Purpose
The objective of this work was to characterise the dimensions of knee focal cartilage defects, generate a heat map of cartilage defect locations, and characterise the native articular surface morphology at the most common defect sites to aid in the design of generic focal knee resurfacing implants (FKRIs).

Methods
Healthy femoral cartilage was segmented from knee magnetic resonance imaging scans of 70 patients eligible for FKRI surgery. Cartilage defects were manually reconstructed to create three-dimensional (3D) native cartilage and cartilage defect models. A 3D statistical shape model of the native cartilage was created and 200 artificial models with varying shapes were generated. A heat map showing the frequency of defect occurrence location was created, and the size and aspect ratio of the defects were determined. Local radii of curvature were calculated at five locations that exhibit high defect occurrence for the medial and lateral condyles and in the trochlear groove.

Results
The median defect size and aspect ratio were 2.7 and 1.6, respectively. Cartilage defect location frequency heat maps were successfully generated for the medial and lateral condyles. The median mediolateral and anteroposterior radius of curvature at the medial condyle hotspot were 17.5 mm and 40.8 mm respectively, and 22.6 mm and 33.8 mm at the lateral condyle hotspot.

Conclusions
Characteristics of knee focal cartilage defect properties such as the size, their spatial distribution and local articular cartilage surface morphology offer valuable insights for the design of generic FKRIs.

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.