Development and Manufacturing of Scaffold-less Constructs for Tendon/Ligament Repair.

Soft tissues, such as rotator cuff tendons and the anterior cruciate ligament (ACL), integrate with the subchondral bone through a complex multi-tissue interface that functions to minimize the formation of stress concentrations and enable the efficient transfer of load between tendon or ligament and bone. Current rotator cuff tendon and ACL repair techniques, requiring the reattachment of the tendon/ligament to its original bony footprint, fail to regenerate this interface. Instead, the repaired insertion site transitions from tendon/ligament to bone through a disorganized, fibrovascular scar tissue with weak mechanical properties, leaving it prone to failure and compromising long-term clinical outcomes. To improve tendon-bone integration following rotator cuff repairs, the objective of this thesis was to utilize a scaffold-less tissue engineered construct to promote the regeneration of the tendon-bone interface and develop a reproducible, automated manufacturing system to facilitate the advancement of the construct towards clinical use. Matrix organization and mechanical properties of the regenerated enthesis were evaluated in both acute (immediate repair) and chronic (repair 4 weeks post injury) supraspinatus tear rat models. Utilization of tissue-engineered constructs resulted in superior enthesis regeneration compared to current mechanical fixation techniques. Next, to enhance the reproducibility and uniformity of existing multi-phasic scaffold-less construct fabrication methodologies, protocol standards and a novel delamination system were developed and later extrapolated for use with human derived constructs. The novel construct fabrication methods yielded an increased number of engineered constructs of consistent size and mechanical properties. Temporal gene expression confirmed the commitment of human derived constructs toward tendon and bone-like tissues. Lastly, to facilitate the eventual large-scale commercial production of our multiphasic tissues, a novel semi-closed bioreactor system was developed and validated. The use of the bioreactor successfully facilitated the co-culture and integration of two distinct tissue types in a single chamber without any direct user manipulation. The findings described in this thesis will lead to the development of a new soft-tissue-to-bone repair strategy to improve functional tendon/ligament repair outcomes and provide the framework for expediting the clinical and commercial translation of our tissue engineering technologies.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108984/1/mikesmee_1.pd

Nucleoside analogues synthesis using natural phosphate doped with I2 (NP/I2) in HMDS

Several D-ribonucleosides are prepared from1-O-acetyl-2,3,5-tri-O-benzoyl-?-D-ribofuranoside and trimethylsilylated nucleobases under mild conditions by using natural phosphate doped with I2 as catalyst Keywords: Natural Phosphate doped with I2, one-step seynthesis. ?/?-D-ribonucleoside

Catalytic And Economic Method Using As Catalyst the I-Si(CH3)3 Generated During The Condensation Of Silylated Base With Natural Phosphate Doped With KI (NP/KI)

Several D-ribonucleosides are prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-?-D-ribofuranoside  and trimethylsilylated nucleobases under mild conditions by using as catalyst I-Si(CH3)3 generated during the condensation of  silylated base with NP/KI. Keywords: I-Si(CH3)3 generated during the condensation of silylated base with NP/KI,one-step synthesis, and D-ribonucleosides

A New Selective Synthesis Of D-Ribonucleosides In Acetonitrile With BSA Using NP/KI As Catalyst

The one-step synthesis of several ?-D-ribonucleosides was performed in good yields under reflux. Keywords: D-nucleosides, natural phosphate, catalyst, one-step seynthesis

Novel approach to Synthesis of Pentofurano nucleoside assisted natural phosphate doped with CF3SO3H as catalyst

This article describes a method for the preparation of ribonucleosides using the solid-phase approach. Several D-ribonucleosides are prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-?-D-ribofuranoside and trimethylsilylated nucleobases under mild conditions by using natural phosphate doped with CF3SO3H (NP/ CF3SO3H) as catalyst. Keywords: Natural phosphate, CF3SO3H, D-Ribonucleoside

Heterogeneous Solid-Liquid Catalysis Of N-glycosylation By Natural Phosphate Doped With Potassium Iodide

Theone-step synthesis of several ?-D-ribonucleosides was performed in good yields under reflux in HMDS. Keywords: N-Glycosylation D-ribonucleosides,Natural phosphate doped with Potassium iodide KI (NP/KI), heterogeneous solid-liquid catalysi

Applications of Microwave in Organic Synthesis: A One-step Synthesis of Ribonucleosides using natural phosphate as solid catalyst

A clean, efficient and fast method for synthesis of ribonucleosides was developed, using  natural phosphate as solid catalyst catalysts supported in a solid phase  promoted by MW irradiation. Keywords: Natural phosphate, Ribonucleosides , Micowave irradiation

Activation of NRF2 and ATF4 Signaling by the Pro-Glutathione Molecule I-152, a Co-Drug of N-Acetyl-Cysteine and Cysteamine

I-152 combines two pro-glutathione (GSH) molecules, namely N-acetyl-cysteine (NAC) and cysteamine (MEA), to improve their potency. The co-drug efficiently increases/replenishes GSH levels in vitro and in vivo; little is known about its mechanism of action. Here we demonstrate that I-152 not only supplies GSH precursors, but also activates the antioxidant kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (KEAP1/NRF2) pathway. The mechanism involves disulfide bond formation between KEAP1 cysteine residues, NRF2 stabilization and enhanced expression of the γ-glutamil cysteine ligase regulatory subunit. Accordingly, a significant increase in GSH levels, not reproduced by treatment with NAC or MEA alone, was found. Compared to its parent compounds, I-152 delivered NAC more efficiently within cells and displayed increased reactivity to KEAP1 compared to MEA. While at all the concentrations tested, I-152 activated the NRF2 pathway; high doses caused co-activation of activating transcription factor 4 (ATF4) and ATF4-dependent gene expression through a mechanism involving Atf4 transcriptional activation rather than preferential mRNA translation. In this case, GSH levels tended to decrease over time, and a reduction in cell proliferation/survival was observed, highlighting that there is a concentration threshold which determines the transition from advantageous to adverse effects. This body of evidence provides a molecular framework for the pro-GSH activity and dose-dependent effects of I-152 and shows how synergism and cross reactivity between different thiol species could be exploited to develop more potent drugs

Three-Dimensional Engineered Bone–Ligament–Bone Constructs for Anterior Cruciate Ligament Replacement

The anterior cruciate ligament (ACL), a major stabilizer of the knee, is commonly injured. Because of its intrinsic poor healing ability, a torn ACL is usually reconstructed by a graft. We developed a multi-phasic, or bone?ligament?bone, tissue-engineered construct for ACL grafts using bone marrow stromal cells and sheep as a model system. After 6 months in vivo, the constructs increased in cross section and exhibited a well-organized microstructure, native bone integration, a functional enthesis, vascularization, innervation, increased collagen content, and structural alignment. The constructs increased in stiffness to 52% of the tangent modulus and 95% of the geometric stiffness of native ACL. The viscoelastic response of the explants was virtually indistinguishable from that of adult ACL. These results suggest that our constructs after implantation can obtain physiologically relevant structural and functional characteristics comparable to those of adult ACL. They present a viable option for ACL replacement.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98477/1/ten%2Etea%2E2011%2E0231.pd

Fresh Versus Frozen Engineered Bone–Ligament–Bone Grafts for Sheep Anterior Cruciate Ligament Repair

Surgical intervention is often required to restore knee instability in patients with anterior cruciate ligament (ACL) injury. The most commonly used grafts for ACL reconstruction are tendon autografts or allografts. These current options, however, have shown failure rates requiring revision and continued instability in the long term. The mismatched biomechanical properties of the current tendon grafts compared with native ACL tissue are thought to contribute to these poor outcomes and potential risk of early onset osteoarthritis. As a possible solution to these issues, our laboratory has fabricated tissue-engineered ligament constructs that exhibit structural and functional properties similar to those of native ACL tissue after 6 months implantation. In addition, these tissue-engineered grafts achieve vascular and neural development that exceeds those of patellar tendon grafts. However, the utility of our tissue-engineered grafts is limited by the labor-intensive method required to produce the constructs and the need to use the constructs fresh, directly from the cell culturing system. Ideally, these constructs would be fabricated and stored until needed. Thus, in this study, we investigated the efficacy of freezing our tissue-engineered constructs as a method of preservation before use for ACL reconstruction. We hypothesized that frozen constructs would have similar histological and biomechanical outcomes compared with our fresh model. Our results showed that 6 months postimplantation as an ACL replacement graft, both our tissue-engineered fresh and frozen grafts demonstrated similar mechanical and histological outcomes, indicating that freezing is a suitable method for preserving and storing our graft before ACL reconstruction. The ability to use frozen constructs significantly increases the versatility of our graft technology expanding the clinical utility of our graft.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140250/1/ten.tec.2014.0542.pd