Biofabrication of customized bone grafts by combination of additive manufacturing and bioreactor knowhow

This study reports on an original concept of additive manufacturing for the fabrication of tissue engineered constructs (TEC), offering the possibility of concomitantly manufacturing a customized scaffold and a bioreactor chamber to any size and shape. As a proof of concept towards the development of anatomically relevant TECs, this concept was utilized for the design and fabrication of a highly porous sheep tibia scaffold around which a bioreactor chamber of similar shape was simultaneously built. The morphology of the bioreactor/scaffold device was investigated by micro-computed tomography and scanning electron microscopy confirming the porous architecture of the sheep tibiae as opposed to the non-porous nature of the bioreactor chamber. Additionally, this study demonstrates that both the shape, as well as the inner architecture of the device can significantly impact the perfusion of fluid within the scaffold architecture. Indeed, fluid flow modelling revealed that this was of significant importance for controlling the nutrition flow pattern within the scaffold and the bioreactor chamber, avoiding the formation of stagnant flow regions detrimental for in vitro tissue development. The bioreactor/scaffold device was dynamically seeded with human primary osteoblasts and cultured under bi-directional perfusion for two and six weeks. Primary human osteoblasts were observed homogenously distributed throughout the scaffold, and were viable for the six week culture period. This work demonstrates a novel application for additive manufacturing in the development of scaffolds and bioreactors. Given the intrinsic flexibility of the additive manufacturing technology platform developed, more complex culture systems can be fabricated which would contribute to the advances in customized and patient-specific tissue engineering strategies for a wide range of applications.This work was supported by the NHMRC, the Australian Research Council and Hans Fischer Senior Fellowship, IAS-TUM. Pedro Costa acknowledges the Portuguese Foundation for Science and Technology for his PhD grant (SFRH/BD/62452/2009)

Local delivery of hydrogel encapsulated vascular endothelial growth factor for the prevention of medication-related osteonecrosis of the jaw

The anti-angiogenic effects of bisphosphonates have been hypothesized as one of the major etiologic factors in the development of medication-related osteonecrosis of the jaw (MRONJ), a severe debilitating condition with limited treatment options. This study evaluated the potential of a gelatine-hyaluronic acid hydrogel loaded with the angiogenic growth factor, vascular endothelial growth factor (VEGF), as a local delivery system to aid in maintaining vascularization in a bisphosphonate-treated (Zoledronic Acid) rodent maxillary extraction defect. Healing was assessed four weeks after implantation of the VEGF-hydrogel into extraction sockets. Gross examination and histological assessment showed that total osteonecrosis and inflammatory infiltrate was significantly reduced in the presence of VEGF. Also, total vascularity and specifically neovascularization, was significantly improved in animals that received VEGF hydrogel. Gene expression of vascular, inflammatory and bone specific markers within the defect area were also significantly altered in the presence of VEGF. Furthermore, plasma cytokine levels were assessed to determine the systemic effect of locally delivered VEGF and showed similar outcomes. In conclusion, the use of locally delivered VEGF within healing extraction sockets assists bone healing and prevents MRONJ via a pro-angiogenic and immunomodulatory mechanism

Tissue integration and biodegradation of soft tissue substitutes with and without compression: an experimental study in the rat

OBJECTIVES To analyze the influence of compression on tissue integration and degradation of soft tissue substitutes. MATERIAL AND METHODS Six subcutaneous pouches in twenty-eight rats were prepared and boxes made of Al$_{2}$O$_{3}$ were implanted and used as carriers for soft tissue substitutes: a collagen matrix (MG), two volume-stable collagen matrices (FG/MGA), and a polycaprolactone scaffold(E). The volume-stable materials (FG/MGA/E) were further implanted with a twofold (2) and a fourfold (4) compression, created by the stacking of additional layers of the substitute materials. The samples were retrieved at 1, 2, and 12 weeks (10 groups, 3 time points, n = 5 per time point and group, overall, 150 samples). The area fraction of infiltrated fibroblasts and inflammatory cells was evaluated histologically. Due to within-subject comparisons, mixed models were conducted for the primary outcome. The level of significance was set at 5%. RESULTS The area fraction of fibroblasts increased in all groups over time. At 12 weeks, the densely compressed materials FG4 (1.1%), MGA4 (1.7%), and MGA2 (2.5%) obtained lower values as compared to the other groups, ranging between 4.7 (E2) and 6.5% (MG). Statistically significant differences (p ≤ 0.05) were observed between groups FG4 vs MG/FG2/E/E4 as well as between MGA4 vs MG/FG2/E/E4 and E vs MGA2. CONCLUSIONS Higher levels of compression led to delayed tissue integration. The effect of different compression levels was more distinct when compared to the differences between the materials. CLINICAL RELEVANCE All biomaterials demonstrated tissue integration and a minimal concomitant inflammatory reaction. Clinically, it might be more favorable to obtain a sufficient flap release or to reduce the material size to improve the tissue integration processes

3D‐printed cryomilled poly(ε‐caprolactone)/graphene composite scaffolds for bone tissue regeneration

In this study, composite scaffolds based on poly(caprolactone) (PCL) and non-covalently functionalized few-layer graphene (FLG) were manufactured by an extrusion-based system for the first time. For that, functionalized FLG powder was obtained through the evaporation of a functionalized FLG aqueous suspension prepared from a graphite precursor. Cryomilling was shown to be an efficient mixing method, producing a homogeneous dispersion of FLG particles onto the PCL polymeric matrix. Thereafter, fused deposition modeling (FDM) was used to print 3D scaffolds and their morphology, thermal, biodegradability, mechanical, and cytotoxicity properties were analysed. The presence of functionalized FLG demonstrated to induce slight changes in the microstructure of the scaffold, did not affect the thermal stability and enhanced significantly the compressive modulus. The composite scaffolds presented a porosity of around 40% and a mean pore size in the range of 300 μm. The cell viability and proliferation of SaOs-2 cells were assessed and the results showed good cell viability and long-term proliferation onto produced composite scaffolds. Therefore, these new FLG/PCL scaffolds comprised adequate morphological, thermal, mechanical, and biological properties to be used in bone tissue regeneration.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT), the European program FEDER/COMPETE for the financial support through project LA ICVS/3Bs - 2015-2017 and to IPC (UID/CTM/50025/2013 and UID/CTM/50025/2016), and the scholarship SFRH/BD/87214/2012 granted to Eunice Cunha. Daniela Dias acknowledges the mobility grant from the BEAM project- Biomedical Engineering-EU Australian cooperation at master level, ICIECP Education Cooperation Programme (388414-EM-January 1, 2014-IT-ERA MUNDUS-ICIJMP). We also acknowledge Prof. Dietmar W. Hutmacher that kindly hosts Daniela Dias in the IHBI laboratory

Additively manufactured polyethylene terephthalate scaffolds for Scapholunate Interosseous Ligament Reconstruction

The regeneration of the ruptured scapholunate interosseous ligament (SLIL) represents a clinical challenge. Here, we propose the use of a Bone-Ligament-Bone (BLB) 3D-printed polyethylene terephthalate (PET) scaffold for achieving mechanical stabilisation of the scaphoid and lunate following SLIL rupture. The BLB scaffold featured two bone compartments bridged by aligned fibres (ligament compartment) mimicking the architecture of the native tissue. The scaffold presented tensile stiffness in the range of 260+/-38 N/mm and ultimate load of 113+/-13 N, which would support physiological loading. A finite element analysis, using inverse finite element analysis for material property identification, showed an adequate fit between simulation and experimental data. The scaffold was then biofunctionalized using two different methods: injected with a Gelatin Methacryloyl solution containing human mesenchymal stem cell spheroids or seeded with tendon-derived stem cells and placed in a bioreactor to undergo cyclic deformation. The first approach demonstrated high cell viability, as cells migrated out of the spheroid and colonised the interstitial space of the scaffold. These cells adopted an elongated morphology suggesting the internal architecture of the scaffold exerted topographical guidance. The second method demonstrated the high resilience of the scaffold to cyclic deformation and the secretion of a fibroblastic related protein was enhanced by the mechanical stimulation. This process promoted the expression of relevant proteins, such as Tenomodulin, indicating mechanical stimulation may enhance cell differentiation and be useful prior to surgical implantation. In conclusion, the PET scaffold presented several promising characteristics for the immediate mechanical stabilisation of disassociated scaphoid and lunate and, in the longer-term, the regeneration of the ruptured SLIL

The effect of biomimetic mineralization of 3D-printed mesoporous bioglass scaffolds on physical properties and in vitro osteogenicity

Three-dimensional Mesoporous bioactive glasses (MBGs) scaffolds has been widely considered for bone regeneration purposes and additive manufacturing enables the fabrication of highly bioactive patient-specific constructs for bone defects. Commonly, this process is performed with the addition of polymeric binders that facilitate the printability of scaffolds. However, these additives cover the MBG particles resulting in the reduction of their osteogenic potential. The present work investigates a simple yet effective phosphate-buffered saline immersion method for achieving polyvinyl alcohol binder removal while enables the maintenance of the mesoporous structure of MBG 3D-printed scaffolds. This resulted in significantly modifying the surface of the scaffold via the spontaneous formation of a biomimetic mineralized layer which positively affected the physical and biological properties of the scaffold. The extensive surface remodeling induced by the deposition of the apatite-like layer lead to a 3-fold increase in surface area, a 5-fold increase in the roughness, and 4-fold increase in the hardness of the PBS-immersed scaffolds when compared to the as-printed counterpart. The biomimetic mineralization also occurred throughout the bulk of the scaffold connecting the MBGs particles and was responsible for the maintenance of structural integrity. In vitro assays using MC3T3-E1 pre-osteoblast like cells demonstrated a significant upregulation of osteogenic-related genes for the scaffolds previously immersed in PBS when compared to the as-printed PVA-containing scaffolds. Although the pre-immersion scaffolds performed equally towards osteogenic cell differentiation, our data suggest that a short immersion in PBS of MBG scaffolds is beneficial for the osteogenic properties and might accelerate bone formation after implantation

Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs

Decellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity.Human periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/- DNase besides Freeze-thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays.DNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention.This study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention