicin, IGF-I or BMP-2, was used for two- or three-layer coatings of K-wires. To control the release kinetics, the polymer concentrations in the solvent were varied. The activity of early released gentamicin from a two-layer coating was confirmed microbiologically and BMP-2 stimulated the metabolic activity and alkaline phosphatase activity of C2C12 cells after 2 weeks. From the three-layer coated wires, IGF-I continuously stimulated the cell proliferation, whereas BMP-2 enhanced ALP between 1 and 3 weeks. The sequential release of growth factors revealed an additive effect on the metabolic activity and ALP of primary osteoblast-like cells compared to the single coated controls. The controlled delivery of different factors from one implant might prevent infections and subsequently stimulate the different phases of bone healing.

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

BackgroundBony 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).MethodsTo 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.ResultsThe 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-β1, VEGF, and PDGF. Comparing the three extraction methods, significant differences were measured for the individual growth factor content.ConclusionPES sterilized bony allografts contain several growth factors. Depending on the extraction method, the quantity of the analyzed growth factors varies.

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-κB 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.

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-β1 (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-β1 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-β1 or BMP-2. These results indicate a differentiation-dependent effect of growth factors on osteoclasts. TGF-β1 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.

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.

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.

A Focused Low-Intensity Pulsed Ultrasound (FLIPUS) System for Cell Stimulation: Physical and Biological Proof of Principle

Quantitative ultrasound (QUS) is a promising technique for bone tissue evaluation. Highly focused transducers used for QUS also have the capability to be applied for tissue-regenerative purposes and can provide spatially limited deposition of acoustic energy. We describe a focused low-intensity pulsed ultrasound (FLIPUS) system, which has been developed for the stimulation of cell monolayers in the defocused far field of the transducer through the bottom of the well plate. Tissue culture well plates, carrying the cells, were incubated in a special chamber, immersed in a temperature-controlled water tank. A stimulation frequency of 3.6 MHz provided an optimal sound transmission through the polystyrene well plate. The ultrasound was pulsed for 20 min daily at 100-Hz repetition frequency with 27.8% duty cycle. The calibrated output intensity corresponded to ISATA = 44.5 ± 7.1 mW/cm2, which is comparable to the most frequently reported nominal output levels in LIPUS studies. No temperature change by the ultrasound exposure was observed in the well plate. The system was used to stimulate rat mesenchymal stem cells (rMSCs). The applied intensity had no apoptotic effect and enhanced the expression of osteogenic markers, i.e., osteopontin (OPN), collagen 1 (Col-1), the osteoblast-specific transcription factor-Runx-2 and E11 protein, an early osteocyte marker, in stimulated cells on day 5. The proposed FLIPUS setup opens new perspectives for the evaluation of the mechanistic effects of LIPUS.

Low-Intensity Pulsed Ultrasound Improves the Functional Properties of Cardiac Mesoangioblasts

Cell-based therapy is a promising approach for many diseases, including ischemic heart disease. Cardiac mesoangioblasts are committed vessel-associated progenitors that can restore to a significant, although partial, extent, heart structure and function in a murine model of myocardial infarction. Low-intensity pulsed ultrasound (LIPUS) is a non-invasive form of mechanical energy that can be delivered into biological tissues as acoustic pressure waves, and is widely used for clinical applications including bone fracture healing. We hypothesized that the positive effects of LIPUS on bone and soft tissue, such as increased cell differentiation and cytoskeleton reorganization, could be applied to increase the therapeutic potential of mesoangioblasts for heart repair. In this work, we show that LIPUS stimulation of cardiac mesoangioblasts isolated from mouse and human heart results in significant cellular modifications that provide beneficial effects to the cells, including increased malleability and improved motility. Additionally, LIPUS stimulation increased the number of binucleated cells and induced cardiac differentiation to an extent comparable with 5′-azacytidine treatment. Mechanistically, LIPUS stimulation activated the BMP-Smad signalling pathway and increased the expression of myosin light chain-2 together with upregulation of β1 integrin and RhoA, highlighting a potentially important role for cytoskeleton reorganization. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore in the field of heart cell therapy.

Slight changes in the mechanical stimulation affects osteoblast- and osteoclast-like cells in co-culture.

SummaryBackground: Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Methods: Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1-5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. Results: In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. Conclusion: These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells.

Effect of β-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 β-tricalcium phosphate (β-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. β-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 β-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 β-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.