Pen-hsiu Grace Chao

Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).

Electrical stimulation systems for cardiac tissue engineering

We describe a protocol for tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cells with the application of pulsatile electrical fields designed to mimic those present in the native heart. Tissue culture is conducted in a customized chamber built to allow for cultivation of (i) engineered three-dimensional (3D) cardiac tissue constructs, (ii) cell monolayers on flat substrates or (iii) cells on patterned substrates. This also allows for analysis of the individual and interactive effects of pulsatile electrical field stimulation and substrate topography on cell differentiation and assembly. The protocol is designed to allow for delivery of predictable electrical field stimuli to cells, monitoring environmental parameters, and assessment of cell and tissue responses. The duration of the protocol is 5 d for two-dimensional cultures and 10 d for 3D cultures.

Silk hydrogel for cartilage tissue engineering

Cartilage tissue engineering based on cultivation of immature chondrocytes in agarose hydrogel can yield tissue constructs with biomechanical properties comparable to native cartilage. However, agarose is immunogenic and nondegradable, and our capability to modify the structure, composition, and mechanical properties of this material is rather limited. In contrast, silk hydrogel is biocompatible and biodegradable, and it can be produced using a water-based method without organic solvents that enables precise control of structural and mechanical properties in a range of interest for cartilage tissue engineering. We observed that one particular preparation of silk hydrogel yielded cartilaginous constructs with biochemical content and mechanical properties matching constructs based on agarose. This finding and the possibility to vary the properties of silk hydrogel motivated this study of the factors underlying the suitability of hydrogels for cartilage tissue engineering. We present data resulting from a systematic variation of silk hydrogel properties, silk extraction method, gel concentration, and gel structure. Data suggest that silk hydrogel can be used as a tool for studies of the hydrogel-related factors and mechanisms involved in cartilage formation, as well as a tailorable and fully degradable scaffold for cartilage tissue engineering.

Engineering custom-designed osteochondral tissue grafts

Tissue engineering is expected to help us outlive the failure of our organs by enabling the creation of tissue substitutes capable of fully restoring the original tissue function. Degenerative joint disease, which affects one-fifth of the US population and is the countrys leading cause of disability, drives current research of actively growing, functional tissue grafts for joint repair. Toward this goal, living cells are used in conjunction with biomaterial scaffolds (serving as instructive templates for tissue development) and bioreactors (providing environmental control and molecular and physical regulatory signals). In this review, we discuss the requirements for engineering customized, anatomically-shaped, stratified grafts for joint repair and the challenges of designing these grafts to provide immediate functionality (load bearing, structural support) and long-term regeneration (maturation, integration, remodeling).

Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.

Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair

Cartilage tissue lacks an intrinsic capacity for self-regeneration due to slow matrix turnover, a limited supply of mature chondrocytes and insufficient vasculature. Although cartilage tissue engineering has achieved some success using agarose as a scaffolding material, major challenges of agarose-based cartilage repair, including non-degradability, poor tissue-scaffold integration and limited processing capability, have prompted the search for an alternative biomaterial. In this study, silk fiber-hydrogel composites (SF-silk hydrogels) made from silk microfibers and silk hydrogels were investigated for their potential use as a support material for engineered cartilage. We demonstrated the use of 100% silk-based fiber-hydrogel composite scaffolds for the development of cartilage constructs with properties comparable to those made with agarose. Cartilage constructs with an equilibrium modulus in the native tissue range were fabricated by mimicking the collagen fiber and proteoglycan composite architecture of native cartilage using biocompatible, biodegradable silk fibroin from Bombyx mori. Excellent chondrocyte response was observed on SF-silk hydrogels, and fiber reinforcement resulted in the development of more mechanically robust constructs after 42 days in culture compared to silk hydrogels alone. Thus, we demonstrate the versatility of silk fibroin as a composite scaffolding material for use in cartilage tissue repair to create functional cartilage constructs that overcome the limitations of agarose biomaterials, and provide a much-needed alternative to the agarose standard.

Chondrocyte Translocation Response to Direct Current Electric Fields

Using a custom galvanotaxis chamber and time-lapse digital video microscopy, we report the novel observation that cultured chondrocytes exhibit cathodal migration when subjected to applied direct current (DC) electric fields as low as 0.8 V/cm. The response was dose-dependent for field strengths greater than 4 V/cm. Cell migration appeared to be an active process with extension of cytoplasmic processes in the direction of movement. In some cells, field application for greater than an hour induced elongation of initially round cells accompanied by perpendicular alignment of the long axis with respect to the applied field. Antagonists of the inositol phospholipid pathway, U-73122 and neomycin, were able to inhibit cathodal migration. Cell migration toward the cathode did not require the presence of serum during field application. However, the directed velocity was nearly threefold greater in studies performed with serum. Studies performed at physiologic temperatures (approximately 37 degrees C) revealed a twofold enhancement in migration speed compared to similar studies at room temperature (approximately 25 degrees C). Findings from the present study may help to elucidate basic mechanisms that mediate chondrocyte migration and substrate attachment. Since chondrocyte migration has been implicated in cartilage healing, the ability to direct chondrocyte movement has the potential to impact strategies for addressing cartilage healing/repair and for development of cartilage substitutes.

Effects of Applied DC Electric Field on Ligament Fibroblast Migration and Wound Healing

Applied electric fields (static and pulsing) are widely used in orthopedic practices to treat nonunions and spine fusions and have been shown to improve ligament healing in vivo. Few studies, however, have addressed the effect of electric fields (EFs) on ligament fibroblast migration and biosynthesis. In the current study, we applied static and pulsing direct current (DC) EFs to calf anterior cruciate ligament (ACL) fibroblasts. ACL fibroblasts demonstrated enhanced migration speed and perpendicular alignment to the applied EFs. The motility of ligament fibroblasts was further modulated on type I collagen. In addition, type I collagen expression increased in ACL fibroblasts after exposure to pulsing EFs. In vitro wound-healing studies showed inhibitory effects of static EFs, which were alleviated with a pulsing EF. Our results demonstrate that applied EFs augment ACL fibroblast migration and biosynthesis and provide potential mechanisms by which EFs may be used for enhancing ligament healing and repair.

Engineering cartilage and bone using human mesenchymal stem cells.

Cartilage and bone defects are leading causes of disability. The economic burden of orthopedic repair exceeds 28 billion dollars per year in the United States alone,1 and the situation is similar in many other countries. Although artificial joints or metal inserts are widely utilized and in most cases work well, cell-based therapies based on tissue-engineered cartilage and bone beckon a new frontier for clinical treatment owing to their biocompatibility and long-term prognosis. Functional tissue engineering2 involves an integrated use of three components — cells, scaffold material, and a bioreactor (Fig. ​(Fig.1),1), — in settings that mimic some elements of the in vivo environment. Synergistic interactions of biomimetic cues applied with temporal and spatial regulation influence cell growth and biosynthesis and guide cellular development into functional replacement tissue constructs. This article discusses the design criteria and parameters essential for engineering cartilage and bone grafts as well as the current status and future perspective of the field. Fig. 1 Main components of tissue engineering Tissue engineering Cells Cells are the actual “tissue engineers,” and several considerations guide the choice of cell sources. The cells are ideally immunocompatible, such as autologous chondrocytes for cartilage repair. The use of chondrocytes, however, is limited by their availability and their capacity for expansion in culture, as well as the need for a separate surgery to harvest the cells. The proliferative characteristics of autologous adult mesenchymal stem cells (MSCs) and the less invasive procedures for their procurement are thus considered important advantages. Although MSCs have better expandability than differentiated cells, extensive expansion is known to decrease their biosynthetic capacity and multipotency.3 Clearly, cells used for tissue engineering need to be biosynthetically active. Mauck and coworkers reported that when bovine chondrocytes and bone-marrow derived MSCs from the same animals were cultured under identical conditions chondrocytes not only created a significantly more functional tissue than MSCs they also maintained the improvement in biosynthetic and mechanical properties after 10 weeks of culture, by which time the MSCs had already reached a plateau.4 Therefore, optimizing the methods for MSC isolation, expansion, and differentiation remain critical for their effective use in tissue engineering and regenerative medicine.

Electrical stimulation enhances cell migration and integrative repair in the meniscus

Electrical signals have been applied towards the repair of articular tissues in the laboratory and clinical settings for over seventy years. We focus on healing of the meniscus, a tissue essential to knee function with limited innate repair potential, which has been largely unexplored in the context of electrical stimulation. Here we demonstrate for the first time that electrical stimulation enhances meniscus cell migration and integrative tissue repair. We optimize pulsatile direct current electrical stimulation parameters on cells at the micro-scale, and apply these to healing of full-thickness defects in explants at the macro-scale. We report increased expression of the adenosine A2b receptor in meniscus cells after stimulation at the micro- and macro-scale, and propose a role for A2bR in meniscus electrotransduction. Taken together, these findings advance our understanding of the effects of electrical signals and their mechanisms of action, and contribute to developing electrotherapeutic strategies for meniscus repair.