Stem cell therapies have shown promising capabilities in regaining the functionality of scar tissue following a myocardial infarction. Biological sutures composed of fibrin have been shown to more effectively deliver human mesenchymal stem cells (hMSCs) to the heart when compared to traditional cell delivery mechanisms. While the biological sutures do show promise, improvements can be made. To enhance the fibrin sutures, we propose to incorporate native cardiac extracellular matrix (ECM) into the fibrin microthreads to produce a more in vivo-like environment. This project investigated the effects that ECM incorporation has on fibrin microthread structure, mechanics, stem cell seeding, and pro-angiogenic potential. Single microthreads composed of fibrin or fibrin and ECM were subjected to uniaxial tensile testing. It was found that the microthreads consisting of both fibrin and ECM had significantly high elastic moduli than fibrin only microthreads. Cell seeding potential was evaluated by performing a 24-hour hMSC seeding experiment using sutures of the varying microthread types. A CyQuant cell proliferation assay was used to determine the number of cells seeded onto each suture type. The results determined that there was no statistical difference between the numbers of cells seeded on the types of sutures. To examine the pro-angiogenic potential the microthreads had, a 24-hour endothelial progenitor outgrowth cell (EPOC) outgrowth assay was used. Fibrin and 15% ECM-fibrin microthreads were placed within the scratch of an EPOC culture and evaluated every 6 hours for 24 hours. We found that the 15% ECM microthreads had significantly increased the EPOC outgrowth, approximately 16% more distance travelled than fibrin microthreads and 18% more than no microthreads. Our combined results suggest that ECM does not affect hMSC attachment to biological sutures but does increase the pro-angiogenic potential of the microthreads due to their increase in guiding EPOC outgrowth.

"Engineered muscle tissue offers a promising solution for the treatment of large muscle defects. Three-dimensional tissue engineered matrices, such as microthreads, can be used to grow new myofibers that will reduce scar formation and integrate easily into native myofibers. We hypothesize that adsorbing growth factors to the surface of braided collagen scaffolds using crosslinking strategies will promote muscle derived fibroblastic cell (MDFC) attachment and growth, which will serve as a platform for delivering cells to large muscle defects for muscle regeneration. To test this hypothesis, self-assembled type I collagen threads were braided and crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with and without heparin and 5 ng/mL, 10 ng/mL, or 50 ng/mL fibroblast growth factor (FGF-2) bound to the surface. Using immunhistochemistry, braided collagen scaffolds showed the presence of FGF-2 on the surface, and braiding the microthreads increased the mechanical properties compared to single threads. To determine the effect of FGF-2 on MDFC attachment, growth, and alignment, scaffolds were seeded with a MDFC cell suspension for 4 hours using a PDMS mold with a sealed 1 mm by 12 mm channel and cultured for 1, 5, or 7 days. After 1 day of culture, the results show a significant increase in cell attachment on braids crosslinked with EDC/NHS with heparin and no significant difference in attachment between the different concentrations of FGF-2 and EDC/NHS crosslinked scaffolds. After 7 days in culture, the MDFCs responded to FGF-2 with a positive linear correlation between growth rate and concentration of FGF-2 on the surface. Additionally, all control scaffolds showed cellular alignment after 7 days, while MDFCs on FGF-2 modified scaffolds showed limited alignment. These results show braided collagen scaffolds crosslinked with EDC/NHS with heparin delivering a controlled quantity of FGF-2 can support MDFC attachment and growth, which may serve as an exciting new approach to facilitate the growth and ultimately the delivery of cells to large defects in muscle regeneration."

Myocardial infarction is a prominent medical problem in the world today. Current treatments are limited and do not strive to regenerate the myocardial tissue that is lost post-infarction. Human mesenchymal stem cells (hMSCs) have been shown to improve cardiac function when implanted post-infarction. The effectiveness of stem cell therapy largely depends on the delivery method. Current delivery methods are insufficient due to their low cell engraftment rate and inability to target the endocardium, where most myocardial infarctions occur. Biological microthreads are a promising new local cell delivery method that may improve upon these current limitations. We hypothesize that biological microthreads will increase efficiency of hMSC delivery to the beating rat heart compared to intramyocardial injection. To test our hypothesis we seeded biological microthreads in vitro with 100 ìL of cell suspension (100,000 hMSCs). After one day, an average of 11,806 ± 3,932 hMSCs were counted on the biological microthreads. The biological microthreads were attached to suture needles to allow targeted delivery to the rat heart (in the left ventricular wall). Human mesenchymal stem cells were loaded with quantum dots prior to seeding the biological microthread bundles or delivery to the rat heart via injection. For intramyocardial injection, a cell suspension containing 10,000 hMSCs (35 ìL) was injected into the myocardial wall using a 100 ìL syringe. The delivery efficiency of each method was determined by sectioning the heart into 8 µm thick sections and analyzing three sections every sixty sections (24 µm every 480 µm) for quantum dot loaded hMSCs. These sections were stained with Hoechst dye and quantum dot loaded cells in the heart sections were manually counted. The delivery efficiency of each biological microthread implantation was calculated by dividing the number of counted quantum dot loaded hMSCs in the heart wall by the average number of hMSCs on the biological microthread bundles (normalized to the length that was implanted in the heart wall) after 24 hours. The delivery efficiency of intramyocardial injection was calculated by dividing the number of counted quantum dot loaded hMSCs in the heart wall by 10,000 (the number of cells injected). Biological microthread mediated hMSC delivery had a significantly higher delivery efficiency (66.6 ± 11.1%) compared to intramyocardial injection (11.8 ± 6.25%) after 1 hour (p < 0.05). Biological microthread implantation tracking illustrated that we were able to deliver hMSCs to the myocardium and endocardium of the left ventricular wall for hMSC delivery. This study illustrates that biological microthreads can serve as an efficient means of delivering hMSCs to the infarcted heart. Unlike the currently utilized delivery methods, biological microthreads can target the infarcted layer of the left ventricular wall and maximize hMSC engraftment to that layer.

"Recent data show that 7.6 million Americans have survived a myocardial infarction (MI), and 5.1 million Americans suffer from severe heart failure. Stem cell therapy has the potential to improve cardiac function after MI. Two promising cells for cardiovascular regeneration therapies include human mesenchymal stem cells (hMSCs) and pluripotent stem cell derived cardiomyocytes (hPS-CM) each with their own unique method for improving cardiac function post-infarct. However, a limiting factor to cell therapies is that the methods currently used to deliver cells to the myocardium, including intramyocardial injection (considered the gold standard), suffer from low retention rates. To promote localization of delivered cells to the infarct and increase retention rates, our lab has developed a fibrin biological suture that can deliver human mesenchymal stem cells (hMSCs) with an efficiency of 64% compared to just 11% with intramyocardial injection in the normal rat heart. In this dissertation we sought to examine the functionality of hMSC and hPS-CM seeded sutures and their impact on cardiovascular regeneration applications. We began by delivering hMSC seeded fibrin sutures to an infarcted rat heart and found that the sutures are an effective method to deliver cells to the infarcted myocardium and demonstrated a trend towards improved regional mechanical function in the infarct region over infarct alone. Next, we transitioned to using hPS-CM and developed methods to seed the sutures, as well as a method to measure hPS-CM contractility with high spatial and temporal resolution, while concurrently capturing calcium transients. This technique allowed us to examine the contractile behavior in terms of contractile strain and conduction velocity of hPS-CM seeded on fibrin microthreads over 21 days in culture. We found that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction and that extended culture promotes cell alignment along the length of the suture as well as improvements in contractile function in terms of increases in contractile strain and conduction velocity. Finally, we delivered the hPS-CM seeded microthreads to an uninjured rat heart and found a delivery efficiency of 67%. Overall, we further demonstrated the technology of the fibrin suture to deliver cells to an infarct as well as the ability to support the attachment, contraction and delivery of hPS-CM to cardiac tissue. "

A wide variety of techniques have been explored to synthesize small diameter tissue engineered blood vessels. Toward this end, we are exploring direct cell seeding and culture on tubular mandrels to create engineered vascular tissues. In the present study, v-shaped channels cast from polydimethyl siloxane (PDMS) were used as cell seeding wells. Fibrin microthreads placed in the chamber were used as model tubular seeding mandrels. Human mesenchymal stem cells (hMSCs) were seeded onto fibrin microthreads in v-shaped channels for 4 hours. Cell attachment to the microthreads was confirmed visually by Hoechst nuclear staining and a cell quantification assay showed that 5,114 ± 339 cells attached per 1 cm fibrin microthread sample (n = 6). Fibrin microthreads were completely degraded by hMSCs within 5 days of culture, therefore UV crosslinking was used to increase their mechanical strength and prolong the amount of time cells could be cultured on fibrin microthreads and generate tubular tissue constructs. Cell attachment was unaffected on UV-crosslinked microthreads compared to uncrosslinked microthreads, resulting in a count of 4,944 ± 210 cells per 1 cm of fibrin microthread sample (n = 3). Long term culture of the hMSCs on the UV-crosslinked fibrin microthreads showed an increase in cell number over time to 11,198 ± 582 cells per cm of microthread after 7 days with 92% cell viability (CYQUANT NF/DEAD staining) and evidence of cell proliferation. The results show that the v-well cell seeding technique was effective in promoting rapid hMSC attachment on UV-crosslinked fibrin microthreads and encouraged their growth, maintained viability and also promoted their proliferation over the culture period. In conclusion, the technique could serve as an efficient model system for rapid formation of tissue engineered vascular grafts.

"Recent evidence suggests that delivering human mesenchymal stem cells (hMSCs) to the infarcted heart reduces infarct size and improves ventricular performance. However, cell delivery systems have critical limitations such as inefficient cell retention and poor survival, and lack targeted localization. Our laboratories have recently developed a method to produce discrete fibrin microthreads that can be attached to a needle and delivered to a precise location within the heart wall. We hypothesize that fibrin microthreads will support hMSC proliferation, survival and retention of multipotency, and may therefore facilitate targeted hMSC delivery to injured tissues such as infarcted myocardium. To test this hypothesis, we bundled 100 μm diameter microthreads to provide grooves to encourage initial cell attachment. We seeded hMSCs onto the microthread bundles by applying 50,000 cells in 100 μL of media. The number of cells adhered to the microthreads was determined up to 5 days in culture. Cell density on the fibrin microthreads increased over time in culture, achieving an average density of 730 ± 101 cells/mm2. A LIVE/DEAD assay confirmed that the cells were viable and Ki-67 staining verified the increase in cell number over time was due to proliferation. Additionally, functional differentiation assays proved that the hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that delivering 1 to 4 cell seeded microthread bundles to the infarcted rat myocardium has the potential to produce a positive improvement in mechanical function and these microthreads support hMSC proliferation and survival. Additionally these findings suggest that cell-seeded microthreads may serve a platform technology to improve localized delivery of viable cells to infarcted myocardium to promote functional tissue regeneration. "

Volumetric muscle loss (VML) typically results from traumatic incidents; such as those presented from combat missions, where soft-tissue extremity injuries account for approximately 63% of diagnoses. These injuries lead to a devastating loss of function due to the complete destruction of large amounts of tissue and its native basement membrane, removing important biochemical cues such as hepatocyte growth factor (HGF), which initiates endogenous muscle regeneration by recruiting progenitor cells. Clinical strategies to treat these injuries consist of autologous tissue transfer techniques, requiring large amounts of healthy donor tissue and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient’s cells to align and differentiate into muscle tissue in situ. In this thesis, we developed fibrin microthreads, scaffolds composed of aligned fibrin material that direct cell alignment along the longitudinal axis of the microthread structure, with specific structural and biochemical properties to recreate structural cues lost in VML injuries. We hypothesized that fibrin microthreads with an increased resistance to proteolytic degradation and loaded with HGF would enhance the functional, mechanical regeneration of skeletal muscle tissue in a VML injury. We developed a crosslinking strategy to increase fibrin microthread resistance to enzymatic degradation, and increased their tensile strength and stiffness two- to three-fold. This crosslinking strategy enhanced the adsorption of HGF, facilitated its rapid release from microthreads for 2 to 3 days, and increased the chemotactic response of myoblasts twofold in 2D and 3D assays. Finally, we implanted HGF-loaded, crosslinked (EDCn-HGF) microthreads into a mouse model of VML to evaluate tissue regeneration and functional recovery. Fourteen days post-injury, we observed more muscle ingrowth along EDCn-HGF microthreads than untreated controls, suggesting that released HGF recruited additional progenitor cells to the injury site. Sixty days post-injury, EDCn-HGF microthreads guided mature, organized muscle to replace the microthreads in the wound site. Further, EDCn-HGF microthreads restored the contractile mechanical strength of the tissue to pre-injured values. In summary, we designed fibrin microthreads that recapitulate regenerative cues lost in VML injuries and enhance the functional regeneration of skeletal muscle.