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HOW TO CULTURE CARTILAGE CELLS ON BIOLAMININ SUBSTRATES
High laminin expression in the cartilage microenvironment
Cartilage is a specialized connective tissue with a multi-component extracellular matrix (ECM) that regulate cartilage repair and regeneration and maintain its functionality. The chondrocytes, the major cartilage resident cells, produce laminins (LN) α1, α2, α3, α4, α5, β1 and γ1 chains, collagen type IV, proteoglycans, elastin, nidogen and perlecan and these that forms a surrounding thin pericellular matrix (Kvist, 2008; Sun, 2017). There is increasing evidence showing that laminins, secreted by chondrocytes and primarily located in the PCM in cartilage and cartilage-like tissues, are involved in the regulation of chondrocyte activities, such as adhesion, migration, and survival. Furthermore, laminins play a major role in stem cell proliferation and chondrogenic differentiation. Laminins are mainly located in the PCM of human adult articular cartilage with most prominent staining in cartilage superficial layer, a niche for chondroprogenitor cells (Kvist, 2008; Sun, 2017; Chen, 2009; Lee, 1997). The influence of the pericellular matrix on the chondroprogenitor cells has been shown to be important for the expression of the major regulator transcription factors, SOX9, and RUNX2 (Schminke, 2016). Similar to the location pattern in the superficial layer of adult articular cartilage, most laminins are detected in the resting zone in epiphyseal cartilage and expression decreases in the proliferating and hypertrophic zones (Kvist, 2008; Sun, 2017; Kruegel and Miosge, 2010).
Laminin expression changes during human cartilage development
Laminins are continuously expressed during all developing stages of cartilage and cartilage-like tissues and show a region-specific expression pattern. In cartilage from newborn mice, basement membrane components are widespread in the territorial and interterritorial matrix, while in mature cartilage of adult mice the basement membrane components are localized mainly to a narrow pericellular zone around the chondrocytes (Kvist, 2008). Of the laminin subunits surveyed, α1, α5, β1, and γ1 have shown the highest staining intensity (Kvist, 2008). Lee and colleagues found that laminin chains (α1, α2, β1, β2, and γ1), produced by chicken embryo sternal chondrocytes, exhibited an increased expression in aggregated cells during the maturation stage (Lee, 1997). In newborn mouse knee cartilage, laminin 111 showed staining in the extracellular matrix, while laminin α5 (511 and 521) stained weakly at this point (Kvist, 2008). The laminin β1 chain is expressed from gestational weeks (gw) 10 onwards but not during gw 8 and 9, whereas the detection of the laminin β2 chain was limited to gw 8 and 9. This indicates a developmental switch in the laminin β chain and suggests that the laminin β1 chain does not play a role in human cartilage development until the fetal stage. Later at gw 17, a strong pericellular immunohistochemical reaction for LN111 can be detected (Roedinger, 2010). Laminin chains β1 and β2 are also expressed in the cytoplasm of chondrocytes in other species, such as chicken and mouse (Roedinger, 2010). Laminin 332 has also been shown to be expressed in embryonic cartilage (Kruegel and Miosge, 2010; Roedinger et al. 2010). The transiently expressed laminin g2 chain in embryonal cartilage suggests a possible role of laminin 332 in chondrogenesis (Uehara, 2017; Hashimoto, 2006; Lu, 2001).
During the development of intervertebral disc (IVD), laminin is distributed pericellular in developing nucleus pulposus (NP), annulus fibrosus (AF) and vertebral bodies of rats. In general, a gradual shift from diffuse generalized staining of basement membrane components in the newborn towards a pericellular localization in the mature cartilage has been observed (Kvist, 2008). Laminin interactions with nucleus pulposus (NP) cells are distinct from that of the annulus fibrosus (AF) cells. The laminin chain α5 (LN511 or LN521) and corresponding receptors CD239, integrin subunits α3, α6, and β4, are to a higher level expressed in the nucleus pulposus (NP) regions and the α1 chain (LN111 or LN121) shows higher expression in AF cells (Chen, 2009).
Laminin chains are also differentially expressed during chondrocyte differentiation. With the used an in vitro chick chondrocyte differentiation mode, the expression and localization of fibronectin, laminin, and their receptors were investigated. The results show that fibronectin contributes to the initial cell-cell interactions but is downregulated and replaced by laminin in the in vitro cell condensation process predominantly in the central areas of the cell aggregates, that correspond to differentiating cells. Laminin 111 is maximally expressed at the higher level of cell aggregation and progressively decrease during the differentiation to stage I chondrocytes. The increase in a1, b1, and g1, LN mRNAs is paralleled by a switch in the isotype of a6 integrin subunits synthesized, from B to A (Tavella, 1997).
The role of laminin in cartilage repair and regeneration
It has been suggested that laminins regulate the fate and functions of chondroprogenitors and chondrocytes in cartilage repair and regeneration. Laminins exist in developing and normal cartilage but many studies show that the expression of laminins significantly decreased or disappears in degenerative, traumatically-damaged cartilage. Interestingly, the diverse expression of laminins in degenerative cartilage indicate their role in cartilage degeneration and suggests that laminin could serve as an early marker for cartilage degeneration. An investigation of cartilage tissue and isolated chondrocytes obtained from patients with late-stage knee osteoarthritis (OA) showed significantly higher expression α1 and α5 laminins compared to healthy cartilage specimens (Schminke, 2016). In addition, chondrogenic progenitor cells (CPCs) in culture produced high levels of laminin chain α1 and α5 and it’s been shown that these laminins promote chondrogenesis and restoration of the chondrocyte phenotype by enhancing collagen type II, COMP and aggrecan expression, and by down-regulating collagen type II (Schminke, 2016), indicating that laminins have essential roles in promoting chondrogenesis of cartilage-forming cells. In degenerated cartilage sites, the laminin chain α4 has also shown to be highly expressed and has been suggested to play a deleterious role in cartilage degeneration and might aggravate cartilage damage in osteoarthritis (Sun, 2017). The altered laminin expression pattern and/or decreased in quantity in conjunction with the degeneration of the cartilage-like tissues, implicate the role of laminin in cartilage degeneration.
LN511, LN332, and LN111 increase the attachment of chondrocytes in culture
The dynamic expression of various laminin isoforms and their functions during chondrogenesis has not been fully delineated. It is likely that the expression of laminins is highly regulated during proliferation and differentiation and specific laminin isoforms could be involved in lineage-specific differentiation. Laminin 511 and 521 are the most abundant isoforms in the bone marrow and bone marrow-derived mesenchymal stem cells (BM-MSCs) cultured in vitro has been shown to synthesize α5, α4, α3, α1 and β2 at the significant amount (Seeger, 2015; Siler, 2000; Hashimoto, 2006). Naturally, laminin 511 and 521 have been shown to have strong adhesive interactions with human CD34+ cell lines (Jiang, 2016; Siler, 2000; Yang and Xiao, 2016). However, MSCs does not do not attach well to laminin 111, 211 and 221 (Sun, 2017). Laminin 511, 521 and 332 promote the strongest BM-MSC growth and proliferation rate and affect the mitogenic activity and migration of these cells via binding to integrin α6β1 and α3β1 (Siler, 2000; Sun, 2017, Hashimoto, 2005; Hashimoto, 2006).
Chondrogenic culture of bone marrow-derived mesenchymal stem cells (MSCs) in hydrogels and tissue-engineered cartilaginous constructs reveals extensive expression of laminin throughout the ECM (Sun, 2017). Chondrocytes and rat nucleus pulposus (NP) cells attach strongly to plates coated with laminin isoforms 521, 511, 332 or 111, mainly via the interaction with integrin receptor α6β1 (Sun, 2017). Primary osteoblasts in culture, express laminin 332 which promotes attachment and osteogenic differentiation (Mittag, 2012). Laminin 332 expression, negatively regulated by osteoclastogenic factors (Uehara, 2017), has been shown to suppresses chondrogenic differentiation of BM-MSC and mouse ATDC5 cells, without inducing apoptosis or inhibiting cell growth (Hashimoto, 2005; Hashimoto, 2006). These results suggest that laminin 332 may contribute to the development of bone tissues by promoting proliferation and by suppressing chondrogenic differentiation.
BIOLAMININ KEY ADVANTAGES
Laminin 511and 521 are the most abundant isoforms in the bone marrow. Laminin isoforms 111, 331 and 332 are also expressed.
Laminins, secreted by chondrocytes and primarily located in the PCM in cartilage and cartilage-like tissues, are involved in the regulation of chondrocyte activities, such as adhesion, migration and survival and chondrogenic differentiation.
Laminins regulate the fate and functions of chondroprogenitors and chondrocytes in cartilage repair and regeneration.
Region- and age-specific laminin expression pattern.
Chondrocytes and rat nucleus pulposus cells attach strongly to plates coated with laminin isoforms 521, 511, 332 or 111, mainly via the interaction with integrin receptor α6β1.
- Laminin 511 and 521 have been shown to have strong adhesive interactions with human CD34+ cell lines and promote BM-MSC growth and proliferation.
Defined and animal component-free culture.
No lot-to-lot variability for standardized experiments with less variation.