Full length articleTowards the scale up of tissue engineered intervertebral discs for clinical application
Graphical abstract
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.
Section snippets
Disc cell isolation and DAPS fabrication
NP and AF tissues were isolated from three adult bovine caudal spines, purchased from a local abattoir with institutional approval, and incubated overnight at 37 °C in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 2% penicillin/streptomycin/fungizone (PSF, Antibiotic-Antimycotic; Gibco) and 10% fetal bovine serum (FBS). Tissues were then digested, first in 2.5 mg/mL pronase for 1 h, followed by 0.5 mg/mL collagenase (Type IV, Sigma-Aldrich, St. Louis, MO) for 4 h (NP tissue)
Viability and metabolic activity
Medium and large DAPS were successfully fabricated and cultured for up to 15 weeks in vitro (Fig. 1). In medium DAPS, cell viability in the NP region as a whole was maintained over the 15-week culture duration (Fig. 2A); however, a significant reduction in cell number in the center region of the NP was observed between 5 and 10 weeks of culture. Mean percent viability of cells in the center of the NP remained high, however, stabilizing at 79.1% at 15 weeks. In contrast, for large DAPS, cell
Discussion
IVD tissue engineering is a promising treatment strategy for end-stage disc degeneration, with the potential to restore structure and function compared with standard spinal fusion techniques. While several groups have developed composite engineered IVDs composed of tissue engineered NP and AF analogs, the size of such constructs remain a fraction of the size of human cervical or lumbar IVDs [11], [13], [14], [15], [16], [17]. In this study, we fabricated and characterized tissue-engineered IVDs
Conclusions
In conclusion, we fabricated and evaluated the maturation of tissue-engineered IVDs at clinically relevant size scales during in vitro culture and following in vivo subcutaneous implantation. While medium DAPS outperformed large DAPS, those sized for the goat and human cervical disc space did functionally and compositionally mature with increasing culture duration. Future work will seek to optimize the properties of these large size DAPS in vitro, as well as to evaluate the performance of both
Acknowledgements
This work was supported by the Department of Veterans Affairs (IK1 RX002445, IK2 RX001476 & I01 RX002274) and the Penn Center for Musculoskeletal Disorders (National Institutes of Health P30 AR069619). The authors would like to acknowledge Debra Pawlowski and Jeffrey House from the Corporal Michael J. Crescenz VA Medical Center Animal Research Facility for their assistance with the animal studies.
Disclosures
The authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this manuscript.
Role of the Funding Source
The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Veteran’s Affairs. No funding source had a role in the study design, collection, analysis and interpretation of data, writing of the manuscript, or the decision to submit the manuscript for publication.
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