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An optimized 3D-printed perfusion bioreactor for homogeneous cell seeding in bone substitute scaffolds for future chairside applications

ABSTRACT: A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailor-made bioreactor design. A perfusion system for efficient, homogeneous, and rapid seeding with human adipogenic stem cells in bone substitute scaffolds was designed. Variants concerning medium inlet and outlet port geometry, i.e. cylindrical or conical diffuser, cell concentration, perfusion mode and perfusion rates were simulated in silico. Cell distribution during perfusion was monitored by dynamic [18F]FDG micro-PET/CT and validated by laser scanning microscopy with three-dimensional image reconstruction. By iterative feedback of the in silico and in vitro experiments, the homogeneity of cell distribution throughout the scaffold was optimized with adjustment of flow rates, cell density and perfusion properties. Finally, a bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode. During this short period of time, the cells initially adhere within the entire scaffold and stay viable. After two weeks, the formation of several cell layers was observed, which was associated with an osteogenic differentiation process. This newly designed bioreactor may be considered as a prototype for chairside application.

SUBMITTER: Engel N

PROVIDER: S-EPMC8593024 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Extracellular vesicles (EVs) have garnered significant interest in the biotechnology field due to their intrinsic therapeutic properties as well as their ability to serve as vehicles for bioactive cargo. However, the lack of an established biomanufacturing platform and limited potency of EVs in vivo remain critical bottlenecks for clinical translation. In this study, we utilized a 3D-printed scaffold-perfusion bioreactor system to assess the response of dynamic culture on extracellular vesicle production from endothelial cells (ECs). We also investigated whether ethanol conditioning, which was previously shown to enhance vascularization bioactivity of EC-derived EVs produced in standard 2D culture conditions, could be employed successfully for the same purpose in a 3D production system. Our results indicate that dynamic culture in a perfusion bioreactor significantly enhances EV production from human ECs. Moreover, the use of ethanol conditioning in conjunction with dynamic culture induces pro-vascularization bioactivity of EC-derived EVs that is correlated with increased EV levels of pro-angiogenic lncRNAs HOTAIR and MALAT1. Thus, this study represents one of the first reports of rationally-designed EV potency enhancement that is conserved between static 2D and dynamic 3D EV production systems, increasing the potential for scalable biomanufacturing of therapeutic EC-derived EVs for a variety of applications. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) have substantial therapeutic potential in a variety of applications. However, translation of EV-based therapies may be hindered by biomanufacturing challenges. EV production to date has predominantly involved the use of tissue culture flasks. Here, we report, for the first time, the use of a tubular perfusion bioreactor system with an integrated 3D-printed biomaterial scaffold for EV production from human endothelial cells. This system increases EV yield by over 100-fold compared to conventional tissue culture systems. Further, we show that an ethanol-conditioning approach that our group previously developed in 2D culture for enhancing EV potency is compatible with this new system. Thus, potency enhancement of EVs for vascularization applications is possible even with significantly increased production rate.

Project description:OBJECTIVE:Design of bioactive scaffolds with osteogenic capacity is a central challenge in cell-based patient-specific bone tissue engineering. Efficient and spatially uniform seeding of (stem) cells onto such constructs is vital to attain functional tissues. Herein we developed heparin functionalized collagen gels supported by 3D printed bioceramic scaffolds, as bone extracellular matrix (ECM)-mimetic matrices. These matrices were designed to enhance cell seeding efficiency of mesenchymal stem cells (MSCs) as well as improve their osteogenic differentiation through immobilized bone morphogenic protein 2 (BMP2) to be used for personalized bone regeneration. METHODS:A 3D gel based on heparin-conjugated collagen matrix capable of immobilizing recombinant human bone morphogenic protein 2 (BMP2) was synthesized. Isolated dental pulp Mesenchymal stem cells (MSCs) were then encapsulated into the bone ECM microenvironment to efficiently and uniformly seed a bioactive ceramic-based scaffold fabricated using additive manufacturing technique. The designed 3D cell-laden constructs were comprehensively investigated trough in vitro assays and in vivo study. RESULTS:In-depth rheological characterizations of heparin-conjugated collagen gel revealed that elasticity of the matrix is significantly improved compared with freely incorporated heparin. Investigation of the MSCs laden collagen-heparin hydrogels revealed their capability to provide spatiotemporal bioavailability of BMP2 while suppressing the matrix contraction over time. The in vivo histology and real-time polymerase chain reaction (qPCR) analysis showed that the designed construct supported the osteogenic differentiation of MSCs and induced the ectopic bone formation in rat model. SIGNIFICANCE:The presented hybrid constructs combine bone ECM chemical cues with mechanical function providing an ideal 3D microenvironment for patient-specific bone tissue engineering and cell therapy applications. The implemented methodology in design of ECM-mimetic 3D matrix capable of immobilizing BMP2 to improve seeding efficiency of customized scaffolds can be exploited for other bioactive molecules.

Project description:BackgroundPeriodontitis often leads to progressive destruction and loss of alveolar bone, the reconstruction of which remains difficult in periodontal therapy. As a novel bone graft material, tooth-derived bone substitute (TDBS) processed from extracted teeth has been previously reported about its osteoconductivity and promising results in bone regeneration. This study was to investigate the biological effects and bone regeneration properties of TDBS in vitro and in vivo using rat periodontal bone defect model.MethodsThree groups of materials were used in the experiments: TDBS, TDBS treated with ethylene diamine tetraacetic acid (EDTA) (TDBS-E), and allogeneic bone materials. Calcium (Ca) and phosphate (P) ion dissolutions were quantified by spectrophotometer for seven days. The releases of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) were identified by enzyme-linked immunosorbent assay (ELISA). Human osteoblast proliferation, migration, and differentiation were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell counting, alkaline phosphatase activity (ALP), and alizarin red staining (ARS), respectively. Furthermore, the osteogenic effects of TDBS on periodontal furcation bone defects were evaluated at eight weeks postoperatively using micro-computed tomography (Micro-CT) and histological analysis.ResultsThe dissolution of both Ca and P ions in TDBS increased over time. The BMP-2 released from TDBS was significantly higher than that from TDBS-E and allografts, while the TGF-β1 release from TDBS and TDBS-E groups was higher than that in the allografts. The TDBS-E group could induce the highest level of osteoblast proliferation compared to other groups. Cell migration with allografts co-culture was significantly induced compared to the blank control. However, all groups demonstrated similar positive effects on osteoblast differentiation. Furthermore, in the periodontal model, all materials could effectively enhance bone regeneration in the furcation defect.ConclusionsThe TDBS prepared chairside as an autogenous bone graft, demonstrating osteoinductivity, which enhances the osteogenic biological characteristics. Therefore, TDBS is suggested as an economical and biocompatible material for periodontal bone regeneration.

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An optimized 3D-printed perfusion bioreactor for homogeneous cell seeding in bone substitute scaffolds for future chairside applications

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