Wan-Ju Li

Cartilage tissue engineering : its potential and uses

Purpose of reviewThe prevalent nature of osteoarthritis, a cartilage degenerative disease that results in the erosion of joint surfaces and loss of mobility, underscores the importance of developing functional articular cartilage replacement. Recent research efforts have focused on tissue engineering as a promising approach for cartilage regeneration and repair. Tissue engineering is a multidisciplinary research area that incorporates both biological and engineering principles for the purpose of generating new, living tissues to replace the diseased/damaged tissue and restore tissue/organ function. This review surveys and highlights the current concepts and recent progress in cartilage tissue engineering, and discusses the challenges and potential of this rapidly advancing field of biomedical research. Recent findingsCartilage tissue engineering is critically dependent on selection of appropriate cells (differentiated or progenitor cells); fabrication and utilization of biocompatible and mechanically suitable scaffolds for cell delivery; stimulation with chondrogenically bioactive molecules introduced in the form of recombinant proteins or via gene transfer; and application of dynamic, mechanical loading regimens for conditioning of the engineered tissue constructs, including the design of specialized biomechanically active bioreactors. SummaryCell selection, scaffold design and biological stimulation remain the challenges of function tissue engineering. Successful regeneration or replacement of damaged or diseased cartilage will depend on future advances in our understanding of the biology of cartilage and stem cells and technological development in engineering.

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.

Braided nanofibrous scaffold for tendon and ligament tissue engineering.

Tendon and ligament (T/L) injuries present an important clinical challenge due to their intrinsically poor healing capacity. Natural healing typically leads to the formation of scar-like tissue possessing inferior mechanical properties. Therefore, tissue engineering has gained considerable attention as a promising alternative for T/L repair. In this study, we fabricated braided nanofibrous scaffolds (BNFSs) as a potential construct for T/L tissue engineering. Scaffolds were fabricated by braiding 3, 4, or 5 aligned bundles of electrospun poly(L-lactic acid) nanofibers, thus introducing an additional degree of flexibility to alter the mechanical properties of individual scaffolds. We observed that the Youngs modulus, yield stress, and ultimate stress were all increased in the 3-bundle compared to the 4- and 5-bundle BNFSs. Interestingly, acellular BNFSs mimicked the normal tri-phasic mechanical behavior of native tendon and ligament (T/L) during loading. When cultured on the BNFSs, human mesenchymal stem cells (hMSCs) adhered, aligned parallel to the length of the nanofibers, and displayed a concomitant realignment of the actin cytoskeleton. In addition, the BNFSs supported hMSC proliferation and induced an upregulation in the expression of key pluripotency genes. When cultured on BNFSs in the presence of tenogenic growth factors and stimulated with cyclic tensile strain, hMSCs differentiated into the tenogenic lineage, evidenced most notably by the significant upregulation of Scleraxis gene expression. These results demonstrate that BNFSs provide a versatile scaffold capable of supporting both stem cell expansion and differentiation for T/L tissue engineering applications.

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.

Fibroblast Growth Factor-2 Primes Human Mesenchymal Stem Cells for Enhanced Chondrogenesis

Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.

Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

Abstract Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression.

Polymer/Alginate Amalgam for Cartilage- Tissue Engineering

Abstract: Marrow stroma‐derived cells (MSC) are highly proliferative, multipotential cells that have been considered as ideal candidate cells for autologous tissue engineering applications. In this study, we have characterized the chondrogenic potential of human MSCs in both a PLA/alginate amalgam and pure PLA macrostructure as model three‐dimensional constructs to support both chondrogenic differentiation and proliferation following TGF‐β treatment. MSCs were seeded in experimental groups that consisted of PLA‐loaded constructs and PLA/alginate amalgams with and without recombinant human TGF‐β1. Chondrogenesis of the PLA and the PLA/alginate amalgam cultures was assessed at weekly intervals by histology, immunohistochemistry, scanning electron microscopy, sulfate incorporation, and RT‐PCR. Chondrogenic differentiation occurs within a polymeric macrostructure with TGF‐β1 treatment as indicated by histological, immunohistochemical, sulfate incorporation, and gene expression profiles. This macrostructure can be further encased in an alginate gel/solution to optimize cell shape and to confine growth factors and cells within the polymer construct, while the polymeric scaffold provides appropriate mechanical/tissue support. The stable three‐dimensional PLA/alginate amalgam represents a novel candidate system of mesenchymal chondrogenesis, which is amendable to investigation of mechanical and biological factors that normally modulate cartilage development and formation as well as a potential tissue engineering construct for cartilage repair.

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.

Thermoplastic polyurethane/hydroxyapatite electrospun scaffolds for bone tissue engineering: Effects of polymer properties and particle size

Thermoplastic polyurethane (TPU)/hydroxyapatite (HA) scaffolds were fabricated via electrospinning. The effects of TPU properties and HA particle size on scaffold physical properties and osteoblast-like cell performance were investigated. It was found that the addition of micro-HA (mHA), which was inlayed in the fiber, decreased the electrospun fiber diameter. On the contrary, nano-HA (nHA), which was either embedded or existed inside of the fiber, increased the fiber diameter for both soft and hard TPUs. The soft TPU had a much lower Youngs modulus and higher strain-at-break than the hard TPU. The addition of both mHA and nHA decreased the tensile properties; this decrease was more significant with mHA. The cells on the hard scaffolds actively proliferated and migrated compared to those on the soft scaffolds. On the other hand, cells on the soft scaffolds more effectively induced osteogenesis of human mesenchymal stem cells (hMSCs) than those on the hard scaffolds. In addition, our data suggest that the soft scaffolds with supplementation of nHA further enhanced osteogenesis of hMSCs compared to those without nHA. The soft TPU scaffolds containing nano-HA have the potential to be used in bone tissue engineering applications.

Tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells dictated by properties of braided submicron fibrous scaffolds

Tendon and ligament (T/L) engineering is a growing area of research with potential to address the inadequacies of current T/L defect treatments. Our group previously developed braided submicron fibrous scaffolds (BSMFSs) and demonstrated the viability of BSMFSs for T/L tissue engineering. The objective of this study was to investigate the effect of fiber chemistry and braiding angle on BSMFS mechanical properties and in turn, tenogenic differentiation of human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) seeded on BSMFSs subjected to cyclic tensile stimulation in the absence of tenogenic medium. By varying fiber chemistry and/or braiding angle, BSMFSs with a range of mechanical properties were produced. We found that fiber chemistry dictated cell adhesion while braiding angle dictated the tissue-specific lineage commitment of hiPSC-MSCs. Scaffolds braided with large angles better supported hiPSC-MSC tenogenic differentiation as evidenced by the production of T/L-associated markers, downregulation of osteogenic markers, and expression of fibroblast-like, spindle cell morphology compared to scaffolds braided with small angles. Our results demonstrate the importance of substrate properties and mechanical stimulation on tenogenic differentiation. These results also demonstrate the versatility of BSMFSs and the potential of hiPSC-MSCs for T/L tissue engineering.