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Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_6 | Pages 24 - 24
2 May 2024
Lawrence J Woods S Roberts K Tuck E Balogh P Predeus A He P Polanski K Prigmore E Zhou D Webb S Jardine L
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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.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_20 | Pages 39 - 39
1 Dec 2017
Alsinan Z Cieslak M He P Rupertus N Spinelli C Vives M Hacihalioglu I
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In recent years, there has been a growing interest to incorporate ultrasound into computer assisted orthopaedic surgery procedures in order to provide non-ionizing intra-operative imaging alternative to traditional fluoroscopy. However, identification of bone boundaries still continues to be a challenging process due low signal to noise ratio and imaging artifacts. The quality of the collected images also depends on the orientation of the ultrasound transducer with respect to the imaged bone surface. Shadow region is an important feature indicating the presence of a bone surface in the collected ultrasound data. In this work, we propose a framework for the enhancement of shadow regions from extended field of view spine ultrasound data. First bone surfaces are enhanced using a combination of local phase based image features. The combination of the phase features provides a more compact representation of vertebrae bone surfaces with supressed soft tissue interfaces. These enhanced features are used as an input to a L1 norm based regularisation method which emphasised uncertainty in the shadow regions. Validation on phantom and in vivo experiments achieve a mean dice coefficient value of 0.93 and 0.9 respectively.