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TRACK 3 TRACK 3 Technical Program

Altogether, these results indicate that a high level of structural mimicry of the Injuries associated with tendons are among the most common trauma, with
scaffold to the composition and structure of the human osteogenic niche over 250,000 rotator cuff tendon repairs performed annually in the US
can translate in a faster and more efficient osteoinduction in vivo. Our study [1]. The native tendon-bone (TB) interface is comprised of diverse tissues,
represents a significant step in the development of acellular off-the-shelf namely, tendon, fibrocartilage and bone, containing multiple cell phenotypes
substitutes for applications of bone augmentation, not requiring growth fac- such as fibroblasts, fibrochondrocytes and osteoblasts in respective zones
tors, whose use is still highly controversial. [2]. Current clinical approach, mechanical fixation, for tendon reconstruction
grafts often fail to reestablish this hierarchical transition post-surgery leading
5:10pm A Shear Gradient Activated Microfluidic Device for Re- to as high as 90% recurrence rates [3]. Therefore, there is a need for new
al-Time Quantitative Assessment of Blood Hemostasis in vitro augmentation matrices to improve the biological fixation to obtain a scarless
and ex vivo healing at the TB interface.
Our approach to TB integration focuses on the use of biomimetic, nanofi-
Technical Presentation. NEMB2016-6015 brous scaffolds incorporated with bioactive agents. Growth factors injected
into the zone of injury facilitates restoration of the normal function of TB
Abhishek Jain, Amanda Graveline, Anna Waterhouse, Andyna interface [4]. In this regard, transforming growth factor beta3 (TGF-b3) was
Vernet, Wyss Institute for Biologically Inspired Engineering at Har- found to be upregulated during TB insertion development [5]. Similarly, con-
vard University, Boston, MA, United States, Robert Flaumenhaft, nective tissue growth factor (CTGF) was shown to be sufficient to differenti-
Beth Israel Deaconess Medical Center at Harvard Medical School, ate mesenchymal stem cells into tendon specific cells [6]. Hydroxyapatite is
Boston, MA, United States, Donald E. Ingber, Wyss Institute for Bi- known as the major mineral-based component of the bone tissue and poly-
ologically Inspired Engineering at Harvard University, Boston, MA, caprolactone (PCL) is a versatile biomaterial utilized for the engineering of
multiple tissues including tendon, ligament, cartilage as well as bone [7]. The
United States objectives of this study are to 1) fabricate PCL-based scaffolds containing
TGF-b3, CTGF and nano-HA (nHA), where concentrations of CTGF and nHA
Rapid rise in cardiac procedures and use of extra-corporeal devices have change in opposite directions, while TGF-b3 is located in the middle portion
increased the need for integrated instruments for more accurate monitor- of the nanofibrous composites [such organization is expected to contribute
ing of whole blood hemostasis and platelet function in real-time. Here, we to generation of tendon (in CTGF rich zone), fibrocartilage (by TGF-b3) and
describe a microfluidic device that is composed of a biomimetic arteriolar bone (in nHA rich zone) upon positioning the scaffold in proper zones], 2)
blood vessel network including the architecture of stenosis, leading to ap- establish controlled release of TGF-b3 and CTGF from nanofiber scaffolds,
plication of pathophysiological shear rates and shear gradients to flowing and 3) to investigate stem cell behavior on these scaffolds. Such a design is
whole blood, which act as hemodynamic activators to cause blood clotting proposed for the first time, represents a significant departure from the con-
inside the device. Imaging of fluorescently-labeled fibrinogen in whole blood ventional stratified approach, and is expected to contribute to scar-free TB
perfused through this device revealed that clot formation follows a sigmoidal interface regeneration.
trend (S-curve) in this device. Building on this observation, we created an
analytical model that predicted the temporal dynamics of blood clotting that Our findings show that linearly varying nHA distribution can be accom-
was based on a simple pressure measurement using an inline pressure plished across the scaffold thickness that is also the case in native TB in-
sensor. We validated this model at clinically-relevant heparin doses (0.1-2.5 terface[8]. Incorporation of nHA into PCL nanofibers led to increased mean
IU/ml) at various shear rate gradients and found that the parametric fitting fiber diameter from 361±9nm to 459±21nm, and a decrease in contact angle
parameter can be used as a characteristic clotting time (τCT) with sensitivity from 120.01±2.77 ° to 115.24±1.17 °. We have also demonstrated that TGF-b3
higher than standard assays. Then, to rapidly form clots (<30 mins) and be can be incorporated into nanofiber scaffolds with electrospinning and re-
able to work with low blood volume (<1 ml), we coated the device with type leased in a sustained manner. We are to investigate the response of stem
I fibrillar collagen and then showed that the ?CT is sensitive to antiplatelet cells on the proposed scaffolds for interface-related matrix formation (colla-
drug dose, abciximab in its clinical range (0-10 µg/ml). We have tested the gen and glycosaminoglycans) and expression of relevant markers such as
performance of this device in clinical settings in vitro, and found that τCT collagen types I, II, and X.
was increased by ~58% when we perfused blood from patients who were
regularly taking antiplatelet medication. Further, blood from patients who This study not only reveals the importance of design and use of biomimetic
have Hermansky-Pudlak syndrome (HPS) congenital bleeding disorder did scaffolds in tissue engineering but also yields new insights into the effect of
not clot in this device, thus confirming that this device could also act as a bioactive molecules on interface regeneration by controlling their local avail-
more sensitive diagnostic tool of platelet function. Finally, to test the efficacy ability. These discoveries will serve as the foundation for the development of
of this device as an ex vivo monitor or as direct attachment to extracorpo- biomimetic tissue engineering technologies aimed at promoting biological
real systems, we tested this on an endotoxemia porcine model and found graft fixation.
that as the coagulopathy progressed in the pig over 8 hours and blood
became hypercoagulated, the τCT also reduced and trend correlated well This research is funded by TUBITAK.
with changes in plasma thrombin-antithrombin time (TAT) and fibrinogen. In [1]Gulotta+ Am J Sports Med 2009; [2]Benjamin+ J Anat 1986, [3] Galatz+
a second study, when we injected different concentrations of unfractionated J Bone Joint Surg 2004; [4]Gulotta+ Clin Sports Med 2009; [5]Galatz+ J
heparin sequentially into a pig, we found that in addition to displaying a lin- Orthop Res 2007; [6]Lee+ J Clin Invest 2010; [7] Woodruff&Hutmacher Prog
ear dose response, the microfluidic clotting times we determined ex vivo us- Polym Sci 2010; [8]Genin+ Biophys J 2009.
ing the microfluidic device were also significantly more sensitive than those
measured using standard lab tests (aPTT/ACT). Therefore, this bioinspired
shear gradient incorporating global hemostasis and platelet function moni-
toring device, when used in vitro, can be more sensitive and accurate than TUESDAY, FEBRUARY, 23
current gold standard lab tests, and when used ex vivo, can allow real-time
surveillance of antithrombotic therapy using native blood.
3-6
5:30pm Biomimetic Nanofibrous Composites for Tendon-Bone BIOSENSORS
Interface Regeneration

Technical Presentation. NEMB2016-5924 Harris 9:30am - 11:00am

Ece Bayrak, Burak Ozcan, Cevat Erisken, TOBB University of Eco- Session Organizer: Jungkyu Kim, Texas Tech University, Lubbock, 39
nomics and Technology, Ankara, Select State/Province,Turkey TX, United States