Use of FGF-2 and FGF-18 to direct bone marrow stromal stem cells to chondrogenic and osteogenic lineages

Aim: Intervertebral disc degeneration/low back pain is the number one global musculoskeletal condition in terms of disability and socioeconomic impact. Materials & methods Multipotent mesenchymal stem cells (MSCs) were cultured in micromass pellets ± FGF-2 or -18 up to 41 days, matrix components were immunolocalized and gene expression monitored by quantitative-reverse transcription PCR. Results: Chondrogenesis occurred earlier in FGF-18 than FGF-2 cultures. Lower COL2A1, COL10A1 and ACAN expression by day 41 indicated a downregulation in chondrocyte hypertrophy. MEF2c, ALPL, were upregulated; calcium, decorin and biglycan, and 4C3 and 7D4 chondroitin sulphate sulfation motifs were evident in FGF-18 but not FGF-2 pellets. Conclusion: FGF-2 and -18 preconditioned MSCs produced cell lineages which promoted chondrogenesis and osteogenesis and may be useful in the production of MSC lineages suitable for repair of cartilaginous tissue defects.

The capacity of mesenchymal stem cells (MSCs) for self-renewal and directed differentiation [1,2] makes them attractive candidates for a range of cell-based therapies [3][4][5], and considerable interest has centered around their use in regenerative medicine and in intervertebral disc (IVD) repair [6][7][8]. MSCs have been sourced from bone marrow [9,10], adipose tissue [11], synovium [12], olfactory [13] and fetal spinal tissues [14]. In the present study we utilized MSCs isolated from adult ovine bone marrow and stimulated these with FGF-2 and -18 to examine if we could direct the chondrogenic and osteogenic differentiation of the MSCs in vitro. FGF-2 upregulates Sox9 during cellular expansion of chondroblasts and early activation of chondrogenesis, and augments extracellular matrix (ECM) synthesis [15]. FGF-18 signaling through FGFR3 modulates the expression profiles of established chondrocytes during chondrogenesis, delaying hypertrophy but enhancing anabolic ECM gene expression during early chondrogenesis [16,17].
The FGF family currently has 23 members, which signal through four FGFRs that each occur as three alternatively spliced isoforms and are important in skeletogenesis [18]. FGFR1c, FGFR2c and FGFR3c are the isoforms used by mesenchymal cells in cartilage and bone development whereas epithelial cells in the ectoderm signal through FGFR2b. In early limb bud development prior to the establishment of the cartilaginous rudiment, FGFR1c is widely expressed, however, as the limb bud develops FGFR2c is expressed in the central mesenchymal condensation. Chondrocyte differentiation ensues as the limb bud develops into the cartilaginous rudiment along with expression of FGFR3c [19]. During joint development, chondroprogenitor cells express FGF-1, -2, -9 and -18 and utilize FGFR1c, FGFR2c, FGFR3c and perlecan to undertake cell signaling and initiate cell proliferation and ECM production, which enlarges the cartilaginous rudiment in vivo [18]. Studies with Baf-32 engineered cells expressing individual FGFR isoforms have demon strated FGF-2 and -18 utilize FGFR1c and FGFR3c to promote chondrogenesis in vitro [20,21]. Widespread FGF-2 expression occurs in human fetal spinal development at 12-20 weeks gestation, FGF-2 primes the discoprogenitor cells for chondrogenesis, which is a major driving force in early spinal development [22][23][24]. FGF-18 signals through FGFR3c within the cartilaginous vertebral rudiment promoting hypertrophy and establishment of the primary ossification center in the vertebral rudiments. These subsequently undergo ossification by a process similar to endo chondral ossification to form the spinal vertebrae [23].
Cartilaginous tissues such as articular cartilage (AC), knee-joint meniscus, IVD and tendon, all contain collagens and proteoglycans to variable degree as functional ECM components equipping these tissues with the ability to withstand tensional stresses or to act as weight-bearing structures located at strategic points in the axial skeleton. With aging, trauma and disease these ECM components are degraded by a family of 26 MMPs, ADAMTS and ADAMS metallo protease families [25]. This can compromise the normal biomechanical properties of these tissues and lead to secon dary changes in adjacent tissues in joint structures resulting in impaired articulation, diminished mobility, pain generation and a reduction in the quality of life. The IVD is a particularly important supportive structure in the axial skeleton. It is a composite tissue consisting of an outer annulus fibrosus containing annular lamellae rich in type I collagen, which provides tensile strength [24,26]. The annulus fibrosus also contains type II collagen in its inner regions. The central region of the IVD is the nucleus pul posus (NP). This region is rich in aggrecan, which forms massive ternary complexes with hyaluronan (HA) and link protein which are entrapped in the NP within a network of type II collagen fibers. The aggrecan-HA macroaggregates have impressive water regain properties equipping the NP and composite IVD with hydrodynamic weight-bearing properties. Thus the IVD has both the ability to withstand tension and compressive loading of the spine and its properties are coordinated with other components in the axial skeleton critical to the provision of a supportive framework. Articular cartilage (AC) is another important supportive weight-bearing tissue in human joints and also provides joint articulation. Type II collagen and aggrecan are significant functional ECM components in AC [27]. The semi-lunar fibrocartilaginous menisci of the knee joint contains type I collagen and lesser amounts of type II collagen and aggrecan, and has important weight-bearing properties and also protects the weight-bearing regions of the femoral and tibial AC from damaging focal overload [28]. The tendons and ligaments in the human body interconnect bone to muscle and bone to bone and are rich in type I collagen, which provides tensional strength. Ligaments and tendons have important roles in the stabilization of joint structures and in the transmittance of forces from the muscles that facilitate joint movement. As already mentioned, all of the aforementioned tissues can deteriorate due to excessive proteolytic attack in disease processes and with traumatic loading. Cells of a chondrogenic background are responsible for the assembly of these tissues and are also important in tissue homeostasis in maturity, however, they have a limited ability to affect tissue-repair processes. Adult stem cells represent a means of overcoming this shortcoming and show considerable therapeutic promise in these tissues. It was from this background that we embarked on the present study using FGF-2 and -18 to develop adult stem cell lineages with chondrogenic and osteogenic capability. This work follows on from earlier basic studies on human fetal spinal development where FGF-2, -18, the heparan sulphate proteoglycan perlecan and FGFR1c and FGFR3c were all prominently expressed by the chondroprogenitor cell populations [23]. Chondrogenesis is an active driving force promoting early spinal development. It was, therefore, logical to follow-up on these findings by determining if adult stem cells could be primed to develop into chondrogenic and osteogenic cell lineages, which may be applicable to the repair of cartilaginous tissues including the IVD. Besides its ability to act as a low-affinity co-receptor for FGFs, FGF2, FGF18 promote chondrogenesis/osteogenesis in mesenchymal stem cells Research Article perlecan is also an early chondro genic marker with an extensive repertoire of interactive matrix components through which it provides ECM stabilization. Thus the expression of perlecan by the adult stem cells was of interest in our strategic plans aimed at disc repair. The IVD is a particularly important tissue to look at in such repair strategies. Degeneration of the IVD is associated with low back pain (LBP), which is now considered to be the number one musculoskeletal global condition in terms of years lived with disability, loss of working days, impairment in the quality of life and detrimental socioeconomic impact of global significance. The American Academy of Pain Medicine stated in 2016 that chronic pain costs in the USA were $560-635 billion/annually and that 53% of all chronic pain patients had LBP with 31 million people suffering from this condition at any one time [7]. Crow and Willis (2009) quoted US$317 billion costs for LBP for the USA in 2009 [8]. The WHO indicated that the development of MSCs and bioscaffolds to promote IVD repair should be made high-priority research objectives and many scaffold and cell-based treatment strategies have subsequently been developed for the treatment of LBP [29]. The present study has developed adult stem cell lineages which deserve further investigation in such strategies for their ability to promote disc repair, alleviation of LBP and the repair of cartilaginous tissues in general.

Tissues
Ovine knee and hip tissues were harvested from newborn lambs supplied by a local abattoir. Adult (18 month) male wethers were used for bone marrow harvest.

Bone marrow harvest
Ovine iliac crest bone marrow aspirates of three pooled ovine male wether donors were collected under general anesthesia into Na 2 EDTA. All animal procedures were approved by our Institutional Animal Care and Ethics Committee.

Isolation, expansion & authentication of ovine bone marrow MSCs
The MSCs isolated complied with the minimal criteria for defining multipotent MSCs as described by The International Society for Cellular Therapy [30]. That is, they adhered to tissue culture plastic; they future science group future science group Research Article Shu, Smith, Little & Melrose expressed high levels of CD44 and CD106, but were negative for CD11b, CD34, CD45; and they were multi potent cell types with the capability under appro-priate growth conditions of differentiating into osteoblasts, adi pocytes and chondroblasts and displayed multipotency up to passage 11. The findings reported  in the present study were from passage 8 cells. Ovine iliac crest bone marrow aspirates were collected into Na 2 EDTA and the buffy coat cells were collected by centrifugation at 2500 × g for 10 min [31] and resuspended in phosphate buffered saline (PBS; 1.5 ml), then filtered through a cell sieve (70 μm) to remove cellular and tissue debris and the clarified cell suspension layered on to a Ficoll gradient (10 ml). The Ficoll gradient was centrifuged at 1200 × g for 20 min in a swing-out rotor and the cells at the interface were collected and washed twice in sterile PBS. The buffy coat cells were allowed to attach overnight to 75 cm 2 canted neck polystyrene tissue culture flasks in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), antibiotics and 2 mM L-glutamine (DMEM-FBS culture medium). The flasks were subsequently washed with PBS to remove nonadherent cells and the adherent cells were further cultured in DMEM-FBS till confluent then serially passaged up to passage 11 to expand cell numbers.
The expanded cells were examined by flow cytometry using the anti-ovine markers CD44, CD34, CD45, CD11b and antihuman CD106. The MSCs isolated were CD44 and CD106 positive, but CD34, CD45 and CD11b negative. The MSCs were frozen down in liquid nitrogen in aliquots of 5 million MSCs/cryovial in DMEM + 20% FBS + 10% v/v DMSO.

Demonstration of MSC multipotency
The MSCs were grown in micromass pellet and monolayer culture in chondrogenic, adipogenic and osteogenic selection medias to demonstrate chondrogenesis, adipogenesis and osteogenesis.

Monolayer culture of MSCs
The MSC numbers were initially expanded in 75 cm 2 canted neck flasks in DMEM + 10% FBS till 75-80% confluent then the cells were harvested by trypsinization and cell numbers determined on a hemocytometer using trypan blue exclusion.

Monolayer adipogenesis cultures
The MSCs were seeded into 12-well plates (13,150 cells/cm 2 ) and cultured in DMEM + 10% FBS with a media change once per week. When the cells had reached 70-80% confluence, the media was changed to DMEM supplemented with 10% FCS, dexamethasone (0.5 μM), indomethacin (50 μM) and 3-isobutyl-1-methylxanthine (0.5 μM), and culturing continued for a further 7 days. The monolayers were fixed in 10% neutral buffered formalin for 30 min and washed in PBS. The fixed cells were stained with Oil red-O stain (5 μg/ml) in 70% isopropanol for 1 h at 37°C.

Monolayer MSC osteogenesis cultures
The MSCs were cultured in 24-well plates at a density of 13,150 cells/cm 2 and cultured in NH OsteoDiff selection media with media changes every 3 days. The cultures were terminated on day 18 and stained with Alizarin Red S to demonstrate calcium deposition. The cells were initially washed in calcium-free cold PBS then fixed in 10% neutral buffered formalin for 15 min, rinsed in PBS then distilled water and stained with a 1% w/v solution of Alizarin Red S pH 4.2 for 15 min followed by rinsing in several changes of distilled water [32]. 15 ml conical test tube. The cells were spun down and cultured in ChondroDiff selection media at 37°C under an atmosphere of 5% CO 2 in air with 98% humidity, media was changed every 3 days. After 21 days, the cultures were either continued in ChondroDiff selection media or in ChondroDiff media supplemented with FGF-2 or -18 (20 ng/ml) for an additional 20 days and the cultures were terminated on day 41. Chondrocyte pellets were removed after various culture periods, washed in PBS and fixed in 10% neutral-buffered formalin then processed to paraffin and microtome sections attached to positively charged microscope slides. These were stained with toluidine blue-fast green to visualize tissue proteoglycans. Type I and II collagen and aggrecan were also immunolocalized to confirm the chondrogenic status of the cells [33]. Staining patterns in the monolayer and pellet cultures were documented on a Leica DFC 450 digital photo microscope system (Leica Microsystems Pty Ltd, North Ryde, Australia).

Quantitative-reverse transcription PCR gene profiling of MSC pellets
Chondrocyte pellets (n = 5-7) from micromass MSC cultures in ChondroDiff media; or in ChondroDiff media supplemented with FGF-2; or FGF-18 were collected on days 31 and 41. On day 21 the media was supplemented with FGF-2 or -18 and culturing continued. Pellets from the FGF cultures were harvested on days 31 and 41. The pellets were homogenized using pellet pestles driven by a cordless motor. Total RNA was extracted using Trizol (Invitrogen) and purified using a Qiagen RNA extraction kit and quantitated as previously described [34]. RNA (1 μg) from each sample was reverse transcribed (Omniscript; Qiagen) using random pentadecamers (50 ng/ml, Sigma-Genosys) and RNase inhibitor (10 U per reaction, Bioline, Sydney, Australia). The resulting cDNA was subjected to real-time PCR in a Rotorgene 6000 (Corbett Life Science, New South Wales, Australia)    (Table 1). Standard curves were constructed using a range of dilutions of total ovine MSC cDNA, and a relative copy number determined for each gene of interest. Melt curves were obtained following quantitative-reverse transcription (qRT)-PCR to check for single products, specificity was confirmed by sequencing at Sydney University Prince Alfred Macromolecular Analysis Centre. The genes investigated, primers, annealing temperatures used for qRT-PCR are listed in Table 1. The qRT-PCR data are presented as box plots showing interquartile (25-75%) range with whiskers indicating the maximum-minimum range and the mean values indicated by a horizontal line within the box.

Histochemical processing of cell pellets
Pellets from six cultures for each growth condition were fixed in 10% neutral buffered formalin for 12 h, dehydrated through sequential alcohols, cleared in chloroform then infiltrated and embedded in paraffin by our standard procedures [23,26,35]. The pellets were sectioned at 4 μm and immunolocalizations undertaken for type I and II collagen, perlecan, decorin, biglycan and the 4C3 and 7D4 CS sulphation motifs. Pellet sections were also stained with toluidine blue-fast green to visualize anionic proteoglycans and with Alizarin Red S to visualize calcium deposition.

Histological processing of ovine knee & hip tissues
Ovine newborn knee and hip joints were fixed en bloc in 10% neutral buffered formalin and decalcified in 10% formic acid/5% formalin then 4 μm sections prepared. The hip joints were sectioned in the midsaggital plane while a mid-coronal section through the main weight-bearing regions of the femoral condyle and tibial plateau were also prepared.

Histochemistry
Anionic proteoglycans were localized in tissue sections by staining for 10 min with 0.04% w/v toluidine blue in 0.1 M sodium acetate buffer, pH 4.0 followed by a 2-min counterstain in 0.1% w/v fast green FCF.

Immunohistochemistry
Incubations with primary antibodies were performed using a Sequenza vertical cover-plate immunostaining system (Thermo Scientific Shandon, Cheshire, UK). Endogenous peroxidase activity was blocked by incubating the tissue sections with 0.3% H 2 O 2 for 5 min and after washing in water nonspecific binding sites were blocked with 10% swine serum for 10 min. Sections destined for localization of type I, II collagen were predigested with bovine testicular hyaluronidase (1000 U/ml) for 1 h at 37°C in phosphate buffer pH 5.0 prior to incubation with primary antibody. The primary, anti-type I and II collagen (1/200 dilution) antibodies were diluted in Dako (Dako, Sydney, Australia) diluent (cat no. S202230) and applied to the slides and incubated at 4°C overnight. The primary antibodies were subsequently localized using Dako Envision+, an horse radish peroxidase-labeled polymer and anti-mouse (cat no. K4001) and -rabbit (cat no. K4004) antibodies for the visualization of the tissue immune complexes using Nova RED substrate for color development. Negative control sections were also prepared where the authentic primary antibody was replaced with an irrelevant isotype-matched mouse IgG directed against Aspergillus niger glucose oxidase, an enzyme which is neither present nor inducible in mammalian tissues or to a concentration-matched rabbit nonimmune serum sample.

Statistical analyses
RNA was isolated from a total of five to seven pellets for each gene. Non-Gaussian qRT-PCR data were analyzed by Mann-Whitney U ranked tests. The α-level was set at 0.05. Gene expression data were presented as box plots showing interquartile (25-75%) range, with whiskers indicating the maximum-minimum range and mean values were indicated with a horizontal line within the bar. Values that were significantly higher or lower than the basal culture data on day 21 were labeled with an asterisk (p < 0.05).

Results
The MSCs isolated by the Ficoll procedure had typical fibroblastic morphologies in monolayer culture ( Figure 1A). The multipotency of the MSC pre parations was established in pellet cultures in ChondroDiff differentiation media (    Comparative gene profiling of cells from each of the culture conditions (Figure 8) showed that decorin and biglycan gene expression were elevated in the FGF-2 and -18 cultures, ACAN was maintained and type II collagen gene expression markedly decreased by 41 days of culture. Type X collagen expression was elevated in the basal and FGF-2 cultures on day 41 but undetectable in the FGF-18 cultures over days [31][32][33][34][35][36][37][38][39][40][41] indicating that FGF-18 downregulated chondrocyte hypertrophy. Perlecan expression was maintained in the FGF-2 cultures but downregulated by FGF-18. Expression of SOX9 and TGFB1/TGFBRI were maintained or were slightly elevated in the FGF-2 and -18 cultures, consistent with induction of a chondrogenic phenotype. Myocyte-specific enhancer factor 2C also known as MADS box transcription enhancer factor 2, polypeptide C (MEF2C), a transcriptional regulator of chondrocyte hypertrophy and osteogenic differentiation was upregulated in the FGF-18 cultures on days 31  A schematic of the morphology of the epiphyseal growth plate cartilage and its constituent chondrocytes and the marker genes operative at various stages of chondrocyte differentiation was prepared to sum-  Figure 9B). A table of the anabolic and catabolic genes, and transcription factors regulated in prechondro genic and chondrogenic cells by FGF-2 and -18 was assembled to summarize the major findings of the present study ( Figure 9C). Anabolic chondrogenic genes such as ACAN, COL2A1, HSPG2 and SOX9 were all upregulated by FGF-2 consistent with its role as a promoter of chondrogenesis. In contrast, while FGF-18 promoted early chondrogenic gene expression on day 31, by day 41, FGF-18 also significantly downregulated Coll2A1 and the hypertrophy marker genes COL10A1, COL2A1 and MMP13, and upregulated genes which promote osteogenesis (MEF2C, ALPL, TGFB1). DEC and BGN were also upregulated by FGF-18 and their deposition in the cell pellets in areas of calcium deposition supported potential roles in biomineralization. Decorin and biglycan also control the bioavailability of TGF-β in tissues thus they may have a role in the regulation of chondrogenesis and promotion of osteogenesis.
Comparative immunolocalization of FGF-2 and -18 in newborn ovine stifle and hip joints (Figures 10 & 11) demonstrated different distribution patterns. FGF-2 was strongly localized in the marrow space and the pericellular matrix of articular chondrocytes in the hip and knee (Figure 10). In contrast, FGF-18 was predominantly expressed by the columnar pre-and hypertrophic chondrocytes of the growth plates with relatively weak expression by the articular chondrocytes and little or no expression in the marrow space ( Figure 11). This was consistent with FGF-2 promoting and maintaining chondrogenesis and FGF-18 promoting early chondrogenesis, chondrocyte maturation and the promotion of osteogenesis in joint tissues.

MSCs & tissue repair
Adult multipotent stem cells hold tremendous promise in regenerative medicine and developmental biology and specifically in joint repair [2,4,5,36,37]. A major challenge in their therapeutic application lies in how their differentiation is controlled to facilitate production of cell lineages of defined properties, engraftment at the therapeutic site of action and maintenance of their viability throughout their therapeutic period. The aim of the present study was to examine whether FGF-2 and -18 could be used to select chondrogenic and osteogenic cell lineages. These experiments extended our earlier observations on spatiotemporal changes in ECM composition and the immunolocalization of FGF-2 and -18 in human fetal spinal development [23]. In the present study, FGF-2 and -18 stimulated ECM production in the pellet cultures; the FGF-2 pellets were larger than the FGF-18 pellets, however, both were significantly larger than the pellets grown under basal conditions. FGF-2 has been reported to stimulate human bone marrow-derived MSC proliferation and delay the loss of chondrogenic potential through the transient expression of JNK P13K-Akt and ERK-1, -2 cell-signaling pathways [38,39]; FGF-2 also inhibits lineage differentiation of MSCs, by delaying Erk-1 and -2 phosphorylation and represses osteogenic gene   [38]. This may explain the absence of Alizarin Red staining in the FGF-2 stimulated cell pellets in the present study.

Induction of TGF-β1/TGFBR1 by FGF-2 & -18 & their roles in chondrogenesis & bone formation
The micromass pellet culture system is commonly used to examine the chondrogenic differentiation of MSCs. Initial studies involved the use of TGF-β1 as an induction agent for in vitro chondrogenesis of MSCs [40], subsequent studies showed that TGF-β2 and -β3 were even more effective chondrogenic agents [41]. An analysis of molecular markers of in vitro chondrogenesis by bone marrow MSCs demonstrated distinct stages of chondrocyte differentiation toward full chondrocyte commitment (Chen et al.). The earliest stages of in vitro chondrogenesis were an up regulation of TGF-β1, -β2 and -β3 followed by upregulation of the BMP and SMAD pathways to reach the final stages of chondro cytic commitment [42,43]. This is followed by maturational changes in the chondrocytes leading to hypertrophy, apoptosis, cartilage calcification, penetration of blood vessels and bone formation. The initial stages of TGF-β-induced chondrogenesis involve the rapid deposition of cartilage-specific ECM components such as collagen II, aggrecan and perlecan. TGF-β is synthesized in the endoplasmic reticulum attached to a prodomain latency-associated peptide, which prevents its interaction with TGF-β receptors. The latency-associated peptide domain of TGF-β interacts with the RGD-binding domains of latent TGF-β-binding proteins (LTBP-1, -3, -4) to form an inactive TGF-β complex which is deposited in the tissues [44] and is also interactive with the Arg-Gly-Aspbinding integrins, α β1, α β3, α β5, α β6, α β8 and α8β1. Fibrillin-1, -2, -3 also share structural homology with the latent transferring growth factor β-binding proteins (LTBPs) and are critical for the placement of latent TGF-β within tissues in close proximity to the cells that assemble these polymers. This is a critical step in tissue homeostasis, evidenced by mutations in fibrillin-1, which result in Marfan syndrome.   Fibrillin/LTBP assemblies have roles in the delivery of TGF-β to cells in appropriate amounts in a spatiotemporal-directed manner, which is important not only in tissue development but also in the homeostasis of mature tissues facilitating cross-talk among TGF-β signaling pathways, integrins and the ECM [44].
In the present study, FGF-2 and -18 significantly upregulated TGFB1 and TGFBR1 gene expression on days 31-41. This was accompanied by the deposition of chondrocyte-specific ECM components, which were immunolocalized in the MSC cell pellets. FGF-18 elicited an earlier chondrogenic response than FGF-2, but by day 41 had downregulated COL2A1 and COL10A1 expression to virtually undetectable levels. FGF-18 also downregulated MMP13 on day 41, thus after an initial chondrogenic phase, chondrocyte hypertrophy was also downregulated. Preconditioning of MSCs with TGF-β3 also induces a downregulation in these hypertrophic markers [42]. Parathyroid hormone and parathyroid hormone-related peptide (PTHrP) also delay chondrocyte hypertrophy through their regulatory actions on the RUNX2 gene, which normally promotes hypertrophy [45,46]. In the present study, FGF-18 also upregulated MEF2C, a key osteogenic regulatory transcription factor, and osteogenic genes such as ALPL, COL1A1 and BGLAP. Osteocalcin (BGLAP) is a highly abundant bone protein secreted by osteoblasts, FGF-2 also stimulates production of BGLAP by chondro cytes [47]. The Gla domain of BGLAP interacts with calcium and hydroxyapatite and promotes bone mineralization and represents a useful early marker of bone formation. In contrast, FGF-2 promoted chondro genesis through ACAN, COL2A1, TGFB/TGFBR1 and SOX9 in the present investigation, but did not induce osteogenesis. In the present study DEC expression was strongly upregulated by FGF-2 and to a lesser extent by FGF-18 while BGN was strongly upregulated by FGF-18. Decorin and biglycan were immunolocalized in regions of the cell pellets where deposition of calcium and the novel CS sulphation motifs 7D4 and 4C3 were also evident. Decorin and biglycan have known roles in the regulation of collagen fibrillogenesis [48][49][50] and matrix stabilization by acting as linking and anchoring modules in collagenous matrices [51,52]. Decorin and biglycan have diverse functions in musculoskeletal tissues as modulators of tissue organization, cellular proliferation, matrix adhesion and response to growth factors and cytokines, and are key signaling molecules with an extensive repertoire of molecular interactions with growth factors and a number of receptors, which regulate cell growth and tissue morphogenesis [53][54][55]. Decorin regulates TGF-β1 bioavailability [56] and may have regulatory roles to play over TGF-β-dependant chondrogenesis [57]. Decorin and biglycan also have roles in biomineralization, which may explain their deposition in the central regions of the cell pellets in the present study [58,59]. Osteoblastic differentiation is accompanied by a decreased expression of decorin, but continuous expression of biglycan. TGF-β1 inhibits decorin expression in osteoblasts at varying stages of differentiation, but not biglycan [60]. Decorin and biglycan have differing roles to play in mineralization and are widely distributed in bone where they are present as CS substituted forms of these SLRPs, whereas in looser connective tissues the D-glucuronic acid of the GAG side chains is epimerized to L-iduronic acid; this confers differing interactive properties to the glycosaminoglycan (GAG) side chains on small leucine-rich proteoglycans (SLRPs) [52,58].

Deposition of perlecan in pellet cultures
FGF-2 and -18 both upregulated perlecan expression in the present study. Immunolocalization studies on the cell pellets indicated that in basal cultures perlecan had  [61][62][63]. Perlecan is an early chondrogenic molecule [24] and has important cell regulatory [64] and matrix-stabilizing properties [28]. FGF-2 and -18 both signal cells through perlecan which acts as a low-affinity FGF receptor in cartilage [20,64]. In the FGF-2 and -18 pellets, the distribution of perlecan was predominantly around the cells of the outer regions of the pellets with significantly lower perlecan levels in the central regions where deposition of calcium, decorin and biglycan was evident.
Genes operative in chondrogenesis & osteogenesis & their regulation by FGF-2 & -18 Figure 9 summarizes information on chondrogenic and osteogenic genes operative in chondrocyte differentiation. This figure also shows that FGF-2 was a prominent pericellular proteoglycan expressed by articular chondrocytes whereas FGF-18 was produced by the prehypertrophic columnar and hypertrophic chondrocytes adjacent to the calcified cartilage of the growth plates. The major findings with genes which were upor downregulated by FGF-2 and -18 are also provided. The primary effect of FGF-2 was to promote chondrogenesis, while FGF-18 promoted early chondrogenesis, delayed chondrocyte hypertrophy and expression of proosteogenic genes. Calcium deposition was evident centrally in the FGF-18 pellets in regions, which also displayed the novel CS sulphation motifs 7D4 and 4C3. Decorin and biglycan were also immuno localized in close association with these components but were not positively shown to be substituted with these CS motifs although evidence exists that decorin and biglycan can bear these motifs [65], which are prominently expressed in the chondrocostal growth plate cartilage as it transitions to bone [61,66].
Immunolocalization of FGF-2 & -18 in developmental joint tissue identifies the cell types responsible for FGF production & their principle areas of action Immunolocalization of FGF-2 in ovine newborn knee and hip demonstrated a strong localization of FGF-2 in the bone marrow and pericellular matrix of the articular chondrocytes. FGF-2 has known roles in the maintenance of stromal stem cells as a slowly recycling quiescent cell type and promotes stem cell viability. FGF-2 also has well-established roles in the regulation of chondrocyte metabolism and regulates cell proliferation and matrix production to ensure tissue homeostasis. While FGF-2 and -18 have similar properties in terms of the stimulation of chondrocyte proliferation and matrix production, FGF-18 has a more prominent expression pattern in the columnar prehypertrophic and terminally differentiated hypertrophic chondrocytes of the epiphyseal growth plate, which is consistent with its roles in chondrocyte maturational processes. Thus the immunolocalization patterns of FGF-2 and -18 in newborn and hip joint tissues observed in the present study were consistent with FGF-2 having a prominent role in AC chondrogenesis while FGF-18 had chondrogenic properties but also regulated growth plate chondrocytes, stimulating terminal chondrocyte differentiation and the expression of osteogenic regulatory genes in the transitional costochondral tissues at the bone-growth plate cartilage interface. These findings are consistent with our proposal that FGF-2 and -18 can be used to precondition bone marrow MSCs to select for chondrogenic and osteogenic cell lineages, which may have improved efficacy for the repair of cartilage and cartilage-bone defects. Studies already support the concept that FGF-2 primes adult bone marrow MSCs for enhanced chondrogenesis [22,67,68], however, the use of FGF-18 in similar studies is relatively un explored. The present study has hopefully provided

Conclusion
The major conclusions from this study were that FGF-2 and -18 promoted chondrogenic differentiation of marrow-derived chondroprogenitor cells early in pellet culture with FGF-2 promoting matrix production and cellular proliferation, while FGF-18 delayed hypertrophy and also supported expression of osteogenic genes. Immunolocalization of FGF-18 in newborn ovine articular and growth plate cartilages clearly showed that FGF-18 was a pericellular matrix component of articular chondrocytes but was particularly strongly expressed by the hypertrophic growth plate chondrocytes of hip and knee joints. A recent study from our laboratory supported a prominent role for FGF-18 in the establishment and maturation of the vertebral ossification centers during fetal human spinal development [69]. FGF-2 and -18 also supported chondrogenesis but delayed hypertrophy, these therefore represent two useful growth factors which can be used to selectively target the marrow-derived stromal cells to develop chondrogenic and osteogenic cell lineages. Selective induction of such MSC cell lineages may well be of application in regenerative strategies maximizing matrix replenishment (FGF-2 lineages) and osteointegration/osteogenesis (FGF-18 lineages) in tensional and weight-bearing connective tissues such as the spine.

Future perspective
MSCs show tremendous potential in cell-based tissue regenerative strategies. Cartilaginous tissues, including the intervertebral disc suffer from a poor spontaneous repair capability. The application of MSCs in repair strategies in these tissues has shown promise in preclinical in vitro and experimental animal models of tissue repair. The development of MSC cell lineages directed to chondrogenesis and osteogenesis should improve the efficacy of these cells in specific regenerative strategies on cartilaginous tissue defects, which otherwise represent a considerable clinical problem.  No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

The requirement for improved therapeutic options in the treatment of IVD degeneration
• IVD derangement leading to the generation of LBP is a particularly debilitating musculoskeletal condition which deserves better therapeutic treatment methods. mesenchymal stem cells have shown tremendous promise for the treatment of IVD degeneration in laboratory based animal studies and in preclinical trials indicating that the cell lineages developed in this study should be evaluated further in appropriate large animal models and preclinical trials for the treatment of this condition.