1. In-vivo induction of chondrocytes from articular cartilage stem cells by defined factors
Linkai Zhu
4/6/16
Targeting OA

2. Osteoarthritis
Image credit :

3. Osteoarthritis (OA) vs. Rheumatoid arthritis (RA)
Pap et al., 2015

4. Molecular mechanism of osteoarthritis
Loss of chondrocyte phenotypic stability is major step in OA progression.
OA stress: genetics, injury, mechanical load and inflammation
Increased MMPs and ADAMTS (extracellular protease enzymes), ECM degradation and release of mediators
Epigenetic changes, loss of CXCR2 and FGF-2
Activation of innate immunity via TLRs
Sensitization to inflammation via IL-1
Pap et al., 2015

5. Molecular mechanism of osteoarthritis
TLR4 mediated responses decrease ECM synthesis of chondrocytes.
Increased MMPs (matrix metalloproteinases) expression
Decreased aggrecan (integral part of the ECM in cartilagenous tissue) and type II collagen synthesis
Gómez et al., 2014

6. Molecular mechanism of osteoarthritis
Cytokines production in OA
TNF, IL‑1β and IL‑6 produced by chondrocytes, macrophages, T cells and osteophytes
Pro-MMPs released by synoviocytes and macrophages
Chevalier et al., 2013

7. Articular cartilage stem cells
Under injury, the cartilage would regenerate the lost chondrocytes by articular cartilage stem cells.
intact cartilage may be barrier and ECM should be degraded to induce migration of embedded cells.
Jiang et al., 2015

8. Progression of OA
An imbalance between
anabolism (chondrogenesis and migration)
catabolism (ECM breakdown for chondrocytes migration)
of the articular cartilage, particularly by an increase in catabolism.
The local microenvironment break down (overwhelming immune response).

9. Hypothesis:
The low regenerative ability of articular cartilage stem cells could be because of poor cellular signaling communication for ACSCs (lack of blood vessels). By ectopically generating suitable microenvironment with gradients of defined factors (Needs further screen), we would be able to guide the migration of both chondrocytes and ACSCs and promote their proliferation rate.

10. In-vitro induction of chondrocytes

11. Isolation and preparation of cartilage explant specimens
Methods
Isolation and preparation of cartilage explant specimens
femoral trochlea (8 human donors, knee arthroplasty due to primary osteoarthritis; mean age years and range years)
central groove created to expose deeper cartilage layers
briefly washed (native cartilage) and thorough washed (cleansed cartilage, minimize cartilage attaching cells)

12. Superficial zone facing up in 500 µl DMEM, 10% FCS and 1% Pen-Strep
Methods
Culturing
Superficial zone facing up in 500 µl DMEM, 10% FCS and 1% Pen-Strep
5 µl DMEM containing either 200 µg/ml HMGB-1, 10 mg/ml TFF3, 10 µg/ml BMP-2 or 10 µg/ml TGF-β1 applied to the groove of the explants (100X bioactive concentration)
Unspecific stimulus: 50 µl/ml cell lysate from primary human articular chondrocytes
Co-culture with pieces of the synovial membrane

13. Results
cleansed
naive
Attaching cells
Retained integrity

14. Cartilage-adjoining cells: fibroblastic morphology
Results
No cell on the surface
Cartilage-adjoining cells: fibroblastic morphology
Neither elicited outgrowth of matrix-embedded chondrocytes, nor significantly increased the number of cells adjoining “native cartilage” specimens.

15. Results
Influence of three-dimensional matrices on the number of cartilage-adjoining cells.
No change in cellular outgrowth and the number of cartilage-adjoining cells.

16. Results
Influence of three-dimensional matrices on the number of cartilage-adjoining cells.
Without an additional matrix, the co-culture with synovial membrane significantly increased the number of cartilage-adjoining cells oriented in a cell layer (b). In co-cultures with application of a fibrin matrix (c) or a collagen matrix (d), the adjoining cells were distributed three-dimensionally within matrices.
From this study, the attaching mesenchymal cells may contribute to cartilage regeneration in normal in-vivo environment.

17. In-vivo induction of chondrocytes
Existence of articular cartilage stem cells (ACSCs) in vivo and in situ for the first time;
ACSCs were activated and exhibited a transient proliferative response in early OA as an initial attempt for self-repair;
ACSCs’ activation were maintained by NF-jB pathway inhibitor, which induced cartilage regeneration, and protected articular cartilage from injury inan OA animal model.

18. Methods
Labeling and detection of ACSCs in-vivo using BrdU (quiescent) and Ki67 (active)
OA animal model generated by medial collateral ligament (MCL) transfection and medial meniscal tear of the knee joints
NF-ĸB pathway inhibitor (10µM, treatment) and DMSO (control) 1 day after the surgery (10 µl per joint per rat twice a week)
The portion of ACSCs characterization was excluded.

19. ACSCs were existent and quiescent in normal mature mouse cartilage
Results
ACSCs were existent and quiescent in normal mature mouse cartilage

20. Results

21. Results
ACSCs were activated in early OA, but the active ACSCs loss gradually as the development of OA progresses.
The highest frequencies of Ki671/BrdU1 ACSCs were found in the superficial zone (SZ) of the cartilage
Articular cartilage stem cells (ACSCs) were activated in the context of osteoarthritis (OA). (A): BrdU1 ACSCs were detected
on the cartilage of the sham group, but no Ki67 expression was observed in ACSCs. (B–D, H): In OA group, Ki67 was gradually detected
in BrdU1 ACSCs as OA progressed and maximized at 14 days after the OA-inducing surgical operation. (E–G, H): The number of transient
proliferating ACSCs (Ki671/BrdU1 ACSCs) visualized as cartilage degeneration significantly decreased. (A–G, I): The cartilage degeneration
and The Osteoarthritis Research Society grade showed the OA development. Cyan nuclear represent BrdU1 ACSCs and white
nuclear represent Ki671/BrdU1 ACSCs. *, p<.05; **, p<.01; ***, p<.001 between two groups.

22. IL-1β Suppressed the TGF-β1-Induced Chondrogenesis in Rat ACSCs
Results
IL-1β Suppressed the TGF-β1-Induced Chondrogenesis in Rat ACSCs
The proinflammatory cytokines IL-1β inhibited the TGF-b1-induced ACSCs chondrogenesis by suppressing the expression of the transcription factor (SOX9), extracellular matrix (ECM) (COL Ila and aggrecan), and related synthetase (hyaluronansynthetase 2), and these inhibitory effects on ACSCs chondrogenesis were dose-dependent.
Figure 4. IL-1β suppressed the TGF-β1 induced chondrogenesis in rat articular cartilage stem cells. (A): Dose dependence of the IL-1β-inhibited effects on aggregates cultured with increasing concentrations of IL-1β in the presence and absence of TGF-β1, which was also demonstrated by (B) pellet diameter. (C): Toluidine blue staining. (D): qPCR analysis of the four cartilage markers, namely, SOX9, COL IIa, aggrecan, and Has 2. The values are the mean and SEM of triplicate experiments. *, p < .05; ***, p < .001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

23. Results
NF-ĸB Pathway Plays an Essential Role in IL-1β -Induced Inhibition of ACSCs Chondrogenesis
NF-κB pathway has a key role in IL-1β-induced inhibition of articular cartilage stem cells chondrogenesis. (A): PAI-1 was significantly stimulated by TGF-β1 and suppressed by IL-1β in a dose-dependent manner. (B): TGF-β1 downregulated the Smad7 expression, whereas IL-1β significantly reversed the effect of TGF-β1 in a dose-dependent manner. Notably, 10 ng/ml of IL-1β completely offset 10 ng/ml of TGF-β1 suppression on Smad7 and significantly increased the Smad7 expression compared with the control (no TGF-β1) group (approximately 1.4-fold) and the TGF-β1 treatment-only group (approximately 2.6-fold). (C): Conversely, TGF-βRII was significantly induced by TGF-β1 and suppressed by IL-1β in a dose-dependent manner. The results are expressed as the percentage of controls after normalization against GAPDH. (D): Western blot results of Smad7 and TGF-βRII showed the same trend as that of qPCR results. (E): The statistics of the Western blot results. (F, G): The NF-κB pathway was inhibited by BAY (F): The suppression of p65 transport into the nucleus induced by IL-1β. (G): MMP13 downregulation. The values are the mean and SEM of triplicate experiments. *, p < .05; **, p < .01; ***, p < .001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

24. Results
NF-ĸB Pathway Inhibitor Could Restore TGF-β1-Induced Chondrogenesis Inhibited by IL-1β
NF-κB pathway inhibitor restored TGF-β1-induced chondrogenesis inhibited by IL-1β. (A–C): BAY distinctly restored TGF-β1-induced chondrogenesis inhibited by IL-1β in a dose-dependent manner. (A): Naked eye pellet size, (B) pellet diameter, and (C) qPCR analysis of SOX9, COL IIa, aggrecan, and Has 2. (D): The level of PAI-1 suppressed by IL-1β, and restored by BAY in a dose-dependent manner. (E): TGFβ-1 downregulated Smad7 expression and IL-1β markedly reversed the effect of TGF-β1. However, the IL-1β effect could be reversed by BAY  (F): Conversely, TGF-βRII was significantly induced by TGF-β1, suppressed by IL-1β, and restored by BAY in a dose-dependent manner. (G, H): Western blot results of Smad7 and TGF-βRII, which showed the same trend as that of the qPCR results. (H): The statistics of the Western blot results. The values are the mean and SEM of triplicate experiments. *, p < .05; **, p < .01; ***, p < .001 between two groups. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

25. Results
Inhibition of NF-ĸB Signaling Can Induce ACSCs Activation and Retard the Progression of Experimental OA
The NF-ĸB pathway is crucial for regulating ACSCs activity and blocking the NF-ĸB pathway to induce ACSCs activation may be a possible method for OA therapy.
Inhibition of NF-κB signaling induced articular cartilage stem cells (ACSCs) activation and retarded the progression of experimental osteoarthritis (OA). (A–I): The BrdU+ ACSCs were gradually activated (Ki67+/BrdU+ ACSCs) from the resting state in early OA, maximized in about the 14th day (A, B) in the DMSO group and the 30th day (E–G) in the BAY treatment group after the OA-inducing surgical operation. (B–D, G–J): The Ki67+/BrdU+ ACSCs in the DMSO group (after 14 days postoperation) and in the BAY treatment group (after 30 days postoperation) gradually reduced with the progression of OA. The Ki67+/BrdU+ ACSCs maintained approximately 25% on the 90th day in the BAY group (H, I) in contrast to 7% in the DMSO group (D, I). (A–H, J): The cartilage degeneration and The Osteoarthritis Research Society grade were more improved in the BAY treatment group compared with the DMSO group. Cyan nuclear represent BrdU+ ACSCs and white nuclear represent Ki67+/BrdU+ ACSCs. ***, p < .001 between two groups.

26. Specific aims
Down regulate IL-1β, IL-6 and TNF to slow the process of ECM degradation in OA.
Induce the migration of chondrocytes and ACSCs to the superficial zone.

27. References
Pap, T. & Korb-Pap, A. Cartilage damage in osteoarthritis and rheumatoid arthritis-two unequal siblings. Nat. Rev. Rheumatol. 11, 606–15 (2015).
Gómez, R., Villalvilla, A., Largo, R., Gualillo, O. & Herrero-Beaumont, G. TLR4 signalling in osteoarthritis-finding targets for candidate DMOADs. Nat. Rev. Rheumatol. 11, 1–12 (2014).
Zingler, C. et al. Limited evidence of chondrocyte outgrowth from adult human articular cartilage. Osteoarthr. Cartil. 24, 124–128 (2016).
Chevalier, X., Eymard, F. & Richette, P. Biologic agents in osteoarthritis: hopes and disappointments. Nat. Rev. Rheumatol. 9, 400–10 (2013).
Jiang, Y. & Tuan, R. S. Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat. Rev. Rheumatol. 11, 206–12 (2015).