1. TO EXPAND OR NOT TO EXPAND?
An In Vitro Comparison Of Culture Expanded Mesenchymal Stem Cells Versus Freshly Isolated Bone Marrow Cells For Cartilage Repair
Nupur Kohli

2. CHONDRAL DEFECTS
Wear & tear, trauma or injury
Ends of bones rubbing against
each other
Chondral defects if left untreated may progress to complete degeneration of cartilage tissue and result in osteoarthritis.

3. Autologous Chondrocyte Implantation
Advantages: improves the lifestyle of patient, helps in pain relief.
Disadvantages: Fibrous cartilage, donor site morbidity and a second surgery is often required.
This procedure involves taking a biopsy of the patient’s cartilage which is then transferred to a laboratory and the cartilage cells isolated. These cells are then multiplied many times and then re-implanted into the patient.
The classical ACI (first generation) was originally described in 1994 (N Engl J Med. 1994 Oct 6;331(14):
Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.
Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L.)Source
Department of Orthopedic Surgery, University of Göteborg, Sahlgrenska University Hospital, Sweden.
a second-generation ACI was developed, in which bioengineered bilayer collagen or synthetic membranes were used in order to avoid spill-over and asymmetric distribution of chondrocytes following implantation
third-generation ACI, where chondroconductive or inductive matrices are used in a 1 step procedure(i.e. 3D environment) for cell growth.
Brittberg et al.1994

4. Whether or not stem cells can repair cartilage?
The big question..
Whether or not stem cells can repair cartilage?

5. James hui –msc for cartilage

6. Which ones??
Evidence suggests that a population of stem cells called mesenchymal stem cells (MSCs) can mature into chondrocytes and osteoblasts.
Studies also suggest that uncultured/freshly isolated population of mononuclear cells may serve to be a better alternative to MSCs.
and therefore, are lead candidates for their use in the repair of cartilage and bone defects.

7. Mesenchymal Stem cells – Isolation, expansion & Culture
Following ethical approval and informed consent, MSCs were isolated from bone marrow and infrapatellar fat pad of the knee. These cells were then culture expanded and characterised as per the standards proposed by the ISCT.
Isolation of mononucleated cells
Collagenase digested

8. One step procedure- Freshly isolated mononucleated cells
BMMNCs, bone marrow was aspirated from iliac crest and cells were isolated using  MarrowStim™ Concentration System.
Aspirate is loaded onto the system and centrifuged to isolate mononucleated cells

9. MSC characterisation Find a better picture for osteogenesis
Chondrogenesis
Osteogenesis
adipogenesis
CD34
CD45
CD73
CD90
CD105
Find a better picture for osteogenesis
Figure 1. Characterisation of culture expanded MSCs. Fig1(above- left) Differentiation of MSCs into osteoblasts, adipocytes and chondrocytes indicated by the presence of alkaline phosphatase positive pink osteoblasts (arrowed), oil redo positive orange lipid vacules (arrowed) and puple metachromatic staining of cartilage ECM (arrowed) respectively. Fig.1(above-right) Flow cytometry for CD-cell surface antigen characterisation of MSCs. Greater than 95% of cells tested (n=1) were CD34 –ve, CD45 –ve (top panel) and more than 84% of cells were CD73 +ve, CD90 +ve and CD105 +ve (bottom panel). The grey lined histogram shows immunopositivity for each indicated marker whereas the black histogram indicates immunolabelling with an isotype-matched control antibody.

10. Scaffolds/cell carriers
Chondro-Gide® - Collagen type I/III
Alpha Chondro Shield®- polyglycolic acid
Hyalofast™- hyaluronic acid
All three are currently used surgically for treating chondral defects.

11. Aim
Primary aim was to assess incorporation and viability of culture expanded versus freshly isolated cells within the 3D scaffolds.

12. Methods
50,000 cells were seeded per 3mm2 scaffold and fed with DMEM/10%FCS and 1%P/S.
All cultures were maintained at 37˚C in 5% CO2 for 4 weeks.
At day1, 7, 14 and 28 scaffolds were scored for cell viability staining and imaged using confocal microscopy.

13. RESULTS : Culture expanded MSCs
Chondro-Gide®
Alpha Chondro Shield®
Hyalofast™
Day 1
Day 7
Culture expanded MSCs adhere to all three scaffolds and proliferate the best in Chondro-Gide® during short term (7 days) and long term (28 days) cultures !
Day 14
Figure1. (Left) MSC incorporation was greatest in Chondro-Gide® compared to Alpha Chondro Shield® and Hyalofast™. In all three scaffolds, MSCs remained over 90% viable up to 14 days in culture. Cell viability scoring results correlated with the live/dead imaging where Chondro-Gide® showed a higher MSC incorporation than Alpha Chondro Shield® and Hyalofast™ at all time points. After 7 days in culture Chondro-Gide® showed significantly higher (p value < 0.05) cellular incorporation than the other two scaffolds and this remained so after 14 and 28 days in culture.
Day 28

14. Freshly isolated cells
Chondro-Gide®
Chondro shield®
Hyalofast™
Day 7
Day 14
Day 28
Day 1
Freshly isolated BM-MNCs do not adhere and grow in any of the three scaffolds during short term or long term cultures!
Figure 2. BMMNCs on all the three scaffolds incorporated and remained viable (green fluorescence) only up to day 1 in culture after which the presence of BMMNCs diminished and no cells were seen to be incorporated in either of the three scaffolds during long term cultures.

15. Aim
To assess chondrogenesis of culture expanded MSCs within these three scaffolds.

16. Methods 50,000 cells were seeded per 3mm2 scaffold.
Treated with and without chondrogenic inducers of differentiation i.e. 100nm dexamethasone, 37.5ug/ml ascorbate, ITS-X, 10ng/ml TGF-β1 in DMEM/2%FCS and 1%P/S.
500,000 cells were formed into micromass pellet cultures as control for chondrogenesis.
All cultures were maintained at 37˚C in 5% CO2 for 4 weeks.
At the end of 4 weeks:
Cell viability
Soluble proteoglycan synthesis
Extracellular matrix deposition

17. Articular cartilage composition
Components are arranged in a way that is maximally adapted for biomechanical functions
•Chondrocytes(~ 1%)
•Collagen (15%) (Type II in articular cartilage)
•Proteoglycans(15%)
•Water (70 %)
Craig & Machado.1995

18. Chondrogenesis – Cell viability staining
+ CM
- CM
+ CM
- CM
Chondro gide®
Alpha Chondro Shield®
Hyalofast™
Micro mass pellets
Clarify what is a nodule
Change colour of the arrows
Change the label

19. Chondrogenesis – Cell viability staining

20. Histology Toluidine blue stain Type II collagen immunolocalization +CM
Chondro-Gide®
Alpha Chondro Shield®
Hyalofast™
Toluidine blue stain
Type II collagen immunolocalization

21. Soluble glycosaminoglycans
Glycosaminoglycan content measured using DMMB assay. Only Chondro-Gide® (p=0.0013) and Hyalofast™ (p= 0.040) showed a significant increase in the amount of soluble proteoglycan content under chondrogenic induction media as compared to scaffolds treated without chondrogenic inducers of differentiation. Data are presented as mean+SEM. Statistical analysis was undertaken using an Unpaired t test with welsh correction.

22. Conclusion & Future work
These preliminary findings suggest that human culture expanded MSCs incorporate, adhere and proliferate better in clinically utilized scaffolds compared to MNCs.
The long term incorporation and growth of MSCs enabled the induction of chondrogenesis with cartilage specific matrix deposition.
Future work would include selectively isolating MSC progenitors from the mono-nucleated cell population and compare with MSCs both in vitro and in vivo.

23. Acknowledgement Dr. Eustace Johnson Mr. Martyn Snow Jono Sheard
Htoo Wai
Merlin Walter
ARCHA – confocal faciltity
DSHB
Friends and Family!