Injectable Biomaterials for Cell Delivery

Although NP tissue engineered scaffolds grown in vitro before being implanted into the disc space are advantageous in that cells would be able to generate appropriate ECM in a controlled, favorable environment, implanting the scaffold would cause significant damage to the AF. Cell delivery to the disc space via injection would limit damage to the adjacent AF tissue; however, calcification of the cartilaginous end plates severely limits diffusion of glucose and oxygen into the disc, creating a hostile extracellular environment not well suited to maintain cell viability or promote matrix synthesis. Additionally, a recent study of mesenchymal stem cells (MSCs) delivered to rabbit IVDs raised concern that delivered cells can migrate out of the nucleus and induce

undesirable osteophyte formation [129]. Therefore, it is likely that functional regeneration of NP tissue via cells delivered to the disc space will require an appropriate biomaterial carrier to retain cells in the IVD space and to promote NP cell survival and phenotype.

Cell delivery to the IVD has been performed in animal models for IVD regeneration both with and without the use of a biomaterial carrier, and shown that reinsertion of autologous disc cells or stem cells delays degeneration in experimental models of degeneration [111, 130-134]. Ganey and colleagues studied a canine model, in which autologous disc cells were delivered without any carrier matrix to injured discs, and showed significantly better maintenance of disc height and structure in dogs receiving transplants as compared to controls [130]. Meisel and colleagues have brought autologous disc cell transplantation to clinical study, comparing safety and efficacy of cell transplantation plus discectomy with discectomy alone [135]. Although these studies have shown some maintenance of disc height and reduction in patient pain compared to discectomy alone, it is thought that a biomaterial carrier will improve cell retention and survival in the disc space, and may lead to the generation of new matrix. A study by Bertram and colleagues provided strong evidence for this, in which luciferase expressing cells were injected into a nucleotomized rabbit disc space either in media or within a two component fibrin matrix that polymerizes upon injection in to the disc space [136]. Results showed that approximately 30-50% of the cells injected within the fibrin biomaterial remained 30 minutes after injection, whereas media injected cells were rapidly lost from

the disc space. While the fibrin carrier improved efficiency of cell delivery to the disc space, it may not be the ideal biomaterial for generating an NP-like matrix. Crevensten and colleagues isolated bone marrow derived MSCs, stained the cells with a CM-DiI membrane stain and delivered them into rat coccygeal discs with a hyaluronan carrier [111]. Results showed the injected cells survived and proliferated, and could be detected within the disc space over the 28-day period; however, there was a marked decrease in cell number over the first 7 days in culture. In a more recent study by Sakai and colleagues [109], autologous MSCs were transplanted from bone marrow into a rabbit model of disc degeneration within an atelocollagen carrier. Results showed that 24 weeks post MSC-transplantation, degenerated discs that received MSCs regained a disc height of approximately 91%, as compared to about 67% for the sham operated group.

Additionally, X-gal staining for LacZ expressing transplanted MSCs showed increased staining at 24 weeks as compared to 2 weeks, suggesting that injected cells survived and proliferation within the disc space. Since this study did not include a control group of cells without biomaterial, it is not clear exactly what therapeutic role was played by the atelocollagen carrier; however, these results combined with previous findings [101]

suggests that an atelocollagen carrier may help promote an NP-like phenotype.

For detecting cells within the region of interest at various time points, much of the work using animal models of disc degeneration has relied on histological techniques subsequent to animal sacrifice at specific time-points, which precludes in vivo assessment

and longitudinal tracking of cell therapy. To address this, Saldanha and colleagues labeled MSCs with iron oxide particles, delivered the cells within a fibrin gel to the rat IVD, and demonstrated initial detection of the transplanted cell population via magnetic resonance imaging (MRI) [123]. Although in vivo longitudinal cell tracking was not performed, this study introduced a new technique that may be useful for evaluating biomaterial carriers’

ability to promote cell survival and retention at the injection site. More recently, luciferase expressing porcine MSCs were delivered within a fibrin matrix to the disc space of minipigs following partial nucleotomy to assess the persistence and activity of delivered cells [137]. Results demonstrated that only 7% of the injected cells could be detected 3 days post injection, suggesting a need for improved biomaterial carriers and increasing focus on anulus reconstruction to reduce cell loss following delivery.

Towards developing improved injectable carriers, a number of groups have modified natural materials and created hybrid natural-synthetic materials to obtain greater control over material properties. Halloran and coworkers compared uncrosslinked atelocollagen to enzymatically crosslinked, atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan [108]. Bovine NP cells were cultured within the scaffolds in vitro for 7 days, with results showing that crosslinking did not reduce cell viability and improved proteoglycan retention within the scaffolds when compared to uncrosslinked atelocollagen. The authors did not assess the effects of crosslinking on gelation time, which is an important parameter to control in

order to prevent cells from leaking back out through the injection site following delivery.

An injectable biomaterial for NP regeneration was recently developed in which type II collagen was crosslinked with a 4-arm PEG derivative, enriched with hyaluronan and used to entrap adipose derived stem cells (ADSCs) [119]. Gelation occurred in less than 10 minutes under physiological conditions, and NP cell viability remained high (>80%) over 14 days in culture. In another recent study, Peroglio and colleagues developed injectable, thermoreversible hyaluronan-based hydrogels for NP cell encapsulation and showed that the materials gelled upon heating to approximately 30°C [114]. Short term in vitro studies demonstrated that scaffolds could induce human MSC differentiation towards the disc phenotype [115]. Although these studies represent steps towards the development of novel injectable biomaterials for cell delivery, additional in vitro studies are needed to further investigate cell phenotype and matrix production within these biomaterials, as well as in vivo studies to assess their ability to promote NP cell retention and survival in the disc space.