Towards the scale up of tissue engineered intervertebral discs for clinical application

Abstract

Replacement of the intervertebral disc with a viable, tissue-engineered construct that mimics native tissue structure and function is an attractive alternative to fusion or mechanical arthroplasty for the treatment of disc pathology. While a number of engineered discs have been developed, the average size of these constructs remains a fraction of the size of human intervertebral discs. In this study, we fabricated medium (3 mm height × 10 mm diameter) and large (6 mm height × 20 mm diameter) sized disc-like angle ply structures (DAPS), encompassing size scales from the rabbit lumbar spine to the human cervical spine. Maturation of these engineered discs was evaluated over 15 weeks in culture by quantifying cell viability and metabolic activity, construct biochemical content, MRI T2 values, and mechanical properties. To assess the performance of the DAPS in the in vivo space, pre-cultured DAPS were implanted subcutaneously in athymic rats for 5 weeks. Our findings show that both sized DAPS matured functionally and compositionally during in vitro culture, as evidenced by increases in mechanical properties and biochemical content over time, yet large DAPS under-performed compared to medium DAPS. Subcutaneous implantation resulted in reductions in NP cell viability and GAG content at both size scales, with little effect on AF biochemistry or metabolic activity. These findings demonstrate that engineered discs at large size scales will mature during in vitro culture, however, future work will need to address the challenges of reduced cell viability and heterogeneous matrix distribution throughout the construct.

Statement of Significance

This work establishes, for the first time, tissue-engineered intervertebral discs for total disc replacement at large, clinically relevant length scales. Clinical translation of tissue-engineered discs will offer an alternative to mechanical disc arthroplasty and fusion procedures, and may contribute to a paradigm shift in the clinical care for patients with disc pathology and associated axial spine and neurogenic extremity pain.

Introduction

The intervertebral discs (IVD) of the spine are fibrocartilaginous composite structures comprised of an inner nucleus pulposus (NP) composed primarily of proteoglycans, type II collagen and water, and an outer annulus fibrosus (AF) composed of aligned types I and II collagen organized into concentric lamellae [1]. When healthy IVDs are loaded, hydrostatic pressure develops in the NP, placing the AF in tension and allowing the tissue to bear load while permitting motion [2]. This structure-function relationship is disrupted with degeneration of the IVD, a complex and multifactorial cascade of biochemical, cellular and structural changes that is commonly associated with low back pain [3], [4].

Low back pain is a top three cause of disability in developed nations, and in the United States is associated with a yearly economic burden of approximately $200 billion due to medical costs and lost wages [5], [6]. For patients with end-stage IVD degeneration who are unresponsive to conservative treatment, the gold standard surgical treatment is fusion to immobilize the affected motion segment. Fusion does not restore native IVD structure or function, is associated with degeneration of adjacent segments, and randomized controlled trials show similar clinical outcomes compared to non-operative treatment [7], [8]. Artificial total disc arthroplasty devices are also utilized as an alternative to fusion, in an effort to preserve spinal motion and prevent degeneration of adjacent segments [9]. The use of mechanical arthroplasty in the lumbar spine for treatment of back pain is somewhat controversial, with some evidence that current lumbar artificial total disc arthroplasty devices offer no significant clinical benefit over fusion [10]. There is greater enthusiasm for cervical mechanical disc arthroplasty, but uncertainty regarding potential for mechanical wear debris and the long-term revision options limits application. Thus, there is a significant need to develop new treatment strategies for patients with IVD degeneration and associated axial spine pain and neurogenic extremity pain.

Tissue engineering offers considerable promise for the treatment of end stage disease, as replacement of the degenerative IVD with a viable engineered construct may restore healthy spine function with the capacity to remodel in response to the in vivo environment. Towards this end, composite IVDs with tissue-engineered NP and AF analogs have been developed by several groups and evaluated in vitro and in vivo in small and large animal models [11], [12], [13], [14], [15], [16], [17], [18]. Our group in particular has developed tissue-engineered disc-like angle ply structures (DAPS), composed of engineered NP and AF analogs. The AF region is comprised of cell-seeded concentric layers of electrospun, nanofibrous poly (ε-caprolactone) (PCL), with alternating fiber alignments of ±30°, to recapitulate the native hierarchical structure of the AF. The PCL AF is combined with a hyaluronic acid or agarose hydrogel that serves as the NP region to form a complete composite tissue-engineered IVD [19]. We have previously fabricated DAPS sized for the rabbit lumbar spine and rat caudal disc space, and shown compositional equivalence of the DAPS with native tissue with extended in vitro culture times [19], [20] and stability upon in vivo implantation [21].

Despite the progress over the past decade in the field of IVD tissue engineering, engineered constructs have thus far only been fabricated at length scales that are a fraction of the size of human IVDs. The average width and height of engineered IVDs reported in the literature are approximately 11 mm and 3 mm, respectively, while human cervical IVDs are an average 30 mm in lateral width and 6 mm in height, and human lumbar IVDs are an average 55 mm in lateral width and 11 mm in height [22], [23], [24]. Fabrication of tissue-engineered IVDs at larger size scales is therefore a critical next step towards the clinical translation of this technology. The purpose of this study was therefore to fabricate and culture DAPS at multiple length scales, the largest of which mimics the scale of a human cervical IVD. At each length scale, we evaluated construct viability, matrix distribution and mechanical properties over 15 weeks culture in vitro, as well as after a 5 week period of in vivo subcutaneous implantation.