The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4).

We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes.

We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells.

We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to hypertrophic cells at full transcriptomic breadth. Using this atlas as the ground truth for evaluating _in vitro_ cells, we found substantial variability in cell states produced by each protocol, with many showing little similarity to _in vivo_ cells, and all exhibiting off-target differentiation.

Trajectory alignment between _in vivo_ and _in vitro_ single-cell data revealed key differences in gene expression dynamics between _in vitro_ and _in vivo cells,_ with several osteoblastic transcription factors erroneously unregulated _in vitro,_ including _FOXO1._

Using this information, we inhibited _FOXO1_ in culture to successfully increase chondrocyte yield _in vitro._

This study presents a new framework for evaluating tissue engineering protocols, using single-cell data to drive improvement and bring the prospect of true engineered cartilage closer to reality.

Introduction

Decreases in trainees' working hours, coupled with evidence of worse outcomes when hip arthroscopies are performed by inexperienced surgeons, mandate the development of additional means of arthroscopic training. Though virtual reality simulation training has been adopted by other surgical specialities, its slow uptake in arthroscopic training is due to a lack of evidence as to its benefits. These benefits can be demonstrated through learning curves associated with simulator training – with practice reflecting measurable increases in validated performance metrics.

Hip arthroscopy is a rapidly expanding technique that has a steep learning curve. Simulation may have a role in helping trainees overcome this. However there is as yet no validated hip arthroscopy simulator. This study aimed to test the construct validity of a virtual reality hip arthroscopy simulator.

Nineteen orthopaedic surgeons performed a simulated arthroscopic examination of a healthy hip joint in the supine position. Surgeons were categorized as either expert (those who had performed 250 hip arthroscopies or more) or novice (those who had performed fewer than this). Twenty-one targets were visualized within joint; nine via the anterior portal, nine via the anterolateral and three via the posterolateral. This was followed by a task testing basic probe examination of the joint in which a series of eight targets were probed via the anterolateral portal. Each surgeon's performance was evaluated by the simulator using a set of pre-defined metrics including task duration, number of soft tissue & bone collisions, and distance travelled by instruments. No repeat attempts at the tasks were permitted. Construct validity was then evaluated by comparing novice and expert group performance metrics over the two tasks using the Mann–Whitney test, with a p value of less than 0.05 considered significant.

On the visualization task, the expert group outperformed the novice group on time taken (P=0.0003), number of collisions with soft tissue (P=0.001), number of collisions with bone (P=0.002) and distance travelled by the arthroscope (P=0.02). On the probe examination, the two groups differed only in the time taken to complete the task (P=0.025).

Increased experience in hip arthroscopy was reflected by significantly better performance on the VR simulator across two tasks, supporting its construct validity.

This study validates a virtual reality hip arthroscopy simulator and supports its potential for developing basic arthroscopic skills.

To quantify the risk posed to the Lateral Femoral Cutaneous Nerve (LFCN) during Total Hip Arthroplasty using the Minimally Invasive Anterior Approach (MIAA), and during placement of the Anterior Portal (AP) in Supine Hip Arthroscopy (SHA).

Forty-five hemipelves from thirty-nine cadavers were dissected. The LFCN was identified proximal to the inguinal ligament (IL), and its path in the thigh identified. The positions of the nerve and its branches in relation to the MIAA incision and the site for AP placement were measured using Vernier Callipers.

44% of nerves crossed the incision line used in the MIAA, at an average distance of 47 ± 28mm from the proximal end of the incision. Of those that did not cross the incision line, the average minimum distance between the nerve and incision was 14.4 ± 7.4mm, occurring on average 74.0 ± 37.3mm from the proximal end of the incision. In addition, the AP was placed in the path of the nerve on 38% of occasions. The nerve took an oblique path, and when found not to intersect with the AP portal, was located 5.7 ± 4.5mm from the portal's edge. We found a reduction in risk if the portal is moved medially or laterally by 15mm from its current location.

The LFCN is at high risk of injury during both THA using the MIAA and SHA using the AP. Our study emphasises the need for meticulous dissection during these procedures, and thorough explanation of these risk whilst consenting patients. We suggest that relocation of the AP 15mm more laterally or medially will reduce the risk posed to the LFCN.