Download Optimization and Characterization of Decellularized Adipose Tissue

Optimization and Characterization of Decellularized Adipose Tissue
Malik J. Snowden1; Christopher M. Mahoney, M.S.1; Kacey G. Marra, Ph.D.1-4; J. Peter Rubin, MD1-4
Dept. of Bioengineering1, Dept. of Plastic Surgery2, School of Medicine3, McGowan Institute of
Regenerative Medicine4, University of Pittsburgh, Pittsburgh, PA 15213
INTRODUCTION
There is currently a clinical need for the
improvement of soft tissue repair. Soft tissue repair is
required after soft tissue loss which can be caused by
congenital deformities, traumatic wounds and tumor
resections. A common type of soft tissue repair is
autologous fat grafting.1 However, the problem with
grafted fat is that it tends to succumb to fat ischemia
caused by a lack of vascular integration with the
implant site. This fat ischemia leads to adipocyte
necrosis and the reabsorption of the graft back into
the body.2 In this, the graft volume will be reduced
40-60% within the first six months of the grafting
procedure.3 In order to combat the volume reduction
that is associated with autologous fat grafting, studies
have shown that a suitable regenerative technique
may be to develop scaffolds derived from the
extracellular matrices (ECM) of human adipose
tissue.4 Adipose tissue is used to produce the
extracellular matrices because it contains high
concentrations of proteins that contribute to
adipogenesis.4
The approach taken in the laboratory requires two
protocols, one being to decellularize human adipose
tissue and the other being to derive a hydrogel from
the decellularized adipose tissue. The purpose of the
decellularization protocol is to remove all of the cell,
DNA, and lipid content from the tissue. However,
after decellularization, there is still remaining cell and
lipid content in the ECM. The purpose of the gelation
protocol is to form a thermosensitive hydrogel
derived from the decellularized adipose tissue,
however hydrogels formed do not always maintain
thermosensitive properties. Steps have been taken
towards optimizing these protocols so that the goal of
an off-the-shelf biomaterial scaffold can be achieved.
METHODS
Non-diabetic abdominal whole fat was donated
from patients undergoing elective abdominoplasty at
the University of Pittsburgh Medical Center. The
decellularization process used to produce the ECM
includes four main stages, consisting of alcohol rinses,
delipidization, and disinfection of the adipose matrix.
After processing, the matrix is to be snap frozen
through the use of liquid nitrogen, or frozen overnight
in a -80C freezer, and then lyophilized. Following the
freeze drying process, the matrix is broken down into
a powder in preparation for digestion by pepsin
through the use of a Mini Wiley Mill. A 1:10 ratio of
pepsin to ECM is used. The pH of the ECM-pepsin
solution is neutralized (7.4) through the use of sodium
hydroxide and gelation is tested by heating the
hydrogel to body temperature (37°C).
In order to begin optimizing the protocol used for
tissue decellularization, familiarization with the
protocol first occurred, in which the protocol was
repeated multiple times. Following this, observations
were made that could potentially be reasons for why
end results of the decellularization process still have
lipid and cell content. The alteration that was made in
attempt to optimize the protocol was changing the
volumes and concentrations of the alcohols and
detergents used so that the tissue would be fully
covered during the decellularization rinses. To
optimize the protocol used for the gelation of a
hydrogel derived from adipose tissue, the ratio of
pepsin to ECM was changed from 1:10 to 2:15, so that
more pepsin would be present to digest the ECM.
RESULTS
By altering the concentrations and volumes of the
alcohols and detergents used during the
decellularization protocol, the protocol was
effectively optimized as there was a decrease in the
lipid and cell content. As seen in figure 1 below,
Hematoxylin and Eosin staining indicates decreased
cell content following the optimization of the
decellularization, as seen by the decreased amount of
purple dots and the decreased amount of overall
purple color. Figure 2 indicates decreased lipid
content following the optimization of the
decellularization protocol. This is indicated by the lack
of brown color in the matrix, as the brown color
indicates lipid content.
Figure 1: Hematoxylin and Eosin stains nucleic acid to detect cells. Left
image represents post-optimization and right image represents preoptimization. ECM sample on the right indicates high cell content,
characterized by the higher concentration of purple.
Figure 2: ECM prior to optimization (left) and after optimization (right).
The brown color of the ECM indicates high lipid content while the white
color of the right ECM indicated minimal lipid content.
By altering the ratio of pepsin to ECM in the
hydrogel gelation protocol, the protocol was
effectively optimized. The ratio was changed from
1:10 to 2:15 in order to increase the amount of
pepsin present to digest the ECM. This allowed the
hydrogel to successfully gel at 37°C (the font here
isn’t the same as the rest of the paper).
DISCUSSION
The goal of this project is to optimize two protocols
used throughout the laboratory, one being to
decellularize adipose tissue and the other to derive an
injectable thermosensitive hydrogel form the
decellularized adipose tissue. The problem faced
when using the decellularization protocol is that
tissue still contains cell and lipid content after
decellularization. The problem faced when using the
hydrogel gelation protocol is that the hydrogels made
don’t always possess the thermosensitive properties
that we expect them to. The problems initially faced
with the decellularization protocol were caused by a
lack of interaction between the reagents and the
tissue during the decellularization rinses. In order to
solve this problem, the surface area of the tissue was
increased by adding in additional manual processing
of the tissue, as well as by increasing the amount of
the reagents used. The problems faced with the
hydrogel gelation protocol were caused by a lack of
ECM digestion by the pepsin. This was combated by
increasing the pepsin concentration to increase the
digestion of the pepsin.
CONCLUSION
The decellularization protocol and the hydrogel
gelation protocol were both successfully optimized.
This is important as they are necessary to reaching
the project’s end goal, which is to develop an
injectable biomaterial. Optimizing these protocols
allows for ECM suitable for hydrogel gelation and a
hydrogel suitable to be an injectable biomaterial.
ACKNOWLEDGEMENTS
I would like to acknowledge my lab’s primary
investigators Dr Kacey Marra, PhD and Dr Peter
Rubin, MD, FACS, as well as my graduate mentor
Christopher Mahoney and the other members of the
Adipose Stem Cell Center as well as our funders.
REFERENCES
1. Choi, JH et al. “Adipose Tissue Engineering for
Soft Tissue Regeneration.” Tissue Engineering
(2010) 413-428.
2. Kelmendi-Doko, A et al. “Adipogenic FactorLoaded Microspheres Increase Retention of
Transplanted Adipose Tissue.” Tissue Engineering
(2014) 1-8.
3. Wang, W et al. “Adipose tissue engineering with
human adipose tissue-derived adult stem cells
and a novel porous scaffold.” Society For
Biomaterials (2012) 68-75.
4. Sano, H et al. “Acellular adipose matrix as a
natural scaffold for tissue engineering.” Journal of
Plastic, Reconstructive & Aesthetic Surgery (2014)
99-106.