The aim of this study was to investigate the role of an antichondrogenic factor, MIR221 (miR-221), in intervertebral disc degeneration (IDD), and provide basic information for the development of a therapeutic strategy for the disc repair based on specific nucleic acid based drugs, such as miR-221 silencing. We established a relatively quick protocol to minimize artifacts from extended in vitro culture, without selecting the different types of cells from intervertebral disc (IVD) or completely disrupting extracellular matrix (ECM), but by using the whole cell population with a part of resident ECM. During the de-differentiation process miR-221 expression significantly increased. We demonstrated the effectiveness of miR-221 silencing in driving the cells towards chondrogenic lineage. AntagomiR-221 treated cells showed in fact a significant increase of expression of typical chondrogenic markers including COL2A1, ACAN and SOX9, whose loss is associated with IDD. Moreover, antagomiR-221 treatment restored FOXO3 expression and increased TRPS1 expression levels attenuating the severity grade of degeneration, and demonstrating in a context of tissue degeneration and inflammation not investigated before, that FOXO3 is target of miR-221. Data of present study are promising in the definition of new molecules useful as potential intradiscal injectable biological agents.

Mesenchymal stem cells (MSCs) hold great promise for the treatment of cartilage related injuries. However, selectively promoting stem cell differentiation in vivo is still challenging. Chondrogenic differentiation of MSCs usually requires the use of growth factors that lead to the overexpression of hypertrophic markers. In this study, for the first time the effect of stiffness on MSC differentiation has been tested without the use of growth factors. Three-dimensional collagen and alginate scaffolds were developed and characterised. Stiffness significantly affected gene expression and ECM deposition. While, all hydrogels supported chondrogenic differentiation and allowed deposition of collagen type II and aggrecan, the 5.75 kPa hydrogel showed limited production of collagen type I compared to the other two formulations. These findings demonstrated for the first time that stiffness can guide MSCs differentiation without the use of growth factors within a tissue engineering scaffold suitable for the treatment of cartilage defects.

Focal cartilage defects are a prevalent knee problem affecting people of all ages. Articular cartilage (AC) possesses limited healing potential, and osteochondral defects can lead to pain and long-term complications such as osteoarthritis. Autologous chondrocyte implantation (ACI) has been a successful surgical approach for repairing osteochondral defects over the past two decades. However, a major drawback of ACI is the dedifferentiation of chondrocytes during their in vitro expansion. In this study, we isolated ovine chondrocytes and cultured them in a two-dimensional environment for ACI procedures. We hypothesized that 3D scaffolds would support the cells' redifferentiation without the need for growth factors so we encapsulated them into soft collagen and alginate (col/alg) hydrogels. Chondrocytes embedded into the hydrogels were viable and proliferated. After 7 days, they regained their original rounded morphology (aspect ratio 1.08) and started to aggregate. Gene expression studies showed an upregulation of COL2A1, FOXO3A, FOXO1, ACAN, and COL6A1 (37, 1.13, 22, 1123, and 1.08-fold change expression, respectively) as early as day one. At 21 days, chondrocytes had extensively colonized the hydrogel, forming large cell clusters. They started to replace the degrading scaffold by depositing collagen II and aggrecan, but with limited collagen type I deposition. This approach allows us to overcome the limitations of current approaches such as the dedifferentiation occurring in 2D in vitro expansion and the necrotic formation in spheroids. Further studies are warranted to assess long-term ECM deposition and integration with native cartilage. Though limitations exist, this study suggests a promising avenue for cartilage repair with col/alg hydrogel scaffolds.

This study evaluated the use of silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO₂/PTHF/PCL-diCOOH) 3D-printed scaffolds, with channel sizes of either 200 (SC-200) or 500 (SC-500) µm, as biomaterials to support the chondrogenesis of sheep bone marrow stem cells (oBMSC), under in vitro conditions. The objective was to validate the potential use of SiO₂/PTHF/PCL-diCOOH for prospective in vivo ovine studies. The behaviour of oBMSC, with and without the use of exogenous growth factors, on SiO₂/PTHF/PCL-diCOOH scaffolds was investigated by analysing cell attachment, viability, proliferation, morphology, expression of chondrogenic genes (RT-qPCR), deposition of aggrecan, collagen II, and collagen I (immunohistochemistry), and quantification of sulphated glycosaminoglycans (GAGs). The results showed that all the scaffolds supported cell attachment and proliferation with upregulation of chondrogenic markers and the deposition of a cartilage extracellular matrix (collagen II and aggrecan). Notably, SC-200 showed superior performance in terms of cartilage gene expression. These findings demonstrated that SiO₂/PTHF/PCL-diCOOH with 200 µm pore size are optimal for promoting chondrogenic differentiation of oBMSC, even without the use of growth factors.

Articular cartilage (AC) regeneration remains a major challenge in musculoskeletal tissue engineering. Cartilage extracellular matrix (ECM)‐derived scaffolds have shown promise as tissue‐specific biomaterials, but the extent to which the fabrication process can be tuned to modulate scaffold pore architecture and mechanics and how this might influence the chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) remains incompletely defined. The aim of this study was to develop solubilized porcine AC‐ECM scaffolds with controlled differences in pore size and matrix stiffness, and to evaluate how these factors regulate chondrogenesis of MSCs. Scaffolds with different pore sizes were fabricated by modifying the freezing cycle during lyophilization through the inclusion of an annealing step, while scaffold stiffness was varied by adjusting the AC‐ECM concentration from 10 (1% ECM) to 50 mg/mL (5% ECM). Incorporation of an annealing step led to a ~2.5‐fold increase in the mean scaffold pore size and was associated with improved construct elasticity and greater early cellular penetration. Increasing the AC‐ECM concentration increased scaffold stiffness, with the ramp modulus increasing from approximately 5 kPa in the 1% scaffolds to approximately 60 kPa in the 5% scaffolds. All scaffold groups supported cartilage‐matrix deposition during chondrogenic culture. Higher ECM concentrations were associated with greater total collagen accumulation, while the stiffest 5% scaffolds supported increased Type X collagen staining relative to the softer scaffolds. The expression of key chondrogenic genes SOX9 , ACAN , and COL2A1 was higher in the 2% scaffolds compared to 5% scaffolds, while the expression of the hypertrophic marker COL10A1 was higher in the 5% scaffolds. In summary, scaffold processing provided a practical means to tune pore architecture and bulk mechanical behavior in solubilized AC‐ECM scaffolds, and these differences were associated with changes in cell distribution and phenotype. These findings can potentially be leveraged to engineer scaffolds supportive of zonal cartilage tissue engineering or for osteochondral defect repair.

Focal cartilage defects are a prevalent knee problem affecting people of all ages. Due to its avascular nature, cartilage has limited self-repair capacity, and osteochondral defects can lead to pain and long-term complications such as osteoarthritis. Autologous chondrocyte implantation (ACI) has been a successful surgical approach for repairing osteochondral defects over the past two decades. However, a major drawback of ACI is the de-differentiation of chondrocytes during their in vitro expansion. In this study, we isolated ovine chondrocytes and cultured them in a two-dimensional environment as for ACI procedures. We hypothesised that the 3D scaffolds would support the cells re-differentiation without the need for growth factors and so we encapsulated them into soft collagen and alginate (col/alg) hydrogels. Chondrocytes embedded into hydrogels were viable and proliferated. After 7 days they acquired a rounded morphology and started to aggregate. Gene expression studies showed that the genes associated with chondrogenesis started to be up regulated as early as day one. At 21 days chondrocytes had extensively colonized the hydrogel, forming large cell clusters and started to deposit collagen II and aggrecan with limited collagen type I deposition. These findings highlight the potential of soft col/alg hydrogels to enhance ACI outcomes by creating a favourable microenvironment for chondrocyte reprogramming and re-differentiation, eliminating the dependency on growth factors.