Harnessing Adipose - Derived Stem Cells to Catalyze Tissue Regeneration by Neonatal Chondrocytes in Fully Biodegradable Hydrogels Krista Chew | Heather Rogan | Fan Yang Stanford University | Bioengineering – Orthopaedic Surgery Background and Rationale Methodology Conclusions and Discussion • Cartilage injury is a leading cause of disability among adults. Cartilage loss is largely irreversible, due to its low cellularity and avascular nature. • Allogeneic neonatal articular chondrocytes (NChons) are a promising cell source for cartilage regeneration because they are highly proliferative, immune-privileged, and readily produce abundant cartilage matrix. However, scarce donor availability for NChons greatly hinders their broad clinical application. Stem cells, aside from their ability to differentiate into different tissue types, may contribute to tissue regeneration through the secretion of paracrine factors. Our lab has recently reported the potential to use a minimal number of NChons to catalyze cartilage tissue formation by co-culturing them with adipose-derived stem cells (ADSCs) in 3D biomimetic hydrogels (5% poly [ethylene glycol] diacrylate [PEGDA] / 3% chondroitin sulfate [CS] [Lai et. al., Scientific Reports, 2014]). • The goals of this study are: (1) to evaluate the potential of a CS-based, fully biodegradable, cartilage matrix–mimicking hydrogel in supporting catalyzed cartilage formation by ADSCs and NChons; and (2) to determine whether transforming growth factor beta (TGF–β) is required for such synergy to occur. • We chose a methacrylated CS hydrogel: a fully degradable hydrogel made from components of native cartilage matrix. We chose a 9% (weight/volume) CS concentration for cell encapsulation, which is comparable to the Young’s modulus of previously used PEGDA/CS composite hydrogels that supported catalyzed cartilage formation (30 kPa). • ADSCs were isolated from human liposuction aspirates. NChons were isolated from juvenile bovine articular cartilage. The cells were encapsulated at a density of 13 million cells/mL. CS was cross-linked under UV light for 5 minutes at 4 mW/cm 2 . The cells were cultured in chondrogenic media for 21 days. ADSC (100A) NChon (100N) Co-Culture (75A:25N) - + + + - - TGF-β Groups Cell Type Results ADSC Co-Culture NChon - TGF-β + TGF-β - TGF-β + TGF-β A B C Figure 1 | Co-culture in CS hydrogels led to enhanced cell proliferation and catalyzed collagen deposition; TGF-β is required for such synergy to occur. (A) Cell proliferation is quantified by DNA content. (B) Hydroxyproline content per wet weight at day 21. (C) Compressive moduli of cell/hydrogel constructs at day 21. The red line represents the average hydrogel stiffness at day 1. Figure 2 | CS hydrogels supported catalyzed neocartilage formation, as shown by immunofluorescence staining of cartilage marker type II collagen (A) and Safranin O staining (B). (A) Distribution of newly- deposited type II collagen. Green: collagen II; blue: cell nuclei. Scale bar = 100μm. (B) Safranin O imaging. Red: sGAG; black dots: cell nuclei. The insets portray a 2X image of the hydrogel. See Acellular (right) for depiction of background (hydrogel and extracellular matrix). Scale bar = 100μm. A B Figure 3 | Cell viability assay at day 21 confirms high viability and formation of larger neocartilage nodules in the co-culture group only in the presence of TGF-β. Green: live cells; red: dead cells. Scale bar = 200μm. • Outcome analyses: live/dead assays, mechanical testing, histological analysis, and biochemical analysis (PicoGreen assay for DNA content, hydroxyproline assay for collagen content). Preparation for biochemical assays included acquisition of wet weight, lyophilization, acquisition of dry weight, and digestion in papainase solution. Samples for histology were paraffin fixed, embedded in OCT, frozen in liquid nitrogen, and cryosectioned prior to staining. NChon Co-Culture - TGF-β + TGF-β ADSC [email protected] • The results demonstrate that fully degradable, CS-based hydrogels are capable of supporting ADSCs in catalyzing NChons to form cartilage in 3D. Such synergy requires the presence of TGF-β. • The outcomes of this study justify the feasibility of using fully biodegradable hydrogels to achieve catalyzed cartilage formation, which is preferable for clinical translations. • Future studies will test the synergy and efficacy in vivo using the cartilage defect model. Krista Chew would like to thank Heather Rogan and Professor Fan Yang for mentoring her throughout the summer. We would like to thank the Stanford Bioengineering REU program for supporting this work. Acknowledgements
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Harnessing Adipose-Derived Stem Cells to Catalyze Tissue Regenerationby Neonatal Chondrocytes in Fully Biodegradable Hydrogels
Krista Chew | Heather Rogan | Fan YangStanford University | Bioengineering – Orthopaedic Surgery
Background and Rationale
Methodology
Conclusions and Discussion
• Cartilage injury is a leading cause of disability among adults. Cartilage loss is largely irreversible, due toits low cellularity and avascular nature.
• Allogeneic neonatal articular chondrocytes (NChons) are a promising cell source for cartilageregeneration because they are highly proliferative, immune-privileged, and readily produce abundantcartilage matrix. However, scarce donor availability for NChons greatly hinders their broad clinicalapplication. Stem cells, aside from their ability to differentiate into different tissue types, may contributeto tissue regeneration through the secretion of paracrine factors. Our lab has recently reported thepotential to use a minimal number of NChons to catalyze cartilage tissue formation by co-culturing themwith adipose-derived stem cells (ADSCs) in 3D biomimetic hydrogels (5% poly [ethylene glycol] diacrylate[PEGDA] / 3% chondroitin sulfate [CS] [Lai et. al., Scientific Reports, 2014]).
• The goals of this study are: (1) to evaluate the potential of a CS-based, fully biodegradable,cartilage matrix–mimicking hydrogel in supporting catalyzed cartilage formation by ADSCs andNChons; and (2) to determine whether transforming growth factor beta (TGF–β) is required for suchsynergy to occur.
• We chose a methacrylated CS hydrogel: a fully degradablehydrogel made from components of native cartilage matrix. Wechose a 9% (weight/volume) CS concentration for cellencapsulation, which is comparable to the Young’s modulus ofpreviously used PEGDA/CS composite hydrogels that supportedcatalyzed cartilage formation (30 kPa).
• ADSCs were isolated from human liposuction aspirates.NChons were isolated from juvenile bovine articular cartilage.The cells were encapsulated at a density of 13 million cells/mL.CS was cross-linked under UV light for 5 minutes at 4 mW/cm2.The cells were cultured in chondrogenic media for 21 days.
ADSC (100A)
NChon (100N)
Co-Culture (75A:25N)
- +
+
+
--
TGF-β
Groups
Cell Type
Results
ADSC
Co-Culture
NChon
- TGF-β + TGF-β- TGF-β + TGF-β
A B C
Figure 1 | Co-culture in CS hydrogels led to enhanced cell proliferation and catalyzed collagen deposition; TGF-β is required for such synergy to occur. (A) Cell proliferation is quantified by DNA content. (B) Hydroxyproline content per wet weight at day 21. (C) Compressive moduli of cell/hydrogel constructs at day 21. The red line represents the average hydrogel stiffness at day 1.
Figure 2 | CS hydrogels supported catalyzed neocartilage formation, as shown by immunofluorescence staining of cartilage marker type II collagen (A) and Safranin O staining (B). (A) Distribution of newly-deposited type II collagen. Green: collagen II; blue: cell nuclei. Scale bar = 100µm. (B) Safranin O imaging. Red: sGAG; black dots: cell nuclei. The insets portray a 2X image of the hydrogel. See Acellular (right) for depiction of background (hydrogel and extracellular matrix). Scale bar = 100µm.
A B
Figure 3 | Cell viability assay at day 21 confirms high viability and formation of larger neocartilage nodules in the co-culture group only in the presence of TGF-β. Green: live cells; red: deadcells. Scale bar = 200µm.
• Outcome analyses: live/dead assays, mechanical testing, histological analysis, and biochemicalanalysis (PicoGreen assay for DNA content, hydroxyproline assay for collagen content). Preparation forbiochemical assays included acquisition of wet weight, lyophilization, acquisition of dry weight, anddigestion in papainase solution. Samples for histology were paraffin fixed, embedded in OCT, frozen inliquid nitrogen, and cryosectioned prior to staining.
NChonCo-Culture
- TGF-β
+ TGF-β
ADSC
• The results demonstrate that fully degradable, CS-based hydrogels are capable of supporting ADSCs incatalyzing NChons to form cartilage in 3D. Such synergy requires the presence of TGF-β.
• The outcomes of this study justify the feasibility of using fully biodegradable hydrogels to achievecatalyzed cartilage formation, which is preferable for clinical translations.
• Future studies will test the synergy and efficacy in vivo using the cartilage defect model.
Krista Chew would like to thank Heather Rogan and Professor Fan Yang for mentoring her throughoutthe summer. We would like to thank the Stanford Bioengineering REU program for supporting thiswork.
Acknowledgements
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