Porosity and cell preseeding influence electrospun scaffold maturation and meniscus integration in vitro.

Electrospinning generates fibrous scaffolds ideal for engineering soft orthopedic tissues. By modifying the electrospinning process, scaffolds with different structural organization and content can be generated. For example, fibers can be aligned in a single direction, or the porosity of the scaffold can be modified through the use of multi-jet electrospinning and the removal of sacrificial fibers. In this work, we investigated the role of fiber alignment and scaffold porosity on construct maturation and integration within in vitro meniscus defects. Further, we explored the effect of preseeding expanded meniscus fibrochondrocytes (MFCs) onto the scaffold at a high density before in vitro repair. Our results demonstrate that highly porous electropun scaffolds integrate better with a native tissue and mature to a greater extent than low-porosity scaffolds, while scaffold alignment does not influence integration or maturation. The addition of expanded MFCs to scaffolds before in vitro repair improved integration with the native tissue, but did not influence maturation. In contrast, preculture of these same scaffolds for 1 month before repair decreased integration with the native tissue, but resulted in a more mature scaffold compared to implantation of cellular scaffolds or acellular scaffolds. This work will inform scaffold selection in future in vivo studies by identifying the ideal scaffold and seeding methods for meniscus tissue engineering.

Growth factor supplementation improves native and engineered meniscus repair in vitro.

Few therapeutic options exist for meniscus repair after injury. Local delivery of growth factors may stimulate repair and create a favorable environment for engineered replacement materials. In this study we assessed the effect of basic fibroblast growth factor (bFGF) (a pro-mitotic agent) and transforming growth factor ß3 (TGF-ß3) (a pro-matrix formation agent) on meniscus repair and the integration/maturation of electrospun poly(ε-caprolactone) (PCL) scaffolds for meniscus tissue engineering. Circular meniscus repair constructs were formed and refilled with either native tissue or scaffolds. Repair constructs were cultured in serum-containing medium for 4 and 8weeks with various growth factor formulations, and assessed for mechanical strength, biochemical content, and histological appearance. Results showed that either short-term delivery of bFGF or sustained delivery of TGF-ß3 increased integration strength for both juvenile and adult bovine tissue, with similar findings for engineered materials. While TGF-ß3 increased proteoglycan content in the explants, bFGF did not increase DNA content after 8weeks of culture. This work suggests that in vivo delivery of bFGF or TGF-ß3 may stimulate meniscus repair, but that the time course of delivery will strongly influence success. Further, this study demonstrates that electrospun scaffolds are a promising material for meniscus tissue engineering, achieving comparable or superior integration compared with native tissue.

Maturation state-dependent alterations in meniscus integration: implications for scaffold design and tissue engineering.

The knee meniscus is a crucial component of the knee that functions to stabilize the joint, distribute load, and maintain congruency. Meniscus tears and degeneration are common, and natural healing is limited. Notably, few children present with meniscus injuries and other related fibrocartilaginous tissues heal regeneratively in immature animals and in the fetus. In this work, we evaluated fetal, juvenile, and adult bovine meniscus properties and repair capacity in vitro. Although no changes in cell behavior (migration and proliferation) were noted with age, drastic alterations in the density and distribution of the major components of meniscus tissue (proteoglycan, collagen, and DNA) occurred with development. Coincident with these marked tissue changes, the in vitro healing capacity of the tissue decreased with age. Fetal and juvenile meniscus formed a robust repair over 8 weeks on both a histological and mechanical basis, despite a lack of vascular supply. In contrast, adult meniscus did not integrate over this period. However, integration was improved significantly with the addition of the growth factor transforming growth factor-beta 3. Finally, to evaluate engineered scaffold integration in the context of aging, we monitored cellular infiltration from native tissue into engineered nanofibrous constructs. Our findings suggest that maturation processes that enable load bearing in the adult limit endogenous healing potential and identify new metrics for the development of tissue-engineered meniscus implants.

An anisotropic nanofiber/microsphere composite with controlled release of biomolecules for fibrous tissue engineering.

Aligned nanofibrous scaffolds can recapitulate the structural hierarchy of fiber-reinforced tissues of the musculoskeletal system. While these electrospun fibrous scaffolds provide physical cues that can direct tissue formation when seeded with cells, the ability to chemically guide a population of cells, without disrupting scaffold mechanical properties, would improve the maturation of such constructs and add additional functionality to the system both in vitro and in vivo. In this study, we developed a fabrication technique to entrap drug-delivering microspheres within nanofibrous scaffolds. We hypothesized that entrapping microspheres between fibers would have a less adverse impact on mechanical properties than placing microspheres within the fibers themselves, and that the composite would exhibit sustained release of multiple model compounds. Our results show that microspheres ranging from 10 - 20 microns in diameter could be electrospun in a dose-dependent manner to form nanofibrous composites. When delivered in a sacrificial PEO fiber population, microspheres remained securely entrapped between slow-degrading PCL fibers after removal of the sacrificial delivery component. Stiffness and modulus of the composite decreased with increasing microsphere density for composites in which microspheres were entrapped within each fiber, while stiffness did not change when microspheres were entrapped between fibers. The release profiles of the composite structures were similar to free microspheres, with an initial burst release followed by a sustained release of the model molecules over 4 weeks. Further, multiple model molecules were released from a single scaffold composite, demonstrating the capacity for multi-factor controlled release ideal for complex growth factor delivery from these structures.

New directions in nanofibrous scaffolds for soft tissue engineering and regeneration.

This review focuses on the role of nanostructure and nanoscale materials for tissue engineering applications. We detail a scaffold production method (electrospinning) for the production of nanofiber-based scaffolds that can approximate many critical features of the normal cellular microenvironment, and so foster and direct tissue formation. Further, we describe new and emerging methods to increase the applicability of these scaffolds for in vitro and in vivo application. This discussion includes a focus on methods to further functionalize scaffolds to promote cell infiltration, methods to tune scaffold mechanics to meet in vivo demands and methods to control the release of pharmaceuticals and other biologic agents to modulate the wound environment and foster tissue regeneration. This review provides a perspective on the state-of-the-art production, application and functionalization of these unique nanofibrous structures, and outlines future directions in this growing field.