Adipose-Derived Stem Cells Enhance the Angiogenic and Adipogenic Potential of Decellularized Adipose Tissue in vivo

Han, Tim Tian Y. (Queen's Unversity)
Toutounji, Sandra (Queen's Unversity)
Amsden, Brian G. (Queen's Unversity)
Flynn, Lauren E. (Western University, Queen's Unversity)

Introduction

Bio-scaffolds derived from the extracellular matrix (ECM) may provide an ideal 3D environment for the natural regeneration of damaged or deficient subcutaneous soft tissues, which would have many applications in plastic and reconstructive surgery. Adipose tissue is not only an excellent source of ECM for bio-scaffold preparation, but it is also abundantly available and commonly discarded following routine lipo-reduction surgeries. To generate a bio-scaffold from adipose tissue, our group has pioneered a process that removes cellular content from human fat to generate decellularized adipose tissue (DAT)(1). Previous studies indicate that DAT contains pro-angiogenic and pro-adipogenic factors, and is capable of enhancing the adipogenic differentiation of seeded human adipose-derived stem cells (ASCs) in culture(2). ASCs are a population of multipotent mesenchymal stem cells that are capable of differentiating into adipocytes, but can also secrete paracrine factors that promote angiogenesis, mediate inflammation, and enhance endogenous stem cell recruitment(3). Since both DAT and ASCs have the potential to promote soft tissue regeneration via angiogenesis and adipogenesis, the goal of the current study was to assess whether a combination of ASCs and DAT would further accelerate these two processes in vivo.

Materials and Methods

Human adipose tissue was collected following lipo-reduction surgery at the Kingston General Hospital or Hotel Dieu Hospital in Kingston, ON (REB Approval CHEM-002-07), and decellularized using published methods (1). Primary rat ASCs were isolated from the epididymal fat pad of male Wistar rats and cultured until passage 2 (P2). The P2 ASCs were seeded onto 50 mg DAT samples. Triplicate ASC-seeded and unseeded DAT samples were implanted into the subcutaneous tissue of female Wistar rats via a small surgical incision on the back of the animal. The rats (n=3) were sacrificed at 72 hours, 1 week, 4 weeks, 8 weeks and 12 weeks post-surgery. The implanted scaffold as well as the adjacent tissue was collected, embedded in paraffin and sectioned. To visualize the exogenous ASCs from the male rats, fluorescence in situ hybridization (FISH) analysis of the Y-chromosome (IDlabs) was used. The host macrophage response was analyzed by immunohistochemistry (IHC) with antibodies against the pan-macrophage marker CD68 (AbD Serotec) and the M2 macrophage marker CD163 (AbD Serotec). Angiogenesis was evaluated with antibodies for CD31 (Abcam) and VEGF(Abcam), to identify mitotic endothelial cells. Adipogenesis was assessed with anti-peroxisomal proliferator-activated receptor gamma (PPAR, Abcam) antibodies. Masson's trichrome staining was performed to quantify the area of adipose tissue formation with respect to the implanted region. Canadian Council on Animal Care (CCAC) guidelines for the care and use of laboratory animals were followed and all protocols were approved by the University Animal Care Committee (UACC). Numerical differences are statistically significant for P values ɤ 0.05 using one-way ANOVA with a Tukey’s post-hoc test.

Results

In the ASC-seeded group, FISH demonstrated that the exogenous cells were present within the implant region throughout the course of the study. Further, the percentage of M2 macrophages increased from 44±13% at 1 week to over 95±2% at 12 weeks, while the unseeded DAT cohort reached a maximum of 67±6% at 12 weeks. ASC seeding also significantly accelerated blood vessel formation in the DAT implants. At 4 weeks post implantation, approximately 62±6% of all CD31+ blood vessels were also VEGF+ in the ASC-seeded DAT, as compared to 38±1% in the unseeded DAT. This difference in VEGF expression diminished at 8 and 12 weeks post-implantation. In terms of adipogenesis, although no significant difference in PPAR expression was detected between the ASC-seeded and unseeded DAT at 1 and 4 weeks, the percentage of PPAR+ cells in the implant and surrounding areas increased dramatically at 8 weeks. At this time point, the number of PPAR+ cells in the seeded DAT group was 24±4% of total cells quantified, while the unseeded DAT cohort was only 12±5%. This enhancement in adipogenesis was confirmed by Masson's trichrome staining, which showed that at 12 weeks post implantation 56±9% of the original DAT implant had become converted into adipose tissue in the ASC-seeded group, as compared to only 4±2% in the unseeded DAT scaffold group (Fig.1).

Discussion and Conclusion

Our results indicate that exogenous ASCs present in the implanted DAT bio-scaffolds mediated the macrophage profile, accelerated blood vessel formation and enhanced remodelling of the scaffolds into adipose tissue. This data strongly supports the notion that the incorporation of ASCs enhances the natural regenerative capacity of our bio-scaffolds. Seeding with exogenous ASCs may be particularly important for enabling the stable and predictable augmentation of large volume soft tissue defects, where the implanted material alone may not successfully revascularize within the critical time frame required to support the desired soft tissue regeneration.


Figure 1. The effect of ASC seeding on adipogenesis in DAT. a) Representative Masson's trichrome staining at 12 weeks. b) The relative area of adipose tissue within the implant region over time. * Statistical significance was found between seed and unseeded DAT at 12 weeks. (n=3, p ”¹ 0.05)

Acknowledgements

We would like to acknowledge funding from the CIHR and the Ontario Centres of Excellence, as well as Dr. Frederick Watkins and Mrs. Karen Martin for clinical collaborations to support this work. We would like to thank Cody Brown, Lydia Fuetterer and Dr. Juares Bianco for their technical assistance with the implant preparation and surgeries.

References

1. 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(17), 4715-24 2. Zhao Y.; Waldman S. D.; and Flynn L. E. Multilineage co-culture of adipose-derived stem cells for tissue engineering, J of Tissue Eng and Reg Med. 2012, DOI: 10.1002/term.1643 3. Zuk P. A.; Zhu M.; Ashjian P.; De Ugarte D. A.; Huang J. I.; Mizuno H.; Alfonso Z. C.; Fraser J. K. Benhaim P.; Hendrick M. H. Human adipose tissue is a source of multipotent stem cells, Mol Biol of cell. 2002 13(12) 4279-95

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