Kristen L. Moffat

Novel Nanofiber-Based Scaffold for Rotator Cuff Repair and Augmentation

The debilitating effects of rotator cuff tears and the high incidence of failure associated with current grafts underscore the clinical demand for functional solutions for tendon repair and augmentation. To address this challenge, we have designed a poly(lactide-co-glycolide) (PLGA) nanofiber-based scaffold for rotator cuff tendon tissue engineering. In addition to scaffold design and characterization, the objective of this study was to evaluate the attachment, alignment, gene expression, and matrix elaboration of human rotator cuff fibroblasts on aligned and unaligned PLGA nanofiber scaffolds. Additionally, the effects of in vitro culture on scaffold mechanical properties were determined over time. It has been hypothesized that nanofiber organization regulates cellular response and scaffold properties. It was observed that rotator cuff fibroblasts cultured on the aligned scaffolds attached along the nanofiber long axis, whereas the cells on the unaligned scaffold were polygonal and randomly oriented. Moreover, distinct integrin expression profiles on these two substrates were observed. Quantitative analysis revealed that cell alignment, distribution, and matrix deposition conformed to nanofiber organization and that the observed differences were maintained over time. Mechanical properties of the aligned nanofiber scaffolds were significantly higher than those of the unaligned, and although the scaffolds degraded in vitro, physiologically relevant mechanical properties were maintained. These observations demonstrate the potential of the PLGA nanofiber-based scaffold system for functional rotator cuff repair. Moreover, nanofiber organization has a profound effect on cellular response and matrix properties, and it is a critical parameter for scaffold design.

Characterization of the structure-function relationship at the ligament-to-bone interface.

Soft tissues such as ligaments and tendons integrate with bone through a fibrocartilaginous interface divided into noncalcified and calcified regions. This junction between distinct tissue types is frequently injured and not reestablished after surgical repair. Its regeneration is also limited by a lack of understanding of the structure–function relationship inherent at this complex interface. Therefore, focusing on the insertion site between the anterior cruciate ligament (ACL) and bone, the objectives of this study are: (i) to determine interface compressive mechanical properties, (ii) to characterize interface mineral presence and distribution, and (iii) to evaluate insertion site-dependent changes in mechanical properties and matrix mineral content. Interface mechanical properties were determined by coupling microcompression with optimized digital image correlation analysis, whereas mineral presence and distribution were characterized by energy dispersive x-ray analysis and backscattered scanning electron microscopy. Both region- and insertion-dependent changes in mechanical properties were found, with the calcified interface region exhibiting significantly greater compressive mechanical properties than the noncalcified region. Mineral presence was only detectable within the calcified interface and bone regions, and its distribution corresponds to region-dependent mechanical inhomogeneity. Additionally, the compressive mechanical properties of the tibial insertion were greater than those of the femoral. The interface structure–function relationship elucidated in this study provides critical insight for interface regeneration and the formation of complex tissue systems.

Orthopedic Interface Tissue Engineering for the Biological Fixation of Soft Tissue Grafts

Interface tissue engineering is a promising new strategy aimed at the regeneration of tissue interfaces and ultimately enabling the biological fixation of soft tissue grafts used in orthopedic repair and sports medicine. Many ligaments and tendons with direct insertions into subchondral bone exhibit a complex enthesis consisting of several distinct yet continuous regions of soft tissue, noncalcified fibrocartilage, calcified fibrocartilage, and bone. Regeneration of this multi-tissue interface will be critical for functional graft integration and improving long-term clinical outcome. This review highlights current knowledge of the structure-function relationship at the interface, the mechanism of interface regeneration, and the strategic biomimicry implemented in stratified scaffold design for interface tissue engineering and multi-tissue regeneration. Potential challenges and future directions in this emerging field are also discussed. It is anticipated that interface tissue engineering will lead to the design of a new generation of integrative fixation devices for soft tissue repair, and it will be instrumental for the development of integrated musculoskeletal tissue systems with biomimetic complexity and functionality.

Scaffold Fiber Diameter Regulates Human Tendon Fibroblast Growth and Differentiation

The diameter of collagen fibrils in connective tissues, such as tendons and ligaments is known to decrease upon injury or with age, leading to inferior biomechanical properties and poor healing capacity. This study tests the hypotheses that scaffold fiber diameter modulates the response of human tendon fibroblasts, and that diameter-dependent cell responses are analogous to those seen in healthy versus healing tissues. Particularly, the effect of the fiber diameter (320 nm, 680 nm, and 1.80 μm) on scaffold properties and the response of human tendon fibroblasts were determined over 4 weeks of culture. It was observed that scaffold mechanical properties, cell proliferation, matrix production, and differentiation were regulated by changes in the fiber diameter. More specifically, a higher cell number, total collagen, and proteoglycan production were found on the nanofiber scaffolds, while microfibers promoted the expression of phenotypic markers of tendon fibroblasts, such as collagen I, III, V, and tenomodulin. It is possible that the nanofiber scaffolds of this study resemble the matrix in a state of injury, stimulating the cells for matrix deposition as part of the repair process, while microfibers represent the healthy matrix with micron-sized collagen bundles, thereby inducing cells to maintain the fibroblastic phenotype. The results of this study demonstrate that controlling the scaffold fiber diameter is critical in the design of scaffolds for functional and guided connective tissue repair, and provide new insights into the role of matrix parameters in guiding soft tissue healing.

Characterization of the mechanical properties and mineral distribution of the anterior cruciate ligament-to-bone insertion site.

The anterior cruciate ligament (ACL) connects the femur to the tibia through direct insertion sites and functions as the primary restraint to anterior tibial translation. The ACL-to-bone insertion sites exhibit a complex structure consisting of four zones of varied cellular and matrix components, consisting of ligament, non-mineralized fibrocartilage, mineralized fibrocartilage and bone, which allow for the effective load transfer from ligament to bone, thereby minimizing stress concentrations and preventing failure. The mineral content and distribution within the fibrocartilage region may be an important structural component of the insertion site which may influence the mechanical properties. The goals of this study are to characterize the compressive mechanical properties of the fibrocartilage region of the ACL-to-bone insertion site and evaluate how the mineral distribution at the interface relates to these compressive properties. In order to determine the compressive mechanical properties we have utilized a novel microscopic mechanical testing method combined with digital image correlation and employed energy dispersive X-ray analysis (EDAX) in order to evaluate the mineral content and distribution across the femoral and tibial insertion sites. The results reveal that a regional mineral gradient is observed across the fibrocartilage which corresponds to depth-dependent variations in compressive mechanical properties. This depth- dependent mechanical inhomogeneity strongly correlates to the increase in mineral content of the mineralized fibrocartilage (MFC) region compared to the non-mineralized fibrocartilage (NFC). Additionally, the tibial NFC and MFC mechanical properties are greater than those of the femoral NFC and MFC which corresponds to a greater mineral content in the NFC and MFC regions of the tibial insertion. The findings of this study suggest that a structure-function relationship exists at the ACL-to-bone interface

Effect of hydroxyapatite particles on stem cell response in nanofiber scaffolds

Rotator cuff tears are among the most common shoulder injuries that require surgery. High failure rates of biological graft-based repairs underscore the need for functional alternatives. Specifically, functional grafts must incorporate the gradient of mineralization from tendon to bone in order to be biomimetic. In this study, effects of varying concentrations of mineral content in aligned nanofiber scaffolds on human mesenchymal stem cells (hMSC) are evaluated. It is hypothesized that mineral content will regulate cell response, matrix deposition, and integrin gene expression. hMSC were seeded on aligned nanofiber scaffolds of polylactide-co-glycolide with 0%, 10%, and 15% hydroxyapatite content, and were maintained in chondrogenic medium. Cell proliferation (n=5), collagen deposition (n=5), and gene expression (n=5) for Collagen X, Sox9, osteopontin, osteonectin, and osteocalcin were determined over 42 days. Cell number was found to differ between the non-mineralized and mineralized groups. Significant increase in collagen deposition over time was observed in mineralized scaffold groups, and the 15% group showing significantly higher deposition than the 0% group by day 42. Lower expressions of chondrocyte hyperotrophy marker Collagen X and chondrogenic marker Sox9 and maintained high expressions of osteogenic markers osteopontin, osteonectin, and osteocalcin suggest an osteogenic lineage for the stem cells. In conclusion, addition of HA particles influenced hMSC proliferation, matrix deposition, and may induce an osteogenic differentiation response from the stem cells.

Nanofiber alignment regulates adhesion and integrin expression of human mesenchymal stem cells and tendon fibroblasts

Rotator cuff tears are among the most common shoulder injuries that require surgery. High failure rates of biological graft-based repairs underscore the need for functional alternatives. In this study, effects of nanofiber organization on adhesion of cuff fibroblasts (hRCF) and human mesenchymal stem cells (hMSC) are evaluated. It is hypothesized that fiber alignment will regulate cell morphology and integrin gene expression. hRCF and hMSC were seeded on aligned and unaligned nanofiber scaffolds of polylactide-co-glycolide. Cell morphology (n=3) and gene expression (n=5) for integrins α2, αV, α5, and β1 were determined over 14 days. Cell morphology was found to differ between groups. Higher α2 and β1 expressions in hRCF and hMSC on aligned scaffolds suggest aligned scaffolds a more biomimetic structure to native tendon, since integrin α2β1 facilitates cell attachment to collagenous matrices. Expressions for αV and α5, which are associated with tendon healing, were significantly higher on unaligned scaffolds, and suggest a healing response by the cells. In conclusion, the cells may recognize differences in matrix organization, and fiber alignment regulates cell adhesion. Moreover, the aligned nanofiber matrix may promote a more biomimetic fibroblast response than the unaligned scaffold.

Innovative Scaffold Design for Soft Tissue-to-Bone Interface Tissue Engineering

Soft tissue-based ACL reconstruction grafts are limited by their inability to reestablish a functional interface with bone tissue[1–2]. The native ACL-bone interface consists of three regions: ligament, fibrocartilage, and bone[3–5]. Graft integration is a critical factor governing its clinical success, and the regeneration of an anatomic interface on synthetic or biological ACL grafts will improve clinical outcome. Our interface tissue engineering effort has focused on biomimetic scaffold design to recapitulate the inherent complexity of the ligament-to-bone interface and ultimately, to guide interface regeneration. To this end, we have designed a tri-phasic scaffold comprised of three distinct yet continuous phases, each designed for the formation of a specific tissue type found at the ACL-to-bone interface, as well as a bi-phasic collar to promote the formation of fibrocartilage on ACL reconstruction grafts and also enhance osteointegration.Copyright

In vitro characterization of polymer-ceramic nanofiber scaffolds

Rotator cuff tears often occur at the tendon-bone insertion site, a fibrocartilaginous interface that is not regenerated during current repairs. Our aim is to regenerate the insertion site through use of extracellular matrix based scaffolds. In this study, we evaluated the response of mineralized and non-mineralized nanofiber scaffolds to cell culture environment without the influence of cell growth and matrix deposition. We observed minimal change in scaffold mineral content, composition, and chemistry over culture time. Structural properties were influenced, in particular, fiber diameter increased throughout culture. This data is essential for scaffold characterization and understanding cell-material interactions.