In this study, we report the successful production of a biphasic cartilaginous tissue construct, using a novel HANFS biomaterial amalgam seeded with adult human MSCs. The biphasic architecture and ECM distribution and composition of the constructs resemble those of native IVD, suggesting that these constructs have the potential to develop and mature into IVD-like tissues. While cell-based tissue engineering has been considered a promising approach to tissue repair and regeneration, applications to IVD have been limited. While other investigators have engineered portions of the IVD,34
this is the first construct to be developed as a complete unit, possessing both AF and NP regions. Since the IVD fails as a unit, an ideal biological solution must address the entire disc, not just one component. Furthermore, the fundamental challenge for partial IVD constructs has been their inability to restore the complete IVD as a functionally integrated unit.34,35
In this study, we have demonstrated in vitro
that MSCs loaded on a HANFS scaffold can produce a bioengineered cartilaginous tissue that exhibits the distinct anatomical architecture and biosynthetic features of the native human IVD.
While multiple biological approaches have been considered for the treatment of DDD, including biologics- and gene-based therapies, we will focus our discussion on tissue-engineered solutions and their comparison to current TDR options.36–38
The current tissue engineering approaches for IVD replacement primarily focus on regenerating a specific component of the IVD.39–43
Specifically, the NP was the first and continues to be the most frequent target for cell-based tissue-engineered solutions for IVD pathology.44,45
The aim of the approach reported here is the introduction of in vitro
–cultured IVD using an MSC-based tissue engineering approach to restore function of the degenerated IVDs. Sato et al.
have developed a true tissue-engineered construct to regenerate injured portions of the IVD, in that it is composed of allogeneic AF cells loaded on a three-dimensional collagen scaffold, rather than simply injecting a slurry of cultured cells.46
The biocompatibility and successful integration of this experimental construct provide optimism for the more challenging endeavor presented here to regenerate an entire IVD, rather than restoring regions resected or lost during treatment of herniated NP. Meisel et al.
have taken cell-based therapies to the next step using autologous disc chondrocyte transplantation (ADCT), reporting results at 2-year follow-up in the first 28 patients (12 received ADCT) enrolled in an unblinded randomized (no control) multi-center clinical trial (EuroDisc Study).44
This study has suggested that transplanted ADCTs remain viable and restore proteoglycan and collagen type II production, thus correlating to the findings from their previous canine model.47
While the results of this clinical study may not be dramatic, the fact that cell-based IVD therapies are being clinically tested shows promise for future cell-based therapies.
Bioengineered disc replacements should outperform traditional metal and synthetic devices currently used in spine surgery. Unlike metal and synthetic polymer devices, a bioengineered replacement disc will not release exogenous wear particles over the life of the implant. Although newer TDRs have adopted a metal-on-metal design with significantly lower wear rates than those for metal-on-polyethylene prostheses, the long-term biological effects, both local and systemic, of metal ions created when this type of prosthesis wears are still unknown. Additionally, metal ions from spinal implants, while to a significantly lower degree, have been demonstrated to incite wear-induced osteolysis.48,49
In contrast, bioengineered IVDs have the potential for minimal to no antigenicity and, most importantly, can biointegrate. Thus, unlike metal prosthesis that begin to wear from the time they are implanted, a biointegrated disc may hold the potential to maintain and improve its function with time.
Bioengineering an IVD requires the combination of an ideal cell source, biomaterial scaffold, and enabling biological signals. As noted above, previous IVD tissue engineering studies have investigated the proliferative and regenerative potential of mature NP and AF cells.41
These cell types have the disadvantage of being scarce and difficult to safely harvest.34
The amount of harvestable NP cells is greatly reduced in degenerated IVDs, and harvesting requires injury to the annulus (needle puncture, typically). Most pragmatic of concerns is that autologous harvesting may not be financially and commercially sustainable. A very viable option is thus the use of MSCs. A number of studies have demonstrated the potential use of MSCs in IVD tissue engineering with promising results.50–53
MSCs may provide a more ideal cell source for the regeneration of the two distinct regions of the IVD for the following reasons: (1) they are easily accessible for harvest, with bone marrow being a frequently used source; (2) they possess extensive self-renewal and expansion capability; (3) they possess little to no immunogenicity; and (4) most importantly, as demonstrated in this study, they are capable of differentiating into cells phenotypically and biosynthetically similar to those found in AF and NP regions of the IVD. These characteristics make MSCs well suited for cell-based tissue engineering of an IVD.
In this study, the MSCs were seeded into a novel HANFS construct; the cells proliferated, differentiated, and produced a proteoglycan-rich ECM with a protein expression profile similar to that of a native IVD. While there are many biocompatible polymers used in IVD tissue engineering,46,54,55
we have found that electro-spun PLLA nanofibers, with the unique characteristic of nanofiber size that exponentially increases the surface area of the scaffold, allow for efficient cell loading and generation of an environment highly conducive to chondrogenesis. The efficient production of a proteoglycan-rich matrix within the NFS is critical for IVD tissue engineering, since diminished proteoglycan production and disc dehydration are central to DDD.
We performed RT-PCR for several genes that are thought to be important for IVD ECM composition. Full interpretation of the temporal expression of these genes listed is not possible at this time as there is no clear order that has been established in the developing IVD. Additionally, we have found that there is asymmetric loading of our construct that may result in differences in the quantity of ECM deposition in various regions of the disc. For example, while Col II is expected to be seen in both the AF and NP of a native disc, its concentration in the NP should be greater. Our construct initially demonstrates higher Col II expression in the AF, but progressive increases in NP and AF are seen throughout the culture period and equivalent production in both regions is seen by 28 days. We are currently investigating new cell loading techniques that may improve the distribution of cells and associated ECM production.
The biphasic HANFS scaffold and IVD growth medium directed region-specific differentiation of MSCs into cells with phenotypes and biosynthetic activities resembling those of the two distinct regions in the native IVD. Our preliminary studies reported here thus demonstrate the potential for this specific construct to be a prototype for further research into the creation of an ideal tissue-engineered IVD. Future studies will evaluate the mechanical properties of the MSC-seeded HANFS constructs and to optimize the culture conditions. It is likely that exposure of the constructs to mechanical stimulation during culture will improve ECM production, metabolism, and the mechanical characteristics of the tissue-engineered IVD.56,57
In addition, a number of growth factors have been shown to play a role in IVD development, maintenance, and repair,58–61
and the importance of these growth factors in IVD tissue engineering will be studied to optimize culture conditions and construct function.