Investigation of Scaffold Processing Methods and Dynamic Seeding Techniques to Enhance Cell Infiltration in Decellularized Adipose Tissue Foams

Curet, Marjorie A (University of Western Ontario)
Turco, Bryen A (Queen's University)
Flynn, Lauren E (University of Western Ontario)

Introduction

Decellularized adipose tissue (DAT) foams have shown promise as a natural bioscaffolds for adipose-derived stem cell (ASC) delivery in soft tissue engineering applications [1]. However, in previous studies, there was limited cell infiltration into the foams following static seeding and in vitro culture [1]. To further enhance the potential of this cell delivery platform, our aims in the current study were to investigate (i) the effects of scaffold processing methods and (ii) dynamic cell seeding on ASC distribution within a range of DAT foams. More specifically, we characterized the effects of mincing versus cryomilling the DAT during scaffold fabrication on the structure, composition and physical properties of the DAT foams. Subsequently, we assessed the effects of dynamic cell seeding using an orbital shaker on ASC infiltration, proliferation and contraction over 28 days in culture.

Materials and Methods

Adipose tissue samples were obtained from patients undergoing lipo-reduction procedures at the Hotel Dieu Hospital in Kingston, Ontario, with research ethics board approval from Queen’s University (REB# CHEM-002-07). DAT was prepared using established methods [2] Foams were synthesized using lyophilized DAT that was either minced into fragments (~0.2 cm3) or cryo-milled using a ball mill, and further processed through α-amylase digestion and homogenization as previously described [1]. The DAT suspensions (DATsus) were chloroform-sterilized [3] and minced and milled DAT foams (10, 15, 25, 50 mg/mL) were fabricated by freezing (-20°C) and lyophilization. SEM was used to visualize the micro-architecture of the foams and immunohistochemistry was used to assess the presence of collagen I and IV, fibronectin and laminin. Equilibrium water content (EWC), swelling, porosity and protein release were measured. In a pilot study of cell seeding methods, human ASCs were seeded using: i) static dry seeding, ii) static hydrated seeding, and iii) orbital shaker hydrated seeding (n=3). Masson’s trichrome staining was used to assess cell infiltration and collagen architecture in the seeded foams at 7 days. In a follow up study, cell-induced foam contraction was assessed in dynamically-seeded minced and milled foams (10, 25, and 50 mg/mL) (n = 3), with surface area measurements at 4, 7, 14 and 28 days using ImageJ. Finally, the effects of scaffold concentration, cell seeding density and orbital shaking speed were probed in the milled foams using DAPI staining to assess distribution, quantification of total cell number using the PicoGreen assay (LifeTechnologies) and Ki67 staining to assess cell proliferation.

Results

Fabrication protocols yielded foams with a porous 3-D architecture that were soft and pliable. Minced DAT foams had a more heterogeneous structure as compared to the milled DAT foams (Fig. 1). Similar to human fat and intact DAT controls, the foams incorporated collagen I and IV, fibronectin and laminin, distributed throughout a porous network. The foams retained their shape following rehydration, with minimal swelling in aqueous medium. EWC was greater than 96% for all foams, with higher levels in the minced group for all concentrations. The scaffolds were highly porous (>96%), with reduced porosity in the milled foams fabricated at lower concentrations. Protein release was most notable within the first 3 days for all foams. The pilot seeding study indicated that cell infiltration was enhanced through dynamic orbital shaker cell seeding (Fig. 2). The contraction study revealed there was a significant reduction of foam surface area in the foams fabricated at lower concentrations (10, 25 mg/mL) within 4 days in culture, with the minced foams demonstrating reduced contraction. DAPI staining showed the highest levels of ASC infiltration in the milled foams fabricated with lower DATsus concentrations. Further analysis of cell seeding with the milled foams suggested there were increased total cell numbers at early time points in the 25 and 50 mg/mL scaffolds that were dynamically seeded at 120 RPM at a density of 1,000,000 ASCs/foam. However, reduced cell numbers were observed at 14 days, potentially due to diffusion limitations caused by scaffold contraction. Ki67 staining of proliferating ASCs showed localization within the surface regions of the foams at both 7 and 14 days.

Discussion and Conclusion

Our results indicate that DAT foam structure and physical properties can be modulated by varying the methods for processing the lyophilized DAT prior to enzymatic digestion. Both mincing and milling protocols yielded foams that were stable in culture without the need for chemical crosslinking. For applications in cell culture and delivery, dynamic cell seeding using an orbital shaker is a simple approach that can be used to enhance cell infiltration into the porous foams. Further, we observed improved cell distribution in the milled foam group, highlighting the importance of tuning the scaffolds for increased efficacy in ASC delivery. In further developing this platform towards in vivo applications, it will be critical to consider the balance between cell infiltration and scaffold contraction, as well as culture time, to achieve a predictable dose of highly viable ASCs.

Figure 1: Top: Representative images of a lyophilized DAT foam (50 mg/mL). Bottom: SEM images of minced and milled DAT foam cross-sections. Scale bars represent 1 mm. SEM of 50 mg/mL foams.

Figure 2: Representative Masson’s trichrome staining from the 7 day ASC seeding methods pilot study showing cross-sections of 35 mg/mL DAT foams (n = 3). ASC nuclei in black. a. Static seeding - dry foam; b. Static seeding - hydrated foam; c. Orbital shaker seeding

Acknowledgements

Operational funding for this study was provided by the CIHR. Scholarship support for M.C. was provided through the Joint Motion Program (JuMP), a CIHR Strategic Training Program in Musculoskeletal Health Research and Leadership. The authors would like to thank Dr. J.F. Watkins and Mrs. K. Martin for their clinical collaborations.

References

1. Yu, C.; Bianco, J.; Brown, C.; Fuetterer, L.; Watkins, J.F.; Samani, A.; Flynn, L.E. Porous decellularized adipose tissue foams for soft tissue regeneration, Biomaterials 2013, 34, 3290–3302. 2. Flynn, L.E. The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells, Biomaterials 2010, 31, 4715-4724. 3. Rajan, N.; Habermehl, J.; Coté, M.F.; Doillon, C.J.; Mantovani, D. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications, Nat Protoc. 2006, 1, 2753-2758.

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