Stephan Zeiter

Augmentation of bone defect healing using a new biocomposite scaffold: an in vivo study in sheep.

Previous studies support resorbable biocomposites made of poly(L-lactic acid) (PLA) and beta-tricalcium phosphate (TCP) produced by supercritical gas foaming as a suitable scaffold for tissue engineering. The present study was undertaken to demonstrate the biocompatibility and osteoconductive properties of such a scaffold in a large animal cancellous bone model. The biocomposite (PLA/TCP) was compared with a currently used beta-TCP bone substitute (ChronOS, Dr. Robert Mathys Foundation), representing a positive control, and empty defects, representing a negative control. Ten defects were created in sheep cancellous bone, three in the distal femur and two in the proximal tibia of each hind limb, with diameters of 5 mm and depths of 15 mm. New bone in-growth (osteoconductivity) and biocompatibility were evaluated using microcomputed tomography and histology at 2, 4 and 12 months after surgery. The in vivo study was validated by the positive control (good bone formation with ChronOS) and the negative control (no healing with the empty defect). A major finding of this study was incorporation of the biocomposite in bone after 12 months. Bone in-growth was observed in the biocomposite scaffold, including its central part. Despite initial fibrous tissue formation observed at 2 and 4 months, but not at 12 months, this initial fibrous tissue does not preclude long-term application of the biocomposite, as demonstrated by its osteointegration after 12 months, as well as the absence of chronic or long-term inflammation at this time point.

Accuracy of Three Techniques to Determine Cell Viability in 3D Tissues or Scaffolds

Several different assays are commonly used to evaluate survival of cells inside tissues or three-dimensional carriers, but their accuracy and reliability have not been evaluated. Here, we compare three methods for cell viability (CV) determination: (i) lactate dehydrogenase (LDH) staining on cryosections, (ii) calcein AM/ethidium homodimer-1 (CaAM/EthH) staining, and (iii) carrier digestion and trypan blue (TB) assay. Living and dead cell populations were generated from bovine chondrocytes and combined to produce approximately 0%, 25%, 50%, 75%, and 100% CV mixtures. CV ratios were measured with TB assay (MIX) before seeding cells into fibrin carriers. CV was then determined using the three methods (n = 5/method). Custom-written macros were used to process LDH- and CaAM/EthH-stained images, and hand counting with hemocytometer was used for the TB method. Absolute error and intraclass correlation (ICC) were used for accuracy and reliability evaluation. All methods estimated CV values close to MIX values. TB method was the most accurate (ICC = 0.99) followed by CaAM/EthH (ICC = 0.98) and LDH (ICC = 0.97). As for absolute quantification of living and dead cells, TB and LDH methods performed well (ICC = 0.75-0.96), whereas CaAM/EthH largely overestimated cell numbers (living, ICC = 0.30; dead, ICC = 0.30). Although TB was the most accurate, LDH and CaAM/EthH provide valuable information on cell shape and spatial distribution of cells in tissue or a scaffold.

Effect of TGF β1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells

Bioactive factors, such as TGF beta and BMP-2, as well as mechanical factors i.e. compressive loading and hydraulic pressure, have been shown to induce and/or modulate chondrogenesis of bone marrow derived mesenchymal stromal cells (BMSCs). Since these factors are intracellularly transduced through different mechanisms, it is hypothesized that TGF beta, BMP-2 and hydraulic pressure may act synergistically on chondrogenic differentiation of BMSCs. Aggregates of bovine BMSC were cultured in the presence of 10 ng/ml TGF beta1, 50 ng/ml BMP-2 or both. Half of the samples were loaded for 4 hours per day with 0.5-3 MPa cyclic hydraulic pressure at 1 Hz. After 14 days of culture/loading, gene expression of chondrogenic genes was assessed. DNA as well as glycosaminoglycan (GAG) content of the pellets were analysed. Neither pressure nor BMP-2 had an influence on GAG/DNA content. However, cells responded to the presence of TGF beta1 with an up-regulation of chondrogenic genes and GAG/DNA of the aggregates increased compared to controls demonstrating the cells ability to respond to external stimuli. The used concentrations of BMP-2 and parameters for pressure were neither able to induce nor modulate chondrogenesis of bovine BMSCs and thus no synergistic effects were observed.

Comparison of locking and conventional screws for maintenance of tibial plateau positioning and biomechanical stability after locking tibial plateau leveling osteotomy plate fixation.

OBJECTIVE To compare locking screws with conventional screws inserted in the tibial plateau fragment for reduction and stability of the construct after tibial plateau leveling osteotomy (TPLO), using a locking TPLO plate. STUDY DESIGN Experimental biomechanical study. ANIMALS Cadaveric canine pelvic limbs (n=8 pairs). METHODS TPLO was stabilized with either conventional cortical screws or locking screws in a compressed osteotomy model. Titanium pins inserted into the tibial plateau and proximal metaphysis were used to track bone fragment location by computed tomography (CT) imaging. CT imaging was performed after osteotomy reduction, after plate stabilization, and after 30,000 cycles of axial compression testing. After 30,000 cycles, cyclic loading was continued with monotonically increasing peak-load until failure. RESULTS The magnitude of rotation about the sawing axis was significantly greater for the conventional screw group because of plate application (P=.009). Translational movement of the tibial plateau fragment toward the plate was significantly greater for the conventional screw group (P=.006). There were no significant differences between groups in stiffness or number of cycles to failure. CONCLUSION Maintenance of tibial plateau position was significantly superior for the locking screw group during plate application; however, screw type had no effect on fixation stability under cyclic loading. CLINICAL RELEVANCE These results suggest that conventional screws and careful contouring of the TPLO plate can provide comparable mechanical stability to fixation with locking screws in the tibial plateau under load-sharing conditions, but potentially at the expense of osteotomy reduction.

CD34/CD133 enriched bone marrow progenitor cells promote neovascularization of tissue engineered constructs in vivo

Vascularization is critical for 3D tissue engineered constructs. In large size implants the ingrowth of vessels often fails. The purpose of this study was to identify an easily accessible, clinically relevant cell source able to promote neovascularization in engineered implants in vivo and to establish an autologous culture method for these cells. MSCs (mesenchymal stem cells) and an endothelial progenitor containing cell (EPCC) population were obtained from human bone marrow aspirates. The expression of endothelial-markers, uptake of acetylated low density lipoprotein (acLDL) and tube-like structure formation capability of EPCCs were analyzed after expansion in endothelial growth medium or medium supplemented with autologous platelet lysate (PL). EPCCs were co-seeded with MSCs on hydroxyapatite-containing polyurethane scaffolds and then implanted subcutaneously in nude mice. Human EPCCs displayed typical characteristics of endothelial cells including uptake of acLDL and formation of tube-like structures on Matrigel™. In vivo, EPCCs cultured with PL triggered neovascularization. MSC/EPCC interactions promoted the maturation of newly formed luminal structures, which were detected deep within the scaffold and partly perfused, demonstrating a connection with the host vascular system. We demonstrate that this population of cells, isolated in a clinically relevant manner and cultured with autologous growth factors readily promoted neovascularization in tissue engineered constructs in vivo enabling a potential translation into the clinic.

A Standardized Critical Size Defect Model in Normal and Osteoporotic Rats to Evaluate Bone Tissue Engineered Constructs

Tissue engineered constructs should be tested for their efficacy not only in normal but also in osteoporotic bone. The rat is an established animal model for osteoporosis and is used often for bone healing studies. In this study a defined and standardized critical size defect model in the rat suitable for screening new tissue engineered constructs in normal and osteoporotic bone is described and validated. Normal and ovariectomised Wistar rats received a unilateral middiaphyseal 5 mm defect in the femur, which was instrumented with a radiolucent PEEK plate fixed with angular stable titanium screws and left untreated. All animals were euthanized eight weeks after defect surgery and the bone healing was evaluated using radiographs, computed tomography measurements, and histology. The developed fixation system provided good stability, even in osteoporotic bone. The implants and ancillary instruments ensured consistent and facile placement of the PEEK plates. The untreated defects did not heal without intervention making the model a well-defined and standardized critical size defect model highly useful for evaluating tissue engineered solutions in normal and osteoporotic bone.

The fate of bovine bone marrow stromal cells in hydrogels: a comparison to nucleus pulposus cells and articular chondrocytes

Bone marrow‐derived stromal cells (BMSCs) are good candidates for cell‐based tissue regeneration. For such purposes, cell survival within three‐dimensional (3D) scaffolds is often desirable. We hypothesize that undifferentiated BMSCs will have difficulties thriving within these gels, in contrast to articular chondrocytes (ACs) and nucleus pulposus cells (NPCs), but that early chondrogenic differentiation of the former will increase their survival. BMSCs, ACs and NPCs cast in 1.2% alginate or 2% agarose were cultured for 21 days in serum‐containing media. BMSCs were also cultured in medium with 10 ng/ml TGF‐β1. By day 21, NPCs and ACs proliferated, maintained upregulation of aggrecan and collagen type II, produced glycosaminoglycans and stained positively for collagen type II in both scaffolds. In contrast, the number of living BMSCs and the DNA content of their constructs decreased in both scaffolds. Addition of TGF‐β1 resulted in cell survival and behaviour more similar (gene expression, glycosaminoglycan production and collagen type II synthesis) to ACs and NPCs. This study demonstrated that, unlike ACs and NPCs, undifferentiated BMSCs have more difficulty thriving within hydrogels, but that this can be improved by chondrogenic induction. Hence, immediate conditioning of BMSCs could be a worthwhile strategy. Copyright

Injectable gentamicin-loaded thermo-responsive hyaluronic acid derivative prevents infection in a rabbit model

UNLABELLED Despite the use of systemic antibiotic prophylaxis, the surgical fixation of open fractures with osteosynthesis implants is associated with high infection rates. Antibiotic-loaded biomaterials (ALBs) are increasingly used in implant surgeries across medical specialties to deliver high concentrations of antibiotics to the surgical site and reduce the risk of implant-associated infection. ALBs which are either less or not restricted in terms of spatial distribution and which may be applied throughout complex wounds could offer improved protection against infection in open fracture care. A thermo-responsive hyaluronic acid derivative (hyaluronic acid-poly(N-isopropylacrylamide) (HApN)) was prepared by a direct amidation reaction between the tetrabutyl ammonium (TBA) salt of hyaluronic acid and amine-terminated poly(N-isopropylacrylamide) (pN). The degree of grafting, and gelation properties of this gel were characterized, and the composition was loaded with gentamicin. The rheological- and release properties of this gentamicin-loaded HApN composition were tested in vitro and its efficacy in preventing infection was tested in a rabbit model of osteosynthesis contaminated with Staphylococcus aureus. The gentamicin-loaded HApN composition was able to prevent bacterial colonization of the implant site as shown by quantitative bacteriology. This finding was supported by histopathological evaluation of the humeri samples where no bacteria were found in the stained sections. In conclusion, this gentamicin-loaded HApN hydrogel effectively prevents infection in a complex wound, simulating a contaminated fracture treated with plating osteosynthesis. STATEMENT OF SIGNIFICANCE Fracture fixation after trauma is associated with high infection rates. Antibiotic loaded biomaterials (ALBs) can provide high local concentrations without systemic side effects. However, the currently available ALBs have limited accessibility to contaminated tissues in open fractures because of predetermined shape. Thus, a novel thermo-responsive hyaluronan based hydrogel with control over gelation temperature is reported. The efficacy of this gentamicin loaded hyaluronan derivative is demonstrated in an in vivo fracture model in the presence of fracture fixation hardware. The bacterial burden is cleared in all of the inoculated rabbits in the presence of the ALB. Thus, the proposed injectable thermo-responsive hyaluronan presents an effective ALB for the prevention of infection.

Evaluation of an injectable thermoresponsive hyaluronan hydrogel in a rabbit osteochondral defect model

Articular cartilage displays very little self-healing capabilities, generating a major clinical need. Here, we introduce a thermoresponsive hyaluronan hydrogel for cartilage repair obtained by covalently grafting poly(N-isopropylacrylamide) to hyaluronan, to give a brush co-polymer HpN. The gel is fluid at room temperature and becomes gel at body temperature. In this pilot study HpN safety and repair response were evaluated in an osteochondral defect model in rabbit. Follow-up was of 1 week and 12 weeks and the empty defect served as a control, for a total of four experimental groups. At 12 weeks the defect sites were evaluated macroscopically and histologically. Local lymph nodes, spleen, liver, and kidneys were analyzed for histopathological evaluation. HpN could be easily injected and remained into the defect throughout the study. The macroscopic score was statistically superior for HpN versus empty. Histological score gave opposite trend but not statistically significant. A slight tissue reaction was observed around HpN, however, vascularization and subchondral bone formation were not impeded. An upper proteoglycans rich fibro-cartilaginous tissue with fairly good continuity and lateral integration into the existing articular cartilage was observed in all cases. No signs of local or systemic acute or subacute toxicity were observed. In conclusion, HpN is easily injectable, remains into an osteochondral defect within a moving synovial joint, is biocompatible and does not interfere with the intrinsic healing response of osteochondral defects in a rabbit model.

Accuracy of Fragment Positioning After TPLO and Effect on Biomechanical Stability

OBJECTIVE To compare tibial plateau rotation after tibial plateau leveling osteotomy with the radiographically planned rotation and to determine the effect of translations and rotations of the tibial plateau fragment on the biomechanical stability of the construct under cyclic loading. STUDY DESIGN Experimental biomechanical study. ANIMALS Cadaveric canine pelvic limbs (n=10). METHODS Titanium pins were inserted into the tibial plateau and the proximal metaphysis to track the fragment movements by means of computed tomography (CT) imaging. CT scans were performed (1) before osteotomy, (2) after osteotomy and tibial plateau rotation, and (3) after stabilization with plate and screws. The bones were then cyclically loaded in axial compression. RESULTS The radiographically planned tibial plateau rotation correlated significantly with the achieved rotation (r=0.73, P=.016), although deviations of up to 4.7 degrees were observed. A significant positive correlation between the amount of rotation about the sawing axis and the plastic deformation of the construct after 30,000 test cycles could be found (r=0.81, P=.005). CONCLUSION Considerable deviation occurred between planned and achieved rotation of the tibial plateau fragment. Lower degrees of rotation were beneficial for biomechanical stability. CLINICAL RELEVANCE Dogs with larger tibial plateau angles may be at a relatively higher risk for fixation failure, but further studies are needed to establish a safe margin of tibial plateau rotation.