Osteogenic Scaffolds for Enhanced Graft-Bone Integration in Ligament Tissue Engineering
Gadalla, Dina Mohamed Adly
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Among the most common knee ligament injuries are those to the anterior cruciate ligament (ACL). Annually, approximately 350,000 people require surgical ACL reconstruction, accounting for more than $6 billion of health-care costs in the United States alone. An injured ACL loses its functions as it cannot heal with larger injuries and heals slowly with smaller ones. This may introduce complications, such as abnormal joint kinematics and deterioration, prior to complete rupture. Although the use of an autologous graft is the current gold standard for ACL reconstruction surgery, it is associated with donor site morbidity and a decrease in mechanical strength at the donor site. The use of allogenic grafts instead of autografts introduces the risk of disease transmission. Furthermore, integration of soft tissue grafts (e.g., hamstring tendon) to native bone is slow and risks graft pullout. To circumvent these limitations, tissue engineering seeks to fabricate suitable biomaterials that could replace the entire ACL, stimulate regeneration of the ligament tissue, and integrate with host bone tissue. Numerous efforts have led to the development of complex, multi-phased biomaterial scaffold designs that are intended to deliver an array of cell types and biological cues. Particularly, scaffolds that possess bone-regenerating biomaterials at the ends are envisioned to facilitate rapid integration with the femur and tibia. Electrospun fiber scaffolds continue to be regularly utilized for their high tensile strength, flexibility, and ability to bend. Nevertheless, fibrous scaffolds are inert and require the incorporation of trophic factors to guide tissue regeneration. Additionally, electrospun fibers are often densely packed, which can hinder cell infiltration and subsequent tissue formation. The objective of this work was to guide bone remodeling through the incorporation of trophic factors with 1) electrospun fiber scaffolds or 2) nanoparticles that could be combined with electrospun fiber scaffolds, and 3) to develop model three-dimensional fiber-hydrogel composites that support cell viability and proliferation. Two approaches were utilized to present the trophic factor bone morphogenic protein (BMP)-2 to stimulate bone formation. In the first approach, electrospun fibers were modified through the adsorption or covalent conjugation of BMP-2. These fibers exhibited increased BMP-2 concentrations with covalent conjugation over adsorption, and the incorporation of heparin into the fibers improved both adsorption and conjugation. Mesenchymal stem cells (MSCs) – that have the capacity to differentiate into osteoblastic cells – were able to attach and proliferate on all films yet appeared to do so to a greater extent on surfaces with higher heparin contents. Additionally, markers of osteoblastic differentiation were significantly higher on surfaces with covalently conjugated BMP-2 than on those with adsorbed BMP-2. In the second approach, a nanoparticle system was produced to control BMP-2 delivery and release. Importantly, this flexible system can be fabricated separately, and then combined with a scaffold for tissue regeneration. In this approach, BMP-2 was combined with chitosan nanoparticles through adsorption, encapsulation, or covalent conjugation. The particular BMP-2 incorporation technique had no significant effect on BMP-2 incorporation efficiencies, but affected particle size and BMP-2 release kinetics. Specifically, covalent conjugation method caused the aggregation of particles while adsorption method allowed the most sustainable release. MSCs cultured in the presence of the different particles survived and proliferated, but only particles with adsorbed BMP-2 stimulated osteoblastic differentiation. Finally, three-dimensional fiber-hydrogel composites of various models were fabricated to mimic the complexity of full-sized scaffolds for ACL regeneration, and to study cell infiltration, differentiation, and tissue formation. A collagen hydrogel phase was introduced to electrospun fiber scaffolds using different approaches. MSCs seeded within a thin collagen layer were able to proliferate, sense underlying substrate and spread according to fiber orientation, while those within thicker layers were not. Additionally, cells initially present in only the collagen phase infiltrated to the fiber phase. These results demonstrate that minor changes in fabrication steps to combine the two phases could significantly alter cell function during the formation of three-dimensional fiber-hydrogel composites for tissue regeneration.
General Audience Abstract
The anterior cruciate ligament (ACL) is one of four ligaments that connect the thigh bone to the shin bone and stabilize the knee. Injuries to the ACL often occur during high impact sports, and ruptures can necessitate surgical intervention. ACL reconstruction surgery involves drilling tunnels through the ends of leg bones, deploying the tissue graft through the knee joint and bone tunnels, and anchoring it within the bone tunnels. The most common grafts are autografts that use tendons of the patient's own body or allografts that are obtained from cadavers. The complications associated with autografts include pain at the site of tissue harvest, while allografts risk disease transmission. Additionally, directly affixing a soft tissue graft (e.g., the hamstring tendon) to bone within the bone tunnel suffers from slow tissue integration and risk of pull-out. Tissue engineering is a field that seeks to develop devices to direct the regeneration of damaged tissues and organs. In the context of ACL repair, it seeks to achieve a biomaterial device with the properties of ACL, that can both guide the regeneration of ligament tissue and facilitate integration with bone tunnels, eliminating the need for autografts and allografts and their associated risks. Toward the development of an engineered ACL, this work focuses on improving graft-to-bone integration. In the first project, fibrous materials are surface-modified with bone morphogenetic protein (BMP)-2 (a bone-forming protein), and then tested for their ability to stimulate formation of a bone-like tissue in cell culture. In the second project, the deployment of BMP-2 either on the surface of or within nanoparticle delivery vehicles is evaluated as an alternative strategy to stimulate bone-like tissue formation. The third project explores the inclusion of a hydrogel phase to facilitate cell infiltration and bone-like tissue formation within fibrous materials. Together these studies provide insights into how the architecture of the engineered tissue and the deployment of bone-forming proteins can be used to enhance ACL regeneration.
- Doctoral Dissertations