Intervertebral disc tissue engineering using a novel hyaluronic acid-nanofibrous scaffold (HANFS) amalgam.

Degeneration of the intervertebral disc (IVD) represents a significant musculoskeletal disease burden. Although spinal fusion has some efficacy in pain management, spine biomechanics is ultimately compromised. In addition, there is inherent limitation of hardware-based IVD replacement prostheses, which underscores the importance of biological approaches to disc repair. In this study, we have seeded multipotent, adult human mesenchymal stem cells (MSCs) into a novel biomaterial amalgam to develop a biphasic construct that consisted of electrospun, biodegradable nanofibrous scaffold (NFS) enveloping a hyaluronic acid (HA) hydrogel center. The seeded MSCs were induced to undergo chondrogenesis in vitro in the presence of transforming growth factor-beta for up to 28 days. The cartilaginous hyaluronic acid-nanofibrous scaffold (HANFS) construct architecturally resembled a native IVD, with an outer annulus fibrosus-like region and inner nucleus pulposus-like region. Histological and biochemical analyses, immunohistochemistry, and gene expression profiling revealed the time-dependent development of chondrocytic phenotype of the seeded cells. The cells also maintain the microarchitecture of a native IVD. Taken together, these findings suggest the prototypic potential of MSC-seeded HANFS constructs for the tissue engineering of biological replacements of degenerated IVD.

Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies.

Biodegradable nanofibrous scaffolds serving as an extracellular matrix substitute have been shown to be applicable for cartilage tissue engineering. However, a key challenge in using nanofibrous scaffolds for tissue engineering is that the small pore size limits the infiltration of cells, which may result in uneven cell distribution throughout the scaffold. This study describes an effective method of chondrocyte loading into nanofibrous scaffolds, which combines cell seeding, mixing, and centrifugation to form homogeneous, packed cell-nanofiber composites (CNCs). When the effects of different growth factors are compared, CNCs cultured in medium containing a combination of insulin-like growth factor-1 and transforming growth factor-beta1 express the highest mRNA levels of collagen type II and aggrecan. Radiolabeling analyses confirm the effect on collagen and sulfated-glycosaminoglycans (sGAG) production. Histology reveals chondrocytes with typical morphology embedded in lacuna-like space throughout the entire structure of the CNC. Upon culturing using a rotary wall vessel bioreactor, CNCs develop into a smooth, glossy cartilage-like tissue, compared to a rough-surface tissue when maintained in a static environment. Bioreactor-grown cartilage constructs produce more total collagen and sGAG, resulting in greater gain in net tissue weight, as well as express cartilage-associated genes, including collagen types II and IX, cartilage oligomeric matrix protein, and aggrecan. In addition, dynamic culture enhances the mechanical property of the engineered cartilage. Taken together, these results indicate the applicability of nanofibrous scaffolds, combined with efficient cell loading and bioreactor technology, for cell-based cartilage tissue engineering.

Chondrocyte phenotype in engineered fibrous matrix is regulated by fiber size.

A biomaterial scaffold acting as a functional substitute for the native extracellular matrix provides space for cell accommodation. In this study, we seeded chondrocytes, isolated from 4- to 6-month-old calves, in 2 types of poly(L-lactide) scaffolds, composed of micro- and nanofibers, and compared the effects on cellular activities. Scanning electron microscopy revealed a well-spread morphology for chondrocytes grown on microfibers. In contrast, chondrocytes on the nanofibers were found to have a rounded morphology and displayed a disorganized actin cytoskeletal structure compared to the organized cytoskeleton seen in well-spread chondrocytes culture on the microfibrous scaffold. Both scaffolds supported chondrocyte proliferation, with a higher rate seen in cultures in nanofibrous scaffold. Quantitative reverse transcription-polymerase chain reaction analysis showed that both cultures supported expression of collagen types I and II and aggrecan. Biochemical analysis showed a higher level of sulfated glycosaminoglycan in the nanofiber culture, confirmed by more intense alcian blue histologic staining. The nanofiber cultures also showed higher immunostaining for collagen types II and IX, aggrecan, and cartilage proteoglycan link protein. Based on these results, we conclude that chondrocytes respond differently to fibrous scaffolds of varying diameters, and that the scaffolds made of nanofibrous biomaterial promote efficient cell-based cartilage tissue engineering.