Engineering bone-forming biohybrid sheets through the integration of melt electrowritten membranes and cartilaginous microspheroids.

Bone fractures are one of the most common traumatic large-organ injuries and although many fractures can heal on their own, 2-12% of fractures are slow healing or do not heal (non-unions). Autologous grafts are currently used for treatment of non-unions but are associated with limited healthy bone tissue. Tissue engineered cell-based products have promise for an alternative treatment method. It was previously demonstrated that cartilaginous microspheroids of periosteum-derived cells could be assembled into scaffold-free constructs and heal murine critically-sized long bone defects (non-unions). However, the handleability of such scaffold-free implants can be compromised when scaling-up. In this work, cartilaginous spheroids were combined with melt electrowritten (MEW) meshes to create an engineered cell-based implant, able to induce in vivo bone formation. MEW polycaprolactone meshes were tailored to contain pores (116 ± 28 µm) of a size that captured microspheroids (180 ± 15 µm). Periosteum-derived microspheroids pre-cultured for 4 days, were seeded on MEW meshes and gene expression analysis demonstrated up-regulation of chondrogenic (SOX9, COL2) and prehypertrophic (VEGF) gene markers after 14 days, creating a biohybrid sheet. When implanted subcutaneously (4 weeks), the biohybrid sheets mineralized (23 ± 3% MV/TV) and formed bone and bone marrow. Bone formation was also observed when implanted in a murine critically-sized long bone defect, though a high variation between samples was detected. The high versatility of this biofabrication approach lies in the possibility to tailor the scaffolds to shape and dimensions corresponding to the large bone defects and the individual patient using robust bone forming building blocks. These strategies are instrumental in the development of personalized regenerative therapies with predictive clinical outcomes. STATEMENT OF SIGNIFICANCE: Successful treatments for healing of large long bone defects are still limited and 2-12% of fractures do not heal properly. We combined a novel biofabrication technique: melt electrowriting (MEW), with robust biology: bone forming cartilaginous spheroids to create biohybrid sheets able to form bone upon implantation. MEW enabled the fabrication of scaffolds with micrometer-sized fibers in defined patterns which allowed the capturing of and merging with cartilaginous spheroids which had the potency to mature into bone via the developmental process of endochondral ossification. The present study contributes to the rapidly growing field of "Biofabrication with Spheroid and Organoid Materials'' and demonstrates design considerations that are of great importance for biofabrication of functional tissues through the assembly of cellular spheroids.

A platform for automated and label-free monitoring of morphological features and kinetics of spheroid fusion.

Spheroids are widely applied as building blocks for biofabrication of living tissues, where they exhibit spontaneous fusion toward an integrated structure upon contact. Tissue fusion is a fundamental biological process, but due to a lack of automated monitoring systems, the in-depth characterization of this process is still limited. Therefore, a quantitative high-throughput platform was developed to semi-automatically select doublet candidates and automatically monitor their fusion kinetics. Spheroids with varying degrees of chondrogenic maturation (days 1, 7, 14, and 21) were produced from two different cell pools, and their fusion kinetics were analyzed via the following steps: (1) by applying a novel spheroid seeding approach, the background noise was decreased due to the removal of cell debris while a sufficient number of doublets were still generated. (2) The doublet candidates were semi-automatically selected, thereby reducing the time and effort spent on manual selection. This was achieved by automatic detection of the microwells and building a random forest classifier, obtaining average accuracies, sensitivities, and precisions ranging from 95.0% to 97.4%, from 51.5% to 92.0%, and from 66.7% to 83.9%, respectively. (3) A software tool was developed to automatically extract morphological features such as the doublet area, roundness, contact length, and intersphere angle. For all data sets, the segmentation procedure obtained average sensitivities and precisions ranging from 96.8% to 98.1% and from 97.7% to 98.8%, respectively. Moreover, the average relative errors for the doublet area and contact length ranged from 1.23% to 2.26% and from 2.30% to 4.66%, respectively, while the average absolute errors for the doublet roundness and intersphere angle ranged from 0.0083 to 0.0135 and from 10.70 to 13.44°, respectively. (4) The data of both cell pools were analyzed, and an exponential model was used to extract kinetic parameters from the time-series data of the doublet roundness. For both cell pools, the technology was able to characterize the fusion rate and quality in an automated manner and allowed us to demonstrate that an increased chondrogenic maturity was linked with a decreased fusion rate. The platform is also applicable to other spheroid types, enabling an increased understanding of tissue fusion. Finally, our approach to study spheroid fusion over time will aid in the design of controlled fabrication of "assembloids" and bottom-up biofabrication of living tissues using spheroids.

Patterned, organoid-based cartilaginous implants exhibit zone specific functionality forming osteochondral-like tissues in vivo.

Tissue engineered constructs have the potential to respond to the unmet medical need of treating deep osteochondral defects. However, current tissue engineering strategies struggle in the attempt to create patterned constructs with biologically distinct functionality. In this work, a developmentally-inspired modular approach is proposed, whereby distinct cartilaginous organoids are used as living building blocks. First, a hierarchical construct was created, composed of three layers of cartilaginous tissue intermediates derived from human periosteum-derived cells: (i) early (SOX9), (ii) mature (COL2) and (iii) (pre)hypertrophic (IHH, COLX) phenotype. Subcutaneous implantation in nude mice generated a hybrid tissue containing one mineralized and one non-mineralized part. However, the non-mineralized part was represented by a collagen type I positive fibrocartilage-like tissue. To engineer a more stable articular cartilage part, iPSC-derived cartilage microtissues (SOX9, COL2; IHH neg) were generated. Subcutaneous implantation of assembled iPSC-derived cartilage microtissues resulted in a homogenous cartilaginous tissue positive for collagen type II but negative for osteocalcin. Finally, iPSC-derived cartilage microtissues in combination with the pre-hypertrophic cartilage organoids (IHH, COLX) could form dual tissues consisting of i) a cartilaginous safranin O positive and ii) a bony osteocalcin positive region upon subcutaneous implantation, corresponding to the pre-engineered zonal pattern. The assembly of functional building blocks, as presented in this work, opens possibilities for the production of complex tissue engineered implants by embedding zone-specific functionality through the use of pre-programmed living building blocks.

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture.

Xenogeneic-free media are required for translating advanced therapeutic medicinal products to the clinics. In addition, process efficiency is crucial for ensuring cost efficiency, especially when considering large-scale production of mesenchymal stem cells (MSCs). Human platelet lysate (HPL) has been increasingly adopted as an alternative for fetal bovine serum (FBS) for MSCs. However, its therapeutic and regenerative potential in vivo is largely unexplored. Herein, we compare the effects of FBS and HPL supplementation for a scalable, microcarrier-based dynamic expansion of human periosteum-derived cells (hPDCs) while assessing their bone forming capacity by subcutaneous implantation in small animal model. We observed that HPL resulted in faster cell proliferation with a total fold increase of 5.2 ± 0.61 in comparison to 2.7 ± 02.22-fold in FBS. Cell viability and trilineage differentiation capability were maintained by HPL, although a suppression of adipogenic differentiation potential was observed. Differences in mRNA expression profiles were also observed between the two on several markers. When implanted, we observed a significant difference between the bone forming capacity of cells expanded in FBS and HPL, with HPL supplementation resulting in almost three times more mineralized tissue within calcium phosphate scaffolds. FBS-expanded cells resulted in a fibrous tissue structure, whereas HPL resulted in mineralized tissue formation, which can be classified as newly formed bone, verified by µCT and histological analysis. We also observed the presence of blood vessels in our explants. In conclusion, we suggest that replacing FBS with HPL in bioreactor-based expansion of hPDCs is an optimal solution that increases expansion efficiency along with promoting bone forming capacity of these cells. Stem Cells Translational Medicine 2019;8:810&821.

Limb derived cells as a paradigm for engineering self-assembling skeletal tissues.

Mimicking developmental events has been proposed as a strategy to engineer tissue constructs for regenerative medicine. However, this approach has not yet been investigated for skeletal tissues. Here, it is demonstrated that ectopic implantation of day-14.5 mouse embryonic long bone anlagen, dissociated into single cells and randomly incorporated in a bioengineered construct, gives rise to epiphyseal growth plate-like structures, bone and marrow, which share many morphological and molecular similarities to epiphyseal units that form after transplanting intact long bone anlage, demonstrating substantial robustness and autonomy of complex tissue self-assembly and the overall organogenesis process. In vitro studies confirm the self-aggregation and patterning capacity of anlage cells and demonstrate that the model can be used to evaluate the effects of large and small molecules on biological behaviour. These results reveal the preservation of self-organizing and self-patterning capacity of anlage cells even when disconnected from their developmental niche and subjected to system perturbations such as cellular dissociation. These inherent features make long bone anlage cells attractive as a model system for tissue engineering technologies aimed at creating constructs that have the potential to self-assemble and self-pattern complex architectural structures.