Matthias Cornelsen

A concept for scaffold-based tissue engineering in alveolar cleft osteoplasty

BACKGROUND Alveolar cleft osteoplasty (ACO) using autologous bone grafts, is used worldwide as a standard treatment in the management of patients with clefts. Harvesting of the various autologous bone grafts is accompanied by considerable donor-site morbidity. Use of scaffold-based tissue engineering in ACO could potentially provide treatment options with decreased, or no donor-site morbidity. This study aims to demonstrate the technical and cell biological feasibility of using scaffold-based tissue engineering in ACO. MATERIAL AND METHODS Pre-existing cone-beam computed tomography scans were used for 3D printing of custom-made scaffolds (tricalcium phosphate-polyhydroxybutyrate (TCP-PHB)) according to the individual geometry of the alveolar bone in patients with clefts. The scaffolds were seeded with commercially available human mesenchymal stem cells (hMSCs). Cell survival and cell proliferation was monitored by live-dead assay, scanning electron microscopy (SEM) and WST-1 assay. The osteogenic differentiation of hMSCs on the scaffolds was evaluated by alkaline phosphatase (ALP) assay. RESULTS The custom-made scaffolds were nearly identical to the size and shape of the digital master. Approximately 91% of the subsequently applied mesenchymal stem cells could be seeded on the rails. We could demonstrate successful cell proliferation by a factor of 5-7 over the first 3 weeks. SEM showed a pore-border growth of the hMSCs on the scaffolds after 3 weeks of cell proliferation. The successful osteogenic differentiation of the scaffold-seeded cells could be demonstrated. CONCLUSION The concept of scaffold-based tissue engineering provides great potential as an alternative for the present gold standard of autologous bone grafts in ACO. The treatment causes less morbidity and is less invasive for managing young patients with cleft alveolar bone defects. Further in vivo studies and clinical trials are needed to demonstrate the advantages of this novel treatment for ACO in the clinical setting.

Osteoblast Behavior In Vitro in Porous Calcium Phosphate Composite Scaffolds, Surface Activated with a Cell Adhesive Plasma Polymer Layer

Synthetic materials such as bone substitutes are permanently under development for applications in orthopedic and trauma surgery. Our porous scaffolds were produced from ß-tricalcium phosphate (TCP) using the three dimensional (3D)-printing technology. After sintering the porosity and the pore size of the 3D printed scaffolds reached nearly 50 % and 500 µm, respectively. TCP scaffolds were additionally stabilized by infiltration with polylactic acid (PLA). Because PLA usually impeded cell adhesion we activated the composite surface with plasma polymerized allylamine in a low temperature plasma process. For cell investigations inside the scaffold we used a module system, where two porous discs can be horizontally fixed within a clamping ring. Thereby a 3D cell culture module with four levels and a maximal height of 10 mm was generated. Human MG-63 osteoblasts (ATCC) were seeded apically and placed in serum-containing DMEM. After 14 days of a static cell culture the cell ingrowth and mobility was analyzed by scanning electron microscopy. Osteoblasts initial adhesion and short time occupation of the surface is significantly improved on plasma polymer activated TCP surfaces, which could be a precondition for an enhanced colonization inside a calcium phosphate scaffold. Interestingly, the plasma functionalization of the pure TCP scaffold was possible and successful concerning cell acceptance.

Biomechanical behavior of bone scaffolds made of additive manufactured tricalciumphosphate and titanium alloy under different loading conditions.

Purpose The repair of large segmental bone defects caused by fracture, tumor or infection remains challenging in orthopedic surgery. The capability of two different bone scaffold materials, sintered tricalciumphosphate and a titanium alloy (Ti6Al4V), were determined by mechanical and biomechanical testing. Methods All scaffolds were fabricated by means of additive manufacturing techniques with identical design and controlled pore geometry. Small-sized sintered TCP scaffolds (10 mm diameter, 21 mm length) were fabricated as dense and open-porous samples and tested in an axial loading procedure. Material properties for titanium alloy were determined by using both tensile (dense) and compressive test samples (open-porous). Furthermore, large-sized open-porous TCP and titanium alloy scaffolds (30 mm in height and diameter, 700 μm pore size) were tested in a biomechanical setup simulating a large segmental bone defect using a composite femur stabilized with an osteosynthesis plate. Static physiologic loads (1.9 kN) were applied within these tests. Results Ultimate compressive strength of the TCP samples was 11.2 ± 0.7 MPa and 2.2 ± 0.3 MPa, respectively, for the dense and the open-porous samples. Tensile strength and ultimate compressive strength was 909.8 ± 4.9 MPa and 183.3 ± 3.7 MPa, respectively, for the dense and the open-porous titanium alloy samples. Furthermore, the biomechanical results showed good mechanical stability for the titanium alloy scaffolds. TCP scaffolds failed at 30% of the maximum load. Conclusions Based on recent data, the 3d printed TCP scaffolds tested cannot currently be recommended for high load-bearing situations. Scaffolds made of titanium could be optimized by adapting the biomechanical requirements.

Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCPs mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts.

Migration Capacity and Viability of Human Primary Osteoblasts in Synthetic Three-dimensional Bone Scaffolds Made of Tricalciumphosphate

In current therapeutic strategies, bone defects are filled up by bone auto- or allografts. Since they are limited by insufficient availability and donor site morbidity, it is necessary to find an appropriate alternative of synthetic porous bone materials. Because of their osteoconductive characteristics, ceramic materials like tricalciumphosphate (TCP) are suitable to fill up bone defects. Another advantage of TCP implants is the ability of patient-specific engineering. Objective of the present in-vitro study was to analyze the migration capacity and viability of human primary osteoblasts in porous three-dimensional TCP scaffolds in a static cell culture. To obtain data of the cellular supply with nutrients and oxygen, we determined the oxygen concentration and the pH value within the 3D scaffold compared to the surrounding medium using microsensors. After eight days of cultivation we found cells on all four planes. During incubation, the oxygen concentration within the scaffold decreased by approximately 8%. Furthermore, we could not demonstrate an increasing acidification in the core of the TCP scaffold. Our results suggest that osteoblasts could migrate and survive within the macroporous TCP scaffolds. The selected size of the macropores prevents overgrowth of cells, whereby the oxygen and nutrients supply is sufficiently guaranteed.

Mechanical and biological effects of infiltration with biopolymers on 3D printed tricalciumphosphate scaffolds

The aim of this study was to evaluate the influence of infiltrating 3D printed (TCP) scaffolds with different biodegradable polymers on their mechanical and biological properties. 3D printed TCP scaffolds with interconnecting channels measuring 450±50 µm were infiltrated with four different biodegradable copolymers. To determine the average compressive strength, a uniaxial testing system was used. Additionally, scaffolds were seeded with MC3T3 cells and cell viability was assessed by live/dead-assay. Uninfiltrated TCP had an average compression strength of 1.92±0.38 MPa. Mechanical stability was considerably increased in all infiltrated scaffolds up to a maximum of 7.36±0.57 MPa. All scaffolds demonstrated high cell survival rates with a maximum of 94±10 % living cells. In conclusion, infiltration of 3D printed tricalcium phosphate scaffolds with biodegradable polymers significantly improved mechanical properties and biological properties were comparable to those of uninfiltrated TCP scaffolds.