Catrin Strobel

Carrier systems and application of growth factors in orthopaedics

SUMMARY With optimal surgical treatment within an appropriate time frame, bony tissue has the potential to regenerate defects without the formation of scar tissue. However, even under optimal mechanical circumstances and appropriate operative treatment, healing can fail and delayed or non-union occur. In Europe delayed bone healing leads to socio-economic costs of up to 14.7 billion euros per year. In addition to the optimal clinical treatment, the success of bone regeneration depends on the following main aspects: (1) adequate mechanical stabilization and biological competence of the organism, (2) osteogenic cells, (3) osteoconductive structures or scaffolds, and (4) growth factors (Diamond Concept)(1). Further, (5) a sufficient vascularisation is essential for the nutritive supply. Within the last years two growth factors, BMP-2 and BMP-7, were approved for clinical use in orthopaedic and trauma surgery for different indications.(2,3) The establishment of carrier systems and application techniques for growths factors is the focus of current research. The combination of a well established stabilization system and local drug delivery system for bioactive factors could be a therapeutical strategy to optimize bone healing and reduce the complication rate, in the future.

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.

Stimulation of Bone Healing by Sustained Bone Morphogenetic Protein 2 (BMP-2) Delivery

The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (μCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p = 0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p = 0.006) with reduced cartilage at Day 84 (p = 0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p = 0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms.

Local BMP-2 application can rescue the delayed osteotomy healing in a rat model

Delayed healing is still a severe complication in the clinic. The aim of the present study was to investigate the effect of locally delivered BMP-2 incorporated in a poly(d,l-lactide) (PDLLA) implant coating in a rat model with delayed tibial healing. The healing delay in this model is not caused by mechanical instability or additional tissue manipulation and presents therefore a common and challenging clinical situation of impaired healing. Radiological, histological and biomechanical evaluations were performed at days 5, 10, 28, 42, and 84 after tibial osteotomy. The control group showed a delayed healing without complete bridging and without reaching the biomechanical stability of the contralateral tibiae after 84 days. The mechanical stability of the BMP-treated tibiae showed a significant increase at days 28 and 42 compared to the control group and exceeded the stability of the intact contralateral tibiae. Less cartilage was detected at day 28 and the mineralisation was significantly enhanced at day 42 due to the local BMP application. Looking at the early healing phase (day 10) a reduced vascularisation was seen in the BMP group. This reflects the situation seen during normal healing, whereas the delayed healing in the present model had an increased vascularisation. The present study clearly demonstrates that local BMP-2 application can stimulate delayed healing in a clinically relevant animal model.

Changing the release kinetics of gentamicin from poly(D,L-lactide) implant coatings using only one polymer.

Creating orthopedic implants that locally deliver drugs is an appealing approach to induce bone regeneration and prevent or treat infections. In this study, titanium K-wires were coated with poly(D, L-lactide) (PDLLA) solutions with different polymer/solvent/drug ratios to modify the release kinetics of the antibiotic gentamicin. The concentrations of PDLLA ranged from one-fold (100 mg/1.5 mL solvent, 1X) to four-fold (400 mg/1.5 mL solvent, 4X), where the higher concentrations led to the thickening of the drug-loaded coatings and an increase of total coating mass. Coated wires were incubated in PBS buffer at 37°C for up to 32 weeks, and the elution kinetics were analyzed at several time points. Different release profiles were observed: I) a burst release within the first hours for the coatings made out of lower concentrations of PDLLA with higher amounts of gentamicin and II) a sustained release of up to 14 weeks for the different coatings with higher polymer amounts with lower concentrations of gentamicin. Moreover, the amounts of remaining gentamicin on the wires after elution were dependent on the coating composition. Nearly complete gentamicin was released from the 1X PDLLA coatings and approximately one-third with respect to initial gentamicin remained in the 4X coatings. Based on these results, we garnered a better understanding of the parameters that influenced release kinetics in this simple system and described how to realize different release patterns by using only one polymer. Using this knowledge, tailored coated implants that can improve infection prophylaxis or stimulate bone healing may be designed.

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.

Feasibility of using sodium chloride as a tracer for the characterization of the distribution of matter in complex multi-compartment 3D bioreactors for stem cell culture.

The experimental characterization of the distribution of matter in complex multi-compartment three-dimensional membrane bioreactors for human cell culture is complicated by tracer interactions with the membranes and other bioreactor constituents. This is due to the fact that membranes with a high specific surface area often feature a hydrophobic chemical backbone that may adsorb tracers often used to this purpose, such as proteins and dyes. Membrane selectivity, and its worsening caused by protein adsorption, may also hinder tracer transfer across neighboring compartments, thus preventing effective characterization of the distribution of matter in the whole bioreactor. Tracer experiments with sodium chloride (NaCl) may overcome some of these limitations and be effectively used to characterize the distribution of matter in complex 3D multi-compartments membrane bioreactors for stem cell culture. NaCl freely permeates most used membranes, it does not adsorb on uncharged membranes, and its concentration may be accurately measured in terms of solution conductivity. In this preliminary study, the feasibility of complex multi-compartment membrane bioreactors was investigated with a NaCl concentration pulse challenge to characterize how their distribution of matter changes when they are operated under different conditions. In particular, bioreactors consisting of three different membrane types stacked on top of one another to form a 3D network were characterized under different feed conditions.