Cartilage defects can result in pain, disability, and osteoarthritis. Hydrogels providing a chondroregeneration‐permissive environment are often mechanically weak and display poor lateral integration into the surrounding cartilage. This study develops a visible‐light responsive gelatin ink with enhanced interactions with the native tissue, and potential for intraoperative bioprinting. A dual‐functionalized tyramine and methacryloyl gelatin (GelMA‐Tyr) is synthesized. Photo‐crosslinking of both groups is triggered in a single photoexposure by cell‐compatible visible light in presence of tris(2,2′‐bipyridyl)dichlororuthenium(II) and sodium persulfate as initiators. Neo‐cartilage formation from embedded chondroprogenitor cells is demonstrated in vitro, and the hydrogel is successfully applied as bioink for extrusion‐printing. Visible light in situ crosslinking in cartilage defects results in no damage to the surrounding tissue, in contrast to the native chondrocyte death caused by UV light (365–400 nm range), commonly used in biofabrication. Tyramine‐binding to proteins in native cartilage leads to a 15‐fold increment in the adhesive strength of the bioglue compared to pristine GelMA. Enhanced adhesion is observed also when the ink is extruded as printable filaments into the defect. Visible‐light reactive GelMA‐Tyr bioinks can act as orthobiologic carriers for in situ cartilage repair, providing a permissive environment for chondrogenesis, and establishing safe lateral integration into chondral defects.

Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering.

Articular cartilage damage is a major challenge in healthcare due to the lack of long-term repair options. There are several promising regenerative implant-based approaches for the treatment, but the fixation of the implant remains a significant challenge. This study evaluated the potential for repair of an osteochondral implant produced through a novel combined bioprinting-based chondral-bone integration, with and without cells, in an equine model. Implants consisted of a melt electrowritten polycaprolactone (PCL) framework for the chondral compartment, which was firmly integrated with a bone anchor. The bone anchor was produced by extrusion-based printing of a low-temperature setting bioceramic material that had been proven to be effective for osteo-regeneration in an orthotopic, non-load bearing and non-articular site in the same species in an earlier in vivo study. Articular cartilage-derived progenitor cells were seeded into the PCL framework and cultured for 28 days in vitro in the presence of bone morphogenetic protein-9 (BMP-9), resulting in the formation of abundant extracellular matrix rich in glycosaminoglycans (GAGs) and type II collagen. The constructs were implanted in the stifle joints of Shetland ponies with cell-free scaffolds as controls. Clinical signs were monitored, and progression of healing was observed non-invasively through radiographic examinations and quantitative gait analysis. Biochemical and histological analyses 6 months after implantation revealed minimal deposition of GAGs and type II collagen in the chondral compartment of the defect site for both types of implants. Quantitative micro-computed tomography showed collapse of the bone anchor with low volume of mineralized neo-bone formation in both groups. Histology confirmed that the PCL framework within the chondral compartment was still present. It was concluded that the collapse of the osteal anchor, resulting in loss of the mechanical support of the chondral compartment, strongly affected overall outcome, precluding evaluation of the influence of BMP-9 stimulated cells on in vivo cartilage regeneration.

Tissue engineering is a promising approach to address the unmet clinical need for treating cartilage damage. Monitoring the characteristics of tissue-engineered cartilage constructs (TECs) during culture is critical for optimizing culture conditions and assessing TEC development. An important characteristic of TEC development is the ratio of glycosaminoglycans (GAGs) to DNA. However, current methods for quantifying GAGs and DNA are destructive, making them impractical for longitudinal monitoring. This study investigates the potential of optical spectroscopy in the visible and near-infrared (NIR) ranges coupled with machine learning (ML) for non-destructive prediction of GAGs and DNA in TECs. Six types of TECs were incubated for 7 or 28 days (n = 6 × 2 × 3). Spectral data were acquired from the constructs in the NIR and visible-short-NIR (VIS-SNIR) ranges using a fiber-optic probe and a multichannel spectrometer, followed by biochemical quantification of GAGs and DNA. ML models were developed for predicting the GAGs and DNA from the spectra and evaluated by leave-one-sample-out cross-validation after determining the optimal spectral range, preprocessing method, and ML algorithm. The performance of the models for predicting the construct's relative maturity (based on culture duration) using the predicted GAGs/DNA ratio was also evaluated. A NIR-based model was optimal for DNA content prediction (R² = 0.81), while a VIS-SNIR-based model was optimal for GAGs prediction (R² = 0.79). The predicted GAGs/DNA ratios were 100 % accurate in distinguishing immature TECs from more mature ones. Thus, optical spectroscopy combined with ML can enable non-destructive prediction of the maturity of TECs via predicting their DNA and GAG contents.

Introduction: The use of autologous human primary articular chondrocytes (hPACs) for repairing damaged cartilage is the golden standard; however, their 2-dimensional (2D) expansion induces dedifferentiation, making it challenging to create hyaline cartilage. Spinner flasks are efficient for generating cartilage organoids, allowing hPACs to proliferate without dedifferentiation; however, porcine notochordal cell-derived matrix (NCM) is needed for aggregation, limiting clinical application. Human articular chondroprogenitor cells (hACPCs) can be expanded many fold while maintaining chondrogenic potential. Therefore, the scalable production of hACPC cartilage organoids without NCM in spinner flasks was investigated in this study. Methods: hPAC organoids with NCM and hACPC organoids using bone morphogenetic protein 9 (BMP-9) were produced in spinner flasks in 14 days. Thereafter, approximately 20 organoids were fused in low adhesive wells for 21 days. Organoids underwent mechanical testing, and both organoids and fused constructs were evaluated using biochemical, histological, and immunohistochemical analysis. Results: The hACPCs self-assembled and synthesized abundant extracellular matrix once stimulated with BMP-9. The hPAC and hACPC organoids showed similar mechanical properties, but hACPC organoids and fused constructs showed a more uniform matrix and cell distribution. Conclusion: The hACPC organoids fused into a neo-hyaline cartilage-like tissue, demonstrating their potential for improved, scalable cartilage tissue repair.