Repair of bone defects in vivo using tissue engineered hypertrophic cartilage grafts produced from nasal chondrocytes

Abstract

The regeneration of large bone defects remains clinically challenging. The aim of our study was to use a rat model to use nasal chondrocytes to engineer a hypertrophic cartilage tissue which could be remodelled into bone in vivo by endochondral ossification.Primary adult rat nasal chondrocytes were isolated from the nasal septum, the cell numbers expanded in monolayer culture and the cells cultured in vitro on polyglycolic acid scaffolds in chondrogenic medium for culture periods of 5-10 weeks. Hypertrophic differentiation was assessed by determining the temporal expression of key marker genes and proteins involved in hypertrophic cartilage formation. The temporal changes in the genes measured reflected the temporal changes observed in the growth plate. Collagen II gene expression increased 6fold by day 7 and was then significantly downregulated from day 14 onwards. Conversely, collagen X gene expression was detectable by day 14 and increased 100-fold by day 35. The temporal increase in collagen X expression was mirrored by increases in alkaline phosphatase gene expression which also was detectable by day 14 with a 30-fold increase in gene expression by day 35. Histological and immunohistochemical analysis of the engineered constructs showed increased chondrocyte cell volume (31-45 µm), deposition of collagen X in the extracellular matrix and expression of alkaline phosphatase activity. However, no cartilage mineralisation was observed in in vitro culture of up to 10 weeks. On subcutaneous implantation of the hypertrophic engineered constructs, the grafts became vascularised, cartilage mineralisation occurred and loss of the proteoglycan in the matrix was observed.Implantation of the hypertrophic engineered constructs into a rat cranial defect resulted in angiogenesis, mineralisation and remodelling of the cartilage tissue into bone. Micro-CT analysis indicated that defects which received the engineered hypertrophic constructs showed 38.48% in bone volume compared to 7.01% in the control defects. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4Development of tissue engineered hypertrophic cartilage to use as a bone graft substitute is exciting development in regenerative medicine. This is a proof of principal study demonstrating the potential of nasal chondrocytes to engineer hypertrophic cartilage which will remodel into bone on in vivo transplantation. This approach to making engineered hypertrophic cartilage grafts could form the basis of a new potential future clinical treatment for maxillofacial reconstruction.

Perfusion Enhances Hypertrophic Chondrocyte Matrix Deposition, But Not the Bone Formation

“…[2][3][4][5][6][7] Among these strategies, the use of hypertrophic chondrocytes, the cell that orchestrates endochondral ossification, appears particularly promising as the maturation from chondrocytes to hypertrophic chondrocytes resulted in significantly more bone formation. 8 The hypertrophic chondrocyte constructs have shown an ability to create maturing bone in vitro, which subsequently developed vasculature and bone marrow following subcutaneous implantation, 6,9 and facilitated healing of calvarial 10,11 and long bone 12 defects. Robust performance of these constructs and their superiority over traditionally utilized osteoblast constructs 11,12 suggested clinical potential for healing complex bone fractures.…”

Tissue engineering applications in otolaryngology—The state of translation

“…Heatmap representing the proportion of publications by year that utilized specific translational research methodologies from construct characterization to human trial to investigate craniofacial tissue engineering 49‐72,74‐77,79,81‐84,145‐197 …”

A Simple Modification Method to Obtain Anisotropic and Porous 3D Microfibrillar Scaffolds for Surgical and Biomedical Applications

“…These medically approved polymeric meshes are attractive scaffolding material for tissue engineering and surgical applications and have been used in clinical practice for several years. [37][38][39][40][41][42] For instance, in situ tendon regeneration, [43] homeostatic cardiac, [44] and abdominal wall [45] surgeries were reported that use PGA meshes as scaffolding material. However, such melt-spun scaffolds have never been used to guide cells in a preferential direction because of technical limitations in the manufacturing process to produce in-plane fiber alignment.…”

“…These meshes (Biofelt and GORE BIO‐A) are made by melt spinning biodegradable polymers like polyglycolic acid (PGA) into macroscopic sheets, which are further processed by carding and needle punching to increase mesh integrity. These medically approved polymeric meshes are attractive scaffolding material for tissue engineering and surgical applications and have been used in clinical practice for several years . For instance, in situ tendon regeneration, homeostatic cardiac, and abdominal wall surgeries were reported that use PGA meshes as scaffolding material.…”

“…In following sections, we present the detailed stepwise procedure of coating the primary polymer, inducing fiber alignment, seeding, and culturing the cells and the final evaluation of cell alignment and tissue formation. For a proof‐of‐concept demonstration, we utilized commercially available PGA scaffolds (Biofelt) that found several clinical applications in the field of surgery and tissue engineering and processed it without further modifications as described below.…”