3D-Printed PLA-Bioglass Scaffolds with Controllable Calcium Release and MSC Adhesion for Bone Tissue Engineering

Large bone defects are commonly treated by replacement with auto- and allografts, which have substantial drawbacks including limited supply, donor site morbidity, and possible tissue rejection. This study aimed to improve bone defect treatment using a custom-made filament for tissue engineering scaffolds. The filament consists of biodegradable polylactide acid (PLA) and a varying amount (up to 20%) of osteoconductive S53P4 bioglass. By employing an innovative, additive manufacturing technique, scaffolds with optimized physico-mechanical and biological properties were produced. The scaffolds feature adjustable macro- and microporosity (200–2000 µm) with adaptable mechanical properties (83–135 MPa). Additionally, controllable calcium release kinetics (0–0.25 nMol/µL after 24 h), tunable mesenchymal stem cell (MSC) adhesion potential (after 24 h by a factor of 14), and proliferation (after 168 h by a factor of 18) were attained. Microgrooves resulting from the 3D-printing process on the surface act as a nucleus for cell aggregation, thus being a potential cell niche for spheroid formation or possible cell guidance. The scaffold design with its adjustable biomechanics and the bioglass with its antimicrobial properties are of particular importance for the preclinical translation of the results. This study comprehensibly demonstrates the potential of a 3D-printed bioglass composite scaffold for the treatment of critical-sized bone defects

3D-Printing of Hierarchically Designed and Osteoconductive Bone Tissue Engineering Scaffolds

In Bone Tissue Engineering (BTE), autologous bone-regenerative cells are combined with a scaffold for large bone defect treatment (LBDT). Microporous, polylactic acid (PLA) scaffolds showed good healing results in small animals. However, transfer to large animal models is not easily achieved simply by upscaling the design. Increasing diffusion distances have a negative impact on cell survival and nutrition supply, leading to cell death and ultimately implant failure. Here, a novel scaffold architecture was designed to meet all requirements for an advanced bone substitute. Biofunctional, porous subunits in a load-bearing, compression-resistant frame structure characterize this approach. An open, macro- and microporous internal architecture (100 µm–2 mm pores) optimizes conditions for oxygen and nutrient supply to the implant’s inner areas by diffusion. A prototype was 3D-printed applying Fused Filament Fabrication using PLA. After incubation with Saos-2 (Sarcoma osteogenic) cells for 14 days, cell morphology, cell distribution, cell survival (fluorescence microscopy and LDH-based cytotoxicity assay), metabolic activity (MTT test), and osteogenic gene expression were determined. The adherent cells showed colonization properties, proliferation potential, and osteogenic differentiation. The innovative design, with its porous structure, is a promising matrix for cell settlement and proliferation. The modular design allows easy upscaling and offers a solution for LBDT

Entwicklung und Realisierung neuer Designs zur Nutzung als Knochenersatzmaterial mittels Fused-Deposition-Modelling-3D-Druck

Die Therapie langstreckiger Knochendefekte stellt auch weiterhin eine große Herausforderung dar. Dies beruht unter anderem darauf, dass der therapeutische Goldstandard - die Verwendung von autogener Knochensubstanz aus dem Beckenkamm - neben der begrenzten Verfügbarkeit vor allem Komplikationen im Bereich der Entnahmestelle mit sich bringen kann. Es wurde bisher aber noch kein durchschlagendes Ergebnis in der Entwicklung neuer Scaffolds zum Einsatz bei langstreckigen Knochendefekten erreicht. Dies kann eine Vielzahl an Ursachen haben, die sich von der verwendeten Ausgangssubstanz, bis hin zum verwendeten Design erstrecken können. Neben dem Ausgangsmaterial spielen vor allem die Formgebung und physikalische Eigenschaften, wie Porosität und Mikroarchitektur, eine wichtige Rolle. Ein aktueller Ansatz zur Nutzung als alternatives Knochenersatzmaterial ist das Knochen-Tissue-Engineering. Hierbei werden körpereigene, knochen-regenerative Zellen mit einem dreidimensionalen Gerüststoff (Knochenersatzmaterial oder -scaffold) kombiniert und in den Knochendefekt implantiert. In dieser Arbeit wurde der Fokus auf die Designentwicklung eines neuen Kochenersatz-Scaffolds gelegt. Nach Vorbild schon vorgestellter Knochenersatzdesigns und unter Berücksichtigung einer Grundstruktur, die auch Phasen der Knochenheilung wie die Frakturhämatomausbreitung und initiale Nährstoffversorgung einbeziehen sollte, wurden mehrere Designs (Raster, Tempel, Zwiebel) entwickelt. Mithilfe des additiv extrusionsbasierten Schmelzschichtverfahrens (Fused Filament Fabrication) wurden die in Computer-Aided Design entworfenen Scaffolds realisiert. Dieser Ansatz beinhaltet, unter Verwendung des resorbierbaren und biokompatiblen Trägerpolymers Polylaktat, mehrstufige Designs, die kleine biologisch funktionelle Einheiten in eine tragende, kompressionsfeste Rahmenstruktur einbetten. Hierdurch entsteht einerseits die nötige mechanische Belastbarkeit und andererseits eine offene Architektur mit Poren, die Diffusion von Sauerstoff und Nährstoffen in die inneren Bereiche des Implantats ermöglicht. Es wurden verschiedene Designs entwickelt, gedruckt und mechanisch sowie in vitro in den Kernbereichen Zelladhäsion, Zellaktivität und osteogene Differenzierung nach Besiedelung mit Saos-2-Zellen charakterisiert. Ein weiterer Entwicklungsschritt stellte das Einführen eines neuartigen, innerhalb der Designs kompatiblen Baukastensystems dar. Hierdurch wird nicht nur die Anpassbarkeit an den Knochendefekt verbessert, es sind auch weitere Funktionen ergänzbar und die unterschiedlichen Designs untereinander kombinierbar. Die Ergebnisse dieser Dissertationsarbeit dienen als Basis für einen völlig neuen Ansatz von Knochenersatzmaterialien mit positiven biologischen sowie biophysikalischen Eigenschaften

Visualization of complicated fractures by 3D-printed models for teaching and surgery: hands-on transitional fractures of the ankle

Aims: Understanding the orientation of fracture lines and mechanisms is the essential key to sufficient surgical therapy, but there is still a lack of visualization and teaching methods in traumatology and fracture theory. 3D-printed models offer easy approach to those fractures. This paper explains the use of the teaching possibility with 3-dimensional models of transitional fractures of the ankle. Methods and results: For generating 3D printable models, already obtained CT data were used and segmented into its different tissues, especially parts concerning the fracture. After the segmentation process, the models were produced with FFF (fused filament fabrication) printing technology. The fracture models then were used for hands-on teaching courses in AO course (Arbeitsgemeinschaft für Osteosynthesefragen) of pediatric traumatology in 2020 in Frankfurt. In the course fracture anatomy with typical fracture lines, approaches, and screw placement could be shown, discussed and practiced. Conclusion: The study shows the use of 3D-printed teaching models and helps to understand complicated fractures, in this case, transitional fractures of the ankle. The teaching method can be adapted to numerous other use cases

Sterilization of PLA after Fused Filament Fabrication 3D Printing: Evaluation on Inherent Sterility and the Impossibility of Autoclavation

Three-dimensional printing, especially fused filament fabrication (FFF), offers great possibilities in (bio-)medical applications, but a major downside is the difficulty in sterilizing the produced parts. This study evaluates the questions of whether autoclaving is a possible solution for FFF-printed parts and if the printer itself could be seen as an inherent sterilization method. In a first step, an investigation was performed on the deformation of cylindrically shaped test parts after running them through the autoclaving process. Furthermore, the inherent sterility possibilities of the printing process itself were evaluated using culture medium sterility tests. It could be shown that, depending on the needed accuracy, parts down to a diameter of 5–10 mm can still be sterilized using autoclaving, while finer parts suffer from major deformations. For these, inherent sterilization of the printer itself is an option. During the printing process, over a certain contact time, heat at a higher level than that used in autoclaving is applied to the printed parts. The contact time, depending on the printing parameters, is calculated using the established formula. The results show that for stronger parts, autoclaving offers a cheap and good option for sterilization after FFF-printing. However, the inherent sterility possibilities of the printer itself can be considered, especially when printing with small layer heights for finer parts

The Impact of Defect Size on Bone Healing in Critical-Size Bone Defects Investigated on a Rat Femur Defect Model Comparing Two Treatment Methods

Critical-size bone defects up to 25 cm can be treated successfully using the induced membrane technique established by Masquelet. To shorten this procedure, human acellular dermis (HAD) has had success in replacing this membrane in rat models. The aim of this study was to compare bone healing for smaller and larger defects using an induced membrane and HAD in a rat model. Using our established femoral defect model in rats, the animals were placed into four groups and defects of 5 mm or 10 mm size were set, either filling them with autologous spongiosa and surrounding the defect with HAD or waiting for the induced membrane to form around a cement spacer and filling this cavity in a second operation with a cancellous bone graft. Healing was assessed eight weeks after the operation using µ-CT, histological staining, and an assessment of the progress of bone formation using an established bone healing score. The α-smooth muscle actin used as a signal of blood vessel formation was stained and counted. The 5 mm defects showed significantly better bone union and a higher bone healing score than the 10 mm defects. HAD being used for the smaller defects resulted in a significantly higher bone healing score even than for the induced membrane and significantly higher blood vessel formation, corroborating the good results achieved by using HAD in previous studies. In comparison, same-sized groups showed significant differences in bone healing as well as blood vessel formation, suggesting that 5 mm defects are large enough to show different results in healing depending on treatment; therefore, 5 mm is a viable size for further studies on bone healing

3D-Printed PLA-Bioglass Scaffolds with Controllable Calcium Release and MSC Adhesion for Bone Tissue Engineering

Large bone defects are commonly treated by replacement with auto- and allografts, which have substantial drawbacks including limited supply, donor site morbidity, and possible tissue rejection. This study aimed to improve bone defect treatment using a custom-made filament for tissue engineering scaffolds. The filament consists of biodegradable polylactide acid (PLA) and a varying amount (up to 20) of osteoconductive S53P4 bioglass. By employing an innovative, additive manufacturing technique, scaffolds with optimized physico-mechanical and biological properties were produced. The scaffolds feature adjustable macro- and microporosity (200–2000 µm) with adaptable mechanical properties (83–135 MPa). Additionally, controllable calcium release kinetics (0–0.25 nMol/µL after 24 h), tunable mesenchymal stem cell (MSC) adhesion potential (after 24 h by a factor of 14), and proliferation (after 168 h by a factor of 18) were attained. Microgrooves resulting from the 3D-printing process on the surface act as a nucleus for cell aggregation, thus being a potential cell niche for spheroid formation or possible cell guidance. The scaffold design with its adjustable biomechanics and the bioglass with its antimicrobial properties are of particular importance for the preclinical translation of the results. This study comprehensibly demonstrates the potential of a 3D-printed bioglass composite scaffold for the treatment of critical-sized bone defects

Human Acellular Collagen Matrices—Clinical Opportunities in Tissue Replacement

The field of regenerative medicine is increasingly in need of effective and biocompatible materials for tissue engineering. Human acellular dermal matrix (hADM)-derived collagen matrices stand out as a particularly promising candidate. Their ability to preserve structural integrity, coupled with exceptional biocompatibility, positions them as a viable choice for tissue replacement. However, their clinical application has been largely confined to serving as scaffolds. This study aims to expand the horizon of clinical uses for collagen sheets by exploring the diverse cutting-edge clinical demands. This review illustrates the clinical utilizations of collagen sheets beyond traditional roles, such as covering skin defects or acting solely as scaffolds. In particular, the potential of Epiflex®, a commercially available and immediately clinically usable allogeneic membrane, will be evaluated. Collagen sheets have demonstrated efficacy in bone reconstruction, where they can substitute the induced Masquelet membrane in a single-stage procedure, proving to be clinically effective and safe. The application of these membranes allow the reconstruction of substantial tissue defects, without requiring extensive plastic reconstructive surgery. Additionally, they are found to be apt for addressing osteochondritis dissecans lesions and for ligament reconstruction in the carpus. The compelling clinical examples showcased in this study affirm that the applications of human ADM extend significantly beyond its initial use for skin defect treatments. hADM has proven to be highly successful and well-tolerated in managing various etiologies of bone and soft tissue defects, enhancing patient care outcomes. In particular, the application from the shelf reduces the need for additional surgery or donor site defects

3D-Printing of Hierarchically Designed and Osteoconductive Bone Tissue Engineering Scaffolds

In Bone Tissue Engineering (BTE), autologous bone-regenerative cells are combined with a scaffold for large bone defect treatment (LBDT). Microporous, polylactic acid (PLA) scaffolds showed good healing results in small animals. However, transfer to large animal models is not easily achieved simply by upscaling the design. Increasing diffusion distances have a negative impact on cell survival and nutrition supply, leading to cell death and ultimately implant failure. Here, a novel scaffold architecture was designed to meet all requirements for an advanced bone substitute. Biofunctional, porous subunits in a load-bearing, compression-resistant frame structure characterize this approach. An open, macro- and microporous internal architecture (100 µm-2 mm pores) optimizes conditions for oxygen and nutrient supply to the implant's inner areas by diffusion. A prototype was 3D-printed applying Fused Filament Fabrication using PLA. After incubation with Saos-2 (Sarcoma osteogenic) cells for 14 days, cell morphology, cell distribution, cell survival (fluorescence microscopy and LDH-based cytotoxicity assay), metabolic activity (MTT test), and osteogenic gene expression were determined. The adherent cells showed colonization properties, proliferation potential, and osteogenic differentiation. The innovative design, with its porous structure, is a promising matrix for cell settlement and proliferation. The modular design allows easy upscaling and offers a solution for LBDT