Objective: The zone of calcified cartilage (ZCC) connects non-calcified articular cartilage to the subchondral bone, acting as transitional layer. Regeneration of this layer is key for cartilage repair but remains a challenge. Knowledge on the formation of this layer during development is limited. This study describes the use of an ex vivo explant culture model to investigate the formation of the ZCC. Design: Explants were harvested from immature bovine metacarpophalangeal joints and cultured in the presence of β-glycerophosphate for 3 weeks as osteochondral explants, full-thickness cartilage or divided in top and bottom cartilage layers. To investigate cell-driven vs matrix-dependent calcification, explants were devitalized. Calcification was analysed using calcium uptake, micro-computed tomography, gene expression analysis, and histological stainings. Results: A distinct area of calcified cartilage formed in the explants ex vivo. This layer showed similar characteristics to the ZCC in mature bovine tissue. Viable chondrocytes in bottom layers actively contributed to cartilage calcification, while calcification in top layers was only present in devitalized top layer explants. Top layers inhibited cartilage calcification in bottom layers and expressed higher levels of FGF18, PTHLH and MGP, while the bottom layers expressed more ALPL, COL10A1 and IHH. Conclusion: We present the first ex vivo model allowing to study and modulate cartilage calcification and the formation of the ZCC. We demonstrated an inherent zone-specific calcification pattern within the cartilage explants. This model allows future studies investigating mechanisms of ZCC formation in cartilage repair procedures, and the role of the top layer in pathological cartilage calcifications and potential interventions.

Objective: The cartilage-bone interface, including the zone of calcified cartilage (ZCC), is a thin layer of calcified tissue with a low cell number, thus making it technically challenging to study. The aim of this study was to develop methods to selectively study the ZCC for gene expression analysis and transcriptomic studies. Design: We set-up a method to isolate ZCC explants comprising the ZCC and a thin layer of subchondral bone from human osteochondral tissue (n=4 patients) for tissue culture. The cellular response to a pro-inflammatory cytokine cocktail or TGF-ß1 (72 h) was compared to unstimulated explants. Full-thickness non-calcified cartilage (NCC) was used as control group. Next, we have set-up an optical coherence tomography (OCT)-guided 3D-laser microtomy to isolate the calcified ZCC and used FLASH-seq technology (n=4 patients) to characterize the cells in the ZCC compared to donor-matched deep zone of non-calcified cartilage (dNCC). Results: The culture model revealed metabolically active cells in ZCC explants that responded to inflammatory cytokines and to TGF-ß1. The responses were clearly different from the responses of NCC. With 3D-laser microtomy we isolated sufficient RNA from fresh human ZCC. ZCC had similar expression of cartilage-associated genes, lower expression of angiogenesis-related genes, and higher expression of SOST compared to dNCC. Conclusion: The ZCC explant model holds promise to decipher the role of the cartilage-bone interface in disease progression. OCT-guided 3D-laser microtomy and FLASH-seq are innovative technologies overcoming the limitation of standard approaches to phenotypically characterize chondrocytes in the thin ZCC layer and in other calcified tissues.

As a highly specialized shock-absorbing connective tissue, articular cartilage (AC) has very limited self-repair capacity after traumatic injuries, posing a heavy socioeconomic burden. Common clinical therapies for small- to medium-size focal AC defects are well-developed endogenous repair and cell-based strategies, including microfracture, mosaicplasty, autologous chondrocyte implantation (ACI), and matrix-induced ACI (MACI). However, these treatments frequently result in mechanically inferior fibrocartilage, low cost-effectiveness, donor site morbidity, and short-term durability. It prompts an urgent need for innovative approaches to pattern a pro-regenerative microenvironment and yield hyaline-like cartilage with similar biomechanical and biochemical properties as healthy native AC. Acellular regenerative biomaterials can create a favorable local environment for AC repair without causing relevant regulatory and scientific concerns from cell-based treatments. A deeper understanding of the mechanism of endogenous cartilage healing is furthering the (bio)design and application of these scaffolds. Currently, the utilization of regenerative biomaterials to magnify the repairing effect of joint-resident endogenous stem/progenitor cells (ESPCs) presents an evolving improvement for cartilage repair. This review starts by briefly summarizing the current understanding of endogenous AC repair and the vital roles of ESPCs and chemoattractants for cartilage regeneration. Then several intrinsic hurdles for regenerative biomaterials-based AC repair are discussed. The recent advances in novel (bio)design and application regarding regenerative biomaterials with favorable biochemical cues to provide an instructive extracellular microenvironment and to guide the ESPCs (e.g. adhesion, migration, proliferation, differentiation, matrix production, and remodeling) for cartilage repair are summarized. Finally, this review outlines the future directions of engineering the next-generation regenerative biomaterials toward ultimate clinical translation.

Neutrophils play a pivotal role in orchestrating the immune system response to biomaterials, the onset and resolution of chronic inflammation, and macrophage polarization. However, the neutrophil response to biomaterials and the consequent impact on tissue engineering approaches is still scarcely understood. Here, we report an in vitro culture model that comprehensively describes the most important neutrophil functions in the light of tissue repair. We isolated human primary neutrophils from peripheral blood and exposed them to a panel of hard, soft, naturally- and synthetically-derived materials. The overall trend showed increased neutrophil survival on naturally derived constructs, together with higher oxidative burst, decreased myeloperoxidase and neutrophil elastase and decreased cytokine secretion compared to neutrophils on synthetic materials. The culture model is a step to better understand the immune modulation elicited by biomaterials. Further studies are needed to correlate the neutrophil response to tissue healing and to elucidate the mechanism triggering the cell response and their consequences in determining inflammation onset and resolution.

Background: The use of acellular hydrogels to repair osteochondral defects requires cells to first invade the biomaterial and then to deposit extracellular matrix for tissue regeneration. Due to the diverse physicochemical properties of engineered hydrogels, the specific properties that allow or even improve the behaviour of cells are not yet clear. The aim of this study was to investigate the influence of various physicochemical properties of hydrogels on cell migration and related tissue formation using in vitro, ex vivo and in vivo models. Methods: Three hydrogel platforms were used in the study: Gelatine methacryloyl (GelMA) (5% wt), norbornene hyaluronic acid (norHA) (2% wt) and tyramine functionalised hyaluronic acid (THA) (2.5% wt). GelMA was modified to vary the degree of functionalisation (DoF 50% and 80%), norHA was used with varied degradability via a matrix metalloproteinase (MMP) degradable crosslinker and THA was used with the addition of collagen fibrils. The migration of human mesenchymal stromal cells (hMSC) in hydrogels was studied in vitro using a 3D spheroid migration assay over 48h. In addition, chondrocyte migration within and around hydrogels was investigated in an ex vivo bovine cartilage ring model (three weeks). Finally, tissue repair within osteochondral defects was studied in a semi-orthotopic in vivo mouse model (six weeks). Results: A lower DoF of GelMA did not affect cell migration in vitro (p ​= ​0.390) and led to a higher migration score ex vivo (p ​< ​0.001). The introduction of a MMP degradable crosslinker in norHA hydrogels did not improve cell infiltration in vitro or in vivo. The addition of collagen to THA resulted in greater hMSC migration in vitro (p ​= ​0.031) and ex vivo (p ​< ​0.001). Hydrogels that exhibited more cell migration in vitro or ex vivo also showed more tissue formation in the osteochondral defects in vivo, except for the norHA group. Whereas norHA with a degradable crosslinker did not improve cell migration in vitro or ex vivo, it did significantly increase tissue formation in vivo compared to the non-degradable crosslinker (p ​< ​0.001). Conclusion: The modification of hydrogels by adapting DoF, use of a degradable crosslinker or including fibrillar collagen can control and improve cell migration and tissue formation for osteochondral defect repair. This study also emphasizes the importance of performing both in vitro and in vivo testing of biomaterials, as, depending on the material, the results might be affected by the model used. The translational potential of this article: This article highlights the potential of using acellular hydrogels to repair osteochondral defects, which are common injuries in orthopaedics. The study provides a deeper understanding of how to modify the properties of hydrogels to control cell migration and tissue formation for osteochondral defect repair. The results of this article also highlight that the choice of the used laboratory model can affect the outcome. Testing hydrogels in different models is thus advised for successful translation of laboratory results to the clinical application.