Bahar Bilgen

Sequential release of bioactive IGF-I and TGF-β1 from PLGA microsphere-based scaffolds

Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta 1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-beta 1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-beta 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-beta 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-beta 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-beta 1 released continuously, TGF-beta 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-beta 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-beta 1 sequentially makes them very useful for cartilage tissue engineering applications.

FBS suppresses TGF‐β1‐induced chondrogenesis in synoviocyte pellet cultures while dexamethasone and dynamic stimuli are beneficial

In vitro cartilage tissue engineering culture systems benefit from a fine balance of biochemical and mechanical components to maintain the chondrocyte phenotype. This balance, however, can be disrupted by using typical methods for cultivating chondrogenic cells in medium supplemented with fetal bovine serum (FBS) and growth factors. Our goal was to determine the effects of fluid‐dynamic stimuli, fetal bovine serum and dexamethasone on the chondrogenesis of 14‐day synoviocyte pellet cultures in the presence of TGF‐β1. We employed a pellet culture system that provides a highly cellular three‐dimensional structure that permits differentiation and extracellular matrix synthesis. Our results indicated that FBS inhibited glycosaminoglycan (GAG) and type II collagen production. Interestingly, the effect of dynamic stimuli was modulated by the presence of FBS; mixed serum‐free cultures had increased GAG production, whereas mixed cultures with 10% FBS exhibited less GAG production compared with their static counterparts, possibly due to pronounced suppressive effects of FBS via increased transport. Dexamethasone addition during the first week of culture resulted in enhanced extracellular matrix production and increased cellularity. Moreover, the presence of 10% FBS in addition to ITS+ and TGF‐β1 did not significantly increase cell proliferation compared with serum‐free medium. These results indicate the importance of a comprehensive analysis of growth conditions for each cell culture system. Copyright

Hydrodynamic Parameters Modulate Biochemical, Histological, and Mechanical Properties of Engineered Cartilage

Functional engineered cartilage constructs represent a promising therapeutic approach for the replacement of damaged articular cartilage. The in vitro generation of cartilage tissue suitable for repair requires an understanding of the complex interrelationships between environmental cues, such as hydrodynamic forces, and tissue growth and development. In the present study, engineered cartilage constructs were cultivated in four well-defined hydrodynamic environments within a bioreactor, and correlations were established between construct ultrastructural and mechanical properties and key hydrodynamic parameters. Results suggest that even for similar composition, constructs may exhibit different mechanical properties due to differences in their ultrastructure that can be modulated by hydrodynamic parameters. For example, improved mechanical properties were observed in constructs that exhibited a thick fibrous outer capsule as a result of cultivation under increased hydrodynamic shear. In particular, uniformity in the contribution of the fluid velocity vectors (axial, radial, and tangential) to the total fluid velocity and shear stress were the hydrodynamic parameters that most affected the construct properties under investigation. The correlations identified here may be useful in the development of engineered tissue growth models that inform the design of bioreactor cultivation systems toward the production of clinically relevant engineered cartilage.

Tissue growth modeling in a wavy-walled bioreactor.

Bioreactors have played a crucial role in recent approaches to cartilage tissue engineering, providing an environment that promotes efficient cell seeding, nutrient and waste transport, and essential physical stimuli. This study employed a wavy-walled bioreactor to investigate the effects of the hydrodynamic environment on the properties of engineered cartilage. Its unique design provides multiple hydrodynamic environments within one setting. A tissue growth model was developed to characterize the tissue growth and extracellular matrix synthesis by chondrocytes seeded and cultivated on polyglycolic acid scaffolds in the wavy-walled bioreactor for a period of 4 weeks. This model consists of four components: 1) a computational fluid dynamics model, 2) a kinetic growth model, 3) an artificial neural network that empirically correlates hydrodynamic parameters with kinetic constants, and 4) a second artificial neural network that correlates the biochemical composition of constructs with their material properties. In tandem, these components enable the prediction of the dynamics of tissue growth, as well as the final compositional and mechanical properties of engineered cartilage. The growth model methodology developed in this study serves as a tool to predict the optimal bioprocessing conditions required to achieve desired tissue properties.

Effects of Chondroitinase ABC-Mediated Proteoglycan Digestion on Decellularization and Recellularization of Articular Cartilage

Articular cartilage has a limited capacity to heal itself and thus focal defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. Decellularization is a novel strategy that aims to preserve the bioactive factors and 3D biophysical environment of the native extracellular matrix while removing potentially immunogenic factors. The purpose of this study was to develop a procedure that can enable decellularization and recellularization of intact articular cartilage matrix. Full-thickness porcine articular cartilage plugs were decellularized with a series of freeze-thaw cycles and 0.1% (w/v) sodium dodecyl sulfate detergent cycles. Chondroitinase ABC (ChABC) was applied before the detergent cycles to digest glycosaminoglycans in order to enhance donor chondrocyte removal and seeded cell migration. Porcine synovium-derived mesenchymal stem cells were seeded onto the decellularized cartilage scaffolds and cultured for up to 28 days. The optimized decellularization protocol removed 94% of native DNA per sample wet weight, while collagen content and alignment were preserved. Glycosaminoglycan depletion prior to the detergent cycles increased removal of nuclear material. Seeded cells infiltrated up to 100 μm into the cartilage deep zone after 28 days in culture. ChABC treatment enhances decellularization of the relatively dense, impermeable articular cartilage by reducing glycosaminoglycan content. ChABC treatment did not appear to affect cell migration during recellularization under static, in vitro culture, highlighting the need for more dynamic seeding methods.

Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo

Background: Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. Purpose/Hypothesis: The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. Study Design: Controlled laboratory study. Methods: In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase–13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase–1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated glycosaminoglycan (GAG) release and Pi/PPi assay. Results: In vivo guinea pig study: Meniscus size and area as well as intensity of meniscus calcification were significantly increased in the ACLT group compared with the control group. Both calcified area and intensity were correlated with cartilage damage in the ACLT group (meniscus calcified area: r = 0.925, P < .0001; meniscus calcified intensity: r = 0.944, P < .0001). Ihh, MMP-13, Col X, ANKH, ENPP1, and ALP expression were increased in the ACLT group compared with the control group. The Pi level and Pi/PPi ratio increased by 63% and 42%, respectively, in the ACLT group compared with the control group. In vitro bovine meniscus explant study: Cell death was found in the superficial zone of the bovine meniscus explants after loading for 3 hours. The mRNA expression and protein levels of MMP-13, ANKH, ENPP1, and ALP were up-regulated in all 3-hour loaded samples. The Pi/PPi ratio and sulfated GAG content in the culture medium were increased in the 3-hour loaded group. Conclusion: Meniscus hypertrophy and mineralization correlated to cartilage degeneration after ACL injuries. Clinical Relevance: The study data suggest that the suppression of meniscus hypertrophy and calcification may decrease the risk of PTOA after ACL injuries.

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

Modeling of Bioreactor Hydrodynamic Environment and Its Effects on Tissue Growth

The design of optimal bioreactor systems for tissue engineering applications requires a sophisticated understanding of the complexities of the bioreactor environment and the role that it plays in the formation of engineered tissues. To this end, a tissue growth model is developed to characterize the tissue growth and extracellular matrix synthesis by chondrocytes seeded and cultivated on polyglycolic acid scaffolds in a wavy-walled bioreactor for a period of 4 weeks. This model consists of four components: (1) a computational fluid dynamics (CFD) model to characterize the complex hydrodynamic environment in the bioreactor, (2) a kinetic growth model to characterize the cell growth and extracellular matrix production dynamics, (3) an artificial neural network (ANN) that empirically correlates hydrodynamic parameters with kinetic constants, and (4) a second ANN that correlates the biochemical composition of constructs with their material properties. In tandem, these components enable the prediction of the dynamics of tissue growth, as well as the final compositional and mechanical properties of engineered cartilage. The growth model methodology developed in this study serves as a tool to predict optimal bioprocessing conditions required to achieve desired tissue properties.

Current Concepts in Meniscus Tissue Engineering and Repair

The meniscus is the most commonly injured structure in the human knee. Meniscus deficiency has been shown to lead to advanced osteoarthritis (OA) due to abnormal mechanical forces, and replacement strategies for this structure have lagged behind other tissue engineering endeavors. The challenges include the complex 3D structure with individualized size parameters, the significant compressive, tensile and shear loads encountered, and the poor blood supply. In this progress report, a review of the current clinical treatments for different types of meniscal injury is provided. The state-of-the-art research in cellular therapies and novel cell sources for these therapies is discussed. The clinically available cell-free biomaterial implants and the current progress on cell-free biomaterial implants are reviewed. Cell-based tissue engineering strategies for the repair and replacement of meniscus are presented, and the current challenges are identified. Tissue-engineered meniscal biocomposite implants may provide an alternative solution for the treatment of meniscal injury to prevent OA in the long run, because of the limitations of the existing therapies.

Hydrogels of agarose, and methacrylated gelatin and hyaluronic acid are more supportive for in vitro meniscus regeneration than three dimensional printed polycaprolactone scaffolds

In this study, porcine fibrochondrocyte-seeded agarose, methacrylated gelatin (GelMA), methacrylated hyaluronic acid (MeHA) and GelMA-MeHA blend hydrogels, and 3D printed PCL scaffolds were tested under dynamic compression for potential meniscal regeneration in vitro. Cell-carrying hydrogels produced higher levels of extracellular matrix (ECM) components after a 35-day incubation than the 3D printed PCL. Cells on GelMA exhibited strong cell adhesion (evidenced with intense paxillin staining) and dendritic cell morphology, and produced an order of magnitude higher level of collagen (p < 0.05) than other materials. On the other hand, cells in agarose exhibited low cell adhesion and round cell morphology, and produced higher levels of glycosaminoglycans (GAGs) (p < 0.05) than other materials. A low level of ECM production and a high level of cell proliferation were observed on the 3D printed PCL. Dynamic compression at 10% strain enhanced GAG production in agarose (p < 0.05), and collagen production in GelMA. These results show that hydrogels have a higher potential for meniscal regeneration than the 3D printed PCL, and depending on the material used, fibrochondrocytes could be directed to proliferate or produce cartilaginous or fibrocartilaginous ECM. Agarose and MeHA could be used for the regeneration of the inner region of meniscus, while GelMA for the outer region.