Browsing by Subject "Regenerative medicine"

Bone grafting is one of the most commonly used surgical methods to augment bone regeneration in orthopedic procedure. While using natural bones, such as autograft and allograft are considered as the gold standard techniques, they suffer from numerous drawbacks including scarcity, donor site complications, and potential disease transmission. To overcome these limitations, mineralized poly (ethylene glycol) diacrylate-co-N-acryloyl 6-aminocaproic acid (PEGDA-co-A6ACA) composed of an organic phase and an inorganic, biomineralized phase that recapitulates certain aspects of dynamic mineral environment of native has been developed. The real-world application this biomineralized material in treating bone defects in vivo depends upon a myriad of parameters including scaffold structural parameters (e.g. pore size), mechanical properties (e.g. strength and toughness), and host environments (e.g. age of the recipient). In this dissertation, I explored these biomaterial and biological parameters for biomaterials mediated bone regeneration through leveraging endogenous healing mechanism. First of all, I evaluated the potential of mineralized biomaterials to induce bone repair of a critical-sized cranial defect in the absence of exogenous cells and growth factors. I demonstrated that the mineralized biomaterial alone can support complete bone formation within critical-sized bone defects through recruitment of endogenous cells and neo-bone tissue formation in mice. By providing a bone-specific mineral environment, these biomaterials induce osteogenic commitment of recruited host progenitor cells and support the maintenance of cells relevant for the formation and function of bone tissues, including vascularization of the implant during repair. Based on these findings, I further investigated the effect of the scaffold pore size on in vivo ectopic bone formation. Biomineralized PEGDA-co-A6ACA hydrogels were made to have an interconnected macroporous network with different pore size ranges (45-53 μm, 90-106 μm, 160-180 μm, 212-250 μm or 300-355 μm) and similar overall porosity between 65% to 70%. Using these scaffolds, I evaluated their abilities to promote ectopic bone formation upon subcutaneous implantation in wild-type mice as a function of time. I found that scaffolds with pore sizes larger than 100 μm showed similar bone formation abilities, whereas in scaffolds with pore sizes 45-53 μm, cell infiltration only happened at the peripheral region of the scaffolds. Results from this study revealed that pore size of the scaffolds had a prominent influence on the extent of cell infiltration and bone ingrowth. While such biomaterial-mediated in situ tissue engineering is highly attractive, success of this approach relies largely on the regenerative potential of the recruited endogenous cells, which is anticipated to vary with age of the host. To this end, I investigated the effect of the age of the host on mineralized biomaterial-mediated bone tissue repair using critical-sized cranial defects as a model system. Mice of varying ages, 1-month-old (juvenile), 2-month-old (young-adult), 6-month-old (middle-aged), and 14-month-old (elderly), were used as recipients. I showed that the biomineralized scaffolds support bone tissue formation by recruiting endogenous cells for all groups albeit with differences in an age-related manner. The age of the recipient mice had a significant influence on the quantity and quality of the neo-bone tissues characterized in terms of bone mineral deposition and bone tissue-specific markers, where delayed bone formation and decreased quantity of neo-bone tissue formation were observed in older mice. The real-world applications of the biomineralized materials for aiding bone tissue regeneration are greatly limited by the lack of mechanical strength and toughness of the materials. To enhance the mechanical property of the biomineralized scaffold, I further proposed a double network (DN) hydrogel system with an asymmetric network structure, where the first network is tightly cross-linked by A6ACA with crosslinker N, N'-Methylenebisacrylamide (bisacrylamide), and the second network is loosely crosslinked PEGDA. The effects of bisacrylamide crosslinker concentration (2 mol.%, 4 mol.% and 6 mol.%), and molecular weight (Mn: 3.4 kDa, 6 kDa, 10 kDa, and 20 kDa) of 20 w/v % PEGDA on mechanical properties of the resultant DN-hydrogels were investigated and compared to those of single network (SN) hydrogels of the same composition. Findings from this study showed that increase in crosslinker concentration of the first network was correlated with lower ultimate compressive strain, higher compressive strength, toughness and elastic modulus. Furthermore, DN-hydrogels prepared in this work displayed swelling ratios ranging from 569 ± 20% to 1948 ± 12%. Among all compositions, DN-hydrogel with 6 mol.% bisacrylamide and PEGDA 10 kDa demonstrated the highest compressive strength (3.47 ± 0.35 MPa), highest toughness (0.60 ± 0.03 MJ/m3), and elastic modulus (1.04 ± 0.09 MPa). Using this composition, porous DN-hydrogels with interconnected pore architecture were fabricated through polymethylmethacrylate (PMMA) bead leaching method. Resultant porous hydrogels demonstrated potent biomineralization capabilities, and the matrix-bounded CaP minerals were able to undergo dissolutions. Given the high strength and biomineralization capacity, DN-hydrogels reported here could be useful for developments of tissue engineering scaffolds for bone tissue regenerations. Overall, this dissertation explores different biomaterial designs and biological factors in biomaterial-mediated in vivo bone tissue repair, providing materials insights that are useful to researchers and engineers in designs of biomaterials to leverage endogenous healing mechanism for tissue regeneration and repair.

Endothelial dysfunction is the predominant pathophysiological state prior to the onset of atherosclerosis. Currently, treatments for endothelial dysfunction are evaluated in vitro using two-dimensional (2D) cell culture assays or in vivo animal models. Microphysiological systems are small-scale three-dimensional (3D) tissue models that recapitulate the native tissue structure and function. An ideal microphysiological system is comprised of human cells embedded within a 3D matrix introduced to physiological fluid perfusion. Immune challenge in the form of cytokines or immune cells further recapitulates the native microenvironment.

A vascular microphysiological system was developed from a small-diameter tissue engineered blood vessel (TEBV) in a perfusion culture circuit. TEBVs were created from collagen gels embedded with human neonatal dermal fibroblasts and plastically compressed to yield collagen constructs with high fiber densities. TEBVs are rapidly producible and can be directly introduced into perfusion culture immediately after fabrication. Endothelium-independent vasoconstriction in response to phenylephrine and endothelium-dependent vasodilation in response to acetylcholine were used to analyze the health and function of the endothelium non-destructively over time.

Endothelial dysfunction was induced through introduction of the pro-inflammatory cytokine tumor necrosis factor – α (TNF-α). Late-outgrowth endothelial progenitor cells derived from the peripheral blood of coronary artery disease patients (CAD EPCs) were evaluated as a potential endothelial source for autologous implantation in both a two-dimensional (2D) direct co-culture model as well as a 3D model as an endothelial source for a tissue engineered blood vessel. CAD EPCs demonstrated similar adhesive properties to a confluent, quiescent layer of smooth muscle compared to human aortic endothelial cells. Within the TEBV system, CAD EPCs demonstrated the capacity to elicit endothelium-dependent vasodilation. CAD EPCs were compared to adult EPCs from young, healthy volunteers. Both CAD EPCs and healthy volunteer EPCs demonstrated similar endothelium-dependent vasoactivity in response to acetylcholine; however, in response to TNF-α, CAD EPCs demonstrated a reduced response to phenylephrine at high doses.

The treatment of TEBVs with statins was explored to model the drug response within the system. TEBVs were treated with lovastatin, atorvastatin, and rosuvastatin for three days prior to exposure to TNF-α. In all three cases, statins prevented TNF-α induced vasoconstriction in response to acetylcholine within the TEBVs, compared to TEBVs not treated with statins. Overall, this work characterizes and validates a novel vascular microphysiological system that can be tested in situ in order to determine the effects of various patient populations and drugs on endothelial health and function under healthy and inflammatory conditions.

Nucleic acid delivery has applications ranging from tissue engineering to biologics development and manufacturing to vaccines and infectious disease. To improve delivery and extend viable expression over time, we turn to biomaterials as a method for sustained nucleic acid release and enhanced cell culture or tissue interaction. Here, we describe how cationic polymer and lipid condensed nucleic acids can be effectively loaded into injectable granular hydrogel scaffolds by stabilizing the condensed nucleic acid into a lyophilized powder, loading the powder into a bulk hydrogel, and then fragmenting the gel into hydrogel microparticles. The resulting microgels contain non-aggregated nucleic acid particles, can be annealed into an injectable microporous scaffold, and can effectively deliver nucleic acids to cells with a sustained rate of expression. We explore how this technology can improve the production of biologics, like antibodies and viruses, to overcome limitations of current batch processes. Our scaffolds allow for continuous biologics manufacturing, with sustained production upwards of 30 days. We also explore how our platform can improve tissue regeneration in disease models like dermal wounds by delivering nucleic acid drugs, namely DNA, mRNA, and therapeutic viruses. The loaded granular scaffolds allow cells to readily repopulate the missing tissue and drugs be locally released and taken up over time. Overall, our scaffold delivery approach is a customizable platform that can be tuned for many different applications.

Adult mammals have limited regenerative capacities. Lost limbs are replaced by scar tissues. Heart attacks lead to massive cardiomyocyte death without robust proliferative response at the injury sites, resulting in permanently impaired cardiac function. While current treatments mainly rely on drug, tissue transplantation, cell therapy and tissue engineering, there remains an urgent need for new strategies to promote regeneration due to limitations in efficiency and specificity in existing approaches. Previous research in our lab identified vitamin D as a potent pro-regenerative factor in zebrafish. To further determine the role of vitamin D signaling in regeneration, we applied drug treatments, generated transgenic zebrafish, and performed proliferation and regeneration essays to examine the necessity and sufficiency of the pathway in regulating fin and heart regeneration. In addition, we designed a novel system to allow spatial and temporal sensitization of tissue’s response to vitamin D signaling utilizing the tissue regeneration enhancer elements (TREEs) and tested this for its ability to promote regeneration in a tissue-specific way. Key findings from this study are: 1) vitamin D signaling is required for zebrafish fin and heart regeneration; 2) enhanced vitamin D signaling promotes fin and heart regeneration; 3) Temporal overexpression of vitamin D receptor at injury sites regulated by TREEs enables locally sensitized response to vitamin D signaling and improves fin regeneration without global effects in zebrafish.