Triple Culture of Primary Human Osteoblasts, Osteoclasts and Osteocytes as an In Vitro Bone Model.

In vitro evaluation of bone graft materials is generally performed by analyzing the interaction with osteoblasts or osteoblast precursors. In vitro bone models comprising different cell species can give specific first information on the performance of those materials. In the present study, a 3D co-culture model was established comprising primary human osteoblasts, osteoclasts and osteocytes. Osteocytes were differentiated from osteoblasts embedded in collagen gels and were cultivated with osteoblast and osteoclasts seeded in patterns on a porous membrane. This experimental setup allowed paracrine signaling as well as separation of the different cell types for final analysis. After 7 days of co-culture, the three cell species showed their typical morphology and gene expression of typical markers like ALPL, BSPII, BLGAP, E11, PHEX, MEPE, RANKL, ACP5, CAII and CTSK. Furthermore, relevant enzyme activities for osteoblasts (ALP) and osteoclasts (TRAP, CTSK, CAII) were detected. Osteoclasts in triple culture showed downregulated TRAP (ACP5) and CAII expression and decreased TRAP activity. ALP and BSPII expression of osteoblasts in triple culture were upregulated. The expression of the osteocyte marker E11 (PDPN) was unchanged; however, osteocalcin (BGLAP) expression was considerably downregulated both in osteoblasts and osteocytes in triple cultures compared to the respective single cultures.

Tailoring the pore design of embroidered structures by melt electrowriting to enhance the cell alignment in scaffold-based tendon reconstruction.

Tissue engineering of ligaments and tendons aims to reproduce the complex and hierarchical tissue structure while meeting the biomechanical and biological requirements. For the first time, the additive manufacturing methods of embroidery technology and melt electrowriting (MEW) were combined to mimic these properties closely. The mechanical benefits of embroidered structures were paired with a superficial micro-scale structure to provide a guide pattern for directional cell growth. An evaluation of several previously reported MEW fiber architectures was performed. The designs with the highest cell orientation of primary dermal fibroblasts were then applied to embroidery structures and subsequently evaluated using human adipose-derived stem cells (AT-MSC). The addition of MEW fibers resulted in the formation of a mechanically robust layer on the embroidered scaffolds, leading to composite structures with mechanical properties comparable to those of the anterior cruciate ligament. Furthermore, the combination of embroidered and MEW structures supports a higher cell orientation of AT-MSC compared to embroidered structures alone. Collagen coating further promoted cell attachment. Thus, these investigations provide a sound basis for the fabrication of heterogeneous and hierarchical synthetic tendon and ligament substitutes.

Poly(dl-lactide) Polymer Blended with Mineral Phases for Extrusion 3D Printing-Studies on Degradation and Biocompatibility.

A promising therapeutic option for the treatment of critical-size mandibular defects is the implantation of biodegradable, porous structures that are produced patient-specifically by using additive manufacturing techniques. In this work, degradable poly(DL-lactide) polymer (PDLLA) was blended with different mineral phases with the aim of buffering its acidic degradation products, which can cause inflammation and stimulate bone regeneration. Microparticles of CaCO3, SrCO3, tricalcium phosphates (α-TCP, ß-TCP), or strontium-modified hydroxyapatite (SrHAp) were mixed with the polymer powder following processing the blends into scaffolds with the Arburg Plastic Freeforming 3D-printing method. An in vitro degradation study over 24 weeks revealed a buffer effect for all mineral phases, with the buffering capacity of CaCO3 and SrCO3 being the highest. Analysis of conductivity, swelling, microstructure, viscosity, and glass transition temperature evidenced that the mineral phases influence the degradation behavior of the scaffolds. Cytocompatibility of all polymer blends was proven in cell experiments with SaOS-2 cells. Patient-specific implants consisting of PDLLA + CaCO3, which were tested in a pilot in vivo study in a segmental mandibular defect in minipigs, exhibited strong swelling. Based on these results, an in vitro swelling prediction model was developed that simulates the conditions of anisotropic swelling after implantation.

Multimodal additive manufacturing of biomimetic tympanic membrane replacements with near tissue-like acousto-mechanical and biological properties.

The three additive manufacturing techniques fused deposition modeling, gel plotting and melt electrowriting were combined to develop a mimicry of the tympanic membrane (TM) to tackle large TM perforations caused by chronic otitis media. The mimicry of the collagen fiber orientation of the TM was accompanied by a study of multiple funnel-shaped mimics of the TM morphology, resulting in mechanical and acoustic properties similar to those of the eardrum. For the different 3D printing techniques used, the process parameters were optimized to allow reasonable microfiber arrangements within the melt electrowriting setup. Interestingly, the fiber pattern was less important for the acousto-mechanical properties than the overall morphology. Furthermore, the behavior of keratinocytes and fibroblasts is crucial for the repair of the TM, and an in vitro study showed a high biocompatibility of both primary cell types while mimicking the respective cell layers of the TM. A simulation of the in vivo ingrowth of both cell types resulted in a cell growth orientation similar to the original collagen fiber orientation of the TM. Overall, the combined approach showed all the necessary parameters to support the growth of a neo-epithelial layer with a similar structure and morphology to the original membrane. It therefore offers a suitable alternative to autologous materials for the treatment of chronic otitis media. STATEMENT OF SIGNIFICANCE: Millions of people worldwide suffer from chronic middle ear infections. Although the tympanic membrane (TM) can be reconstructed with autologous materials, the grafts used for this purpose require extensive manual preparation during surgery. This affects not only the hearing ability but also the stability of the reconstructed TM, especially in the case of full TM reconstruction. The synthetic alternative presented here mimicked not only the fibrous structure of the TM but also its morphology, resulting in similar acousto-mechanical properties. Furthermore, its high biocompatibility supported the migration of keratinocytes and fibroblasts to form a neo-epithelial layer. Overall, this completely new TM replacement was achieved by combining three different additive manufacturing processes.

Impact of Sr2+ and hypoxia on 3D triple cultures of primary human osteoblasts, osteocytes and osteoclasts.

An in vitro bone triple culture involving human primary osteoblasts, osteocytes and osteoclasts enables the investigation of bone healing factors, drugs or biomaterials in a model system for native bone tissue. The present study analyses the impact of Sr2+ as well as hypoxic cultivation (5% O2 content or chemically induced by Co2+) on bone cells. The three cell types were cultivated together in the presence of 100 µM Sr2+, hypoxic conditions or in the presence of 75 µM Co2+. After cultivation the cell types were separated and analysed on mRNA and protein level individually. In response to Sr2+ osteoblasts showed a downregulation of IBSP expression and a stimulation of ALP activity. Osteocyte gene marker expression of PDPN, MEPE, RANKL, OPG, osteocalcin and likewise the amount of secreted osteocalcin was reduced in the presence of Sr2+. Activity of osteoclast-specific enzymes TRAP and CAII was enhanced compared to the Sr2+ free control. Hypoxic conditions induced by both 5% O2 or a Co2+ treatment led to decreased DNA content of all bone cells and downregulated expression of osteoblast markers ALPL and IBSP as well as osteocyte markers PDPN, RANKL and OPG. In addition, Co2+ induced hypoxia decreased gene and protein expression of osteocalcin in osteocytes. In response to the Co2+ treatment, the TRAP gene expression and activity was increased. This study is the first to analyse the effects of Sr2+ or hypoxia on triple cultures with primary human bone cells. The investigated in vitro bone model might be suitable to reduce animal experiments in early stages of biomaterial and drug development.

3D Plotting of Calcium Phosphate Cement and Melt Electrowriting of Polycaprolactone Microfibers in One Scaffold: A Hybrid Additive Manufacturing Process.

The fabrication of patient-specific scaffolds for bone substitutes is possible through extrusion-based 3D printing of calcium phosphate cements (CPC) which allows the generation of structures with a high degree of customization and interconnected porosity. Given the brittleness of this clinically approved material, the stability of open-porous scaffolds cannot always be secured. Herein, a multi-technological approach allowed the simultaneous combination of CPC printing with melt electrowriting (MEW) of polycaprolactone (PCL) microfibers in an alternating, tunable design in one automated fabrication process. The hybrid CPC+PCL scaffolds with varying CPC strand distance (800-2000 µm) and integrated PCL fibers featured a strong CPC to PCL interface. While no adverse effect on mechanical stiffness was detected by the PCL-supported scaffold design; the microfiber integration led to an improved integrity. The pore distance between CPC strands was gradually increased to identify at which critical CPC porosity the microfibers would have a significant impact on pore bridging behavior and growth of seeded cells. At a CPC strand distance of 1600 µm, after 2 weeks of cultivation, the incorporation of PCL fibers led to pore coverage by a human mesenchymal stem cell line and an elevated proliferation level of murine pre-osteoblasts. The integrated fabrication approach allows versatile design adjustments on different levels.

Development of electrospun, biomimetic tympanic membrane implants with tunable mechanical and oscillatory properties for myringoplasty.

Most commonly, autologous grafts are used in tympanic membrane (TM) reconstruction. However, apart from the limited availability and the increased surgical risk, they cannot replicate the full functionality of the human TM properly. Hence, biomimetic synthetic TM implants have been developed in our project to overcome these drawbacks. These innovative TM implants are made from synthetic biopolymer polycaprolactone (PCL) and silk fibroin (SF) by electrospinning technology. Static and dynamic experiments have shown that the mechanical and oscillatory behavior of the TM implants can be tuned by adjusting the solution concentration, the SF and PCL mixing ratio and the electrospinning parameters. In addition, candidates for TM implants could have comparable acousto-mechanical properties to human TMs. Finally, these candidates were further validated in in vitro experiments by performing TM reconstruction in human cadaver temporal bones. The reconstructed TM with SF-PCL blend membranes fully recovered the acoustic vibration when the perforation was smaller than 50%. Furthermore, the handling, medium adhesion and transparency of the developed TM implants were similar to those of human TMs.

Biomimetic Tympanic Membrane Replacement Made by Melt Electrowriting.

The tympanic membrane (TM) transfers sound waves from the air into mechanical motion for the ossicular chain. This requires a high sensitivity to small dynamic pressure changes and resistance to large quasi-static pressure differences. The TM achieves this by providing a layered structure of about 100µm in thickness, a low flexural stiffness, and a high tensile strength. Chronically infected middle ears require reconstruction of a large area of the TM. However, current clinical treatment can cause a reduction in hearing. With the novel additive manufacturing technique of melt electrowriting (MEW), it is for the first time possible to fabricate highly organized and biodegradable membranes within the dimensions of the TM. Scaffold designs of various fiber composition are analyzed mechanically and acoustically. It can be demonstrated that by customizing fiber orientation, fiber diameter, and number of layers the desired properties of the TM can be met. An applied thin collagen layer seals the micropores of the MEW-printed membrane while keeping the favorable mechanical and acoustical characteristics. The determined properties are beneficial for implantation, closely match those of the human TM, and support the growth of a neo-epithelial layer. This proves the possibilities to create a biomimimetic TM replacement using MEW.