Evaluation of Composite Hydrogel Scaffolds Comprised of Methacrylated Chondroitin Sulphate and Decellularized Adipose Tissue of Varying Particle Size

Brown, Cody (Department of Anatomy and Cell Biology The University of Western Ontario, London, Ontario, Canada)
Yang, Jing (Department of Chemical Engineering, Queen's University, Kingston, Ontario, Canada)
Amsden, Brian (Department of Chemical Engineering, Queen's University, Kingston, Ontario, Canada)
Flynn, Lauren (Department of Chemical and Biochemical Engineering, The University of Western Ontario, London)

Introduction

Engineered 3-D scaffolds seeded with adipose-derived stem cells (ASCs) have shown promise in adipose tissue regeneration. In particular, decellularized adipose tissue (DAT) is unique in that it provides a biochemical and mechanical microenvironment that has been shown to stimulate the in vitro adipogenic differentiation of ASCs without additional supplements (1). An injectable composite scaffold incorporating the unique bioactivity of DAT with in situ polymerization in a hydrogel phase would be advantageous for ASC delivery in the filling of small or irregularly shaped soft tissue defects (2). The current study investigates methods for milling DAT and the effect of DAT particle size on ASC proliferation and differentiation in injectable methacrylated chondroitin sulphate (MCS)-DAT composite hydrogels.

Materials and Methods

DAT Particle Fabrication and Characterization Adipose tissue was acquired from female patients undergoing elective surgery at the Kingston General Hospital in Kingston, Ontario, with Research Ethics Board approval from Queens (CHEM-002-07). The tissue was decellularized using a 5 day protocol (1). The DAT was snap frozen in liquid nitrogen, lyophilized for 48 h, and finely minced. The minced DAT was then loaded into a laboratory ball milling chamber. Three milling conditions were evaluated: 3 min at room temperature; 5 min at room temperature; and 3 min of cryogenic milling using liquid nitrogen. Following milling, the DAT particles were separated using mesh sieves of known pore sizes. The particles were then grouped into 3 size ranges: small (45 μm sieve); medium (collected between 100 μm and 150 μm sieves); and large (collected between 250 μm and 300 μm sieves). The 3 size ranges were analyzed with a Mastersizer2000 to characterize the particle diameter distribution. DAT-MCS Composite Hydrogel Fabrication and in vitro Assessment Small or large DAT particles were added to methacrylated chondroitin sulphate (MCS) prepolymer with adjusted concentrations to obtain equal DAT surface area. The DAT-MCS composite hydrogels were photo-cross-linked with long-wavelength UV light using an established protocol (3). DAT-MCS hydrogels were seeded at two densities with human ASCs (2.5x10^5 and 5x10^5 cells/scaffold) and proliferation was quantified using an MTT assay at 1, 7, and 14 days with an ASC standard. To compare adipogenic differentiation in the MCS-DAT composites, MCS alone and TCPS controls, after 7 days in proliferation medium, samples were induced in adipogenic differentiation medium and glycerol 3-phosphate dehydrogenase (GPDH) enzyme activity was quantitatively assessed at 14 days. Non-induced controls in proliferation medium were included for all groups. Oil red O staining was performed to visualize intracellular lipids. For all assays, (n=3) with statistical analysis by ANOVA (p ɤ 0.05)

Results

DAT Particle Fabrication and Characterization The 3 milling conditions yielded DAT particles of varying sizes. Milling at ambient temperature for 3 and 5 min produced more large particles, with 68 % (w) and 62% (w) of the particles having a diameter > 100 μm respectively, while cryo-milling for 3 min produced smaller particles with only 28% (w) having a diameter > 100 μm. Cryo-milled DAT was sorted into 3 particle size ranges which were found to have an average diameter of 38 ± 6μm, 113 ± 1μm, and 278 ± 3μm respectively (Fig. 1). DAT-MCS in vitro Assessment The MTT analysis of ASCs encapsulated in the MCS-DAT composites indicated that both small (S-DAT) and large DAT (L-DAT) particles enhanced proliferation over the MCS alone controls when seeded at the lower cell density. Proliferation was observed from 1-7 days in the L-DAT group, with a cell density of (4.92 ± 0.66)x10^5 and (4.31 ± 0.18)x10^5 cells/scaffold measured at 7 and 14 days, respectively. In the S-DAT group, proliferation was delayed, with the cell density increasing from (2.28 ± 0.10)x10^5 cells/scaffold at 7 days to (3.70 ± 0.08)x10^5 cells/scaffold at 14 days. At the high seeding density, there were no significant changes observed over 14 days in any of the scaffolds. The GPDH activity for both S-DAT and L-DAT composites demonstrated enhanced adipogenic differentiation in comparison with pure MSC gels and TCPS controls (Fig. 2). The induced S-DAT composite group seeded at the higher density showed the greatest adipogenic activity. L-DAT composites had similar GPDH activity for both initial seeding densities. Consistent with the GPDH results, Oil red O staining showed enhanced cell-cell interactions combined with greater intracellular lipid accumulation in the S-DAT composites.

Discussion and Conclusion

DAT particles were generated by milling lyophilized DAT and particle size could be controlled through milling conditions to develop an optimized injectable composite scaffold with a 3-D microenvironment tuned to enhance adipogenesis. Modifying the size of DAT particles in the MCS-DAT composites altered the proliferative and adipo-inductive effects the scaffold had on ASCs. Larger DAT particles promoted more rapid proliferation, potentially by providing a larger contacting surface to support initial cell attachment and growth. Smaller DAT particles, while also promoting proliferation of encapsulated ASCs, increased adipogenic differentiation likely by promoting greater cell-cell interactions, which is favorable for adipogenesis; this is further supported by the enhanced GPDH activity at the higher cell density for the S-DAT scaffold group.

Figure 1: Cryo-milled DAT particle size distribution analyzed with a particle size analyzer L) Large particle group with an average DAT particle size of 278μm M) Medium particle group with an average DAT particle size of 113μm S) Small particle group with an average particle size of 45μm (n=3)

Figure 2: GPDH enzyme activity of MCS-DAT composite hydrogels with small (S) and large (L) DAT particle sizes seeded with ASCs at 2 densities, 2.5x10^5 and 5x10^5. MCS and TCPS were used as controls, with culturing in adipogenic differentiation medium (I) and proliferation medium (NI). (n=3)

Acknowledgements

Funding for this project was provided from the CIHR. We would like to thank Dr. Frederick Watkins and Mrs. Karen Martin for clinical collaborations to support our work.

References

1. Flynn LE. The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells. Biomaterials. 2010 Jun; 31(17):4715-24. 2. Yu C, Bianco J, Brown C, Fuetterer L, Watkins JF, Samani A, et al. Porous decellularized adipose tissue foams for soft tissue regeneration. Biomaterials. 2013;34(13):3290-302. 3. Cheung HK, Han TTY, Marecak DM, Watkins JF, Amsden BG, Flynn LE. Composite hydrogel scaffolds incorporating decellularized adipose tissue for soft tissue engineering with adipose-derived stem cells. Biomaterials. 2014 Feb; 35(6):1914-23.

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