The development and homeostasis of cartilaginous tissues are regulated by diverse micro-environmental cues including integrin-mediated interactions between chondrocytes and the extracellular matrix (ECM). Integrins are membrane receptors responsible for bi-directional communication between the cells and the surrounding by transmitting physicochemical signals through adhesion complexes (Figure 1). In addition, integrins are involved in sensing mechanical stress signals generated by the ECM and transduce them into the cell interior converting physical stimuli to biochemical signaling. Integrin-activated signaling cascades modulate various chondrocyte functions and play important roles in cartilage morphogenesis, homeostasis and repair. In mammals, 18 α and 8 β subunits have been identified, which can combine into 24 different heterodimers with specific or partially overlapping binding capacity to ECM components (Figure 1A). On chondrocytes, at least 6 integrin receptors contain the β1 subunit, thus forming the biggest integrin subfamily present in cartilage (Figure 1B). Collagen- and fibronectin-binding are implicated in articular cartilage (AC) development, remodelling and repair. The collagen-binding α10β1 integrin is abundantly expressed on chondrocytes and is also present in a sub-population of human mesenchymal stem cells (hMSCs). Our previous studies showed that 1 and 10 integrins are required for skeletal development, the lack of the 1 subunit on chondrocytes leads to severe structural changes of the AC, and the expression of 10 in hMSCs enhances chondrogenesis in vitro.
Figure 1: Integrins. (A) In mammals, 18 α and 8 β subunits can combine into 24 heterodimers. Based on the predominant binding specificity, integrin-ligand interactions are classified into four main classes. (B) Integrin subunits expressed on normal and osteoarthritic chondrocytes. Chondrocyte integrins interact with the major proteins of the cartilage extracellular matrix (ECM) including type II collagen (Col), fibronectin (Fn) and laminin (Lam). Integrins link the ECM to the actin cytoskeleton, and control chondrocyte behavior through signaling cascades initiated at the focal adhesion sites.
The aims of our study are: 1) to further explore the role 1 integrins in AC degeneration, 2) to decipher the role of integrins in response to mechanically induced injury of the joint; and 3) to challenge AC regeneration by the application of 10 overexpressing hMSCs using ex vivo and in vivo repair models. For our studies we have used several transgenic animal models which lack 1 integrin or the collagen binding integrins α1β1, α2β1 or α10β1. In order to assess the consequence of integrin deficiencies on cartilage biomechanics we have utilized indentation-type atomic force microscopy (IT-AFM), which able to monitor structural and biomechanical properties of the cartilage on native tissue samples.
The aims of our study are: 1) to further explore the role b1 integrins in AC degeneration, 2) to decipher the role of integrins in response to mechanically induced injury of the joint; and 3) to challenge AC regeneration by the application of a10 overexpressing hMSCs using ex vivo and in vivo repair models. For our studies we have used several transgenic animal models which lack b1 integrin or the collagen binding integrins α1β1, α2β1 or α10β1. In order to assess the consequence of integrin deficiencies on cartilage biomechanics we have utilized indentation-type atomic force microscopy (IT-AFM), which able to monitor structural and biomechanical properties of the cartilage on native tissue samples.
The role of integrins in skeletal development and articular cartilage function. Our results obtained in the first funding period demonstrated that deletion of Itgb1 in chondrocytes or chondrocyte precursors compromises long bone development in all β1 integrin mutant lines by various severity disrupting the shape, orientation and columnar organization of growth plate (GP) chondrocytes (Figure 2). β1 integrin null (Itgb1 null) proliferative chondrocytes, which lack all collagen binding integrins (α1β1, α2β1, α10β1) and the major fibronectin-binding α5β1 integrin, display rounded geometry and loss of polarity characterized by random orientation of the Golgi-nucleus axis, the mitotic spindle and the division axes. Furthermore, the non-oriented β1-null chondrocytes fail to intercalate into columns. The findings indicate that cell shape anisotropy maintained by β1 integrins-mediated attachment to the cartilage matrix provides a default guiding cue for spindle and division positioning in chondrocytes, which in turn guides growth plate morphogenesis and long bone elongation. In addition, IT-AFM on native GP sections revealed that β1 integrin deficiency leads to significant softening of the interterritorial matrix, primarily influencing the collagen network, implying that loss of cell-matrix interactions impairs the proper mechanical function of the ECM, which controls growth plate cyto-architecture. We also showed that Itga10ko/ko mice lacking α10β1 integrin exhibit milder disorganization of the growth plate and slightly reduced shape index at the newborn stage, which abnormalities are normalized at later postnatal stages suggesting that 1) α10β1 integrins only temporary modulate GP morphogenesis; and 2) collagen-binding integrins likely compensate each other during endochondral bone growth. Importantly, mice lacking the genes Itga1 and Itga2 encoding the α1 and α2 integin subunits, respectively, have normal GP morphogenesis indicating that α1β1and α2β1 are dispensable for structural organization of the cartilage.. Finally, Itga10 deficiency reduced collagen density and, very modestly, the stiffness of the ECM suggesting that α10β1 integrin-mediated chondrocyte-collagen interactions modulate matrix structural and biomechanical properties.
Figure 2: β1 integrin-mediated chondrocyte-matrix interactions are essential for growth plate morphogenesis. (A) While wild type proliferative chondrocytes are organized into longitudinal columns, flattened and oriented perpendicular to the direction of the long axis of the growth plate, Itgb1 null chondrocytes are rounding up and do not form columns. (B) The shape index is decreased in Itgb1 mutant proliferative chondrocytes. (C) Mitotic spindles in control cells form along the long axis (line) and cell division occurs perpendicular to this axis (arrows). Spindle and cleavage plain orientation is randomized in chondrocytes lacking β1 integrins. Random orientation of the Golgi-nucleus axis in Itgb1 null chondrocytes indicates loss of cell polarity. (D) IT-AFM measurement in newborn proliferative zone show a bimodal stiffness distribution representing the collagen fibrils (COL, second peak) and the proteoglycans (PG, first peak). Mice lacking β1 integrins display a unimodal stiffness distribution and a significantly softer matrix compared to control. (E) Mice lacking α10 integrin have mild disorganization of the growth plate at the newborn stage characterized by reduced shape index normal orientation of proliferative chondrocytes, reduced cell number in the GP columns and only slightly decreased stiffness of the cartilage matrix (F). (G) Normal polarity and chondrocyte shape in mice double deficient for the collagen-binding integrin subunits Itga1 and Itga2.
In addition to the growth plate abnormalities, we demonstrated that Itgb1 deficiency severely affect structural and mechanical properties of the articular cartilage (AC) (Figure 3). We have previously reported that β1fl/fl-Prx1cre+ mice have paralyzed legs due to ectopic calcification in the joint and the limb vessels resulting in greatly reduced physical activities and the lack of degradation of the AC despite the structural abnormalities. Because of the milder phenotype of the β1fl/fl-Prx1cre+low line owing to mosaic deletion of Itgb1, the mutant mice have had normal motility, which is mechanically impacted the articular cartilage of the knee joint. We followed articular cartilage degradation associated with aging at various ages, and observed that β1fl/fl-Prx1cre+low mice develop structural disorganization of the AC (Fig. 3A) and display significantly increased cartilage erosion at 7 and 12 months of age compared with wild type animals (Fig. 3B). Applying IT-AFM on the surface of intact hip AC, we observed that the nano-stiffness increased in wild type and decreased in β1fl/fl-Prx1cre+ and β1fl/fl-Prx1cre+low mice (Fig. 3C) during aging. Utilizing AFM on native AC sections (Fig. 3D, E), we found a disorganized collagen network in the superficial zone of the tibial AC, and significantly decreased nano-stiffness values in all AC zones of β1fl/fl-Prx1cre+low mice compared with control. Hence, the loss of β1 integrin heterodimers severely impairs structural and biomechanical properties at various zones of the articular cartilage, implicating their essential role for matrix assembly and stiffness modulation in all adult stages. The findings demonstrate that β1 integrins-mediated chondrocyte-matrix interactions have 1) protective function for age-associated OA induced by mechanical loading; and 2) are essential for the structural and mechanical stability of the articular cartilage. In contrast, ablation of Itga10 alone did not altered AC structure or ECM stiffness at 2 months of age (Fig. 3F), likely due to the presence of compensating expression of Itga1 and Itga2 (Fig. 3G).
Figure 3: Articular cartilage phenotype of β1 integrin mutant mice induced by the Prx1cre transgene. (A) X-ray shows severe chondrodysplasia with vessel calcification, while immunohistochemistry demonstrates the lack of β1 integrin expression on AC chondrocytes in β1fl/fl-Prx1cre mice. β1fl/fl-Prx1crelow mice develop mild chondrodysplasia and the loss of β1 integrin expression in about 50% of AC chondrocytes. Hematoxylin and eosin staining shows the structural disorganization of the AC in both mutant mouse lines. (B) HE stained knee section at 12 months indicates severe AC degradation in β1fl/fl-Prx1crelow mice. Erosion scores demonstrate significantly increased cartilage destruction in β1fl/fl-Prx1cre+low mice at 7 and 12 months of age. (C) Nano-tip indentation AFM shows significant softening of the AC surface in young (4 months) and middle-aged (10 months) β1fl/fl-Prx1cre+ as well as β1fl/fl-Prx1cre+low mice. (D) High-resolution AFM imaging through the superficial zone of native AC section depicts a severely impaired collagen network with disorganized and thickened fibrils in the mutant mice. (E) Whisker plot shows stiffness values and demonstrates that in all AC zone β1fl/fl-Prx1crelow mice display softer ECM compared to wild type. (F) Mice lacking integrin α10 (Itga10 null) exhibit normal articular cartilage structure and matrix stiffness in the deep zone (red rectangles on the phalloidin stained histological sections) at 2 months of age. (G) Semi-quantitative RT-PCR shows the expression of possible compensating alpha subunits (e.g. Itga1 and Itga2) in the AC of wild type (wt) and Itga10 null (m) mice.
Enhancing chondrogenesis of hMSCs by priming α10 integrin. Mesenchymal stem cells (MSCs) isolated from adult tissues have a potential for enhanced skeletogenic differentiation by modulating the expression of integrin heterodimers. In SP1, we have primed human MSCs for chondrogenic differentiation by modulating the expression of the chondrocyte abundant, collagen-binding α10β1 integrin (Figure 4). We could show that bone marrow-derived MSCs (BMSCs) or adipose tissue-derived MSCs (ADMSCs) after growth factor treatments or lentiviral gene transfer of human ITGA10 increase the expression of α10β1 and have superior chondrogenic differentiation potential in pellet culture (Fig. 7A). We have also found that equine ADMSCs cultured in medium supplemented with 5% platelet lysate and sorted for high α10 integrin expression have improved capacity to adhere to chondral and subchondral damaged areas implicating enhanced homing of integrin α10-selected MSCs to cartilage defects. Taken together, these experiments clearly suggest that modulating ITGA10 expression can prime and promote chondrogenic differentiation of MSCs derived from various species and tissue sources.
Figure 4: (A) FGF-2 treatment or lentiviral gene transfer of ITGA10 increases the expression of integrin α10 and primes human bone marrow- or adipose tissue-derived MSCs for better chondrogenic differentiation. (B) α10 integrin-primed MSCs have increased adhesion to AC surface (S), damaged cartilage (C) or subchondral bone (SC) defect.