Stem Cells in Preclinical Spine Studies Brian C

The Spine Journal 14 (2014) 542–551

Review Article Stem cells in preclinical spine studies Brian C. Werner, MD, Xudong Li, MD, PhD, Francis H. Shen, MD* Department of Orthopaedic Surgery, University of Virginia, PO Box 800159, Charlottesville, VA 22908-0159 USA Received 21 January 2013; revised 5 July 2013; accepted 23 August 2013

Abstract BACKGROUND CONTEXT: The recent identiﬁcation and characterization of mesenchymal stem cells have introduced a shift in the research focus for future technologies in spinal surgery to achieve spinal fusion and treat degenerative disc disease. Current and past techniques use allograft to replace diseased tissue or rely on host responses to recruit necessary cellular progenitors. Adult stem cells display long-term proliferation, efﬁcient self-renewal, and multipotent differentiation. PURPOSE: This review will focus on two important applications of stem cells in spinal surgery: spine fusion and the management of degenerative disc disease. STUDY DESIGN: Review of the literature. METHODS: Relevant preclinical literature regarding stem cell sources, growth factors, scaffolds, and animal models for both osteogenesis and chondrogenesis will be reviewed, with an emphasis on those studies that focus on spine applications of these technologies. RESULTS: In both osteogenesis and chondrogenesis, adult stem cells derived from bone marrow or adipose show promise in preclinical studies. Various growth factors and scaffolds have also been shown to enhance the properties and eventual clinical potential of these cells. Although its utility in clinical applications has yet to be proven, gene therapy has also been shown to hold promise in preclinical studies. CONCLUSIONS: The future of spine surgery is constantly evolving, and the recent advancements in stem cell–based technologies for both spine fusion and the treatment of degenerative disc disease is promising and indicative that stem cells will undoubtedly play a major role clinically. It is likely that these stem cells, growth factors, and scaffolds will play a critical role in the future for replacing diseased tissue in disease processes such as degenerative disc disease and in enhancing host tissue to achieve more reliable spine fusion. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Stem cells; Spine fusion; Degenerative disc disease; Scaffold; Growth factors; Intervertebral disc; Bone morphogenic protein; Adipose derived stem cells

Introduction focus for future technologies in spinal surgery to achieve spinal fusion and treat degenerative disc disease. Current The recent identification and characterization of mesen- and past techniques use allograft to replace diseased tissue chymal stem cells has introduced a shift in the research or rely on host responses to recruit necessary cellular progenitors. Adult stem cells display long-term proliferation, FDA device/drug status: Investigational (Adult Mesenchymal Stem efficient self-renewal, and multipotent differentiation. They Cells; Growth Factors [GDF-5, others]). also can be genetically modified to secrete growth factors BCW: XL: Author disclosures: Nothing to disclose. Nothing to dis- important to tissue healing, thereby functioning as implant- close. FHS: Royalties: Elsevier Publishing (B); Consulting: Synthes Spine (B), DePuy Spine (B); Speaking and/or Teaching Arrangements: DePuy able, long-lasting reservoirs for these molecules. Spine (B); Board of Directors: MTF (B, Paid directly to institution); Fel- In spinal surgery, stem cells have great potential applica- lowship Support: AO (D, Paid directly to institution). bility in numerous areas. This review will focus on two im- The disclosure key can be found on the Table of Contents and at www. portant applications: spine fusion and the management of TheSpineJournalOnline.com. degenerative disc disease. The treatment of spinal cord in- * Corresponding author. Department of Orthopaedic Surgery, Univer- sity of Virginia, PO Box 800159, Charlottesville, VA 22908-0159, USA. jury using stem cells is still experimental and outside the Tel.: (434) 243-0291; fax: (434) 243-0242. scope of this review. Relevant preclinical literature regard- E-mail address: [email protected] (F.H. Shen) ing stem cell sources, growth factors, scaffolds, and animal

1529-9430/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.spinee.2013.08.031 B.C. Werner et al. / The Spine Journal 14 (2014) 542–551 543 models for both osteogenesis and chondrogenesis will be to the use of bone marrow clinically include the painful har- reviewed, with an emphasis on those studies that focus on vest as already described and its relatively limited supply re- spine applications of these technologies. quiring lengthy culture expansion. Mesenchymal stem cells from bone marrow were demonstrated to achieve successful spinal fusion more than 10 years Osteogenesis/spine fusion ago. In 2001, Cui et al. [29] found that cloned osteoprogeni- tor cells from bone marrow produced a quicker and more ro- Overview bust posterolateral fusion in an athymic rat model compared Spinal fusion has become a universal procedure since its with mixed marrow cells. Over the ensuing decade, numer- introduction a century ago, with more than 350,000 spinal ous other studies reinforced these findings. Peterson et al. fusions performed each year in the United States represent- [30] demonstrated that bone marrow MSCs transfected with ing a greater than 200% increase over the past decade bone morphogenic protein-2 (BMP-2) ex vivo successful in- [1–5]. Despite significant advances in surgical techniques duced posterolateral spinal fusion in an athymic rat model. and instrumentation, up to 40% of fusion surgeries result In a recent study directly comparing a bone marrow MSC al- in pseudarthrosis, causing significant morbidity and often lograft with autograft, Gupta et al. [31] demonstrated similar a need for reoperation [6–9]. Although failure of spinal fu- fusion rates between the two groups in an ovine posterolat- sions is multifactorial, osteoporosis, poor bone biology, eral lumbar spine fusion model. Similarly, Miyazaki, et al. smoking, diabetes, and decreased autograft cellularity in [32] compared the effectiveness of human MSCs from bone older patient populations all likely contribute [10–13]. So- marrow with adipose-derived MSCs in a posterolateral fu- phisticated techniques to improve fusion rates including sion rat model. The authors found similar fusion rates with modern internal fixation have greatly reduced the incidence both types of MSCs when transfected with BMP-2. of symptomatic pseudarthrosis, but a 10% to 15% incidence Adipose tissue provides an additional well-researched is still reported in recent literature [14–16]. Additionally, and promising source of MSCs for spine applications although autograft remains the gold standard for spinal fu- (Fig. 1). Adipose tissue is easily procured from patients sions, it has limited availability, often low cellularity, and and large numbers of MSCs can be obtained from relatively is associated with up to a 30% donor site morbidity rate small amounts of adipose tissue, in contrast to bone marrow [17–22]. These issues have led spine surgeons and re- [33]. Shen et al. confirmed the osteoblastic differentiation searchers to investigate alternatives for achieving fusion of rat adipose–derived MSCs when cultured with growth and further reducing the pseudarthrosis rate, including the and differentiation factor-5 (GDF-5). [33,34] Subsequent use of mesenchymal stem cells. studies have yielded similar promising results. Hsu et al. For a successful spinal fusion to occur, several vital el- [35] found that MSCs from adipose demonstrate potential ements are necessary. They include the presence of the as cellular delivery vehicles for recombinant proteins such bone-forming cell or its precursor, the appropriate biologi- as BMP-2 using an athymic rat posterolateral fusion model. cal signals directing bone synthesis, and a biocompatible Similarly, Miyazaki et al. [32] demonstrated that BMP-2 scaffold on which the process can occur. The most critical producing human adipose–derived MSCs, created using ad- of these components is the bone forming cell (osteoblast) or enoviral gene transfer, induced spinal fusion in athymic its precursor, the mesenchymal stem cell (MSC), both of rats, demonstrating that adipose-derived MSCs are osteo- which possess the ability to form bone [23]. genic and enhance spinal fusion as effectively as bone marrow MSCs. Gimble et al. [36] reported that syngeneic and In vitro: cells, growth factors, and scaffolds allogeneic rat adipose–derived MSCs on a tricalcium phosphate and collagen I scaffold accelerated spinal fusion in Cells a rat model. Although historical research investigated the use of em- bryonic stem cells, restrictions and various moral and ethi- Growth factors cal obligations on their use have led to mesenchymal stem The complex process of bone formation can be affected cells as the focus of most stem cell research for spinal fu- by a variety of biologic signals, including mechanical loads sion. Mesenchymal stem cells have generated considerable and electromagnetic and chemical factors. The effective- interest because of their ability to self-renew and multipo- ness of stem cells in promoting spinal fusion is heavily tential characteristics. Furthermore, MSCs have been iden- dependent on osteoinductive factors, specifically growth tified in a variety of tissues including bone marrow, muscle, factors that enhance the osteogenic capability of osteopro- periosteum, and adipose tissue [16,24–28]. Most recent and genitors such as MSCs [23]. Although many growth factors promising preclinical studies have focused on MSCs from have been investigated for their role in the osteogenic bone marrow and adipose tissue. differentiation of MSCs, only a select few have been re- Bone marrow is well established as a source of MSCs in searched for their role in inducing spinal fusion with MSCs laboratory animals and humans, with confirmed differentia- or through gene therapy with MSCs. Table 1 provides tion into osteoblasts both in vitro and in vivo. Disadvantages a summary of these growth factors. 544 B.C. Werner et al. / The Spine Journal 14 (2014) 542–551

Fig. 1. Human adipose derived stem cells (Left) undergo effective osteogenic differentiation (Right) as demonstrated by alizarin red staining in this in vitro experiment.

Despite recent controversy regarding negative clinical cells in a rat model was significantly more effective in induc- side effects, BMP-2 has been the subject of considerable re- ing osteoblastic differentiation and spine fusion than indi- search for use as an osteoinductive factor with MSCs for vidual BMP gene transfer. Numerous studies have also spinal fusion. As early as 2003, researchers realized the po- been conducted successfully using BMP-7 as an inductive tential harm in the large doses of BMP required to induce agent without the use of stem cells, which is not the subject a spinal fusion in humans, which suggested that delivery of this review [42,43]. of such osteoinductive proteins could be improved. Wang As an alternative to BMP-2, GDF-5 has also been stud- et al. [37] used ex vivo adenoviral gene transfer to create ied recently for its use as an osteoinductive agent for stem BMP-2–producing bone marrow MSCs, which successfully cells in spine fusion. Shen et al. [34] confirmed that produced an intertransverse fusion in a rat spine model. Pe- adipose-derived stem cells, when treated with GDF-5, are terson et al. [30] reported similar results in their experi- capable of adhering to a bioengineered scaffold while re- ments in which human bone marrow MSCs were infected maining viable and demonstrating the ability to migrate, with a BMP-2 containing adenovirus, yielding sufficient proliferate, and subsequently undergo osteogenic differenti- bone to fuse the lumbar spine in a rat model. Similar results ation. Similarly, Zeng and coauthors [33,44] proved in two with ex vivo gene therapy to overproduce BMP-2 were re- studies that GDF-5 promotes the differentiation of rat adi- ported by Miyazaki et al. [32], Hsu et al. [35], and Sheyn pose–derived stromal cells into osteogenic lineages. et al. [38] Fu et al. [39] also recognized the need to reduce the dose of BMP-2 necessary to achieve fusion. These au- Scaffolds thors found that combining low doses of BMP-2 with bone The importance of an effective osteoconductive scaffold marrow MSCs achieved this goal in a rabbit fusion model. should not be underestimated. Despite the fact that scaf- BMP-7, also known as osteogenic protein-1, is another folds do not contain osteogenic cells or intrinsic biologic clinically available growth factor that has undergone consid- signals, an appropriately designed scaffold can provide erable preclinical evaluation with stem cells. Hidaka et al. the framework for the migration and attachment of osteo- [40] assessed the enhancement of spine fusion using genic cells and a suitable environment for the synthesis bone marrow stem cells modified by an adenovirus vector of extracellular matrix [23]. Allograft is currently the most encoding BMP-7 seeded onto an allograft scaffold in a rat commonly used osteoconductive scaffold and is typically model. The authors found that the addition of adenovirus combined with autograft to provide the necessary osteo- BMP-7–modified marrow stem cells significantly enhanced genic osteoinductive and osteogenic factors and cells. Un- allograft spinal fusion. Zhu et al. [41] found that combined fortunately, the extensive preparation and treatment that gene transfer of BMP-2 and BMP-7 using bone marrow stem allograft is subject to leads to diminished biologic and mechanical properties as well as increased expense, which has Table 1 led researchers to investigate other scaffolds for preclinical Growth factors used with mesenchymal stem cells for spine fusion studies of stem cells for spinal fusion. Growth factor MSC type Model References A scaffold for stem cells should maximize the osteoin- BMP-2 Adipose, BM Rat, rabbit [30,32,35,37–39] ductive and osteogenic effects of cells delivered to the site BMP-6 Adipose, BM Mouse, rabbit [123] of interest, retain growth factors at that site for optimal time BMP-7 BM Rat [40–43] of release, function as an osteoconductive scaffold for bone BMP-9 BM Rat [124] GDF-5 Adipose In vitro [34,44] in-growth with appropriate sized pores for cellular and vas- FGF BM Rabbit [125] cular passage, not compete with or limit bone formation, BM, bone marrow; BMP, bone morphogenic protein; FGF, fibroblast and limit inflammatory response by biocompatibility [45]. growth factor; GDF, growth and differentiation factor; MSC, mesenchymal Important scaffold properties to consider include porosity, stem cell. pore size, geometry, and material. Numerous materials B.C. Werner et al. / The Spine Journal 14 (2014) 542–551 545

Fig. 2. Scaffolds are an integral element in the development of cell-based strategies for spine fusion and disc regeneration. Polylactic-co-glycolic acid (PLGA) microspheres (Left) and a PLGA nanoﬁber scaffold (Right) are two examples used in spine applications. are currently available for scaffolds, including natural or viable scaffold for osteogenic differentiation and delivery synthetic polymers, bioactive materials, and osteophilic of stem cells in preclinical studies [50–52]. materials such as ceramics or composites [46]. Various biomaterials also have served as suitable scaf- Type I collagen is a natural polymer and represents the folds in preclinical studies, the most popular of which are simplest of scaffolds meeting all described criteria. It has calcium phosphate derivatives including hydroxyapatite a long history of use in surgical applications because of its (HA) and beta tricalcium phosphate (b-TCP). Both natural biocompatibility, ease of degradation, and known interac- and synthetic forms of calcium phosphate have been devel- tion with other molecules including proteins. Gabbay et al. oped as materials for bone repair and augmentation. They [47] found a progressively stimulatory effect on adipose- closely resemble the mineral composition, properties, and derived stem cells with regard to osteogenesis when cultured microarchitecture of human cancellous bone and thus have in a three-dimensional collagen I gel compared with a two- a high afﬁnity for binding proteins. Commercially available dimensional monolayer. Hsu et al. [35] successfully used HA is brittle, carries minimal mechanical strength, and is a type I collagen matrix for their study investigating spinal slowly resorbed in vivo, but has still been the subject of fusion in an athymic rat model with BMP-2–producing several preclinical studies involving stem cells. Chistolini adipose-derived stem cells. Similarly, Miyazaki and coau- et al. [53] studied porous HA scaffolds as carriers of bone thors [32] found that both human adipose–derived and bone marrow MSCs. The authors found that cell-loaded implants marrow–derived mesenchymal stem cells on a type I colla- were stronger than cell-free implants with notable bone for- gen sponge induced spinal fusion. Other natural polymers mation. More recently, Minamide et al. [54] and Seo et al. including matrigel [29] and hydrogels [48] have been inves- [55] both reported that bone marrow MSCs on HA scaf- tigated. Although these polymers are suitable for carriers of folds with and without growth factors induced posterolat- cells and growth factors, they do not possess the mechanical eral fusion in rat and rabbit models. strength of stiffer polymers and thus have been used more Beta tricalcium phosphate is another calcium phosphate for intervertebral disc applications. biomaterial that serves as a purely osteoconductive scaffold. Synthetic polymers including polylactic-co-glycolic acid It has greater solubility than HA and is rapidly resorbed. The (PLGA), its derivatives, chitosan, and others have been in- resorption rate of b-TCP closely matches the course of nor- vestigated in preclinical studies as well (Fig. 2). PLGA is mal cancellous bone remodeling, making it an ideal candi- osteoconductive, is able to deliver osteoinductive growth date for stem cell–based scaffolds. It has recently been factors, and has established biocompatibility. Lee et al. studied in rat and ovine models in preclinical studies for [49] and Shen et al. [34] both successfully used PLGA in the delivery of both bone marrow– and adipose-derived their studies. Similarly, numerous studies in the past 5 years MSCs for posterolateral spine fusion with uniformly excel- have established the use of chitosan as an effective and lent results comparable to that of autograft [31,36,56].

Table 2 Animal models of mesenchymal stem cells for spinal fusion Animal model Mesenchymal stem cell type Type of fusion References Rat Bone marrow Posterolateral lumbar spine [29] Rabbit Bone marrow Posterolateral and interbody lumbar spine [39,54,123,125] Sheep Bone marrow Posterolateral lumbar spine [31] Rhesus monkey Bone marrow Interbody lumbar spine [126] 546 B.C. Werner et al. / The Spine Journal 14 (2014) 542–551

Table 3 Animal models of gene therapy with mesenchymal stem cell for spinal fusion Animal model Mesenchymal stem cell type Type of fusion References Mouse Bone marrow, adipose Posterolateral lumbar spine [38] Rat Bone marrow, adipose Posterolateral lumbar spine [30,32,35,37,40,41,124] Rabbit Bone marrow Posterolateral and interbody, lumbar spine [127] Pig Bone marrow Interbody lumbar spine [128]

In vivo: animal models The relationship between the fluid-like NP and the structural lattice of the AF provides the biomechanical properties Although in vitro studies lay the important foundation for necessary for spinal stability [65]. Any disturbance of the the use of stem cells to achieve spine fusion, the transition to NP or AF that compromises this balance leads to disc degen- in vivo studies is a key step in the eventual translation of eration. Degeneration is marked by a loss of disc height and stem cell allografts to clinical use. Various animal models blurring of the gross morphologic characteristics of the AF have been used to investigate the application of mesenchy- and NP, resulting from the inability of the disc to regenerate, mal stem cells in achieving spine fusion. Tables 2 and 3 sum- avascularity, and numerous biochemical changes within the marize various animal models for spine fusion using MSCs disc due to aging [66,67]. and gene therapy using MSCs, respectively. Alterations in the cell population of the IVD have been particularly implicated in the pathogenesis of IVD degeneration [68]. Early disc degeneration is characterized by the Chondrogenesis/intervertebral disc degeneration loss of notochordal cells, which play a key role in maintain- Overview ing the integrity of the disc and matrix stabilization [69]. Loss of chondrogenic NP cells and their replacement by Intervertebral disc (IVD) degeneration is a complex pro- fibroblast-like cells has also been implicated in the later cess that is a result of morphologic and molecular changes stages of IVD degeneration [70]. within the disc itself compounded by progressive structural Although considerable research has been dedicated to failure and instability of the vertebral motion segments that strategies that boost the ability of native disc cells to syn- surround the affected disc [57,58]. The resultant clinical thesize and maintain the ECM, the introduction of exoge- manifestations of degenerative disc disease have a huge im- nous cells such as stem cells to supplement or replenish pact on society and the economy, with estimated direct and the declining disc cell population is gaining popularity as indirect costs of $50 to $90 billion per year in the United a potential treatment for IVD degeneration [68]. States alone [57,59]. Traditional management of low back pain, which is often In vitro and in vivo: cells, growth factors, scaffolds, attributed to IVD degeneration, has focused on conservative and animal models options including lifestyle changes, physical therapy, pain medication, and rehabilitation as well as surgical options Cells that focus on eliminating motion through disc arthroplasty Similar to bone regeneration, preclinical studies of stem or arthrodesis [60,61]. The clinical results of these proce- cells for the IVD focus on MSCs from a variety of sources, dures are often suboptimal. Both nonsurgical and surgical including bone marrow [68,71–74], adipose tissue [75–80], therapies do not deal with the inherent loss of functional muscle tissue [81], synovium [82–84], and even the NP it- native disc tissue and therefore fail to regenerate or cure self (Table 4). the degenerated, painful disc tissue that is the essence of Bone marrow is an excellent source of mesenchymal the disease process [62]. stem cells for most orthopedic applications including chon- The intervertebral disc is a two-part structure composed drogenic differentiation for the diseased intervertebral disc. of the tough annulus fibrosis (AF) on the periphery and Yamamoto et al. [85] investigated the use of bone marrow– the amorphous nucleus pulposus (NP) as the central core derived stromal cells for upregulation of the viability of [60,63]. The cellular content of the AF and NP differ signif- native nucleus pulposus cells in a direct contact coculture icantly. Cells in the AF are fibroblast-like with robust col- system. The authors found significant upregulation of cell lagen fibrils. The extracellular matrix (ECM) in the AF proliferation, DNA synthesis and proteoglycan synthesis is predominantly composed of type I collagen and con- in the NP cells which had direct cell-to-cell contact with tains relatively low amounts of proteoglycan and water bone marrow MSCs. Stoyanov et al. [86] compared stan- [60,64]. Within the NP, cells become more rounded with dard chondrogenic differentiation protocols using trans- a chondrocyte-like shape. ECM in the NP is an amorphous forming growth factor (TGF)-b with a novel technique arrangement of type II collagen. It has a high proteoglycan utilizing hypoxia, GDF-5 and coculture with NP cells. concentration with inverse proportions of collagen and pro- The authors found that both hypoxia and GDF-5 were suit- teoglycan compared with the AF [61,64]. able for directing bone marrow MSCs toward an B.C. Werner et al. / The Spine Journal 14 (2014) 542–551 547 Table 4 phenotype. Tapp et al. [78] revealed that either treatment of Mesenchymal stem cell sources for regeneration of the intervertebral disc TGF-b or co-culture with human disc cells could signifi- Cell source References cantly stimulate expression of proteoglycan and type I colla- Bone marrow [68,71–74] gen in 3D-cultured sand rat ADSCs. Gaetani et al. [75] Adipose tissue [75–80] presented data indicating that co-culture of human NP and Muscle tissue [81] ASCs improved the quality of the invitro reconstructed tissue Synovium [82–84] in term of matrix production and 3D cell organization [62]. Nucleus pulposus [62] Other stem cell sources have been investigated for IVD repair. Vadala et al. [81] investigated the use of adult mus- intervertebral disc-like phenotype. Allon et al. [72] ex- cle tissue as a stem cell source. Others have recently inves- tended these in vitro findings of successful co-culture to tigated the use of synovial cells as a stem cell source for a rat in vivo model. Utilizing a novel bilaminar co-culture IVD tissue engineering. [82–84] In these in vitro and pellet of bone marrow MSCs and NP cells, the authors in vivo studies, the authors found promise with the use of demonstrated prevention of disc degeneration in vivo. synovial stem cells for their inductive capabilities and their Similar to bone applications of MSCs, adipose derived ability to differentiate to a chondrogenic phenotype. Recent stem cells have gained significant attention in recent years, literature has also revealed the potential for human annulus given the ease of procurement and larger number of mesen- fibrosis cells [90] and nucleus pulposus cells [91] to serve chymal stem cells that can be obtained compared to as mesenchymal stem cells. bone marrow or other sources (Fig. 3). Liang et al. [87] recently conducted an in vitro study of both adolescent and adult adipose-derived stem cells and found that despite Growth factors the harsh microenvironment of the IVD, adipose derived Disc cell metabolism is modulated by a variety of growth stem cells are a viable option for IVD regeneration. Choi factors acting in both paracrine and autocrine roles [92,93]. and co-authors [80] investigated the use of human adipose These factors function to increase the synthesis of extracel- derived stem cells co-cultured with human nucleus pulpo- lular matrix components, block their breakdown, or a com- sus cells using a porous membrane. The authors found bination of these roles. Growth factors can be applied in the most ideal culture conditions for chondrogenic differen- IVD tissue engineering via delivery of the ‘‘naked’’ or tiation of the ASCs to be ASC pellets cultured with normal ‘‘embedded’’ proteins as well as prolonged supplement by NP cells. ASCs cultured in monolayer and the use of hu- cell-based gene therapy [62,92]. Numerous growth factors man NP cells from degenerative discs had inferior results. have been used for IVD applications (Table 5), including Yang et al. [88] utilized genetic engineering to induce ec- transforming growth factor-b [94,95], insulin-like growth topic expression of Sox-9 in human ASCs. The authors found factor-1 [96,97], GDF-5 [98–101], platelet-derived growth improved chondrogenic differentiation in the engineered factor [96], osteogenic protein-1/BMP-7 [102–104], ASCs, indicating potential application in the treatment of de- BMP-2 and 12 [105–107], and fibroblast growth factor-2 generative disc disease. Lu et al. [89] reported that co-culture [108]. of human ASCs and NP cells in a micro- mass culture In a 2005 study, Steck, et al. [109] demonstrated the resulted in differentiation of ASCs into an NP cell-like ability of adult bone marrow MSCs to differentiate toward

Fig. 3. Chondrogenic differentiation of human adipose–derived stem cells for disc regeneration. Cells cultured in basal medium demonstrated no positive staining (A). Safranin-O staining indicated deposition of proteoglycan in cells cultured in chondrogenic medium (B). Supplementing the chondrogenic medium with transforming growth factor-b (C), and increasing concentrations of growth and differentiation factor-5 (D–F) augmented chondrogenic differentiation. 548 B.C. Werner et al. / The Spine Journal 14 (2014) 542–551 Table 5 stiffness can influence cell function, proliferation and orien- Growth factors used with mesenchymal stem cells for regeneration of the tation, all of which can have profound effects on the cell intervertebral disc [111]. Numerous biomaterials have been used for scaffolds, Cell source References including PLGA, poly-d-lactide, chitosan, alginate, fibrin, Transforming growth factor-b [94,95] collagens, gels, calcium polyphosphate, demineralized IGF-1 [96,97] bone matrix, and many others [61,112–122]. Growth and differentiation factor-5 [98–101] Platelet-derived growth factor [96] OP-1/BMP-7 [102–104] BMP-2, BMP-12 [105–107] Conclusions FGF-2 [108] The future of spine surgery is constantly evolving, and BMP, bone morphogenic protein; FGF, fibroblast growth factor; IGF, insulin-like growth factor-1; OP, osteogenic protein. the recent advancements in stem cell–based technologies for both spine fusion and the treatment of degenerative disc disease are both promising and indicative that stem cells the molecular phenotype of human IVD cells. Using TGF- will undoubtedly play a major role clinically. In both osteo- b mediated generation, the authors found that MSC spher- genesis and chondrogenesis, adult stem cells derived from oids preferentially differentiated toward an IVD phenotype bone marrow or adipose show promise in preclinical stud- rather than articular cartilage, making them an attractive ies. Various growth factors and scaffolds have also been source to obtain IVD-like cells. shown to enhance the properties and eventual clinical po- Yang et al. [110] strengthened the potential role of TGF- tential of these cells. Although its utility in clinical applica- b and MSCs as therapy for IVD degeneration. In their study, tions has yet to be proven, gene therapy has also been bone marrow MSCs with TGF-b were transplanted into de- shown to hold promise in preclinical studies. It is likely that generative discs of rabbits. The authors found that MSCs can these stem cells, growth factors, and scaffolds will play slow the rate at which the degenerative process occurs, pos- a critical role in the future for replacing diseased tissue in sibly due to the inhibition of apoptosis by the MSCs. disease processes such as degenerative disc disease and in Stoyanov and co-authors [86] utilized bone marrow enhancing host tissue to achieve more reliable spine fusion. MSCs and compared standard chondrogenic differentiation protocols using TGF-b with hypoxia, GDF-5 and co-culture References with bovine NP cells. Their results suggest that hypoxia and GDF-5 may be suitable for directing MSCs toward an IVD- [1] Deyo RA, Gray DT, Kreuter W, et al. United states trends in lumbar like phenotype. fusion surgery for degenerative conditions. Spine 2005;30:1441–5; discussion 1446–7. McCanless et al. [71] investigated the effects of BMP-2 [2] Gray DT, Deyo RA, Kreuter W, et al. 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