Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that.

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Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that enhances expression of type II collagen M. Horie, H. Choi, R.H. Lee, R.L. Reger, J. Ylostalo, T. Muneta, I. Sekiya, D.J. Prockop Osteoarthritis and Cartilage Volume 20, Issue 10, Pages 1197-1207 (October 2012) DOI: 10.1016/j.joca.2012.06.002 Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Intra-articular injection of hMSC promoted regeneration of rat meniscus and inhibited development of osteoarthritis after the meniscectomy. (A) Representative macroscopic findings of the meniscus 2, 4, and 8 weeks after the injection of PBS (left), hMSCs (middle), or rMSCs (right). The white dotted line indicates the regenerated tissue. Scale bar, 1 mm. (B) Area of the total meniscus injected with PBS, hMSCs, or rMSCs at 2, 4, and 8 weeks. The horizontal lines are mean values; n = 5 for each group (one-way ANOVA followed by Bonferroni post-tests. P values less than 0.017 were considered to be statistically significant). (C) Representative sections of the meniscus stained with Toluidine blue (top and middle), and immunostained for type II collagen (bottom) after PBS or hMSCs injection. The staining in the PBS-treated sample was less with Toluidine blue and the antibody for type II collagen. The schema of the meniscus on the left is shown for orientation. Symbols: N, native meniscus; R, regenerated meniscus; TB, Toluidine blue; Col II, type II collagen. Scale bar, 100 μm. (D) Representative gross photographs (top) and sections (bottom) of the joint surface of the tibia at 8 weeks. The cartilage was stained with India ink to identify fibrillation and erosion. The white circle indicates the medial tibial plateau. The tibia was sectioned coronally and stained with safranin-O and fast green to identify cartilage (red). Scale bars, 2 mm (top) or 200 μm (bottom). (E) Quantification of histological analysis using the OARSI cartilage osteoarthritis histopathology grading system. Values are mean with lower and upper limit of 95% CI; n = 5 for each group (Mann–Whitney U test). Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10.1016/j.joca.2012.06.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Intra-articular injected hMSCs were activated to express Ihh, PTHLH, and BMP2. (A) Real-time RT-PCR for human-specific mRNA in regenerated meniscus 3 days after injection. Values are mean with lower and upper limit of 95% CI of fold increase over Con, normalized by ΔΔCt for human-specific GAPDH; n = 4. Symbols: Con, 20,000 hMSCs added to injured meniscus from PBS injected knee before RNA extraction; IA day 3, regenerated meniscus 3 days after intra-articular (IA) injection of hMSCs. (B) Time sequence of human gene expression by real-time RT-PCR for human PTHLH, BMP2, and Ihh in regenerated meniscus. Values are mean with lower and upper limit of 95% CI of the relative quantities (RQ) normalized to 18S rRNA reflecting total levels of both human and rat transcripts. (C–E) Immunohistochemistry of anti-human/rat Ihh (C), PTHLH (D), or BMP2 (E) 3 or 7 days after hMSC injection. Primary antibodies for Ihh and PTHLH reacting with both human and rat were used. Low magnification (top) and higher magnification (bottom) of regenerated meniscus. Right column: merged images (CM-DiI, red; Ihh/PTHLH/BMP2, green; DAPI, blue). The white dotted line indicates border between native and regenerated meniscus, and the white solid line indicates outer edge of regenerated meniscus. CM-DiI labeled hMSCs (red) expressed Ihh, PTHLH, and BMP2 (green) (arrows in merge). Note: CM-DiI negative rat-derived cells surrounding hMSCs also expressed Ihh and PTHLH (arrowhead in merge) at day 7. The schema of the meniscus on the top left (C) is shown for orientation. Symbols: N, native meniscus; R, regenerated meniscus. Scale bars, 50 μm. Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10.1016/j.joca.2012.06.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 hMSCs up-regulated expression of chondrogenic genes in rat cells. Real-time RT-PCR assay for rat-specific mRNA (Sox9, Col2a1, Ihh, PTHLH, PTH-R1, BMP2, and BMPR1A) in the regenerated meniscus 3, 7 and 14 days after PBS or hMSC injection. Values are mean with lower and upper limit of 95% CI of the RQs normalized to rat-specific GAPDH; n = 4 for each group. *P < 0.001 (Mann–Whitney U test). Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10.1016/j.joca.2012.06.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 A large number of PTHLH/PTH-R1 positive cells were found in the regenerated tissue after injection of hMSCs. (A–D) Immunohistochemistry of anti-human/rat PTHLH (A, B) or PTH-R1 (C, D) 7 days after the injection of PBS (A, C) or hMSCs (B, D). Low magnification images are shown in the left and higher magnification of the regenerated tissue is shown in the right. Staining of nuclei (DAPI, blue), CM-DiI (red), and PTHLH or PTH-R1 (green) is shown. The white dotted line indicates the border between the native meniscus and regenerated tissue, and the white solid line indicates the outer edge of the regenerated tissue. A large number of PTHLH or PTH-R1 positive cells (green) were observed in the regenerated meniscus after the injection of hMSCs (B, D). In contrast, few PTHLH or PTH-R1 positive cells were present in the PBS injection group (B, D). Note that not only CM-DiI positive human cells (arrows in merge) but also CM-DiI negative rat cells (arrowheads) expressed PTHLH or PTH-R1. Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10.1016/j.joca.2012.06.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effects of an inhibitor and an agonist of hedgehog signaling. (A) Real-time RT-PCR assay for rat-specific Col2a1 mRNA. RNA was recovered from the regenerated meniscus 2 weeks after the various treatments, including injection of PBS, hMSCs, hMSCs plus 0.1% DMSO (as a control of cyclopamine), hMSCs plus cyclopamine (50 μL of a 20 μM in 0.1% DMSO), 100 nM or 500 nM Smoothened agonist (SAG). Values are mean with lower and upper limit of 95% CI of the RQs normalized to rat-specific GAPDH; n = 4 for each group. +P = 0.005, ++P = 0.002 (one-way ANOVA followed by Bonferroni post-tests. P values less than 0.0033 were considered to be statistically significant). (B, C) Real-time RT-PCR assay for rat-specific Ptc1 (B) and Gli1 (C), downstream targets of hedgehog signaling. RNA was recovered from the regenerated meniscus 3 days after the various treatments. Values are mean with lower and upper limit of 95% CI of the RQs normalized to rat-specific GAPDH; n = 4 for each group. +P = 0.004, ++P = 0.002 in B, ++P = 0.002, +++P = 0.001 in C (one-way ANOVA followed by Bonferroni post-tests. P values less than 0.005 were considered to be statistically significant). (D) Representative macroscopic photographs of the meniscus 2 weeks after the various treatments. Scale bar: 1 mm. (E) Area of the total meniscus 2 weeks after the various treatments. Values are mean with lower and upper limit of 95% CI; n = 4 for each group (one-way ANOVA followed by Bonferroni post-tests. P values less than 0.0083 were considered to be statistically significant). Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10.1016/j.joca.2012.06.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Possible mechanism of rat meniscal regeneration by intra-articular injection of hMSCs. (A) Summary graph of time sequence of human and rat gene expression changes by species-specific real-time RT-PCR. Intra-articular injected hMSCs were activated to express human Ihh, PTHLH, and BMP2 (red) as early as day 1. Values for human genes are expressed as fold increase over values for controls (Con) of cultured hMSCs, normalized by ΔΔCt for 18S to reflect total levels of both human and rat transcripts. Rat Col2a1 gene expression in the regenerated meniscus was increased after the injection of hMSCs (blue). Values for rat genes are expressed as fold increase over values for PBS day 3, normalized by ΔΔCt for rat GAPDH. Values are means from RT-PCR data presented in Figs. 2(B) and 3. Symbols: Con, 20,000 hMSCs added to injured meniscus from PBS injected knee before RNA extraction. (B) Summary diagram of the possible mechanism for rat meniscal regeneration by hMSCs. (1) Intra-articular injected hMSCs were activated to express Ihh, PTHLH, and BMP2 when they were exposed to the in vivo microenvironment. (2) Human Ihh expressed by the hMSCs enhanced expression of rat Col2a1 gene probably through the downstream hedgehog target of rat Gli1 and then through Sox9. hMSCs did not enhance expression of rat Gli1 and Col2a1 in the presence of cyclopamine. In contrast, injection of Smoothened agonist enhanced expression of rat Gli1 and Col2a1. (3) PTHLH and BMP2 may also have enhanced expression of rat Col2a1 through rat Sox9. Symbols: X-factors, unknown factors which activate hMSCs to express Ihh, PTHLH, and BMP2. Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10.1016/j.joca.2012.06.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10. 1016/j. joca Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10. 1016/j. joca Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10. 1016/j. joca Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2012 20, 1197-1207DOI: (10. 1016/j. joca Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions