Altering the architecture of tissue engineered hypertrophic cartilaginous grafts facilitates vascularisation and accelerates mineralisation.

Cartilaginous tissues engineered using mesenchymal stem cells (MSCs) can be leveraged to generate bone in vivo by executing an endochondral program, leading to increased interest in the use of such hypertrophic grafts for the regeneration of osseous defects. During normal skeletogenesis, canals within the developing hypertrophic cartilage play a key role in facilitating endochondral ossification. Inspired by this developmental feature, the objective of this study was to promote endochondral ossification of an engineered cartilaginous construct through modification of scaffold architecture. Our hypothesis was that the introduction of channels into MSC-seeded hydrogels would firstly facilitate the in vitro development of scaled-up hypertrophic cartilaginous tissues, and secondly would accelerate vascularisation and mineralisation of the graft in vivo. MSCs were encapsulated into hydrogels containing either an array of micro-channels, or into non-channelled 'solid' controls, and maintained in culture conditions known to promote a hypertrophic cartilaginous phenotype. Solid constructs accumulated significantly more sGAG and collagen in vitro, while channelled constructs accumulated significantly more calcium. In vivo, the channels acted as conduits for vascularisation and accelerated mineralisation of the engineered graft. Cartilaginous tissue within the channels underwent endochondral ossification, producing lamellar bone surrounding a hematopoietic marrow component. This study highlights the potential of utilising engineering methodologies, inspired by developmental skeletal processes, in order to enhance endochondral bone regeneration strategies

Macrofauna and larvae collected at the Auka hydrothermal vent field in Pescadero Basin in 2017

This data package provides the sampling locations and identifications for macrofauna and larvae collected at the Auka hydrothermal vent field in Pescadero Basin in 2017 and used in a study by Fleming et al. (2022). This data package contains five tables: paired tables for benthic slurps (sampling metadata and specimen counts), paired tables for plankton slurps (sampling metadata and specimen counts), and one table summarizing benthic and plankton specimens with Barcode of Life Data System (BOLD) Barcode Index Numbers (BINs). The paired data tables are partially aligned to Darwin Core event and occurrence tables for future contribution to the Ocean Biodiversity Information System (OBIS). Records for specimens in BOLD are available through the Global Biodiversity Information Facility (GBIF).Dalio Ocean Initiative and E/V Nautilus/Ocean Exploration Trus

H&E staining of constructs post-implantation.

<p>Constructs were stained with H&E at 4 weeks and 8 weeks post-implantation to examine bone formation. (A) Solid construct 4 weeks post-implantation. (B) Channelled construct 4 weeks post-implantation. (C) Solid construct 8 weeks post-implantation. (D) Channelled construct 8 weeks post-implantation. Arrows show lining of osteoblasts laying down new bone, arrowheads show osteocytes embedded within bone matrix, dotted arrows show hematopoietic elements. ‘at’ – adipose tissue, ‘wb’ – woven bone, ‘lb’ – lamellar bone. Main image scale bars are 500 µm. Inset scale bars are 50 µm.</p

Histology and immunohistochemistry of the channels within channelled constructs pre-implantation and 4 weeks post-implantation.

<p>Channels were examined to determine the pathway through which bone formation occurs. (A,E) Alcian blue staining. (B,F) Collagen I staining. (C,G) Collagen II staining. (D,H) Collagen X staining. (A–D) Pre-implantation. (E–H) 4 weeks post-implantation. Scale bars are 50 µm.</p

Histology of constructs pre-implantation and post-implantation.

<p>Solid and channelled constructs were subjected to 5 weeks culture in chondrogenic conditions, followed by 1 week of culture in hypertrophic conditions, and then implanted subcutaneously into nude mice to be harvested at 4 weeks and 8 weeks post-implantation. Constructs were stained with alcian blue, picro-sirius red and alizarin red to assess sGAG, collagen, and calcium accumulation respectively. 8 weeks post-implantation samples were decalcified prior to histological analysis, hence alizarin red staining was not undertaken at this time point. Images show half the construct. Scale bars are 500 µm.</p

Biochemical and µCT analysis of constructs after 10 weeks of <i>in vitro</i> culture.

<p>Solid and channelled constructs were subjected to 5 weeks culture in chondrogenic conditions, followed by an additional 5 weeks culture in hypertrophic conditions. (A) sGAG, (B) collagen, (C) calcium (% wet weight) accumulation of solid and channelled constructs after 5 and 10 weeks of <i>in vitro</i> culture. 3–4 constructs per group were analysed. Significance <i>p</i><0.05: a vs. 5 weeks, b vs. solid constructs. (D) µCT analysis of solid and channelled constructs after 10 weeks <i>in vitro</i> culture. Quarter section corresponds to a region ∼0.75 mm into the depth of the construct. Centre section corresponds to a region ∼1.5 mm into the depth of the construct. Sections correspond to a thickness of 120 µm. Scale bar is consistent across all images. Images are representative of 3 constructs analysed.</p