A large variety of biochemical stimuli have been applied in meniscus tissue engineering investigations. Growth factors are the most prominent biochemical stimuli for tissue engineering the knee meniscus (). Overall, for meniscus cell proliferation, b-FGF in particular has been seen to elicit a strong response [241
]. One group studied the ability of nine growth factors (EGF, b-FGF, TGF-α, PDGF-AB, a-FGF, TGF-β1, PDGF-AA, IGF-I, and NGF) to stimulate proliferation of meniscus cells in monolayer over 4 days [241
]. Of these nine, b-FGF, PDGF-AB, EGF, and TGF-α encouraged proliferation, with b-FGF inducing the greatest effect [241
]. These four growth factors also promoted increased collagen synthesis of meniscus cells [241
]. Another study compared monolayer proliferation of meniscus cells from the different tissue regions (inner/middle/outer) [245
]. An up to 3-fold increase in DNA synthesis was demonstrated when PDGF-AB, HGF, and BMP-2 were applied to these cultured cells, while IGF-1 had no such effect [245
]. Interestingly, cells from different regions responded differently, with BMP-2 having a slightly stronger effect on cells from the middle zone, and HGF exerting a slightly stronger effect on cells from the inner zone [245
]. The effects upon monolayer meniscus cell migration were also examined. PDGF-AB and HGF stimulated migration in cells from all three zones of the meniscus, while EGF, IGF-1, IL-1, and BMP-2 promoted cell migration only in specific zones of the meniscus (outer and inner, middle and inner, outer and middle, and only middle, respectively) [245
Influence of selected growth factors administered to meniscus cells.
Aside from proliferation and migration, another chief function of growth factors in meniscus tissue engineering is to stimulate matrix synthesis. The TGF-β family, regarded as one of the most important for cartilage tissue engineering [246
], has repeatedly demonstrated the ability to heighten meniscus cell synthesis of matrix proteins [102
]. Since the ECM largely confers the mechanical properties which underlie the primary functions of the knee meniscus, this is particularly salient. An early study demonstrated increased proteoglycan synthesis of meniscus cells in monolayer, explant, and scaffold culture when treated with TGF-β1 [103
]. Increased cell proliferation was also observed, in only the monolayer cultures [103
]. Additionally, in scaffold and monolayer studies comparing TGF-β1, IGF-1, b-FGF, and PDGF-AB, only TGF-β1 stimulated significant simultaneous production of both collagens and GAGs over controls [244
]. TGF-β1 has also been seen to up-regulate the expression and secretion of lubricin, or superficial zone protein (SZP) [250
]. This protein is thought to provide essential function to cartilage by aiding in lubrication. By contrast, the same study found that interleukin-1β decreased SZP protein content and gene expression [250
]. Finally, TGF-β has also interestingly been shown to inhibit meniscus cell proliferation [241
]. This highlights the proliferation/production interplay in which meniscus cells are preferentially driven to one function or the other.
One potentially important function of growth factors may be to modulate matrix contraction. Both fibroblasts [251
] and articular chondrocytes [252
] exert local contractile forces on their surrounding matrices. Contiguous tissue constructs may actually benefit from controlled contraction, because ECM compaction and alignment can lead to anisotropy and greater mechanical properties. In fact, inhibition of fibroblast-mediated contraction has been shown to disrupt development of tendon mechanical properties [253
]. Too much contraction, however, can render constructs of incorrect geometry [237
]. However, the use of controlled contraction as a biophysical means of modulating cartilage development is relatively scarce in the literature. Both TGF-β1 and PDGF have been documented as growth factors involved in encouraging matrix contraction by meniscus cells, fibroblasts, and articular chondrocytes [232
]. FGF-2 and IGF-1 can also induce articular chondrocyte-mediated contraction of collagen II/GAG gel scaffolds [257
]. The continued exploration of this topic may lead to interesting advances in the field.
Phenotype maintenance or cell differentiation to fibrochondrocytes is another vital application of growth factors in meniscus tissue engineering. Relatively little work has been done in this area. However, it has been found that meniscus cell phenotype may be salvaged by exposure to FGF-2 during monolayer expansion [258
]. Subsequent 3-D pellet culture of FGF-2 exposed meniscus cells revealed a 200-fold higher expression of collagen II and GAG than controls [258
]. Fibrochondrogenic differentiation of human embryonic stem cells has also been performed [121
]. CDMP-1 has also been explored in a PGA scaffold modality to enhance fibrochondrogenesis of dermal fibroblasts [259
], and has been demonstrated to increase proteoglycan content and collagen II gene expression [260
]. Lastly, exposure to TGF-β1 has also been suggested to push meniscus fibrochondrocytes towards a more chondrocytic phenotype [102
]. Since meniscus fibrocartilage is a tissue with varying regions, either similar to or distinct from the hyaline articular cartilage produced by chondrocytes, this is a relevant result for prospective tissue engineers. These varying results demonstrate considerable potential in, and promise for, further investigations of fibrochondrogenic differentiation of cells.
Chondroitinase ABC (C-ABC) is another biochemical stimuli that has been employed in cartilage tissue engineering. This enzyme cleaves chondroitin and dermatan sulfate from proteoglycan chains while leaving collagen fibers unaffected [261
]. It has been suggested that a dynamic balance between the swelling pressure caused by proteoglycans and the restraining strength of the collagen network exists [263
]. Subsequently, it has been hypothesized that enzymatic depletion of cartilage GAG content (which is afterwards recovered by cellular synthesis) may facilitate increased collagen network alignment and density, leading to heightened tissue tensile properties [232
]. Indeed, serum-free C-ABC treatment of tissue-engineered articular cartilage (in both self-assembled and agarose scaffold forms) has resulted in increased tensile properties versus untreated controls, as well as recovery of GAG content and compressive stiffness after 2 to 4 wks of culture post-treatment [264
]. The repeated beneficial results of C-ABC on tissue-engineered articular cartilage motivate its use for tissue engineering meniscus fibrocartilage. Along these lines, self-assembled meniscus constructs (composed of meniscus cells and articular chondrocytes) treated with C-ABC have been seen to display approximate 2 to 3-fold increases in tensile modulus over untreated controls and GAG recovery after 3 wks of culture post-treatment [232
]. However, more studies using C-ABC for meniscus tissue engineering, especially in conjunction with other stimuli, are necessary.
Biochemical stimulus selection is not clear-cut, and the study of multiple agents (especially growth factors) in conjunction necessitates additional investigations. Culture conditions play a non-trivial role in modulating cell responses to biochemical stimulus administration, whether the treated tissue is arranged in monolayer, scaffold, explant, or self-assembled form. The presence of serum in the media of a study is also a critical variable [245
]. Futures studies may focus on unconventional growth factors, such as the serum-derived phospholipid agent lysophosphatidic acid (LPA). LPA is naturally present in mammalian sera at concentrations ranging from 1-5 μM [268
], and has been studied extensively as an anti-apoptotic factor. Other potent agents may be derived from platelet-rich plasma, which has been shown to increase matrix deposition and proliferation of meniscus cells cultured in monolayer [269
]. Finally, although fibrocartilage is a particularly important soft tissue, research into its generation with biochemical stimuli is nascent, and thus future work along these lines may produce significant medical advances. Much remains to be studied concerning biochemical stimuli used in tissue engineering the knee meniscus.