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Fisher et al published the first attempt at engineering
a full-scale adult human femur head from hMSCs. This
is the largest reported tissue to have been engineered
and took over 700 million hMSCs to fabricate.
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John
Fisher’s laboratory at the University of Maryland, College Park recently
published what can be considered a significant advance for Tissue
Engineering and Regenerative Medicine. Graduate
student Bao Nguyen and her colleagues have engineered a bone construct that is 20 times larger than any
reported previously,
and the size of an adult human femur.
Why is this important?
Critical size bone defects are a significant health problem (resulting in over
$1 billion in annual healthcare costs incurred in the U.S.) and are currently
treated with grafts, decellularized bone, or synthetic bone grafts, with
sometimes unsucessful results. As such, modern medicine has been looking to tissue
engineered bone grafts as future treatments for such defects. Human bone marrow-derived Mesenchymal
Stem Cells (hBM-MSCs)
are a promising cell source for such applications because they efficiently differentiate down the
osteogenic path and
also secrete paracrine factors that may aid survival and
vascularization of engineered bone. Prior to this publication, engineered
constructs have been relatively small due to cell and culture limits. One major challenge has been growing
hBM-MSCs, while maintaining their function, to sufficient numbers needed for an
adult human-sized construct; a challenge adressed by RoosterBio.
In addition, nutrient and O2 transfer are often insufficient
to maintain cell viability and function throughout larger constructs,
especially those of adult human dimension.
To address this cell
culture limitation, the Fisher laboratory developed a Tubular Perfusion System (TPS) bioreactor where cells
and scaffolds are cultured in a cylindrical chamber and subject to circular media
flow. This system has high nutrient and O2 transfer and efficient waste
removal and has been previously used to produce smaller engineered bone and
cartilage constructs (See here and here).
In the study detailed
here, the authors had access to and combined, for the first time, advanced
technologies required for biofabrication: 3D printing, the TPS bioreactor,
and scalable production of hBM-MSCs. The goal of the study was to scale-up bone
constructs to adult human size. A full size mold of the superior portion of a
human femur (the largest bone in the human body) was 3D printed using
information from an opensource database. The mold measured 23 cm long and
10 cm at its widest point with a volume of
200 cm3. The mold was filled with hBM-MSCs in alginate beads
(3 mm beads, 100,000 cells per bead). The entire construct utlillized 7200
aliginate beads containing a total of 7.2 x 108 cells (yes, that is
720 million cells!). The high volume hBM-MSC cell and media systems used were
from RoosterBio, and technical support for the efficient production
of large volumes of hBM-MSCs was provided by our company.
After 8 days of
culture in the TPS, the construct was examined for cell viability and bone
differentiation. High cell viability was seen in all parts of the construct,
both on the outside and the inside (interior). In addition, hBM-MSCs committed
to the osteogenic lineage throughout the construct, demonstrating efficiency of the TPS
culture system. Both early (Alkaline Phosphotase, ALP) and late (Bone
Morphogenic Protein-2, BMP-2) markers of osteogenesis were upregulated relative
to day 0. Interestingly, ALP and BMP-2 expression was 25- to 30-fold higher in
the construct shaft relative to other portions of the construct. The authors
speculate that this is due to shear stress exerted on the parts of the
construct closest to the inlet, which activates hBM-MSC signaling pathways,
causing release of paracrine factors that stimulate osteogenesis of the
“downstream” shaft portion. Taken together, these results demonstrate that the
confluence of cutting-edge technologies such as 3D printing, TPS bioreactors,
and best-in-class hBM-MSC manufacturing processes enable the engineering of
adult human-sized tissue constructs.
While “…this first
foray into full-scale bone engineering provides the foundation for future
clinical applications of bioengineered bone grafts…” the authors point out some
limitations to this study. The 8 day culture period was relatively short, given
the weeks usually needed for high efficiency bone differentiation. Thus,
extended time points and the fabrication of additional large constructs are
needed to fully explore the capabilities of the TPS system. Further, alginate is
a soft material, and its mechanical properties do not render it the best suited
for bone differentiation. In addition,
hBM-MSCs within aliginate beads lack cell:cell contact, which may also limit their
osteogenic differentiation. To address
these limitations of the current system, the Fisher group is developing a 3D
printed shell made of an implantable rigid material better suited for the
engineering of bone constructs. Finally,
the construct lacks a vascular network, which can be overcome by including
endothelial cells (EC) in addition to hBM-MSCs or by incorporating
micro-channels in the engineered constructs through a variety of methods (e.g. biomaterial
fabrication and 3D printing). Despite the aforementioned limitations, the
work presented is a significant advance towards clinical-sized tissue-engineered
bone constructs for use in patients.
In an attempt to
elicit discussion, I will mention other methods that harbor potential for use
in such large-scale tissue engineering applications. For one, hBM-MSC
aggregates could be used in place of cells in alginate beads. These 3D-MSC not only maintain cell:cell contact
but also undergo osteogenic differentiation
more efficiently
than cells grown on tissue culture plastic, are resistant to hypoxia, and secrete angiogenic cytokines. Secondly, factors that
stimulate bone differentiation of hBM-MSCs, and/or alter mechanical properties
of the construct, could be incorporated into the polymer scaffolding, or could
be introduced into 3D-MSC aggregates. Finally, once bio-inks are
developed further, the bone construct could be patterned by 3D printing of cells
(hBM-MSC, EC, 3D-MSC) and materials. Now
that human-sized constructs are possible in terms of cell numbers and O2
and nutrient diffusion, the possibilities are virtually endless.
Finally, we sincerely
thank Bao Nguyen and John Fisher for being early adopters of RoosterBio hBM-MSCs
and joining us in accelerating Regenerative Medicine!
References:
Nguyen BB, Ko H, Moriarty RA, Etheridge
JM, Fisher JP. Dynamic Bioreactor Culture of High Volume Engineered Bone
Tissue. Tissue Engineering Part A. Volume 22, Numbers 3 and 4, 2016, ahead of
print. doi:10.1089/ten.tea.2015.0395. http://online.liebertpub.com/doi/abs/10.1089/ten.tea.2015.0395
I’m sorry that this is paywalled!
Yeatts, A.B., and Fisher, J.P. Tubular
perfusion system for the long-term dynamic culture of human mesenchymal stem
cells. Tissue Eng Part C 17, 337, 2011.
Yeatts, A.B., Choquette, D.T., and
Fisher, J.P. Bioreactors to influence stem cell fate: augmentation of
mesenchymal stem cell signaling pathways via dynamic culture systems. Biochim
Biophys Acta 1830, 2470, 2013.
Ma, X et al. Deterministically
patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting PNAS, Early Edition doi:
10.1073/pnas.1524510113 http://www.pnas.org/content/early/2016/02/04/1524510113
