Evaluation of a co-culture of rapidly isolated chondrocytes and stem cells seeded on tri-layered collagen-based scaffolds in a caprine osteochondral defect model

Cartilage has poor regenerative capacity and thus damage to the joint surfaces presents a major clinical challenge. Recent research has focussed on the development of tissue-engineered and cell-based approaches for the treatment of cartilage and osteochondral injuries, with current clinically available cell-based approaches including autologous chondrocyte implantation and matrix-assisted autologous chondrocyte implantation. However, these approaches have significant disadvantages due to the requirement for a two-stage surgical procedure and an in vitro chondrocyte expansion phase which increases logistical challenges, hospital times and costs. In this study, we hypothesized that seeding biomimetic tri-layered scaffolds, with proven regenerative potential, with chondrocyte/infrapatellar fat pad stromal cell co-cultures would improve their regenerative capacity compared to scaffolds implanted cell-free. Rapid cell isolation techniques, without the requirement for long term in vitro culture, were utilised to achieve co-cultures of chondrocytes and stromal cells and thus overcome the limitations of existing cell-based techniques. Cell-free and cell-seeded scaffolds were implanted in osteochondral defects, created within the femoral condyle and trochlear ridge, in a translational large animal goat model. While analysis showed trends towards delayed subchondral bone healing in the cell-seeded scaffold group, by the 12 month timepoint the cell-free and cell-seeded groups yield cartilage and bone tissue with comparable quality and quantity. The results of the study reinforce the potential of the biomimetic tri-layered scaffold to repair joint defects but failed to demonstrate a clear benefit from the addition of the CC/FPMSC co-culture to this scaffold. Taking into consideration the additional cost and complexity associated with the cell-seeded scaffold approach, this study demonstrates that the treatment of osteochondral defects using cell-free tri-layered scaffolds may represent a more prudent clinical approach

Biomaterial-based endochondral bone regeneration: A shift from traditional tissue engineering paradigms to developmentally inspired strategies

There is an urgent, clinical need for an alternative to the use of autologous grafts for the ever increasing number of bone grafting procedures performed annually. Herein, we describe a developmentally inspired approach to bone tissue engineering, which focuses on leveraging biomaterials as platforms for recapitulating the process of endochondral ossification. To begin, we describe the traditional biomaterial-based approaches to tissue engineering that have been investigated as methods to promote in vivo bone regeneration, including the use of three-dimensional biomimetic scaffolds, the delivery of growth factors and recombinant proteins, and the in vitro engineering of mineralized bone-like tissue. Thereafter, we suggest that some of the hurdles encountered by these traditional tissue engineering approaches may be circumvented by modulating the endochondral route to bone repair and, to that end, we assess various biomaterials that can be used in combination with cells and signaling factors to engineer hypertrophic cartilaginous grafts capable of promoting endochondral bone formation. Finally, we examine the emerging trends in biomaterial-based approaches to endochondral bone regeneration, such as the engineering of anatomically shaped templates for bone and osteochondral tissue engineering, the fabrication of mechanically reinforced constructs using emerging three-dimensional bioprinting techniques, and the generation of gene-activated scaffolds, which may accelerate the field towards its ultimate goal of clinically successful bone organ regeneration

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

A rare case of fournier’s gangrene

We report a rare case that highlights acute pancreatitis as the protagonist of Fournier's Gangrene. This patient was treated with a radical debridement of his perineum at presentation and subsequently reconstructed with split thickness skin grafting. This is an unusual aetiology of necrotizing fasciitis with only one other case reported in the literature. This serves to emphasize to physicians that acute pancreatitis is a potential source when investigating and treating patients with Fournier's Gangrene

A rare case of fournier’s gangrene

We report a rare case that highlights acute pancreatitis as the protagonist of Fournier\u27s Gangrene. This patient was treated with a radical debridement of his perineum at presentation and subsequently reconstructed with split thickness skin grafting. This is an unusual aetiology of necrotizing fasciitis with only one other case reported in the literature. This serves to emphasize to physicians that acute pancreatitis is a potential source when investigating and treating patients with Fournier\u27s Gangrene

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

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