Jasmijn V. Korpershoek

Microtissue-based strategies have gained significant attention for the fabrication of cartilage grafts. Their spatial organization within 3D constructs plays a crucial role in directing tissue formation and maintaining the immediate mechanical stability of the printed structure. Melt-electrowritten (MEW) fibrous scaffolds have been widely used to reinforce cell-laden hydrogels, while also guiding microtissue fusion and self-organization within the constructs. However, current bioprinting methods used for positioning microtissues or spheroids within these structures are limited by insufficient control over spheroid deposition, low throughput, and technical challenges, such as nozzle-clogging. In this study, we leveraged Laser-Induced Forward Transfer (LIFT) to print articular cartilage progenitor cell (ACPC) spheroids of two different sizes (Ø ~80 and 150 μm) into fibrous polycaprolactone scaffolds. We investigated the effects of laser energy, spheroid size, and concentration in the bioink to identify the key parameters for controlled deposition. Furthermore, we assessed print fidelity, post-print spheroid viability, and chondrogenic differentiation capacity. The deposition rate of the spheroids was studied to maximize transfer efficiency, and the resulting optimal parameters were subsequently applied to place the spheroids within the MEW meshes. However, the spheroid transfer efficiency remained limited, not due to shortcomings in the printing process, but because uniform encapsulation becomes challenging when working with discrete and larger entities, such as spheroids. While single cells benefit from homogeneous suspension, enabling random encapsulation, spheroids require precise targeting to be successfully transferred. To address this challenge, an AI-based imaging analysis system was employed, and the amount of bioink on the donor slide was reduced to improve the transfer of larger spheroids further. Here, we demonstrate for the first time the successful convergence between LIFT and MEW for the deposition of ACPC spheroids into reinforcing meshes as the next step towards automated production of tissue constructs.

Premature physeal closure occurs following trauma, cancer, or infection. Current treatments have poor success rates. With recent pediatric donor tissue availability, physeal allograft transfer (PAT) can now be considered. The purpose of this study was to study the safety and feasibility of PAT in a large animal model. The aim of this study is to gather foundational data to inform future studies into the efficacy of PAT. Physeal defects were created in the distal femur of nine female domestic swine and treated with PAT from two male donor pigs, cementation, or bone autograft. Viability was assessed. After 3 months, physes were visualized using CT and MRI. Integration, tissue composition, donor DNA presence, and microscopic appearance were evaluated. Physeal allografts demonstrated 93% viability after procurement and preservation. All animals reached the 3‐months study endpoint without gross deformations. No physeal bars formed in any group. Cystic changes were seen in experimental and control femurs. All groups showed disorganized tissue architecture without growth plate recapitulation. High allografts viability and structural integrity after procurement support the potential of this treatment. Although no gross deformities were found, transferred physes demonstrate poor integration and incomplete repair. The lack of physeal bar formation in the control group limits the reliability of the animal model for studying physeal allograft transfer. Lastly, this study was designed as a feasibility study and lacks power to compare treatment effects statistically. The efficacy of PAT for preventing growth arrest remains undetermined.

Objectives: Damaged articular cartilage cannot regenerate spontaneously. Chondrocyte therapy, the current treatment of choice, requires laboratory expansion, necessitating two surgical procedures. Adding a second cell type to intraoperatively isolated chondrocytes enables single-stage chondrocyte-based repair strategies and has shown promise in vitro . The benefit of this strategy in vivo and in the clinic has not yet been comprehensively assessed. This systematic review assesses the efficacy of cartilage repair by co-implantations of chondrocytes with other cell types over all available in vivo studies. Design: Medline, Embase and Cochrane databases were searched for studies on co-implantations of chondrocytes with other cell types for hyaline cartilage repair. For each study, extracted data were tabulated and reporting quality and risk of bias were assessed. Studies were categorized based on their control groups for qualitative synthesis. Results: The search yielded 48 studies across 46 publications: 44 animal studies (26 ectopic and 18 orthotopic) and four clinical trials. Ectopic studies scored poorly on reporting quality and bias, while orthotopic studies were only moderately better. Twenty-seven of 27 experiments with chondrocyte-only controls demonstrated synergistic cartilage formation through co-implantation. Cartilage formation by co-implantations was also found in 15 of 17 experiments without such control and in four clinical trials. Conclusion: Cartilage repair by articular chondrocytes is improved by adding a second cell type, like mesenchymal stromal cells. This is likely caused by the second cell types’ stimulatory support. Other second cell types and chondrocyte sources have shown promising results too. This is encouraging for further clinical exploration of single-stage co-implantation strategies.

Objective: Allogeneic mesenchymal stromal cells (MSCs) are used in the 1-stage treatment of articular cartilage defects. The aim of this study is to investigate whether transport of mitochondria exists between chondrocytes and MSCs and to investigate whether the transfer of mitochondria to chondrocytes contributes to the mechanism of action of MSCs. Design: Chondrocytes and MSCs were stained with MitoTracker, and CellTrace was used to distinguish between cell types. The uptake of fluorescent mitochondria was measured in cocultures using flow cytometry. Transport was visualized using fluorescence microscopy. Microvesicles were isolated and the presence of mitochondria was assessed. Mitochondria were isolated from MSCs and transferred to chondrocytes using MitoCeption. Pellets of chondrocytes, chondrocytes with transferred MSC mitochondria, and cocultures were cultured for 28 days. DNA content and proteoglycan content were measured. Mitochondrial DNA of cultured pellets and of repair cartilage tissue was quantified. Results: Mitochondrial transfer occurred bidirectionally within the first 4 hours until 16 hours of coculture. Transport took place via tunneling nanotubes, direct cell-cell contact, and extracellular vesicles. After 28 days of pellet culture, DNA content and proteoglycan deposition were higher in chondrocyte pellets to which MSC mitochondria were transferred than the control groups. No donor mitochondrial DNA was traceable in the biopsies, whereas an increase in MSC mitochondrial DNA was seen in the pellets. Conclusions: These results suggest that mitochondrial transport plays a role in the chondroinductive effect of MSCs on chondrocytes in vitro. However, in vivo no transferred mitochondria could be traced back after 1 year.

Over the past two decades, evidence has emerged for the existence of a distinct population of endogenous progenitor cells in adult articular cartilage, predominantly referred to as articular cartilage-derived progenitor cells (ACPCs). This progenitor population can be isolated from articular cartilage of a broad range of species, including human, equine, and bovine cartilage. In vitro, ACPCs possess mesenchymal stromal cell (MSC)-like characteristics, such as colony forming potential, extensive proliferation, and multilineage potential. Contrary to bone marrow-derived MSCs, ACPCs exhibit no signs of hypertrophic differentiation and therefore hold potential for cartilage repair. As no unique cell marker or marker set has been established to specifically identify ACPCs, isolation and characterization protocols vary greatly. This systematic review summarizes the state-of-the-art research on this promising cell type for use in cartilage repair therapies. It provides an overview of the available literature on endogenous progenitor cells in adult articular cartilage and specifically compares identification of these cell populations in healthy and osteoarthritic (OA) cartilage, isolation procedures, in vitro characterization, and advantages over other cell types used for cartilage repair. The methods for the systematic review were prospectively registered in PROSPERO (CRD42020184775).

Objective: Articular cartilage-derived progenitor cells (ACPCs) are a potential new cell source for cartilage repair. This study aims to characterize endogenous ACPCs from healthy and osteoarthritic (OA) cartilage, evaluate their potential for cartilage regeneration, and compare this to cartilage formation by chondrocytes. Design: ACPCs were isolated from full-thickness healthy and OA human cartilage and separated from the total cell population by clonal growth after differential adhesion to fibronectin. ACPCs were characterized by growth kinetics, multilineage differentiation, and surface marker expression. Chondrogenic redifferentiation of ACPCs was compared with chondrocytes in pellet cultures. Pellets were assessed for cartilage-like matrix production by (immuno)histochemistry, quantitative analyses for glycosaminoglycans and DNA content, and expression of chondrogenic and hypertrophic genes. Results: Healthy and OA ACPCs were successfully differentiated toward the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Both ACPC populations met the criteria for cell surface marker expression of mesenchymal stromal cells (MSCs). Healthy ACPCs cultured in pellets deposited extracellular matrix containing proteoglycans and type II collagen, devoid of type I collagen. Gene expression of hypertrophic marker type X collagen was lower in healthy ACPC pellets compared with OA pellets. Conclusions: This study provides further insight into the ACPC population in healthy and OA human articular cartilage. ACPCs show similarities to MSCs, yet do not produce calcified matrix under well-established osteogenic culture conditions. Due to extensive proliferative potential and chondrogenic capacity, ACPCs show potential for cartilage regeneration and possibly for clinical application, as a promising alternative to MSCs or chondrocytes.