Cell Microenvironment Engineering and Monitoring for Tissue Engineering and Regenerative Medicine: The Recent Advances

In tissue engineering and regenerative medicine, the conditions in the immediate vicinity of the cells have a direct effect on cells' behaviour and subsequently on clinical outcomes. Physical, chemical, and biological control of cell microenvironment are of crucial importance for the ability to direct and control cell behaviour in 3-dimensional tissue engineering scaffolds spatially and temporally. In this review, we will focus on the different aspects of cell microenvironment such as surface micro-, nanotopography, extracellular matrix composition and distribution, controlled release of soluble factors, and mechanical stress/strain conditions and how these aspects and their interactions can be used to achieve a higher degree of control over cellular activities. The effect of these parameters on the cellular behaviour within tissue engineering context is discussed and how these parameters are used to develop engineered tissues is elaborated. Also, recent techniques developed for the monitoring of the cell microenvironment in vitro and in vivo are reviewed, together with recent tissue engineering applications where the control of cell microenvironment has been exploited. Cell microenvironment engineering and monitoring are crucial parts of tissue engineering efforts and systems which utilize different components of the cell microenvironment simultaneously can provide more functional engineered tissues in the near future

Kollajen temelli yapay menisküs: tasarım ve uygulaması

Meniscus is a wedge shaped structure, with a convex base attached to a flat tibial surface, and with a concave femoral surface, on which femur and tibia articulate. It has several functions including joint lubrication, shock absorption, load transmission and joint stability. Various methods were tried to treat meniscal tears but each has its own drawbacks. Tissue engineering seems to be a promising solution that avoids all the problems associated with the other approaches. In this study, a three dimensional (3D) collagen-based structure was prepared by tissue engineering to mimic the natural human meniscus. Three different foams prepared under different conditions were combined and nano/microfibrous layers were placed in between them. To mimic the properties of the natural tissue, the top layer was composed of collagen-chondroitin sulfate-hyaluronic acid (Coll-CS-HA) prepared by freezing at -20ºC followed by lyophilization. The middle and bottom layers were made with just collagen after freezing at -20ºC and -80ºC, respectively and lyophilization. Aligned nano/microfibers were prepared using collagen-poly(L-lactic-co-glycolic acid (Coll-PLGA). Various crosslinking procedures such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), genipin (GP), glutaraldehyde (GLU) either alone or in combination with dehydrothermal treatment (DHT) were used and based on both compressive and tensile properties, the best crosslinker was chosen to be DHT+EDC/NHS. Mechanical properties (compressive, tensile and shear) of the dry foams and the final 3D construct were evaluated. The highest mechanical properties were obtained with the 3D construct. Then all these foams and the 3D construct were seeded with human fibrochondrocytes to study the cell behavior such as attachment, proliferation, and extracellular matrix (ECM) and glucosaminoglycan (GAG) production. Furthermore, the influence of cell seeding on the compressive properties of wet individual foams and the 3D construct was observed. As expected, the highest cell proliferation and compressive properties were obtained with the 3D construct. Finally, the 3D constructs, seeded with fibrochondrocytes, were implanted in New Zealand rabbits after meniscectomy. The initial microscopical examination show that the 3D construct has a significant potential as a meniscus substitute.Ph.D. - Doctoral Progra

A Mechanically Functional Collagen-Based Construct Designed as a Meniscus Substitute

In this study, a novel 5-layered meniscus scaffold, constituted of 3 different collagen-based foams interspaced with two electrospun nano/microfibrous mats, was designed to mimic the mechanical and physical properties of the natural meniscus. The top layer was a collagen-chondroitin sulfate-hyaluronic acid (Coll-CS-HA) foam. The middle and bottom layers were collagen foams which had different porosities and compressive properties. The aligned mats in between the foams were a blend of collagen and poly(L-lactic acid-co-glycolic acid). For the 5-layered construct, the compressive, tensile and shear moduli were 445 KPa, 3 MPa and 194 KPa, respectively. In dry state, the construct possessed higher compressive and shear properties than the natural tissue but the tensile properties were much lower. The mechanical properties are expected to be improved when the cells are seeded on the scaffold by their secretion of collagen and GAG

A multilayer tissue engineered meniscus substitute

Various methods have been tried to treat the main meniscus problem, meniscal tears, for which we believe tissue engineering could be a viable solution. In this study, a three dimensional, collagen-based meniscus substitute was prepared by tissue engineering using human fibrochondrocytes and a collagen based-scaffold. This construct was made with 3 different collagen-based foams interspaced with two electrospun nano/microfibrous mats. The top layer was made of collagen type I-chondroitin sulfate-hyaluronic acid (Coll-CS-HA), and the middle and the bottom layers were made of only collagen type I with different porosities and thus with different mechanical properties. The mats of aligned fibers were a blend of collagen type I and poly(l-lactic acid-co-glycolic acid) (PLGA). After seeding with human fibrochondrocytes, cell attachment, proliferation, and production of extracellular matrix and glucoseaminoglycan were studied. Cell seeding had a positive effect on the compressive properties of foams and the 3D construct. The 3D construct with all its 5 layers had better mechanical properties than the individual foams

Collagen Based Multilayer Scaffolds for Meniscus Tissue Engineering: In Vivo Test Results. Biomater Med Appl 2: 1

Meniscus is an important component of the knee joint since it performs several crucial functions such as shock absorption, load bearing and transmission, maintenance of joint stability, and lubrication. The results of common meniscal injury repair approaches are not fully satisfactory with low mechanical properties and long regeneration times. A 3D collagen-based construct consisting of multilayers of lyophilized sponges separated by electrospun fibrous mats was prepared previously to serve as a substitute for meniscus. Mechanical properties of the construct were studied in vitro and a 3 to 4 fold increase was observed when a double crosslinking method was used. Rabbit meniscal cells were cultured in vitro, expanded and seeded onto the polymer scaffolds. 2 weeks later the substitute was implanted to the medial compartment of the rabbit knee joint. The implants were studied 3 and 10 weeks after transplantation. Histological and microscopical characterization showed a significant difference between the groups (Group I: control; Group II: cell free substitute and Group III: cell seeded substitute) with Week3 sample scores. Group III healing score was significantly lower than I and II, which was probably due to the the fibrous tissue surrounding the cell seeded material but this resulted in lower immunological responses. Moreover, the scores decreased from Week3 to Week10 indicating healing. Even though there were no statistically significant differences, the lowest values were observed with the tissue engineered substitute. Therefore, it can be concluded that in vivo studies showed the potential of the cell seeded artificial meniscus

Controlling Incoming Macrophages to Implants: Responsiveness of Macrophages to Gelatin Micropatterns under M1/M2 Phenotype Defining Biochemical Stimulations

Adverse immune reactions to implanted devices can seriously hamper the efficacy of implants. Monocyte derived macrophages play a crucial role in both initiation and resolution of the inflammatory response toward foreign bodies. As the surface microtopography is shown to exert significant effects on cell phenotype, it is hypothesized that the presence of micropatterns on implant/medical device surfaces can attenuate the immune response. To this end, enzymatically crosslinked micropatterned gelatin films of varying groove widths (2, 5, 10, 20, and 40 µm) are tested for their effect on incoming monocyte behavior. In order to distinguish the effect of cytokine microenvironment on pattern presence, monocytes are seeded on micropatterned films in normal culture medium or M1/M2 inducing media and their morphology and cytokine secretions are observed for 6 d. The presence of the patterns induces microenvironment-specific changes on the secretions of the attached cells and also on their size. IL-1ß, IL-4, IL-12, TNF-α, and CCL-18 secretions are significantly affected particularly in M1 induction media by pattern presence. It is demonstrated for the first time that micropatterned surfaces can be used to control the initial attachment and cytokine secretion of incoming macrophages if they are linked with a polarization inducing cytokine microenvironment