This study investigated using microribbon (μRB) scaffolds to support chondrogenesis of adipose-derived stem cells (ADSCs) for cartilage regeneration. The μRB scaffolds were macroporous and gelatin-based, while conventional hydrogel (HG) scaffolds lacked macroporosity. ADSCs encapsulated in μRB scaffolds attached, spread, and proliferated more than in HG scaffolds. After 3 weeks of culture, ADSCs in μRB scaffolds deposited more interconnected type II collagen and sulfated glycosaminoglycans (sGAG), and the resulting neocartilage had a higher compressive modulus than ADSCs in HG scaffolds. The enhanced chondrogenesis and mechanical properties of

1. Enhancing Adipose-derived Stem Cell-based Cartilage Regeneration using Macroporous Microribbon Scaffolds
Heather Rogan1
, MS; Krista Chew1
; Fan Yang1, 2
, PhD
1
Dept. Bioengineering, 2
Dept. Orthopaedic Surgery, Stanford University
DISCLOSURES: The authors declare no competing interests.
INTRODUCTION: Articular cartilage has a limited capacity to repair itself. Cartilage repair by stem cells has been widely explored in order to treat focal
cartilage defects and prevent progression to osteoarthritis. Adipose-derived stem cells (ADSCs) represent a promising autologous cell source for cartilage
repair givens its abundance, ease of isolation, and chondrogenic potential. However, delivery of stem cells alone often leads to fibrocartilage formation with
inferior mechanical properties. Hydrogels are attractive matrices for cell delivery given their injectability and tunable biochemical and mechanical
properties. However, conventional hydrogels generally lack the flexibility when subject to mechanical load, and lack the macroporosity desirable for
efficient cell proliferation, migration and matrix deposition. To overcome these limitations, we have recently reported development of microribbon (μRB)-
based hydrogels with cartilage-mimicking shock-absorbing capacity upon cyclic mechanical compression. Furthermore, how μRB scaffolds influence
chondrogenesis of ADSCs in 3D remains unknown. The goal of this study is to investigate the potential of gelatin-based μRB hydrogels as 3D matrices for
supporting chondrogenesis of ADSCs in vitro, and compare the efficacy to conventional hydrogels for inducing chondrogenesis.
METHODS: Human ADSCs were isolated from liposuction aspirates with informed consent for use in medical research, as otherwise discarded tissues
under an IRB approved protocol by Stanford University. hADSCs were encapsulated in μRB or conventional hydrogel (HG) scaffolds and cultured for 21
days in chondrogenic medium containing 10 ng/ml TGF-β3. μRB scaffolds were synthesized from gelatin; briefly, gelatin was dissolved in dimethyl
sulfoxide, wet spun into ethanol, flattened with acetone, chopped, methacrylated, fixed, washed, and lyophilized prior to use at 7.5% (w/w). HG scaffolds
contained 5% polyethylene (glycol) diacrylate (PEGDA) and 3% methacrylated chondroitin sulfate (CS-MA). Outcomes were analyzed using biochemical
assays (DNA and sGAG), histology (Safranin O), and immunostaining (type II and X collagen). An unpaired t test was used for statistical analysis to
compare μRB and HG scaffolds (n = 3).
RESULTS: Scanning electron microscopy (SEM) showed ribbon morphology with an average ribbon width of ~55 µm. Intercrosslinked μRBs led to the
formation of macroporous scaffolds, whereas no macroporosity was observed in PEG/CS hydrogels (Fig. 1, top row). ADSCs attached quickly and spread
along the surface of μRBs, while remain round and entrapped in the nanoporous HGs (Fig 1). Both μRBs and HG led to high cell viability 48 hrs post
encapsulation (Fig. 1, bottom row). After 21 days of culture, the µRB group showed interconnected matrix deposition of type II collagen (Fig. 2) and sGAG
(data not shown). In contrast, ADSCs produced much less collagen II in HGs, which were constricted to pericellular regions only with no interconnectivity.
Quantitative analysis of cell proliferation and sGAG are in agreement with histological results, showing μRBs led to markedly enhanced ADSC proliferation
and cartilage matrix deposition than HGs (Fig. 3A & B). Such increase in biochemical contents translated to functional improvement of the regenerated
cartilage tissues with a significantly greater increase in Young’s Modulus by µRB than HG (Fig. 2C).
DISCUSSION: Our results show macroporosity within μRB scaffolds substantially enhanced chondrogenesis and new cartilage formation by ADSCs
compared to hydrogels. Macroporous μRBs are more advantageous for cartilage tissue engineering given the enhanced nutrient diffusion, cell attachment
surface area, space for proliferation and matrix deposition, and enhanced shock-absorbing capacity. μRB scaffolds led to interconnected cartilage deposition,
which led to a significant increase in Young’s Modulus improving their long term integrity for in vivo implantation. In comparison to HG scaffolds, in which
the cells must first degrade the HG network before proliferating or depositing new matrix, μRBs provide space for neocartilage formation without requiring
cells to first expend energy degrading their surrounding gel. This accelerates the cartilage formation process and leads to such an increase in mechanical
strength. Our hypothesis was supported by our results indicating that ADSC chondrogenesis should be preferentially performed in μRB scaffolds instead of
HG scaffolds.
SIGNIFICANCE: The outcomes of this study highlight the advantages of novel macroporous μRB-based hydrogels over conventional nanoporous hydrogels
for inducing chondrogenesis of ADSCs with enhanced biochemical and mechanical functions. We envision that this platform will significantly accelerate
clinical translation of stem cell-based therapy for cartilage regeneration by enhancing cell proliferation and interconnected new matrix deposition, thus
improving therapeutic outcomes for treating cartilage defects.
ACKNOWLEDGEMENTS: The authors would like to acknowledge the following funding: NIH R01DE024772 (F.Y), California Institute for Regenerative
Medicine (Grant #TR3-05569, F. Y.), National Science Foundation CAREER award program (CBET-1351289, F.Y.) and NSF predoctoral fellowship (H.R.)
IMAGES AND TABLES:
Figure 3. Quantitative biochemical assays and mechanical testing
showed μRB substantially increased cell proliferation (A), sGAG
deposition (B) and compressive moduli (C) of resulting
neocartilage at day 21. (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 2. Histology showed μRB group led to more
interconnected type II collagen deposition compared to HG
control at day 21. (green = Col II, blue = nuclei, scale bar =
200 μm)
Figure 1. Top: Scanning
electron microscopy shows
macroporosity in μRB-based
scaffold only, but not in
conventional HG. Scale bar:
150 μm; Bottom: Live/dead
staining 48 hr post
encapsulation shows high
cell viability in both groups.
μRB were counterstained as
red. Scale bar: 400 μm.