Hermann Seitz

Ceramic scaffolds produced by computer-assisted 3D printing and sintering: Characterization and biocompatibility investigations

Hydroxyapatite (HAP) and tricalcium phosphate (TCP) are two very common ceramic materials for bone replacement. However, in general HAP and TCP scaffolds are not tailored to the exact dimensions of the defect site and are mainly used as granules or beads. Some scaffolds are available as ordinary blocks, but cannot be customized for individual perfect fit. Using computer-assisted 3D printing, an emerging rapid prototyping technique, individual three-dimensional ceramic scaffolds can be built up from TCP or HAP powder layer by layer with subsequent sintering. These scaffolds have precise dimensions and highly defined and regular internal characteristics such as pore size. External shape and internal characteristics such as pore size can be fabricated using Computer Assisted Design (CAD) based on individual patient data. Thus, these scaffolds could be designed as perfect fit replacements to reconstruct the patients skeleton. Before their use as bone replacement materials in vivo, in vitro testing of these scaffolds is necessary. In this study, the behavior of human osteoblasts on HAP and TCP scaffolds was investigated. The commonly used bone replacement material BioOss(R) served as control. Biocompatibility was assessed by scanning electron microscopy (SEM), fluorescence microscopy after staining for cell vitality with fluorescin diacetate (FDA) and propidium iodide (PI) and the MTT, LDH, and WST biocompatibility tests. Both versions were colonised by human osteoblasts, however more cells were seen on HAP scaffolds than TCP scaffolds. Cell vitality staining and MTT, LDH, and WST tests showed superior biocompatibility of HAP scaffolds to BioOss, while BioOss was more compatible than TCP. Further experiments are necessary to determine biocompatibility in vivo. Future modifications of 3D printed scaffolds offer advantageous features for Tissue Engineering. The integration of channels could allow for vascular and nerve ingrowth into the scaffold. Also the complex shapes of convex and concave articulating joint surfaces maybe realized with these rapid prototyping techniques.

In vitro -Osteoclastic Activity Studies on Surfaces of 3D Printed Calcium Phosphate Scaffolds:

Various biomaterials have been developed for the use as bone substitutes for bone defects. To optimize their integration and functionality, they should be adapted to the individual defect. Rapid prototyping is a manufacturing method to tailor materials to the 3D geometry of the defect. Especially 3D printing allows the manufacture of implants, the shape of which can be designed to fit the bone defect using anatomical information obtained from the patient. 3D printing of calcium phosphates, which are well established as bone substitutes, involves a sintering step after gluing the granules together by a binder liquid. In this study, we analyzed if and how these 3D printed calcium phosphate surfaces can be resorbed by osteoclast-like cells. On 3D printed scaffold surfaces consisting of pure HA and β-TCP as well as a biphasic mixture of HA and TCP the osteoclastic cell differentiation was studied. In this regard, cell proliferation, differentiation, and activation were analyzed with the monocytic cell line RAW 264.7. The results show that osteoclast-like cells were able to resorb calcium phosphate surfaces consisting of granules. Furthermore, biphasic calcium phosphate ceramics exhibit, because of their osteoclastic activation ability, the most promising surface properties to serve as 3D printed bone substitute scaffolds.

Endocultivation: 3D printed customized porous scaffolds for heterotopic bone induction

The aim of this study was to evaluate the ability of computer assisted designed (CAD) synthetic hydroxyapatite and tricalciumphosphate blocks to serve as precise scaffolds for intramuscular bone induction in a rat model. A central channel to allow for vessel pedicle or nerve integration was added. Natural bovine hydroxyapatite blocks served as controls to evaluate and compare biocompatibility of the new matrices. Individually designed 3D-printed rounded and porous hydroxyapatite (HA) and tricalcium phosphate (TCP) blocks were placed in pouches in the Musculus latissimus dorsi in 12 Lewis rats bilaterally. Bovine hydroxyapatite blocks with and without a central channel served as controls. Simultaneously, 200 microg rhBMP-2 in 1 ml sodium chloride was injected on both sides. For 8 weeks, bone generation was monitored by computer tomography and fluorescence labeling. The increase rates of bone density in CT examinations were higher in the HA groups (184-220 HU 8 weeks after implantation) compared to the TCP group (18 HU; p<0.0001). Microradiography and fluorescence microscopy 8 weeks after implantation showed new bone formation for all materials tested. For all scaffolds, toluidine staining revealed vital bone directly on the scaffold materials but also in the gaps between. It can be concluded from our data that the specially shaped hydroxyapatite and tricalcium phosphate blocks tested against the bovine hydroxyapatite blocks showed good biocompatibility and osteoinductivity in vivo. Further studies should explore if the stability of the individually designed blocks is sufficient to cultivate larger replacements without an external matrix for support.

Validation of a Femoral Critical Size Defect Model for Orthotopic Evaluation of Bone Healing: A Biomechanical, Veterinary and Trauma Surgical Perspective

Numerous in vivo studies have been conducted to investigate bone regeneration in orthotopic defect models, but a reliably standardized critical-size defect (CSD) model in small animals is still lacking in tissue-engineering research. Utilizing the expertise of trauma surgeons, veterinary surgeons, and engineers, we evaluated the optimal fixation strategy for in vivo application in terms of surgical suitability and conducted biomechanical studies for 3 fixation devices. Fixation strategies were an external fixation device made of polymethylmethacrylate, widely used in animal care; a self-constructed external clamp-fixation device, designed and manufactured using rapid prototyping techniques; and commercially available 1.2-mm titanium plates used in hand surgery. The CSD was 6 mm in size. Biomechanical testing included compression, 4-point bending, and torsion tests. The surgical procedure was optimized in vitro and validated in a clinical setting in athymic rats in vivo. Despite differences in the results of the biomechanical tests, all fixation devices tested proved suitable for the intended purpose. In conclusion, the evaluated model for stabilizing a CSD in a rats femur can reliably be used for standardized bone regeneration studies in small animals.

Endocultivation: the influence of delayed vs. simultaneous application of BMP-2 onto individually formed hydroxyapatite matrices for heterotopic bone induction

When bone morphogenetic protein (BMP) is delivered to matrices in vivo may affect tissue engineered bone constructs for jaw reconstruction after cancer surgery. This study compared the effects of BMP application at different times after matrix implantation for heterotopic bone induction in a rat model. Hydroxyapatite blocks were implanted unilaterally onto the surface of the latissimus dorsi muscle. A second block was implanted onto the contralateral muscle after 1, 2 or 4 weeks and 200 μg rhBMP-2 was injected into the blocks on both sides. Bone formation and density inside the blocks was analysed by CT and histology. 8 weeks after BMP application increases in bone density within the scaffolds were most pronounced in the simultaneous application group (179 HU). Less pronounced increases were observed for the 1 (65 HU), 2 (58 HU) and 4 (31 HU; p<0.0001) week delay group. Homogeneous bone induction started from the central channel of the blocks. Capillaries and larger vessels were seen in all constructs, samples receiving delayed BMP treatment demonstrated significantly greater neovascularization. Delayed application of BMP was less effective for heterotopic bone formation than simultaneous application. A central channel allows homogeneous bone induction directly from the centre of the blocks.

Modelling of a microfluidic device with piezoelectric actuators

This paper presents the modelling of a given microfluidic device, that is applied as a drop-on-demand printhead for different printing tasks. Piezoelectric bend mode actuators are used to eject a droplet from the respective nozzle on demand. The device is considered as a coupled system consisting of an electric, a mechanical and a fluidic part. The electric and mechanical parts of the piezoelectric actuator are described by conventional lumped elements, while the complex three-dimensional fluid analysis is performed by a commercial computational fluid dynamics (CFD) free-surface modelling package. The model helps one to understand the fluid dynamics and thus to study different design and operating parameter aspects. The general approach to the problem of modelling a microfluidic device as presented here can also be applied in various other modelling tasks.

Characterization and evaluation of a PMMA‐based 3D printing process

Purpose – The purpose of this paper is to characterize and evaluate a new 3D‐printing process based on Poly(methyl methacrylate) (PMMA).Design/methodology/approach – A benchmark part and standard parts were designed, printed by a 3D‐printer and characterized.Findings – 3D printed PMMA parts have a tensile strength of 2.91 MPa and a modulus of elasticity of 223 MPa. The mechanical properties can be improved by infiltrations with epoxy (tensile strength: 26.6 MPa, modulus of elasticity: 1,190 MPa). The surface quality of the parts can be improved by infiltration with wax for usage as lost models. The minimum feature size is 0.3 mm.Research limitations/implications – The PMMA‐based 3D printing process can be used for manufacturing concept models, functional parts and lost models for investment casting.Originality/value – This is the first paper investigating a PMMA‐based 3D printing process.

Non‐toxic flexible photopolymers for medical stereolithography technology

Purpose – To describe the development of a novel polyether(meth)acrylate‐based resin material class for stereolithography with alterable material characteristics.Design/methodology/approach – A complete overview of details to composition parameters, the optimization and bandwidth of mechanical and processing parameters is given. Initial biological characterization experiments and future application fields are depicted. Process parameters are studied in a commercial 3D systems Viper stereolithography system, and a new method to determine these parameters is described herein.Findings – Initial biological characterizations show the non‐toxic behavior in a biological environment, caused mainly by the (meth)acrylate‐based core components. These photolithographic resins combine an adjustable low Youngs modulus with the advantages of a non‐toxic (meth)acrylate‐based process material. In contrast to the mostly rigid process materials used today in the rapid prototyping industry, these polymeric formulations are ...

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.