Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs

Abstract

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx^-/- mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.

ARTICLE

Received 4 Sep 2015 | Accepted 7 Jul 2016 | Published 16 Aug 2016

DOI: 10.1038/ncomms12503 OPEN

Mohawk promotes the maintenance and regeneration of the outer annulus brosus of intervertebral discs

Ryo Nakamichi1,2, Yoshiaki Ito1, Masafumi Inui3, Naoko Onizuka3, Tomohiro Kayama1, Kensuke Kataoka1, Hidetsugu Suzuki1, Masaki Mori1, Masayo Inagawa3, Shizuko Ichinose4, Martin K. Lotz5, Daisuke Sakai6, Koichi Masuda7, Toshifumi Ozaki2 & Hiroshi Asahara1,3,5,8

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus brosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx / mice, the OAF displays a deciency of multiple tendon/ligament-related genes, a smaller OAF collagen bril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen bril formation. Our results indicate a therapeutic strategy for AF regeneration.

1 Department of Systems Biomedicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 2 Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan.

3 Department of Systems Biomedicine, National Research Institute for Child Health and Development, 2-10-1, Okura Setagaya, Tokyo 157-8535, Japan.

4 Research Center for Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 5 Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-161, La Jolla, California 92037, USA. 6 Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1143, Japan. 7 University of California, San Diego, 9500 Gilman Dr Mail Code 0863, La Jolla, California 92093-0863, USA. 8 CREST from Japan Agency for Medical Research and Development, 1-7-1 Ootemachi, Tiyoda, Tokyo 100-0004, Japan. Correspondence and requests for materials should be addressed to H.A. (email: mailto:[email protected]

Web End [email protected] ).

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The spine is composed of vertebral bodies connected by intervertebral discs (IVDs) and plays a central role in human movement, as it enables standing, twisting

and bending positions. Rupture of the exterior section of the IVD known as the annulus brous (AF), especially by degenerative changes in the outer AF (OAF), can cause IVD herniation, which is a common and severe disease associated with pain and disability. Although standard discectomy provides pain relief, this technique reduces the mechanical property of the AF and accelerates progression to IVD degeneration and degenerative spondylosis. To date, tissue regenerative therapy of the IVD has not been achieved, partly because the IVD is the largest avascular tissue and has poor self-healing potential1, and because the specic master transcription factors that regulate IVD development and homeostasis are unknown2,3. A better understanding of the molecular mechanism of IVD development and homeostasis is required for the goal of regenerative therapy of IVD degeneration.

The IVD can be distinguished by its three major components, the nucleus pulposus (NP), the AF and the cartilaginous endplate (EP). The NP is a jelly-like material located in the centre of the disc, and surrounding it is brous tissue of the AF. The EP extends superiorly and inferiorly over the inner AF (IAF) and the NP. The AF comprises the OAF and the IAF. The OAF is a highly organized collagenous structure consisting mainly of type I collagen46. The expansion pressure of the NP and the tensile strength of the AF are balanced and control the mechanical properties of the IVD. The EP, which has specialized capillary beds, provides nutrition to the whole IVD7. The developmental origin of these components is different. At E12.0 in the mouse, sclerotome cells migrate and condense around the notochord8. These cells form a metameric pattern of more-condensed regions, which become vertebral bodies, and less-condensed regions, which form the AF. The notochord expands within the future IVD, to form the NP9. Thus, the molecules that promote disc regeneration are expected to be different between these tissues.

To identify tissue-specic transcription factors present during embryogenesis, we previously generated a whole-mount in situ hybridization (ISH) database, EMBRYS, that covers the expression of B1,500 transcription factors and cofactors during embryogenesis10. Among them, we and others found that homeobox protein Mohawk (Mkx) is specically expressed in tendon-related and ligament-related tissues, and could be used to promote tendon regeneration1116. Mkx is a member of the three-amino-acid loop (TALE) superclass of atypical homeobox genes belonging to the Iroquois family17. The expression of Mkx in the syndetome is detectable at E12.5 and its expression is maintained even in matured ligament cells17. The ligament-like properties of IVDs, especially the AF, which connect the adjacent upper and lower vertebrae, and contribute biomechanically to the stabilization of the spinal motion segment, prompted us to analyse Mkx expression in detail during AF development. By immunohistochemistry (IHC) and ISH, Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx / mice, the OAF displays a deciency of multiple tendon/ligament-related genes, a smaller OAF collagen bril diameter and a more rapid progression of IVD degeneration with age compared with the wild type. Overexpression of Mkx in mesenchymal stem cells (MSCs) promotes tenocyte-like differentiation in vitro and these cells can contribute to functional AF regeneration in a mouse AF defect model with abundant collagen bril formation in vivo. The present work therefore reveals that Mkx is a central transcription factor that regulates AF development, maintenance and regeneration.

ResultsMkx is mainly expressed in the OAF of humans and mice. To investigate the expression of Mkx in IVD, we used Mkx-Venus knock-in mice. The details about these mice were previously reported11. The endogenous expression of Mkx in the IVD, as determined by ISH, shadowed the Venus expression observed in Mkx-Venus knock-in mice by IHC at E14.5. This suggests that the expression of Mkx-Venus is consistent with the endogenous expression pattern of Mkx in the IVD (Fig. 1a). In IHC, Mkx-Venus was strongly expressed at E14.5 in the OAF of the somite, and its expression was continued even at the later stages (Fig. 1a,b). Importantly, the expression of Venus was specically localized in the OAF, even after disc development was completed, in 10-week-old mice (disc formation is completed by approximately postnatal week 8 in mice18). Conversely, Venus expression in the IAF decreased gradually as it approached the NP region (Fig. 1c). We also evaluated the expression of MKX in human lumbar discs (Fig. 1e (details of regions between the OAF and IAF); Supplementary Table 1). Consistent with the observation in mice, more MKX-positive cells were observed in the OAF compared with the IAF (Fig. 1d,f). These results suggest that Mkx is expressed mainly in the OAF.

Mkx deciency affects development of AF. We examined the function of Mkx in the OAF using Mkx / and Mkx / mice.

In haematoxylin and eosin (HE) staining, AF cell number did not differ between Mkx / and Mkx / mice at 10 weeks. For

Mkx / mice, the mean cell number was 213. For Mkx / mice, the mean cell number was 217. The P value was 0.51.

However, HE staining revealed that AF collagen bres were thinner in Mkx / mice than they were in Mkx / mice (Fig. 2a,b). An electron microscopy-based analysis conrmed that the diameter of collagen brils was signicantly narrower in Mkx / mice than in Mkx / mice at 10 weeks (Fig. 2ce).

This difference in phenotype was also conrmed at postnatal day 1 (P1; Supplementary Fig. 1). We next examined the effect of Mkx on gene expression levels using OAF cells isolated from the tail discs (C1/2-8/9) of 10-week-old Mkx / or Mkx / mice (Supplementary Fig. 2). Quantitative real-time reverse transcription PCR (qRTPCR) analyses revealed the downregulation of(i) a set of collagen genes, including Col1a1 and Col1a2, which were the main components of the OAF; (ii) various small leucine-rich proteoglycan (SLRP) family genes (especially Biglycan (Bgn), which is expressed mainly in the OAF and plays an essential role in the maintenance of OAF homeostasis)1921 and (iii) the tendon-related transcription factor Scleraxis (Scx)22 in Mkx /

compared with Mkx / cells (Fig. 3a). The P value of Elastin (Eln) was 0.084 and a signicant difference between Mkx /

mice and Mkx / mice at 10 weeks was not seen. Thus, we

judged that Mkx was not a denitive contributor to Eln expression. In IHC, Bgn and Col14 protein expression were decreased in Mkx / mice compared with Mkx / mice at 10 weeks (Fig. 3c). We also investigated the knockdown effect of

MKX in human AF (HAF) primary cultured cells and found that the expression of tendon/ligament-related genes, such as those encoding SCX, TNMD, COL14A1, TNC, BGN and TNXB, was signicantly downregulated, whereas expression of the cartilage-related genes SOX9 and ACAN was upregulated (Fig. 3b). Western blot analysis conrmed the downregulation of BGN and upregulation of SOX9 following MKX knockdown in HAF cells (Fig. 3d). Taken together, in mice and human, Mkx has an important role in the expression of some collagen and SLRP genes in the OAF. These results also indicate a role for Mkx in collagen bril formation during development, including adulthood.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12503 ARTICLE

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Figure 1 | Mohawk gene (Mkx) is expressed in the outer annulus brosus (OAF). (a) Representative images of intervertebral discs (IVDs) of E14.5 mice. Left: Safranin O fast green stain in a wild-type mouse. Middle: IHC of a Venus knock-in mouse. Green: anti-GFP. Blue: Hoechst. Right: in situ hybridization of Mkx of a Venus knock-in mouse. Scale bars, 200 mm. (b) Representative disc images from embryonic stages to growth stages (upper and middle panels,

Safranin O fast green (S-F) stain and Massons trichrome (M-T) stain in a wild-type mouse; lower panel, IHC of a Venus knock-in mouse; green, anti-GFP; blue; Hoechst). Scale bars, 200 mm. (c) Representative images of IVDs from 10-week-old mice (upper panel, safranin O fast green stain in a wild-type mouse; lower panel, IHC of a Venus knock-in mouse; green; anti-GFP; blue; Hoechst). Scale bars, 200 mm. (d) Immunohistochemistry of the coronal sections of a human lumbar disc IAF and OAF (#1: 20-year-old male, L1/2). Green: anti-MKX. Blue: Hoechst. Scale bars, 100 mm. (e) Overview of the observed area of the IAF and OAF. The section is a haematoxylineosin staining of a human lumbar disc (#1: 20-year-old male, L1/2). Dotted lines indicate borders of the NP, IAF and OAF. Scale bar, 1 cm. (f) Percentages of human IAF and OAF cells that were positive for MKX in each zone. Values are the mean of nine discs from ve donors. Error bars represent s.e.m. ***Po0.001. Statistical differences were assessed with Students t-test. GFP, green uorescent protein; MKX, Mohawk homeobox protein; P1, postnatal day 1.

Next, to test whether the Mkx / phenotype has an effect on the IVD degeneration that occurs during the aging process, we analysed Mkx / and Mkx / mice using HE staining and

Safranin O fast green staining at various stages. The OAF collagen bres in Mkx / mice were thinner than those in Mkx / mice at all ages (Supplementary Fig. 3a). In terms of IVD degeneration, there were no clear differences between Mkx / and Mkx / mice at 10 weeks and 6 months; however, at 12 and 21 months, Mkx / mice showed high levels of degenerative changes in lumbar discs, whereas the lumbar discs of Mkx / mice did not show changes from those observed at 10 weeks. At 12 and 21 months, small round cells, morphologically resembling chondrocytes, were observed in the NP and IAF of Mkx /

mice (Fig. 4a; Supplementary Fig. 3b). The NP and the IAF were stained more strongly with Safranin O, indicating degenerative changes. To further analyse IVD in Mkx / mice, we performed

IHC for Venus, Col1, CD24 and KRT18 using 21-month-old Mkx / mice and Mkx / mice. Col1 is a main component of the AF and Venus shadows Mkx expression. CD24 and KRT18 are well characterized as NP cell markers2327. As expected,

Venus expression is restricted at AF of 21-month-old Mkx / mice. Col1 expression was observed in the AF of both 21-month-old Mkx / and Mkx / mice, but not in the NP region of Mkx / and Mkx / mice (Supplementary Fig. 4a).

KRT18 was observed in the NP region of both 21-month-old Mkx / and Mkx / mice. On the contrary, CD24-positive cells were observed only in the NP of 21-month-old Mkx / mice but not in Mkx / mice (Supplementary Fig. 4b). These results suggested that the cells at NP region of 21-month-old

Mkx / mice do not possess characters of neither normal AF cells nor normal NP cells.

In microcomputerized tomography views of the lumbar spine, the disc height index between L6 and S1 was measured in Mkx / and Mkx / mice28. As a result, there was no signicant difference between the two groups at 10 weeks, but disc height index was clearly reduced in Mkx / mice at 21 months (Supplementary Fig. 5ac). Moreover, at 21 months, bone spur formations were observed in the lumbar spine of Mkx /

mice (Fig. 4b; Supplementary Fig. 5d). This phenotype could be related to the instability of vertebrae in Mkx / mice29.

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Figure 2 | Multiscale analyses of the annulus brosus (AF) from Mkx / mice. (a) Haematoxylineosin staining of IVDs of 10-week-old Mkx/ and Mkx / mice. Black arrow, width of the AF. Scale bars, 300 mm. (b) Width of the AF from 10-week-old Mkx / versus Mkx / mice. For Mkx /, mean width 246.9 mm (s.d. 23.04 mm). For Mkx / , mean width 157.5 mm (s.d. 21.16 mm). (c) Images of transmission electron microscopy

(left column) and scanning electron microscopy (right column) of the OAF of 10-week-old Mkx / and Mkx / mice. Scale bars, 200 nm.(d) Histogram of the diameter of collagen brils from 10-week-old Mkx/ and Mkx / mice. Blue bar, Mkx / ; red bar, Mkx /. (e) The calculated mean of the diameter of 100 collagen brils from 10-week-old Mkx / and Mkx / mice. For Mkx /, mean diameter 72.73 nm

(s.d. 14.5 nm). For Mkx / , mean diameter 53.99 nm (s.d. 10.31 nm). Error bars represent the s.d. ***Po0.001. Statistical differences were assessed

with Students t-test.

The degenerative histological score of Mkx / mice was higher than that of Mkx / mice at 12 and 21 months (Fig. 4c)30. These degenerative changes were also observed in the cervicalthoracic and sacralcaudal junctions in Mkx /

mice. It was predicted that the mechanical stress on IVD was related to the degenerative change of IVD of Mkx / mice because the changes did not occur in all IVDs. These results indicate the essential role of Mkx in collagen bril formation in the OAF during developmental stages, and its function as a stabilizer in IVDs.

The ability of Mkx for the differentiation of MSC. Cell transplantation has been proposed as an effective strategy to treat IVD degeneration. Among the various cell sources available, MSCs either from the bone marrow or adipose tissue have been proved to facilitate IVD repair in animal models31. To test whether MSCs with Mkx induction are a therapeutic option for IVD regeneration, we overexpressed Venus-Mkx in C3H10T1/2 cells using retroviruses (C3H10T1/2 cells induced by Venus-Mkx are referred to as C3H10T1/2-VM or Venus-Mkx, and C3H10T1/2 cells induced only by Venus are referred to as C3H10T1/2-V or

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12503 ARTICLE

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Figure 3 | Molecular analyses of the annulus brosus (AF) cells. (a) Quantitative real-time reverse transcription PCR (qRTPCR) analyses of the expression of ligament-related genes in Mkx/ and Mkx / mice. The mRNA levels of ligament markers in Mkx / mice were normalized to 1. Error bars represent s.e.m. *Po0.05, **Po0.01, ***Po0.001. Statistical differences were assessed with Students t-test. (b) qRTPCR analyses of ligament- and cartilage-related gene expression in human AF cells transfected with a small interfering RNA for MKX (siMKX-a and siMKX-b) or the negative control (siNega). The mRNA levels of ligament markers in human AF (HAF) primary cultured cells transfected with siNega were normalized to 1. Error bars represent s.e.m. *Po0.05, **Po0.01, ***Po0.001. Statistical differences were assessed with Students t-test. (c) Immunohistochemistry (IHC) of the intervertebral disc (IVD) of 10-week-old Mkx/ and Mkx / mice. IHC shows little decreased Bgn and Col14 protein expression in Mkx / mice compared with Mkx / mice. Red: Bgn and Col14. Blue: DAPI. Scale bars, 100 mm. (d) Western blot analyses of human AF cells. Western blotting showed decreased BGN and increased SOX9 protein expression following siMKX induction of HAF cells.

Venus). Venus-Mkx overexpression markedly induced the differentiation of C3H10T1/2 cells into a spindle-shaped cell type, with the upregulation of multiple ligament-related genes, such as those encoding Scx, Col1a1, Col1a2, Col14a1, Tnc and Dcn (Fig. 5a,b). The downregulation of cartilage- and fat-related genes was also observed (Fig. 5c,e), whereas the expressions of bone-related genes were not much changed (Fig. 5d). Western blot analyses of Col1, Dcn and Sox9 were consistent with the results of the qRTPCR (Fig. 5f). We also conrmed the above data observed with C3H10T1/2 using mouse bone marrow-derived MSCs (Supplementary Fig. 6). These data indicate that Mkx promotes differentiation of MSCs to tendon/ligament-like cells in gene expression levels, consistent with the previous reports14,15.

To gain insight into the molecular mechanisms underlying the determination of cell lineage by Mkx, we performed microarray analyses of these C3H10T1/2 cells. In addition to the dynamic upregulation of multiple tendon/ligament-related markers and downregulation of cartilage-related markers, transforming growth factor b (TGFb)-related genes were upregulated and bone morphogenetic protein (BMP)-related genes were downregulated in C3H10T1/2 cells (Supplementary Fig. 7a,b). Previous reports revealed that TGFb functions in IVDs to promote the differentiation of AF from the sclerotome and to prevent chondrocyte differentiation in the presumptive IVD32. Other reports suggested that disc cells continue to respond to TGFb signalling during postnatal growth33 and that BMP signalling was related to IVD

degeneration34. However, the role of Mkx in these pathways remains unclear. Therefore, further analyses were performed. qRTPCR revealed that Smad2, Smad3, TGFbr1 and TGFbr2 were upregulated (Fig. 6a), whereas Smad1, Bmpr1a and Bmpr2 were downregulated (Fig. 6c). Western blot analysis also showed that Smad3 and p-Smad2/3 were increased, whereas Smad1 and p-Smad1/5/8 were decreased (Fig. 6b,d). Luciferase assay using BMP-responsive luciferase reporter revealed that C3H10T1/2-VM exhibited reduced luciferase activity with BMP2 stimulation compared with C3H10T1/2-V (Fig. 6e)35. These results suggest that changes in the Mkx-dependent Smads ratio in C3H10T1/2 account for the promoted signal responses to TGFb and the reduced responsiveness to BMP signals. Furthermore, these changes in signal responsiveness may partly explain the promotive function in the IVD, the ligament differentiation of MSCs36,37 and the suppression of differentiation into other lineages and IVD degeneration22. So we examined the mesodermal-lineage induction in C3H10T1/2-V and C3H10T1/2-VM using BMP2. In C3H10T1/2-VM cells, the induction of differentiation into chondrocytes and osteocytes was severely disrupted (Fig. 6fi). qRTPCR analyses showed either very little or no expression in multiple cartilage- and bone-related markers compared with C3H10T1/2-V cells (Fig. 6j,k). Adipogenic differentiation was also inhibited by Mkx overexpression (Supplementary Fig. 8). These results suggest that Mkx act positively on the property maintenance of the AF cells and suggest that MSCs overexpressing Mkx can be effective as transplanted cells.

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Figure 4 | Mkx / mice develop early onset disc degeneration. (a,b) Histological and computerized tomography (CT) changes with age between Mkx / and Mkx / mice. Safranin O fast green staining of sagittal sections of L3/4 (a) and sagittal CT images of the lumbar spine (b) at 10 weeks, and 6, 12 and 21 months. Scale bars, 300 mm. (c) Histological degenerative scores of intervertebral discs (IVDs) in Mkx/ and Mkx / mice. Error bars represent s.e.m. **Po0.01, ***Po0.001. Statistical differences were assessed with Students t-test.

The possibility of Mkx for therapeutic tools of OAF. To test whether MSCs overexpressing Mkx are capable of producing collagen bres, which can be used for OAF regeneration in vivo, we rst performed an in vitro three-dimensional culture experiment using a chemical gel without animal collagen. After 8 weeks, we performed IHC for Col1a1 and found that C3H10T1/2-VM cells had a higher capacity for type I collagen synthesis compared with C3H10T1/2-V cells (Supplementary Fig. 9). On the basis of these results, we next examined whether C3H10T1/2-VM cells inserted in a type I collagen scaffold, with greater rigidity than a chemical gel, embedded subcutaneously in dorsal skin pockets of mice had the potential to form collagen brils (Fig. 7ac,e). The control group was set to the group of C3H10T1/2-V cells. After 8 weeks, the gels were excised and evaluated. Histologically, thicker tissues were generated in the C3H10T1/2-VM cell group than in the control group (Supplementary Fig. 10). No mesodermal tissues such as cartilage, bone, or fat were observed in either group. IHC revealed abundant type I collagen synthesis in the C3H10T1/2-VM scaffold (Fig. 7d,f).

Transmission electron microscopy (TEM) revealed that the diameter of collagen brils of the C3H10T1/2-VM scaffold was clearly larger compared with that of the C3H10T1/2-V scaffold (Fig. 7gj). TEM also revealed that collagen brils were hardly conrmed in the control group (that is, only transplanted gel was observed) compared with the C3H10T1/2-V group (Supplementary Fig. 11). Although we could not distinguish whether the collagen in the bril is newly synthesized by the cells or originates from the gel, but the amount of collagen increase and the highly organized structure suggest that at least some of

the type I collagen observed newly synthesized as a result of Mkx overexpression. These results demonstrated that MSCs overexpressing Mkx had a strong ability not only to express collagens and proteoglycans, but also to synthesize collagen brils even in an environment different from the OAF.

Next, we investigated whether MSCs overexpressing Mkx have the capacity to repair AF defects. A solution of liqueed collagen gel containing C3H10T1/2-V or C3H10T1/2-VM cells was placed in the cavity of the AF (Fig. 8ad). After 8 weeks, the discs were excised and evaluated. HE staining and IHC revealed that, although the reconstruction of the structure of lamella was not conrmed, abundant tissue was synthesized in the OAF transplanted with C3H10T1/2-VM cells (Fig. 8fi). TEM analysis revealed that there were also abundant collagen brils in the tissues that were transplanted with C3H10T1/2-VM cells in the entire eld, even at low magnication; in contrast, there were few collagen brils in the tissues that were transplanted with C3H10T1/2-V cells (Fig. 8j,k). In addition, the diameter of the collagen brils of C3H10T1/2-VM tissues was larger than that of C3H10T1/2-V tissues (Fig. 8l). Moreover, the diameter of these bres was almost equal in size to those of intact OAF of C3H/HeSlc mice (Fig. 8l). Although the detailed mechanism underlying these observations remains unknown, these results suggest that the environment around the transplant contributes to the regulation of collagen bril formation. Subsequently, to examine whether the newly formed tissue had sufcient physical property against mechanical stress and could inhibit IVD degeneration, we performed a modied tail-looping model, in which the uneven mechanical loading of the IVD was applied to the transplanted site in this experiment38 (Fig. 8e) and compared

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Figure 5 | Mkx promotes the differentiation of mesenchymal stem cells to ligament cells. (a) Images of C3H10T1/2 cells induced by Venus (C3H10T1/2-V or Venus) and Venus-Mkx (C3H10T1/2-VM or Venus-Mkx) in plate culture. Scale bars, 500 mm (top); 100 mm (bottom). (be) Quantitative real-time reverse transcription PCR (qRTPCR) analyses of the expression of ligament, cartilage, bone and fat-related genes in C3H10T1/2-V and

C3H10T1/2-VM cells. mRNA levels in C3H10T1/2-V cells were normalized to 1. Error bars represent s.e.m. *Po0.05, **Po0.01, ***Po0.001. Statistical differences were assessed with Students t-test. (f) Western blot analyses of C3H10T1/2-V and C3H10T1/2-VM cells for lineage markers.

the degenerative changes of the whole AF. Wild-type C3H/HeSlc mice with tail-looping were used as a control group. After 4 weeks, the discs were excised and evaluated. In Safranin O fast green staining, the AF of C3H10T1/2-V group was severely disrupted and the red area, which stained aggrecan in the NP, was reduced (Fig. 8m,n). In contrast, the AF of C3H10T1/2-VM group was only mildly disrupted compared with the control group and NP also kept its structure although the red stain of Safranin O was reduced (Fig. 8m). The histological grading of the AF by Nishimura system showed that the control group and the C3H10T1/2-VM group were not signicantly different and it was signicantly higher in the C3H10T1/2-V group (Fig. 8o)39. These results indicate the newly synthesized tissue of C3H10T1/2-VM cells has nearly equal physical property of an intact AF against compression force.

DiscussionThe biomechanical properties of the IVD are produced by the structure in which AF surrounds the NP to provide enough exibility and alleviate the pressure6,40. Extensive mechanical stress or aging could be a cause of IVD degeneration including disc herniation with serious clinical symptoms, such as lower back pain and leg pain, which restricts the patients quality of life. To prevent degeneration of IVD and to develop regenerative therapies for IVD, it is necessary to understand the molecular program of IVD development and homeostasis. The developmental molecular mechanisms of NP and IAF have recently been revealed to some extent. Sonic Hedgehog (Shh) and transcription factors Fox1a and Fox2a are essential for the formation of the NP in mouse embryos41,42. Sox5, 6 and 9 also play important roles in the formation and maintenance of NP and

IAF43,44. There are also some reports regarding the maintenance of NP: the basic broblast growth factor was required for the maintenance of the properties of NP cells45, and inhibition of tumour necrosis factor a production positively enhanced NP tissue growth46.

In contrast, there has been very limited information about the molecular network regulating OAF development. Here we reveal that Mkx was expressed specically in the AF, and mainly in the OAF from embryonic stages throughout life (Fig. 1ac). In addition, we observed severe degenerative phenotype of the AF in Mkx / mice. In vitro analyses in both mouse and human

AF cells revealed that Mkx regulates the expression of some collagen and SLRPs genes (Fig. 3a,b), the main components of OAF tissues1921,47. Scx expression was downregulated in Mkx / mice (Fig. 3) and was promoted by Mkx overexpression in C3H10T1/2 cells and bone marrow-derived

MSCs (Fig. 5a,b; Supplementary Fig. 6). Scx expression (E12.5) precedes Mkx expression (E13.5) during embryogenesis, suggesting that Mkx is not critical for initiating Scx expression; however, Mkx may support continued expression of Scx in tendon/ligament and IVD development48. In this regard, although the development of the IVD in Scx / mice at E18.5 is grossly normal49, phenotype in intervertebral discs of

Mkx / mice and Mkx ability to regenerate IVD could partly be explained by Scx regulation15,48.

Several gene expression patterns are not in concordance between human and mouse AF cells in these experiments (Fig. 3). The discrepancy between these two systems can be explained as follows. First, we investigated the function of Mkx in the process of differentiation from precursor cells to AF cells in the mouse, whereas in human experiments, we investigated the role of MKX in mature AF cells. Second, the effect of siMKX in

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Figure 6 | Link between Mkx and TGFb and BMP signalling pathway component during ligament differentiation. (a,c) qRTPCR analyses of the expression of TGFb/Smad- and BMP/Smad-related genes in C3H10T1/2-V and C3H10T1/2-VM cells. mRNA levels in C3H10T1/2-V cells were normalized to 1. Error bars represent s.e.m. *Po0.05, **Po0.01, ***Po0.001. Statistical differences were assessed with Students t-test. (b,d) Western blot analyses of C3H10T1/2-V and C3H10T1/2-VM cells. TGFb-dependent phosphorylation of Smads is shown by anti-p-Smad2/3 antibodies. BMP-dependent phosphorylation of Smads is shown by anti-p-Smad1/5/8 antibodies. (e) Luciferase assay to determine responsiveness to BMP2. Id1-Bre luciferase was transfected into C3H10T1/-V and C3H10T1/2-VM cells, followed by treatment with or without BMP2 (100 ng ml 1). Fold-induction values in the presence of BMP2 compared with the basal levels of each cell line are indicated. **Po0.01. Statistical differences were assessed with Students t-test.(fk) C3H10T1/2-V and C3H10T1/2-VM cells were subjected to chondrocyte (f,h,j) and osteocyte (g,i,k) differentiation. (f) Alcian blue staining of

C3H10T1/2-V and C3H10T1/2-VM cells after induction of chondrocyte differentiation. (g) Alizarin red staining of C3H10T1/2-V and C3H10T1/2-VM cells after induction of osteocyte differentiation. Scale bars, 200 mm. (h) Mucopolysaccaride quantication after chondrogenic induction. Error bars represent s.e.m. *Po0.05. Statistical differences were assessed with Students t-test. (i) Calcication evaluation after osteogenic induction. Error bars represent s.e.m. ***Po0.001. Statistical differences were assessed with Students t-test. (j,k) qRTPCR analyses of mesodermal gene expression in C3H10T1/2-V and

C3H10T1/2-VM cells in the presence or absence of induction with BMP2. mRNA levels in C3H10T1/2-V cells without induction were normalized to 1. Error bars represent s.e.m. **Po0.01, ***Po0.001. Statistical differences were assessed with Students t-test. BMP, bone morphogenetic protein; TGFb, transforming growth factor b.

the human experiment is transient while, in mouse experiments, Mkx is genetically deleted.

Egr1 is also an important transcription factor to induce type I collagen tissue50. However, Egr1 did not induce Mkx50 expression and our experiments revealed that Mkx did not induce Egr1 expression (Fig. 5b). These ndings indicate that that the two transcription factors induce type I collagen formation via different pathways. Whether Egr1 is an important factor in the AF is still unknown, and needs to be considered in future research.

Our data also indicated that Mkx is expressed in adult HAF cells, suggesting a potential role for Mkx in adult IVD tissues (Fig. 3b,d). Although the MKX expression in HAF cells during the aging process is unknown so far, we recently reported that MKX expression was decreased with age in the human anterior cruciate ligament13. It is of interest to analyse Mkx expression in AF cells during aging or disease.

The function of AF as spacer and cushion between spines can be established by its multilayer composition. Ligament-like tissues are dominant in the OAF, whereas cartilage-like tissues are more

abundant in the IAF6,40,51. The gradual transition is shadowing the differences between expressional levels of Mkx and Sox9 in AF. In Mkx / mice, this transition between IAF and OAF layers are disturbed and the expression of Sox9 was upregulated in Mkx knockdown OAF cells (Fig. 3b). In addition, Mkx overexpression in C3H10T1/2 cells downregulated the expression of Sox9 (Fig. 5c,f). Consistent with this observation, it is reported that Sox9 expression is downregulated in OAF cells during the differentiation of OAF cells from the Scx /Sox9 progenitor cells44,52. These results suggest that multilayer composition between OAF and IAF might be partly regulated by reciprocal regulation between Mkx and Sox9.

Furthermore, Mkx-induced C3H10T1/2 acquired greater responsiveness to TGFb, but reduced responsiveness to BMP (Fig. 6ae). As TGFb has a function not only to promote the differentiation of AF from the sclerotome but also to prevent chondrocyte differentiation in the presumptive IVD32,53, this shift in signalling responsiveness triggered by Mkx may be among the underlying molecular mechanisms maintaining Mkx-dependent AF homeostasis.

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Figure 7 | Mkx-induced mesenchymal stem cells form tissue consisting of type I collagen. (a,b) Strategy for testing the type I collagen synthesis ability of C3H10T1/2 cells induced by Venus (C3H10T1/2-V or Venus) and Venus-Mkx (C3H10T1/2-VM or Venus-Mkx) after subcutaneous transplantation. (c,e) Gross appearance of products 8 weeks after subcutaneous transplantation (c: C3H10T1/2-V; e: C3H10T1/2-VM). Scale bars, 1 mm. (d,f) IHC of these products (d: C3H10T1/2-V; f; C3H10T1/2-VM). Blue, Hoechst; green, anti-GFP; red, anti-Col1a1. Scale bars, 50 mm. (g,h) Images of transmission electron microscopy (g: C3H10T1/2-V; h: C3H10T1/2-VM). * The area of the collagen gel. Scale bars, 500 nm. (i) Histogram of the diameter of collagen brils from C3H10T1/2-V and C3H10T1/2-VM cells. Blue bar, C3H10T1/2-V; red bar, C3H10T1/2-VM. (j) The calculated mean of the diameter of 100 collagen brils from C3H10T1/2-V and C3H10T1/2-VM cells. The mean diameter of 100 collagen brils was calculated: for C3H10T1/2-V cells, meandiameter 46.99 nm (s.d. 7.409 nm); for C3H10T1/2-VM cells, mean diameter 147.32 nm (s.d. 54.54 nm). Error bars represent the s.d. **Po0.01.

Statistical differences were assessed with Students t-test. GFP, green uorescent protein.

The collapse of either AF or NP may cause the tissue degeneration of the whole IVD. The collapse of NP and loss of NP property will cause a decrease of mechanical resistance of the AF, followed by the degeneration of the entire IVD30,40,54,55. To regain normal NP property, introduction of several growth factors or MSCss into NP has been reported in animal models and in clinical trials5660.

Conversely, in this report, after the initial degradation of AF in Mkx / mice, we also observed the following degenerative

changes of the NP tissue, where Mkx is not expressed (Fig. 1c). This phenotype may be explained as secondary changes due to insufcient mechanical property of the AF, as previous reports19,21. The abolishment of critical matrix molecules in the AF of mice, such as BGN19,21 and secreted protein acidic rich in cysteine (SPARC)61, have been shown to cause the whole IVD degeneration. In these knockout mice, including Mkx / mice, morphologically resembling chondrocytes were seen in the degenerative NP, the origin of which is unclear. Further cell

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Figure 8 | Mkx-induced mesenchymal stem cells promote functional AF regeneration. (a) Strategy for assessing type I collagen synthesis of C3H10T1/2 cells induced by Venus (C3H10T1/2-V or Venus) and Venus-Mkx (C3H10T1/2-VM or Venus-Mkx) after outer AF (OAF) transplantation. (b) OAF before creating a transplantation site. Scale bar, 200 mm. (c) OAF after creating a transplantation site. Scale bar, 200 mm. (d) Cut surface of intervertebral disc (IVD) after creating a transplantation site. Yellow arrow, transplantation site. Scale bar, 200 mm. (e) Schema of the modied tail-looping model. Black arrow, transplanted site. (f,h) Haematoxylin and eosin staining of the transplantation sites of the OAF (f: C3H10T1/2-V; h: C3H10T1/2-VM). * The area in which the collagen gel was dissolved. ** Newly synthesized tissue. *** OAF. Scale bars, 50 mm. (g,i) IHC of these products (g: C3H10T1/2-V; i: C3H10T1/2-VM).

Blue, Hoechst; green, anti-GFP. Scale bars, 50 mm. (j,k) Images of transmission electron microscopy (j: C3H10T1/2-V; k: C3H10T1/2-VM). Scale bars, 500 nm. (l) The calculated mean of the diameter of 100 collagen brils from C3H10T1/2-V and C3H10T1/2-VM cells. The mean diameter of 100 collagen brils was calculated: for C3H10T1/2-V cells, mean diameter 53.72 nm (s.d. 7.281 nm); for C3H10T1/2-VM cells, mean diameter 83.54 nm

(s.d. 16.34 nm); for the intact OAF of C3H/HeSlc mice, mean diameter 77.74 nm (s.d. 24 nm). Error bars represent the s.d. **Po0.01.

(m,n) Representative images of IVDs in each group. Safranin O fast green staining. (m) Whole images of each group. Transplantation sites of C3H10T1/2-V or C3H10T1/2-VM cells are on the left side in the images. Scale bars, 300 mm. (n) Images with a focus on the transplanted side of the AF. Scale bars, 100 mm. (o) Histological grading score of the AF using the Nishimura system. The mean average grade: control 2.4 (s.d. 0.5164); Venus 4

(s.d. 0.4082); Venus-Mkx 2.2 (s.d. 0.7528). **Po0.01. Statistical differences were assessed with Students t-test. GFP, green uorescent protein.

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lineage analysis, such as Shh analysis for notochord lineage41,62,63, is required to understand the phenotype in detail.

Regarding the regeneration of damaged OAF, the transplantation of OAF cells or MSCs into injury sites of IVD in animal models have been reported6466. However, the results of these trials have not been satisfactory at some points. The transplanted OAF cells could not maintain its property as OAF cells and were unable to synthesize sufcient collagen bres64. As for the transplantation of MSCs, the differentiation from MSC to OAF cells is not well directed, resulting in the lack of sufcient collagen bre synthesis65,66. In an attempt to overcome these issues, we applied MSCs expressing Mkx, and successfully directed the MSCs to differentiate into OAF-like cells. These Mkx-induced C3H10T1/2 cells acquired the ability to produce enough number and size of collagen brils and subsequently formed collagenous tissues. Consistent with this nding, we previously observed that MKX overexpression in human bone marrow MSCs induced multiple ligament-related genes including SCX and COL1A1, and SLRPs14. Although the overexpression of SCX in human bone marrow MSCs induced only COL1A1 and TNMD14, examination of the combination of MKX and SCX in the formation OAF cells may be worth trying.

Although the lamellar structure of OAF was not fully reconstituted, the regenerated OAF tissues by transplanted Mkx-induced C3H10T1/2 cells had sufcient physical biomechanical property, which was tested and conrmed by the tail-looping model experiment (Fig. 8mo).

These results support the potential application of Mkx-induced human MSCs transplantation in human disc degenerative diseases, including disc herniation, and may also improve the current OAF regeneration trial by simple MSC introduction60,67. More detailed mechanical evaluation of the intervertebral disc should be performed, using a larger animal model, to test resistance to twisting motion68.

The regulatory mechanisms of specic and consistent expression of Mkx in OAF cells remain unclear. Identication of the enhancer region and its regulatory molecules may provide us with more insight for the application of OAF regeneration therapy via Mkx expression.

Methods

Animals. Venus knock-in Mkx heterozygous mutant mice in a C57BL/6N background carried an insertion of a Venus cassette that inactivates the Mkx gene. We inactivated the Mkx gene by homologous recombination in embryonic stem cells using a targeting vector to replace the Mkx gene from the translation start site to the end of exon 2 with the Venus gene and PGK-neomycin-resistance (PGKneo) cassette11. Male C57BL/6N and C3H/HeSlc mice were purchased from the Sankyo Laboratory (Tokyo, Japan). This animal study was approved by the Tokyo Medical and Dental University ethical committee.

Human samples. The human samples (nine discs from ve human donors; Supplementary Table 1) were obtained from the organ banks through the commercial source (Cosmo Bio USA, Carlsbad, CA). The informed consent was obtained from families of donors through the organ bank.

Cell culture. C3H10T1/2 cells were grown in a-MEM with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin (PS; all Sigma-Aldrich). PLAT-E cells were grown in DMEM plus 10% FBS (all Sigma-Aldrich). HAF cells (Articular Engineering) were grown in DMEM: Nutrient Mixture F-12 HAM (DMEM:F12-HAM) plus 20% FBS, 1% PS and 20 mg ml 1 of ascorbate. Mouse OAF cells were isolated as follows. Mkx / or C57BL/6N mice were killed by cervical dislocation and tail discs were dissected (T1/2-7/8, 10 mice per group). Using a clean bench, the discs were trimmed and pieces of OAF tissues were obtained. These tissues were incubated with Triple Express (Gibco) for 30 min and 0.25% Liberase (Roche) for 75 min (Supplementary Fig. 2ac). Isolated cells were grown in a-MEM with 20% FBS and 1% PS (Invitrogen) until day 5, at which point the medium was changed to a-MEM plus 10% FBS and 1% PS (Invitrogen). Cells were retrieved in passage 1 and processed for qRTPCR analysis. The purity of AF cells was conrmed by examining the number of Venus-positive cells in the isolatedMkx / cells (Supplementary Fig. 2d). All cells were cultured at 37 C in 5% CO2.

Three-dimensional culture. To perform the three-dimensional cell culture, cells were trypsinized and suspended in a-MEM and then mixed with PanaceaGel (Menicon Life Science) solution at a volume ratio of 1:2. The nal concentration of the cells in the hydrogel was 2 106 cells per ml. The cells were grown in a-MEM

plus 10% FBS and 1% PS (Invitrogen), and incubated in 5% CO2 at 37 C. The medium was replaced with fresh medium every 23 days. The gels containing cells were subsequently used for IHC.

Knockdown of MKX in HAF cells. Fifty picomoles of small interfering RNA for MKX (Silencer Select, s49084 and s49085, Thermo Fisher) or negative control (Silencer Select Negative Control No.1, 4390843, Thermo Fisher) was transfected into HAF cells (8 104 cells per well) using 8 ml of Lipofectamine RNAiMAX

(Invitrogen) in six-well plates. After 72 h, total RNAs and cell lysates were extracted and used for qRTPCR and western blotting, respectively.

Retrovirus infection. Venus or Venus-Mkx coding sequences were inserted into the pMIGR vector (Addgene). The pMIGR-Venus construct (as a control) or the pMIGR-Venus-Mkx construct was transfected into PLAT-E cells using FugeneHD (Promega). Forty-eight hours later, media were collected, ltered and transferred to the C3H10T1/2 stable cell line in a medium containing puromycin (5 mg ml 1).

The C3H10T1/2 cells induced with Venus-Mkx are referred to as C3H10T1/2-VM (or Venus-Mkx), and the C3H10T1/2 cells induced only with Venus are referred to as C3H10T1/2-V (or Venus). No clear differences were observed between C3H10T1/2 and C3H10T1/2-V cells in terms of morphology and gene expression. Therefore, C3H10T1/2-V cells were used as control cells.

Two mouse models for transplantation. C3H/HeSlc mice (male, 10 weeks of age, n 3 per group) were anaesthetized with pentobarbital sodium (Somunopentyl;

Kyoritsu Seiyaku). Dorsal skin pockets were made and type I collagen gels (Cellmatrix; Kurabo) containing C3H10T1/2-V or C3H10T1/2-VM cells were embedded. The gels containing the cells were prepared in advance, as follows. Trypsinized cells were resuspended in a collagen solution at a concentration of4 107 cells per ml, and 60 ml per well of this solution was placed in a 96-well plate

(Cellstar; Greiner Bio-One). After incubation at 37 C in 5%CO2 and gelation, the gels were embedded in the dorsal skin pockets and the skin was sutured. After 8 weeks, the mice were killed by cervical dislocation and the gels were excised. The gels were processed for IHC and TEM.

C3H/HeSlc mice (male, 10 weeks of age, n 3 per group) were anaesthetized as

described above. An incision was made in the dorsal skin of the tail, exposing the discs between the tail tendons. An external xator was attached using 20 G needles, and ve discs (C3/47/8) were xed. Using a microscope, the AF of three discs was then cut in a box shape using a 20 G needle and removed while being careful to avoid NP prolapse. A solution of liqueed collagen gel containing C3H10T1/2-V or C3H10T1/2-VM cells (4 107 cells per ml) was placed in the cavity and the skin

was sutured. After 8 weeks, the mice were killed by cervical dislocation and the discs were excised and processed for histochemistry, IHC and TEM.

Modied tail-looping model. We modied the tail-looping model that Sakai et al. created38. C3H/HeSlc mice (male, 10 weeks of age, n 5 per group) were

anaesthetized and mouse models for transplantation of C3H10T1/2-V or C3H10T1/2-VM cells into the OAF were created as described above. After 8 weeks, these mice were re-anaesthetized, external xators were removed and the tails were looped at a xed position between vertebrae 2 and 9 using a 0.88 mm stainless steel wire. The xed distance of vertebrae 2 and 9 was unied to 5 mm. The distal tail end was excised. As a control group, the same treatment was applied to C3H/HeSlc mice (male, 18 weeks of age, n 5) with the AF intact. After 4 weeks, the mice were

killed by cervical dislocation and the discs were excised and processed for histochemistry. C5/6 was analysed. To evaluate statistical differences in this study, a minimum size ove 5 mice per group is needed. Mice were allocated to each group randomly.

Histochemistry and IHC. For histochemistry, mouse spines were obtained after killing by cervical dislocation (E14.5, 18.5, P1 and 4 weeks of age: C57BL/6N; n 3,

10 weeks, 6 months, 12 months and 21 months of age: three groups of littermates, wild type; n 45, Mkx / ; n 5, 18 weeks of age: C3H/HeSlc, n 3 per group).

The samples were xed in 4% paraformaldehyde (PFA) in PBS for 2 days and decalcied with 10% EDTA/PBS (pH 7.17.2) for 14 days. The samples were

then processed for parafn sectioning (10 mm) and stained with Safranin O fast green stain (Wako) or Massons trichrome stain (Promega).

For IHC, mouse spines were obtained after killing by cervical dislocation (E14.5, E18.5, P1, P4 and10-week-old Mkx / , 18-week-old C3H/HeSlc, 10-week-old C57BL6/N or Mkx / , 21-month-old Mkx / or Mkx / ). The samples were xed in 4% PFA/PBS for 22.5 h and processed for frozen sectioning (510 mm). The sections were incubated with 10% EDTA/PBS (pH 7.17.2) for 1 h (GFP and Collagen 1), PBS for 10 min (Collagen 14, Bgn and CD24) or 0.1% Tween 20/PBS for 10 min (KRT18). The samples were blocked in Blocking One solution (Nacalai Tesque) for 3060 min, and then incubated with an anti-GFP antibody (1:500, MBL), anti-Collagen 1 antibody (1:500, ab34710, Abcam),

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anti-Collagen 14 antibody (1:100, LS-C119470, LSBio), anti-Bgn antibody(1:100, LS-C341858, LSBio), anti-CD24 antibody (1:100, 553262, Bioscience) and anti-KRT18 antibody (1:250, ab668, Abcam) overnight at 4 C. The sections were then incubated with an Alexa Fluor 488 donkey anti-rabbit antibody (1:500,Life Technologies), an Alexa Fluor 594 donkey anti-rabbit antibody (1:500;

Life Technologies), an Alexa Fluor 594 donkey anti-mouse antibody (1:500; Life Technologies) or Hoechst 33342 (1:2,000; Life Technologies) for 1 h. The sections for Collagen 14, Bgn, CD24 and KRT18 were mounted with VECTASHIELD Antifade Mounting Medium with 4,6-diamidino-2-phenylindole (H-1200, Vector Laboratories) without Hoechst 33342 incubation.

Human spines were obtained (nine discs from ve humans; Supplementary Fig. 1a) and processed for parafn sectioning (2 mm). All discs were used for IHC analyses. The sections were rst activated with 3% H2O2 for 10 min and with 1%

citric acid buffer at 98100 C for 10 min in a microwave oven. The sections were then blocked with Blocking One solution (Nacalai Tesque) for 60 min and incubated with a rabbit anti-human MKX antibody (1:500, LS-C30267; LifeSpan Biosciences) overnight at 4 C. The sections were then incubated with a rabbit Alexa Fluor 488 donkey anti-rabbit antibody (1:500; Life Technologies) and Hoechst 33342 (1:2000; Life Technologies) for 1 h. Conrmable signals in10 views were recorded in three elds per view, and the sum and percentage

of Hoechst-positive MKX-positive cells were calculated.

In situ hybridization. Mouse embryos were obtained after euthanasia by cervical dislocation (E14.5 of Mkx / mice). The samples were xed overnight in 4%

PFA/PBS and processed for frozen sectioning (16 mm). Sections were treated with 10 mg ml 1 proteinase K (Roche) for 10 min at room temperature (RT) and then acetylated with acetylation solution for 20 min at RT. Pre-hybridization(in 5 SSC (pH 4.5), 50% formamide, 1% SDS, 50 mg ml 1 yeast tRNA (Roche)

and 50 mg ml 1 heparin (Nacalai Tesque)) was performed at 68 C for 2 h; then, a DIGRNA probe (500 ng ml 1) was added and hybridized for 14 h at 68 C.

Subsequently, sections were subjected to a series of post-hybridization washes in wash buffers containing formamide, SSC, SDS and 0.05% CHAPS. After blocking with 2% BM Blocking reagent (Roche) containing 0.1% Tween 20 (TBST) for 30 min at RT, embryos were incubated with anti-DIG-AP Fab antibody fragments (Roche) and 1% sheep serum in TBST for 3 h at RT. After a series of washes with TBST, embryos were equilibrated with NTMT (5 M NaCl, 1 M TrisHCl (pH 9.5), 1 M MgCl2 and 0.1% Tween 20). Colour development reactions were performed at 4 C or RT with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche).

Electron microscopy analysis. Lumbar discs (L4/5 level) from Mkx / or wild-type mice (10 weeks of age, male, one per group of littermates) and tail discs from 8-week postoperative C3H/HeSlc mice (18 weeks of age, n 3 per group)

were dissected and xed in 2.5% glutaraldehyde solution for 2 h and 1% osmium solution (Sigma-Aldrich) for 1 h. After xation, tissues were processed for sectioning (1 mm).

For scanning electron microscopy, the specimens were dried in a critical point drying apparatus (HCP-2; Hitachi, Tokyo, Japan) with liquid CO2 and were spatter-coated with platinum, and then examined by scanning electron microscopy (S-4500; Hitachi, Tokyo, Japan, voltage: 15 kV, current: 10 mA and resolutions:1.5 nm).

For TEM, the specimens were embedded in Epon 812. Sliced at ultrathin 90 nm, it were collected on copper grids and were double-stained with uranyl acetate and lead citrate, and then observed using TEM (H-7100, Hitachi, Tokyo, Japan, voltage: 75 kV, current: 15 mA and resolutions: 0.38 nm).

The Image J software was used for the measurement of collagen bril diameters (100 collagen brils were measured three times using different views. Two blinded investigators measured the results).

Microcomputerized tomography. To evaluate intact spines, microcomputerized tomography (Aloka Latheta LCT 200; Hitachi, Japan, voltage, 80 kV; current,0.5 mA; and resolution, 60 mm) was used after the mice were killed by cervical dislocation (10 weeks, and 6, 12 and 21 months of age: three groups of littermates, wild type; n 45, Mkx / ; n 4, the same samples that were used for his

tochemistry). The scan was done in air.

Chondrogenic and osteogenic differentiation. C3H10T1/2, C3H10T1/2-V and C3H10T1/2-VM cells were stimulated for chondrogenic and osteogenic differentiation. Chondrogenic differentiation was performed as follows. Trypsinized cells were resuspended in a-MEM (Sigma-Aldrich) at a concentration of 2 107 cells per ml 1, and a 20 ml drop of this cell suspension was placed in the

centre of a well in a 12-well plate (Cellstar; Greiner Bio-One). The cells were allowed to adhere for 2 h at 37 C and 5% CO2, and 1 ml of a-MEM (Sigma-Aldrich) containing BMP2 (100 ng ml 1) was added to the culture.

Chondrogenic differentiation was analysed by alcian blue staining, acidic mucopolysaccaride quantication and qRTPCR after 9 days of culture. Acidic mucopolysaccaride quantication was performed using the Acidic Mucopolysaccaride Assay kit (AK03, Cosmo Bio). Osteogenic differentiation was performed as follows. Trypsinized cells were resuspended in a-MEM

(Sigma-Aldrich) at 8 104 cells per well in a 12-well plate (Cellstar; Greiner

Bio-One). The cells were incubated for 24 h, after which the medium was replaced with the induction medium (a-MEM, 10% FBS, 10 nM dexamethasone, 50 mM ascorbic acid, 10 mM b-glycerophosphate and 100 ng ml 1 of BMP2). Osteogenic differentiation was analysed by alizarin red staining, calcication evaluation and qRTPCR after 14 days of culture. Calcication evaluation was performed using the Calcication Evaluation Set (CSR-ARD-SET; Cosmo Bio). PBS-washed cells were xed with 4% PFA/PBS and then stained with Alizarin Red Solution (CSR-ARD-A1; Cosmo bio). After washing the cells, the alizarin red dye was extracted with Calcied Nodule Extraction Solution (CSR-ARD-E1; Cosmo Bio), and the absorbance at 450 nm was determined using a microplate reader.

Adipogenic differentiation. Trypsinized cells were resuspended in medium and added at 1.2 104 per well in a 12-well plate. They were cultured until they

reached 100% conuency, and then placed in induction medium (a-MEM, 10% FBS, 1 mM dexamethasone, 0.5 mM IBMX and 10 mg ml 1 insulin). After 3 days, the medium was changed to maintenance medium (a-MEM, 10% FBS and10 mg ml 1 insulin). Two mediums were then changed alternately every 3 days.

Adipogenic differentiation was analysed by microscopy and qRTPCR after 14 days of culture.

Alcian blue staining. The cells were rst xed in 4% PFA/PBS for 10 min, then incubated in 0.1 N HCl for 5 min and exposed to 0.5% Alcian blue 8GX/0.1 N HCl (pH 1.0) for 3 h. After destaining with 0.1 N HCl, the cells were evaluated for Alcian blue staining using a stereomicroscope. The samples were prepared from three independent clones from each condition.

Alizarin red staining. The cells were rst xed in 4% PFA/PBS for 30 min, then exposed to 2% Alizarin red S (Sigma-Aldrich) for 5 min. After washing with diluted water, the cells were evaluated for Alizarin red staining. The samples were prepared from three independent clones from each condition.

Quantitative real-time PCR. Total RNA was extracted from cell cultures(OAF cells of C57/BL6N, OAF cells of Mkx / , HAF, C3H10T1/2, C3H10T1/2-

V, C3H10T1/2-VM and cells after two mesodermal differentiations). qRTPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). The primer sequences used for qRTPCR are listed in the Supplementary Table 2 (mouse) and Supplementary Table 3 (human). The results are expressed as messenger RNA (mRNA) levels corrected to GAPDH levels in each sample. Total RNAs were prepared at least three independent cell samples from each experiment.

Microarray analysis. DNA microarray analysis was performed using Affymetrix mouse genome 430 2.0 array. RNA samples were obtained from C3H10T1/2, C3H10T1/2 overexpressing Venus and C3H10T1/2 overexpressing Venus-Mohawk. Total RNA (200 ng) was reverse-transcribed and biotinylated using the GeneChip 30 IVT Express kit (Affymetrix). The microarray datawere summarized using the MAS 5.0 method. Microarray data that support the ndings of this study have been deposited in GEO with the primary accession code GSE81276.

Western blotting. Whole-cell proteins were extracted from C3H10T1/2-V and C3H10T1/2-VM cells, and HAF cells. Cellular proteins collected in SDSPAGE sample buffer were electrophoresed on 412% SDS-bis-ris gels and transferred to polyvinylidene diuoride membranes. Membranes were blocked in Blocking One solution (Nacalai Tesque) for 3060 min. After blocking, the membranes were incubated overnight at 4 C with dilutions of anti-Smad1 (1:500, #9743; Cell Signaling Technology), anti-phospho-Smad1/5/8 (1:500, #9511; Cell Signaling Technology), anti-Smad2/3 (1:500 BD #610843), anti-phospho-Smad3 (1:1,000 Abcam Ab52903), anti-Sox9 (1:1,000 Millipore AB5535), anti-Bgn (1:1,000 LSBio LS-C341858), anti-Col1 (1:250 Abcam ab21286), anti-Dcn (1:250 Abcam ab137505), anti-b-actin (1:1,000 SIGMA A5316) and anti-GAPDH (1:2,000,

MAB374; Millipore) antibodies. Rinsed membranes were incubated for 1 h with ECL mouse or rabbit IgG, horseradish peroxidase-Linked Whole antibody(GE Healthcare). The enhanced chemiluminescence immunoblotting detection system (Thermo Scientic) was used to detect the antigenantibody complexes. Extracts were prepared from three independent samples. Full scans of the key immunoblots are indicated to Supplementary Fig. 12.

Luciferase assay. C3H10T1/2-V or C3H10T1/2-VM cells were seeded at 1 104

cells per well in 96-well plates (Cellstar; Greiner Bio-One). On the following day, the ID1-Bre luciferase construct (Addgene)35 was transfected using FugeneHD (Promega). Twenty-four hours later, the medium was changed to a-MEM plus0.5% FBS and 1% PS. Twelve hours later, BMP2 at 100 ng ml 1 was added to the medium. Ten hours later, luciferase activity was quantied using the luciferase assay (Promega) on a luminometer (ARVO 3; PerkinElmer). The assay was

performed three times.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12503 ARTICLE

Scoring system of IVD degeneration. We used the IVD degenerative histological score30 to evaluate the whole IVD of ageing wild-type and Mkx / mice. Two blinded orthopedic surgeons performed each image evaluation and each scoring.

To evaluate the AF of modied tail-looping model mice, we used the Nishimura system39, a histological grading system of degenerative changes in the AF. This system is classied into six grades (grade 0: normal structure; grade 1: mildly serpentine with rupture; grade 2: moderately serpentine with rupture; grade 3: severely serpentine with mildly reversed contour; grade 4: severely reversed contour; grade 5: indistinct).

Statistical analysis. Data are presented as the means.e.m. The Students t-test was used to evaluate differences between groups. A one-tailed t-test was performed for Figs 6e and 8l,o, while a two-tailed t-test was performed for the other gures. P valueso0.05 were considered signicant. *Po0.05. **Po0.01. ***Po0.001.

Data availability. Microarray data that support the ndings of this study have been deposited in GEO with the primary accession code GSE81276. The authors declare that all other data supporting the ndings of this study are available within the article and its les.

References

1. Hansson, E. & Hansson, T. The cost-utility of lumbar disc herniation surgery. Eur. Spine J. 16, 329337 (2007).

2. Wilke, H. J., Heuer, F., Neidlinger-Wilke, C. & Claes, L. Is a collagen scaffold for a tissue engineered nucleus replacement capable of restoring disc height and stability in an animal model? Eur. Spine J. 15 (Suppl 3) S433S438 (2006).

3. Bron, J. L., Helder, M. N., Meisel, H. J., Van Royen, B. J. & Smit, T. H. Repair, regenerative and supportive therapies of the annulus brosus: achievements and challenges. Eur. Spine J. 18, 301313 (2009).

4. Yu, J. et al. Microbrils, elastin bres and collagen bres in the human intervertebral disc and bovine tail disc. J. Anat. 210, 460471 (2007).

5. Eyre, D. R. & Muir, H. Types I and II collagens in intervertebral disc. Interchanging radial distributions in annulus brosus. Biochem. J. 157, 267270 (1976).

6. Kepler, C. K., Ponnappan, R. K., Tannoury, C. A., Risbud, M. V. & Anderson,D. G. The molecular basis of intervertebral disc degeneration. Spine J. 13, 318330 (2013).7. Holm, S., Maroudas, A., Urban, J. P., Selstam, G. & Nachemson, A. Nutrition of the intervertebral disc: solute transport and metabolism. Connect. Tissue Res. 8, 101119 (1981).

8. Hunter, C. J., Matyas, J. R. & Duncan, N. A. The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering. Tissue Eng. 9, 667677 (2003).

9. Aszodi, A., Chan, D., Hunziker, E., Bateman, J. F. & Fassler, R. Collagen II is essential for the removal of the notochord and the formation of intervertebral discs. J. Cell Biol. 143, 13991412 (1998).

10. Yokoyama, S. et al. A systems approach reveals that the myogenesis genome network is regulated by the transcriptional repressor RP58. Dev. Cell 17, 836848 (2009).

11. Ito, Y. et al. The Mohawk homeobox gene is a critical regulator of tendon differentiation. Proc. Natl Acad. Sci. USA 107, 1053810542 (2010).

12. Liu, W. et al. The atypical homeodomain transcription factor Mohawk controls tendon morphogenesis. Mol. Cell. Biol. 30, 47974807 (2010).

13. Nakahara, H. et al. Transcription factor Mohawk and the pathogenesis of human anterior cruciate ligament degradation. Arthritis Rheum. 65, 20812089 (2013).

14. Otabe, K. et al. Transcription factor Mohawk controls tenogenic differentiation of bone marrow mesenchymal stem cells in vitro and in vivo. J. Orthop. Res. 33, 18 (2015).

15. Liu, H. et al. Mohawk promotes the tenogenesis of mesenchymal stem cells through activation of the TGFbeta signaling pathway. Stem Cells 33, 443455 (2015).

16. Kayama, T. et al. Gtf2ird1-dependent Mohawk (Mkx) expression regulates mechanosensing properties of tendon. Mol. Cell. Biol. 36, 12971309 (2016).

17. Anderson, D. M. et al. Mohawk is a novel homeobox gene expressed in the developing mouse embryo. Dev. Dyn. 235, 792801 (2006).

18. Dahia, C. L., Mahoney, E. J., Durrani, A. A. & Wylie, C. Postnatal growth, differentiation, and aging of the mouse intervertebral disc. Spine (Phila Pa. 1976) 34, 447455 (2009).

19. Gotz, W. et al. Immunohistochemical localization of the small proteoglycans decorin and biglycan in human intervertebral discs. Cell Tissue Res. 289, 185190 (1997).

20. Cs-Szabo, G. et al. Changes in mRNA and protein levels of proteoglycans of the anulus brosus and nucleus pulposus during intervertebral disc degeneration. Spine (Phila Pa. 1976) 27, 22122219 (2002).

21. Furukawa, T. et al. Absence of biglycan accelerates the degenerative process in mouse intervertebral disc. Spine (Phila Pa. 1976) 34, E911E917 (2009).

22. Schweitzer, R. et al. Analysis of the tendon cell fate using Scleraxis, a specic marker for tendons and ligaments. Development 128, 38553866 (2001).

23. Tang, X., Jing, L. & Chen, J. Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging. PLoS ONE 7, e52020 (2012).24. Risbud, M. V. et al. Dening the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting. J. Orthop. Res. 33, 283293 (2015).

25. Tang, X. et al. Identifying molecular phenotype of nucleus pulposus cells in human intervertebral disc with aging and degeneration. J. Orthop. Res. doi: 10.1002/jor.23244 (2016).

26. Minogue, B. M., Richardson, S. M., Zeef, L. A., Freemont, A. J. & Hoyland, J. A. Transcriptional proling of bovine intervertebral disc cells: implications for identication of normal and degenerate human intervertebral disc cell phenotypes. Arthritis Res. Ther. 12, R22 (2010).

27. Lv, F. et al. In search of nucleus pulposus-specic molecular markers. Rheumatology (Oxford) 53, 600610 (2014).

28. Masuda, K. et al. Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model. Spine (Phila Pa. 1976) 31, 742754 (2006).

29. Lipson, S. J. & Muir, H. Vertebral osteophyte formation in experimentaldisc degeneration. Morphologic and proteoglycan changes over time. Arthritis Rheum. 23, 319324 (1980).

30. Boos, N. et al. Classication of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine (Phila Pa. 1976) 27, 26312644 (2002).

31. Sakai, D. & Andersson, G. B. Stem cell therapy for intervertebral disc regeneration: obstacles and solutions. Nat. Rev. Rheumatol. 11, 243256 (2015).

32. Sohn, P., Cox, M., Chen, D. & Serra, R. Molecular proling of the developing mouse axial skeleton: a role for Tgfbr2 in the development of the intervertebral disc. BMC Dev. Biol. 10, 29-213X-10-29 (2010).

33. Dahia, C. L., Mahoney, E. J., Durrani, A. A. & Wylie, C. Intercellular signaling pathways active during intervertebral disc growth, differentiation, and aging. Spine (Phila Pa. 1976) 34, 456462 (2009).

34. Tim Yoon, S. et al. The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro. Spine (Phila Pa. 1976) 28, 17731780 (2003).

35. Korchynskyi, O. & ten Dijke, P. Identication and functional characterization of distinct critically important bone morphogenetic protein-specic response elements in the Id1 promoter. J. Biol. Chem. 277, 48834891 (2002).

36. Havis, E. et al. Transcriptomic analysis of mouse limb tendon cells during development. Development 141, 36833696 (2014).

37. Lorda-Diez, C. I., Montero, J. A., Martinez-Cue, C., Garcia-Porrero, J. A. & Hurle, J. M. Transforming growth factors beta coordinate cartilage and tendon differentiation in the developing limb mesenchyme. J. Biol. Chem. 284, 2998829996 (2009).

38. Sakai, D. et al. Migration of bone marrow-derived cells for endogenous repair in a new tail-looping disc degeneration model in the mouse: a pilot study. SpineJ. 15, 13561365 (2015).39. Nishimura, K. & Mochida, J. Percutaneous reinsertion of the nucleus pulposus. An experimental study. Spine (Phila Pa. 1976) 23, 15311538 (1998).

40. Guterl, C. C. et al. Challenges and strategies in the repair of ruptured annulus brosus. Eur. Cell. Mater 25, 121 (2013).

41. Choi, K. S. & Harfe, B. D. Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs. Proc. Natl. Acad. Sci. USA 108, 94849489 (2011).

42. Maier, J. A., Lo, Y. & Harfe, B. D. Foxa1 and Foxa2 are required for formation of the intervertebral discs. PLoS ONE 8, e55528 (2013).

43. Smits, P. & Lefebvre, V. Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and development of the nucleus pulposus of intervertebral discs. Development 130, 11351148 (2003).

44. Barrionuevo, F., Taketo, M. M., Scherer, G. & Kispert, A. Sox9 is required for notochord maintenance in mice. Dev. Biol. 295, 128140 (2006).

45. Tsai, T. T. et al. Fibroblast growth factor-2 maintains the differentiation potential of nucleus pulposus cells in vitro: implications for cell-based transplantation therapy. Spine (Phila Pa. 1976) 32, 495502 (2007).

46. Arana, C. J., Diamandis, E. P. & Kandel, R. A. Cartilage tissue enhances proteoglycan retention by nucleus pulposus cells in vitro. Arthritis Rheum. 62, 33953403 (2010).

47. Ansorge, H. L. et al. Type XIV collagen regulates brillogenesis: PREMATURE COLLAGEN FIBRIL GROWTH AND TISSUE DYSFUNCTION IN NULL MICE. J. Biol. Chem. 284, 84278438 (2009).

48. Huang, A. H., Lu, H. H. & Schweitzer, R. Molecular regulation of tendon cell fate during development. J. Orthop. Res. 33, 800812 (2015).

49. Murchison, N. D. et al. Regulation of tendon differentiation by scleraxis distinguishes force-transmitting tendons from muscle-anchoring tendons. Development 134, 26972708 (2007).

50. Guerquin, M. J. et al. Transcription factor EGR1 directs tendon differentiation and promotes tendon repair. J. Clin. Invest. 123, 35643576 (2013).

51. Pattappa, G. et al. Diversity of intervertebral disc cells: phenotype and function.J. Anat. 221, 480496 (2012).

NATURE COMMUNICATIONS | 7:12503 | DOI: 10.1038/ncomms12503 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 13

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12503

Acknowledgements

We thank all other Asahara lab members and members of Center for Medical Research and Education, Tokai University School of Medicine for their helpful support. This work was supported by the Core Research for the Evolutionary Science and Technology (CREST) funding from Advanced Research and Development Programs for Medical Innovation from Japan Agency for Medical Research and development, AMED, JSPS KAKENHI (grant numbers: 26113008, 15H02560 and 15K15544), and grants from the National Institutes of Health (NIH; grant numbers: AR050631 and AR065379), the Takeda science foundation, Bristol-Myers K.K. RA Clinical Investigation Grant, the Japan Aerospace Exploration Agency (grant number: 14YPTK-005512), and Creation of life innovation materials for interdisciplinary and international researcher development project, Ministry of Education to H.A., grants from the NIH (grant number: AG007996) to M.L.

52. Sugimoto, Y. et al. Scx /Sox9 progenitors contribute to the establishment

of the junction between cartilage and tendon/ligament. Development 140, 22802288 (2013).53. Haschtmann, D., Ferguson, S. J. & Stoyanov, J. V. BMP-2 and TGF-beta3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossication of the annulus brosus. Eur. Spine J. 21, 17241733 (2012).

54. Antoniou, J. et al. The effect of Link N on differentiation of human bone marrow-derived mesenchymal stem cells. Arthritis Res. Ther. 14, R267 (2012).

55. Mwale, F., Wang, H. T., Roughley, P., Antoniou, J. & Haglund, L. Link N and mesenchymal stem cells can induce regeneration of the early degenerate intervertebral disc. Tissue Eng. Part A. 20, 29422949 (2014).

56. Masuda, K., Oegema, Jr T. R. & An, H. S. Growth factors and treatment of intervertebral disc degeneration. Spine (Phila Pa. 1976) 29, 27572769 (2004).

57. Sakai, D. et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials 27, 335345 (2006).

58. Zhang, Y., Chee, A., Thonar, E. J. & An, H. S. Intervertebral disk repair by protein, gene, or cell injection: a framework for rehabilitation-focused biologics in the spine. PM R 3, S88S94 (2011).

59. Leung, V. Y. et al. Mesenchymal stem cells reduce intervertebral disc brosis and facilitate repair. Stem Cells 32, 21642177 (2014).

60. Mochida, J. et al. Intervertebral disc repair with activated nucleus pulposus cell transplantation: a three-year, prospective clinical study of its safety. Eur. Cell. Mater. 29, 202212 (2015).

61. Bradshaw, A. D. & Sage, E. H. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J. Clin. Invest. 107, 10491054 (2001).

62. Harfe, B. D. et al. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118, 517528 (2004).

63. Choi, K. S., Cohn, M. J. & Harfe, B. D. Identication of nucleus pulposus precursor cells and notochordal remnants in the mouse: implications for disk degeneration and chordoma formation. Dev. Dyn. 237, 39533958 (2008).

64. Turner, K. G., Ahmed, N., Santerre, J. P. & Kandel, R. A. Modulation of annulus brosus cell alignment and function on oriented nanobrous polyurethane scaffolds under tension. Spine J. 14, 424434 (2014).

65. Pirvu, T. et al. A combined biomaterial and cellular approach for annulus brosus rupture repair. Biomaterials 42, 1119 (2015).

66. Hoogendoorn, R. J. et al. Adipose stem cells for intervertebral disc regeneration: current status and concepts for the future. J. Cell. Mol. Med. 12, 22052216 (2008).

67. Nerurkar, N. L., Elliott, D. M. & Mauck, R. L. Mechanical design criteria for intervertebral disc tissue engineering. J. Biomech. 43, 10171030 (2010).

68. Gregory, D. E., Bae, W. C., Sah, R. L. & Masuda, K. Anular delamination strength of human lumbar intervertebral disc. Eur. Spine J. 21, 17161723 (2012).

Author contributions

R.N., Y.I. and H.A. designed the research; R.N., Y.I., M.I., K.K., S.I., T.K., H.S., M.M., M.I. and H.A. performed the research; R.N., Y.I., N.O., M.L., D.S., K.M., T.O and H.A. analysed the data; and R.N. and H.A. wrote the paper.

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How to cite this article: Nakamichi, R. et al. Mohawk promotes the maintenance and regeneration of the outer annulus brosus of intervertebral discs. Nat. Commun. 7:12503 doi: 10.1038/ncomms12503 (2016).

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