Biofabrication of articular cartilage: Development of an efficient in vivo repair technique using autologous stem cells

Abstract

Articular cartilage defects represent a major clinical challenge due to the lack of long-term management options available for young patients who present with a symptomatic and functional burden.

Microfracture is the traditional standard treatment of care and has no long-term benefit demonstrated beyond 2 years, with patients reporting symptom relapse and functional compromise. Other techniques used to treat chondral defects include Autologous Chondrocyte Implantation and Matrix-induced Autologous Chondrocyte Implantation, both of which are not superior in comparison to the cheap and easily performed microfracture technique.

Cartilage tissue engineering approaches using stem cells and bioscaffolds have become of significant research focus; additionally, the emergence of bioprinting technology has opened up the ability to efficiently and accurately deliver engineered tissue constructs. Biological tissue can be generated by printing cells and scaffolds together in a ‘bioink’ composition rather than using prefabricated scaffold constructs; this approach is coined ‘Biofabrication’, which is a rapidly growing field.

Biofabrication approaches show promise in treating chondral defects; however, we are no closer today to a human clinical trial. Several hurdles currently prevent the progression of such research; a significant barrier is the use of long periods of laboratory-based cell culture and expansion. This increased culture duration leads to concerns with the use of animal serum-based media, sterility, senescence, loss of differentiation potential, and tumorigenic transformation. To overcome these issues, human tissue harvest, cell isolation and reimplantation should be performed efficiently, thereby reducing the exposure to the risks mentioned above. Furthermore, by establishing a specific timeframe in which a biofabrication procedure can be achieved, surgical planning and patient preparation can be structured and adequately performed.

This thesis aimed to develop an efficient biofabrication procedure for cartilage repair using an autologous cell population, which could produce neocartilage in clinically relevant defects. The chapters in this work present several critical developments concerning the overall aim.

First, the most chondrogenic cell source from those tested within the knee joint was identified to be the human Adipose-Derived Stem Cell (hADSC). A rapid 85-minute hADSCs isolation protocol from the Infrapatellar Fat Pad (IFP) was then developed by optimising the time-consuming aspects of the standard IFP-derived hADSCs isolation protocol (>27 hours) and shown to be comparable.

Secondly, the minimum chondrogenic requirements of rapidly isolated hADSCs before reimplantation were established. It was determined that 5 days is the earliest time point during cellular expansion in which hADSCs could be driven into chondrogenesis. Therefore, the minimum biofabrication turnaround time is roughly 1-week (5 days and 85 minutes to be precise). Next, 5.0 million hADSCs/mL of a biocompatible hydrogel was shown to be the minimum concentration required to produce in vitro neocartilage. Finally, the maximum defect volume treatable in a 1-week turnaround was shown to be 380 uL (mm3) or 760 uL (mm3) using one or two IFPs respectively, representing clinically significant defect volumes.

The next section of this thesis aimed to establish a biofabrication model that could be adapted for surgical use and be implemented in an animal model. In this chapter, a safe, efficient and user-friendly procedure was designed and validated in vitro. First a representative cell source was selected and validated. Next, suitable hydrogel compositions and gelation times were identified, and finally, a safe intraoperative crosslinking set-up was developed.

The final element of this work was a proof of concept study, where the newly devised biofabrication approach was performed on a rabbit model to evaluate chondral repair. This procedure was successfully implemented, and the associated degree of cartilage repair was superior compared to the microfracture (clinical standard) and empty control groups.

In conclusion, an efficient 1-week biofabrication approach was established for chondral repair, and this approach was shown to treat clinically significant defect volumes. The newly developed procedure has been validated for short term repair in vivo and is superior to the existing standard treatment. The next step is to provide mid-long-term efficacy of therapy in vivo using a large animal model, which if successful, paves the way to human translation. This work presents promise in the future management of chondral defects in young patients with a low-risk strategy that could one day treat/halt the progression to early-onset osteoarthritis.