Bone marrow mesenchymal stem cells and cartilage fragments enhance chondrogenesis Gari M1,2,3, Alsehli H 1,2, Abbas M2 , Alkaff M2, Kafienah W2,4, Chaudhary AG1,3, Abuzenadah A1,3, Al Qahtani M1,3, Gauthaman K1,2.

 1Stem Cell Unit, Centre of Excellence in Genomic Medicine Research, King Abdulaziz University, Kingdom of Saudi Arabia 2Department of Orthopaedic Surgery, Faculty of Medicine, King Abdulaziz University, and Sheikh Salem Bin Mahfouz Scientific Chair for Treatment of Osteoarthritis by Stem Cells, Kingdom of Saudi Arabia 3Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Kingdom of Saudi Arabia 4School of Cellular and Molecular Medicine, University of Bristol, United Kingdom

Introduction Cartilage-subchondral bone interphase and the prevailing cellular

and molecular microenvironment play a significant role in

osteoarthritis (OA). To understand whether cellular/local

microenvironmental cues contribute to better cartilage repair, in

the present ex-vivo study, we evaluated the chondrogenic

differentiation and repair of critical size defect in an explant

osteochondral model using bone marrow derived mesenchymal

stem cells (BM-MSCs) and homogenised cartilage.

Informed patient consent was obtained for collecting BM-MSCs,

osteochondral bone, and cartilage from undamaged areas from

patients undergoing total knee arthroplasty. Bone pieces were

trimmed to 1cm (w) x 1cm (b) x 1cm (h) and a central drill defect

(~1mm) was made. Chondrogenic repair was evaluated by

seeding the osteochondral bone defect (OBD) with either BM-

MSCs (1x106cells) pellet (Group II), homogenized cartilage pellet

(Group III) or a combination of both BM-MSCs (0.5x106cells)

and homogenized cartilage as pellet (Group IV). OBD with no

added cells or tissue served as control. Similar sized tissue pellets

were used for all experimental arms. Samples from all four

groups were maintained in standard BM-MSC chondrogenic

medium up to 28 days. The tissue repair was analyzed by

histology, phase contrast imaging and biochemical analysis.

Materials and Methods

References 1.  Chen CC, Liao CH, Wang YH et al. 2011. Cartilage Fragments from Osteoarthritic Knee Promote

Chondrogenesis of Mesenchymal Stem Cells without Exogenous Growth Factor Induction. Journal of Orthopaedic Research, 30(3):393-400.

2.  Leyh M, Seitz A, Durselen et al 2014. Subchondral bone influences chondrogenic differentiation and collagen production of human bone marrow-derived mesenchymal stem cells and articular chondrocytes. Arthritis Res Ther. 16(5):453.

Figure 1: A) Explant culture images (Upper Panel) showing the osetochondral bone with central drill defect (circular dotted lines) with or without cells as mentioned; B) their respective phase contrast images (Middle Panel) taken at the bone defect area on day 14 Central drill defect is represented within the dotted lines. In the control group the defect area is seen empty, whereas the other groups shows the defect area being filled with the respective cell types; C) H&E images (Lower Panel) of the same at low magnification.

Figure 2: H&E staining of demineralized tissue sections of the paraffin embedded bone tissue following 28 days of ex-vivo culture showed neochondrocyte formations (indicated by arrows) that were less in the MSCs group (Group II) and more in the MSCs + cartilage group (Group IV).

Figure 3: a) Toluidine blue staining of demineralized tissue sections of the paraffin embedded bone tissue following 28 days of ex-vivo culture showed positive staining indicative of collagen content; b) Sircol assay showing the secreted collagen levels at days 7, 14 and 21. The amount of secreted collagen is more in Group II (BM-MSCs) and Group IV (BM-MSCs + Cartilage) compared to rest of the groups. The values are shown as mean ± SEM from three independent samples.

a  

We would like to acknowledge the financial support provided by the “Sheikh Salem Bin Mahfouz Scientific Chair for Treatment of Osteoarthritis by Stem Cells”; the Stem Cell Lab at CEGMR and the Orthopedics Stem Cell Research Lab at King Abdulaziz University Hospital for supporting this study.

Acknowledgment

MSCs and cartilage fragments together provided better cartilage

formation and defect filling in a human ex-vivo osteochondral

model. Further analysis of sub-fragments could help design more

effective methods for the delivery of therapeutic chondrogenic

MSCs.

Discussion and Conclusion

Results

May 8th – 11th, 2015