Three-dimensional (3D) titanium-mesh scaffolds offer many advantages over autologous bone grafting for the regeneration of challenging large segmental bone defects. Our study supports the hypothesis that endogenous bone defect regeneration can be promoted by mechanobiologically optimized Ti-mesh scaffolds. Using finite element techniques, two mechanically distinct Ti-mesh scaffolds were designed in a honeycomb-like configuration to minimize stress shielding while ensuring resistance against mechanical failure. Scaffold stiffness was altered through small changes in the strut diameter only. Honeycombs were aligned to form three differently oriented channels (axial, perpendicular, and tilted) to guide the bone regeneration process. The soft scaffold (0.84 GPa stiffness) and a 3.5-fold stiffer scaffold (2.88 GPa) were tested in a critical size bone defect model in vivo in sheep. To verify that local scaffold stiffness could enhance healing, defects were stabilized with either a common locking compression plate that allowed dynamic loading of the 4-cm defect or a rigid custom-made plate that mechanically shielded the defect. Lower stress shielding led to earlier defect bridging, increased endochondral bone formation, and advanced bony regeneration of the critical size defect. This study demonstrates that mechanobiological optimization of 3D additive manufactured Ti-mesh scaffolds can enhance bone regeneration in a translational large animal study.

Three-dimensional (3D) printing technologies are increasingly used to convert medical imaging studies into tangible (physical) models of individual patient anatomy, allowing physicians, scientists, and patients an unprecedented level of interaction with medical data. To date, virtually all 3D-printable medical data sets are created using traditional image thresholding, subsequent isosurface extraction, and the generation of .stl surface mesh file formats. These existing methods, however, are highly prone to segmentation artifacts that either overor underexaggerate the features of interest, thus resulting in anatomically inaccurate 3D prints. In addition, they often omit finer detailed structures and require time-and labor-intensive processes to visually verify their accuracy. To circumvent these problems, we present a bitmap-based multimaterial 3D printing workflow for the rapid and highly accurate generation of physical models directly from volumetric data stacks. This workflow employs a thresholding-free approach that bypasses both isosurface creation and traditional mesh slicing algorithms, hence significantly improving speed and accuracy of model creation. In addition, using preprocessed binary bitmap slices as input to multimaterial 3D printers allows for the physical rendering of functional gradients native to volumetric data sets, such as stiffness and opacity, opening the door for the production of biomechanically accurate models.

BackgroundNew tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone.MethodsRelated to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes.ResultsThe in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %.ConclusionsAs a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site.

Large segmental bone defect reconstruction with sufficient functional restoration is one of the most demanding challenges in orthopaedic surgery. Available regenerative treatment options, as the vascularized bone graft transfer, the Masquelet technique or the Ilizarov distraction osteogenesis, are associated with specific indications and distinct limitations. As an alternative, a hollow cylindrical ceramic-polymer composite scaffold (β-tricalcium phosphate and poly-lactid co-ε- caprolactone), facilitating a strong surface guiding effect for tissue ingrowth (group 1; n = 6) was investigated here. In combination with an additional autologous, cancellous bone graft filling, the scaffold's ability to work as an open-porous membrane to improve the defect healing process was analysed (group 2; n = 6). A novel model of a critical size (40 mm) tibia osteotomy defect stabilized with an external hybrid-ring fixator, was established in sheep. Segmental defect regeneration and tissue organization in relation to the scaffold were analysed radiologically, (immune-) histologically, and with second-harmonic generation imaging 12 weeks after surgery. The scaffold's tubular shape and open-porous structure controlled the collagen fibre orientation within the bone defect and guided the following mineralization process along the scaffold surface. In combination with the osteoinductive stimulus, a unilateral bony bridging of the critically sized defect was achieved in one third of the animals. The external hybrid-ring fixator was appropriate for large segmental defect stabilization in sheep.

To allow bone defect regeneration, autologous bone grafting still represents the gold standard. However, autograft harvesting has limitations, including an additional surgery, donor site morbidity, and limited availability. Demineralized bone matrix (DBM) would represent an alternative, yet lacks sufficient osteoinductive properties. Combining DBM with a potent agent, such as bone morphogenetic protein-2 (BMP-2) might be a feasible alternative approach, optimizing an established grafting material with strong osteoinductive properties. A unique mixing device has been developed that enables perioperative handling to reach a homogeneous and standardized paste for bone defect filling. DBM proved in vitro to be a suitable carrier for BMP-2, with a documented release over 56 days at concentrations sufficient to stimulate osteogenic differentiation. At the end of the elution experiment, 56 days, bioactive BMP was still captured within the DBM. Using a sheep drill hole defect model, DBM perioperatively mixed with BMP-2 showed strong osteoinductive properties comparable to those of autologous bone and outnumbering the one of DBM alone or empty defects. Bone defect healing was enabled at diaphyseal and metaphyseal defects and thus BMP-2-doped DBM represented an easy perioperative enriching method and an efficient carrier for BMP-2. With the comparability to the clinical gold standard autologous bone, DBM mixed with BMP-2 might serve as possible alternative grafting material enabling a controlled osteogenic stimulation.

The 'off-label' use of high-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) in lumbar and cervical fusion leads to heterotopic bone formation and vertebral osteolysis. These radiographically assessed side-effects in patients were frequently associated with an over-dosage of BMP-2. However, little is so far known about the histological, functional or biomechanical tissue consequences of over-dosage of rhBMP-2 in these specific clinical situations. We hypothesized that a high dose of rhBMP-2 in cervical spinal fusion could induce substantial alterations in bone, leading to mechanical impairment. An anterior cervical spinal fusion (C3-C4 ACDF) model in 16 sheep (aged > 2.5 years; n = 8/group) was used to quantify the consequences of a high rhBMP-2 dose (6 mg rhBMP-2) on fusion tissue compared to the 'gold standard' of autologous, cancellous bone graft. The fusion site was assessed by radiography after 0, 8 and 12 weeks. Biomechanical non-destructive testing and (immuno)histological and histomorphometrical analyses were performed 12 weeks postoperatively. Although high-dose rhBMP-2 treatment led to an advanced radiological fusion result compared to autograft treatment, heterotopic bone formation and vertebral bone resorption were induced simultaneously. Histological evaluation unveiled highly active bone-forming processes ventral to the fusion segment after 12 weeks, while radiolucent areas showed still a partial loss of regular trabecular structure, with rare signs of remodelling and restoration. Despite qualitative alteration of the trabecular bone structure within the fusion site, the massive anterior heterotopic bone formation led to a substantial increase in mechanical stiffness compared to the autograft group. Copyright © 2015 John Wiley & Sons, Ltd.

Bioactive coatings have the potential to improve the bony integration of mechanically loaded orthopedic ceramic implants. Using the concept of mimicking the natural bone surface, four different coatings of varying thickness on a zirconia toughened alumina (ZTA) ceramic implant were investigated regarding their osseointegration in a drill-hole model in sheep. The hypothesis that a bioactive coating of ZTA ceramics would facilitate cancellous bone integration was investigated. The bioactive coatings consisted of either a layer of covalently bound multi phosphonate molecules (chemical modification = CM), a nano hydoxyapatite coating (HA), or two different bioactive glass (BG) coatings in micrometer thickness, forming a hydroxyl-carbonate apatite layer on the implant surface in vivo (dip-coated 45S5 = DipBG; sol-gel 70S30C = SGBG). Coated surfaces were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. After 12 weeks, osseointegration was evaluated via mechanical push-out testing and histology. HA enhanced the maximum push-out force (HA: mean 3573.85 ± 1119.91 N; SGBG: mean 1691.57 ± 986.76 N; p = 0.046), adhesive shear strength (HA: mean 9.82 ± 2.89 MPA; SGBG: mean 4.57 ± 2.65 MPA; p = 0.025), and energy release rate (HA: mean 3821.95 ± 1474.13 J/mm2; SGBG: mean 1558.47 ± 923.47 J/mm2; p = 0.032) compared to SGBG. The implant-bone interfacial stiffness increased by CM compared to SGBG coating (CM: mean 6258.06 ± 603.80 N/mm; SGBG: mean 3565.57 ± 1705.31 n/mm; p = 0.038). Reduced mechanical osseointegration of SGBG coated implants could be explained histologically by a foreign body reaction surrounding the implants.

Infections of bone are severe complications, and an optimization of grafting material with antimicrobial drugs might be useful for prevention and treatment. This study aimed to investigate the influence of gentamicin-loaded bone graft on the healing of bone defects in a sheep model. Metaphyseal and diaphyseal drill hole defects (diameter: 6 mm, depth: 15 mm) were filled with graft or gentamicin-loaded graft (50 mg/g graft) or were left untreated. Analysis of regeneration after three and nine weeks, micro-computed tomography (μCT), and histology revealed a significant increase in bone formation in the drill hole defects, which began at the edges of the holes and grew over time into the defect center. The amount of graft decreased over time due to active resorption by osteoclasts, while osteoblasts formed new bone. No difference between the groups was seen after three weeks. After nine weeks, significantly less mineralized tissue was formed in the gentamicin-loaded graft group. Signs of inflammatory reactions were seen in all three groups. Even though the applied gentamicin concentration was based on the concentration of gentamicin mixed with cement, the healing process was impaired. When using local gentamicin, a dose-dependent, compromising effect on bone healing should be considered.