Chondrocyte De-Differentiation: Biophysical Cues to Nuclear Alterations: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Noor Al-Maslamani.

Autologous chondrocyte implantation (ACI) is a cell therapy to repair cartilage defects. In ACI a biopsy is taken from a non-load bearing area of the knee and expanded in-vitro. The expansion process provides the benefit of generating a large number of cells required for implantation; however, during the expansion these cells de-differentiate and lose their chondrocyte phenotype.

  • autologous chondrocyte implantation
  • de-differentiation
  • re-differentiation
  • chondrocyte
  • biophysics
  • mechanobiology

1. Introduction

Cells in the human body experience a variety of mechanical forces. The location of the cells determines what type of forces the cells are exposed to: e.g. cardiomyocytes experience tensile forces by cardiac contractions [1], endothelial cells are exposed to shear forces by fluid flow [2] and chondrocytes reside in an environment that is exposed to compressive, tensile and shear forces [3,4,5][3][4][5].
To examine how cells respond to mechanical forces, researchers have used different approaches, including cell stretchers, growing cells on substrates of difference stiffness, and embedding cells in hydrogels of ranging stiffness. However, less attention is given to how cells from different environments adapt to these artificial culturing environments and how this alters the spatial chromatin architecture and organization. Indeed, the chromatin spatial organization of the liver is different from that of the heart and this organization directly contributes to cell-specific transcriptome [6]. Chromatin compartmentalization is an inherent property of the nuclear architecture, with euchromatin being more accessible to transcriptional factors and heterochromatin less accessible [6,7][6][7]. In the heart, cardiac-specific genes are found in areas of euchromatin, while liver-specific genes are found in heterochromatin areas [6]. This illustrates that the packaging of the genome is not random and has direct implications on gene expression profiles.
Monolayer culture on plastic has been the traditional cell expansion method for cell therapies such as autologous chondrocyte implantation (ACI). Chondrocytes, which normally have a rounded morphology in their native tissue niche adopt an elongated fibroblastic-like morphology on plastic, leading to intracellular and intra-nuclear changes that ultimately result in a loss of chondrocyte phenotype (de-differentiation). On the other hand, culturing chondrocytes in 3D culture systems appears to maintain and/or re-differentiate the chondrocyte phenotype [8]. The main differences between monolayer and three-dimensional cultures are the geometrical constraints impinged on the chondrocyte. The adaptation of chondrocytes to these different constraints leads to not only cytoskeletal changes, but clear alterations in nuclear shape and DNA conformation and organization [9,10][9][10].

2. The ACI Procedure

Autologous chondrocyte implantation (ACI) is a two-stage operative procedure for the treatment of medium to large full thickness defects (2–10 cm2) in cartilage [38][11]. The first generation ACI was first introduced in 1987 by Grande et al. [39][12] and clinically applied in 1994 by Brittberg et al. [40][13]. In these first procedures, cartilage slices were harvested arthroscopically from a healthy non-weight-bearing region of the cartilage [19,21][14][15]. Chondrocytes were released from the cartilage tissue through enzymatic digestion and expanded in monolayer (25 cm2 or 75 cm2 culture flasks) over a period of 11–21 days to yield approximately 2.6 to 5 million cells [38,40,41][11][13][16]. The cultured chondrocytes were then implanted into the affected area and patched using a periosteal patch [42,43][17][18]. Theoretically, the periosteal patch should provide the cells with growth factors that encourage chondrocyte development and differentiation [42,43][17][18]. However, the periosteal patch was found to cause significant graft hypertrophy. This has encouraged researchers to investigate other biomaterials such as collagen patches [42,43][17][18]. One such patch is collagen type I and III that is currently being marketed as Chondro-Gide [42,44][17][19]. An important disadvantage associated with ACI is the accessibility to high cell numbers. Since these cells are harvested from patients via a small tissue biopsy procedure, the number of chondrocytes isolated from native cartilage is limited. However, large quantities of chondrocytes are needed for cartilage repair, which makes ex-vivo expansion essential for any potential use in ACI. When chondrocytes are expanded, they are cultured in a monolayer 2D culturing system, which is an effective method that generates a large number of viable cells [45][20]. Several commercial ACI expansion companies use two-dimensional monolayer systems to provide patients with 4 to 12 million viable cells for implantation. A recent focus in ACI is on priming the chondrocyte phenotype through the transfer of the chondrocytes to a 3D matrix [46][21] that is supplemented with biomolecules. To promote the hyaline forming potential of chondrocytes, current biomolecule cocktails include L-ascorbic acid-2-phosphate, dexamethasone, transforming growth factor β3 (TGF-β3) and insulin-transferrin-selenium (ITS) [45][20]. However, the outcome of this procedure is still inconsistent and a variety of tissues are formed: fibrous, fibrous-hyaline, and hyaline cartilage.

3. Chondrocytes Expansion and the De-Differentiation Effect

At a cellular level, 2D expansion on tissue culture plastic causes chondrocytes to gradually adapt to the monolayer environment by de-differentiating and losing the chondrocyte phenotype. De-differentiation is a process by which a cell goes from a terminally or partially differentiated stage to a less differentiated stage within their own lineage [47][22]. This phenomenon is manifested by a change in cell shape, gene and protein expression and importantly, cellular function [47][22]. The de-differentiation phenomenon is also observed in isolated chondrons grown in monolayer. When chondrons are cultured over a period of 7 days, two populations are observed: a floating population of chondrocytes that has a PCM, and a population of chondrocytes that has lost its PCM and adheres to tissue culture plastic [48][23]. Gene expression profiles of these different populations show the floating population has significantly higher SOX9, collagen type II, aggrecan, COMP, collagen type X, and lower RUNX2, compared to chondrocytes that adhered to the surface [48][23]. These changes highlight the adaptation chondrocytes go through to adhere to tissue culture plastic and that the PCM geometric constraints plays a crucial role in maintaining the chondrocyte phenotype. Only after the loss of the PCM do chondrocytes adhere to the culturing surface [48][23], presenting de-differentiation as a process of adaptation to a new environment. When chondrocytes without their PCM are isolated and expanded on 2D plastic, they change from spherical-shaped chondrocytes to spindle-shaped, fibroblastic-like cells. In addition, collagen type II, the major collagen produced by chondrocytes is switched to the production of type I collagen [49][24] along with a decrease in aggrecan, collagen type XI, and collaged type IX [8,50,51,52][8][25][26][27]. These cells switch from the secretion of a hyaline cartilage ECM to a fibrous cartilage ECM, a matrix that is mechanically inferior. Recent studies have compared the de-differentiation of chondrocytes and their loss of functional in-vitro status to degenerative diseases, such as OA. With the onset of OA, cartilage is degraded and the chondrocytes undergoes a panel of changes, including phenotypic changes. In situ staining of OA cartilage, identified three chondrocyte phenotypes: activated, hypertrophic and fibrotic [49][24]. The de-differentiation-like phenotype that is observed in OA is reflected by a population chondrocytes within the upper middle zone where a shift from quiescent to proliferative state is observed, with a deposition of fibrotic markers such as collagen type I and III [49,53,54][24][28][29]. While middle zone chondrocytes, presented with an activated phenotype, chondrocytes that are producing collagen type II, and the deep zone chondrocytes were hypertrophic, participated in matrix calcification and degradation via the expression of collagen type X and MMP13 [49,55][24][30]. These zonal differences emphasize and suggest the adaptation of chondrocytes to the environmental change is highly dependent on their innate environmental differences.

4. Chondrocytes in Two and Three-Dimensional Culture Compared to Native Tissue

In 2016 A.J. Mueller et al. investigated the transcriptional profile of in-vitro culturing systems both monolayer and three-dimensional systems compared to native cartilage. They found adult cartilage tissue is characterized by the expression of collagen type II, and aggrecan [59][31] as also shown by other studies. Proteoglycans (aggrecan, proteoglycan 2 and 3), tubulins, actin nucleator (Wasp, Arpc5 and Actr2) and kinesins (Kif4a, Kif11, Kif15, Kif20a/b, Kif22, Kif23) are strongly represented in cartilage comparative to monolayer chondrocytes [59][31]. On the other hand, actin assembling units, profilin-2 and cofilin-2, were downregulated in cartilage comparative to monolayer chondrocytes [59][31]. By passage five of 2D expansion, cells exhibited high expression levels of developmental mesenchymal markers: Thy1 (CD90), epithelial-mesenchymal transition regulator Snai1, prion protein encoding gene, Prnp, and bHLH transcription factor Twist1 [59][31]. Thus, monolayer expansion results in significant changes in cellular expression profile with a clear shift towards mesenchymal pre-cursor cell lineage. When comparing cartilage to chondrocytes grown in 3D culture (alginate), 3D culture caused an overexpression of AP-1 (Fos and Junb), the transmembrane glycoprotein osteoactivin gene Gpnmb, clusterin and the bone morphogenetic protein receptor type 1a (Bmpr1a) [59][31]. 3D culture was also found to upregulate genes associated with oxidative stress (Nfe2l2), hypoxia (Hif1a) and antioxidant responses (Sod2, Hmox1) [59][31]. It is therefore clear that while both culture types do not mimic the expression profile of native tissue, the 3D culture appears to have fewer changes than those seen in 2D culture. However, the major limitation in 3D culture, is cellular proliferation is very low [60][32]. This makes 3D culture a preferred method to re-differentiation chondrocytes after monolayer expansion [60][32].

5. The Biophysical Aspects of Monolayer Expansion and De-Differentiation

5.1. Integrin Profile Changes during Expansion

The process of mechanical sensing starts at the cell periphery, where the cell forms a physical connection with its environment [65][33]. Integrins are the main force transducer between the environment and the cell and serve as mechanical linkers between the cytoskeleton and the environment [65,66,67,68][33][34][35][36]. A family of transmembrane proteins that sit in the plasma membrane, integrins are heterodimers composed of α and β subunits. The bulk of these proteins is found in the extracellular domain with 700 aa of the α subunit and 1000 aa of β subunit protruding into the extracellular space [65,69][33][37]. By contrast, the cytoplasmic tail is 40–70 amino acids long. Integrins are maintained in a bent conformation when inactive. The activation process causes conformational changes within the cytoplasmic domain, where the protein talin binds to the β subunit and triggers the activation and conformational change of the α and β subunits [69][37]. Upon activation, the extracellular domains of integrins bind ECM protein such as fibronectin, collagen and others [68,70][36][38]. In the cytoplasm, the β subunit of the integrin heterodimer binds to the actin cytoskeleton through a variety of adaptor proteins. As the ECM ligand binds, the integrins activates further and clusters to initiate the assembly of the focal adhesions (FA) complex that is composed of focal adhesions kinase (FAK), vinculin, paxillin, and tensin, thus forming a linkage between the cell and the environment [68,70][36][38]. In stiff matrices there are increased number of focal adhesions and traction forces generated between FA and ECM, as compared to soft matrices that have fewer focal adhesions [68,70][36][38]. In addition, FA complexes contribute to the reorganization of the actin cytoskeleton in response to mechanical stimuli [66,67][34][35] thus translating the stimuli from the extracellular environment into a cytoskeletal change. Immunophenotyping performed on cartilage tissue showed the expression of α1β1 (collagen type VI, II and matrilin-1), α5β1 (fibronectin) and αVβ5 (fibronectin, vitronectin and osteopontin) and lesser amounts of α3β1 (fibronectin) and αvβ3 (COMP, fibronectin, vitronectin and osteopontin) [71,72,73][39][40][41]. The integrin profile is dependent on ECM proteins that are present. In OA, the ECM structure is altered, which leads to an alteration in integrin profile: α2β1 (collagen type II, VI, and chondroadherin), α4β1 (fibronectin and V-CAM) and α6β1 (laminin) integrins are the predominant integrins expressed in OA [73,74][41][42]. The changes in integrin profiles highlight the adaptation of chondrocyte to the ECM environment. When chondrocytes are harvested for ACI, they are removed from an ECM-rich environment and cultured in an ECM-free environment. In the process of adaptation to an ECM free environment, chondrocytes de-differentiate and exhibit an integrin profile change. Primary chondrocytes cultured over a period of 21 days showed a clear increase in β1 and α2 levels by immunofluorescent analysis. This increase is consistent with the increase in α2β1 complex that is found in OA, suggesting that culturing chondrocytes for prolonged periods (21 days) on plastic can lead to a disease phenotype; however, this does not rule out the formation of other integrin complexes. Furthermore, it is clear evidence that chondrocytes sense and adapt to environmental changes by altering their integrin profile. Such changes will have downstream effects on cytoskeletal arrangement and tensions [75][43] as well as on nuclear responses [76][44].

5.2. Chondrocyte Nuclear Shape and Biomechanical Response to Substrate Rigidity

While mechanical sensing is mediated by integrins [77][45], the intracellular response is driven by the cytoskeleton. Rho GTPases (RhoA, Rac1 and Cdc42) are known as master regulators of actin cytoskeleton dynamics through actin nucleation (WASP/WAVE) and Diaphanous-related formins, affecting cell morphology and cell adhesion [78][46]. Cell morphology and adhesion are two aspects that are influenced by substrate rigidity. On stiff substrates, cells assemble stress fibers that induce high intracellular tension forces, while soft substrates do not promote stress fiber formation [79,80][47][48]. The cytoskeletal forces, mediated in part by these stress fibers, are transduced from the cytoskeleton to the nucleus through the LINC complexes (Linker of Nucleoskeleton to Cytoskeleton) [79,80][47][48]. Several studies have shown the impact of substrate stiffness on chondrocyte de-differentiation [81,82,83,84][49][50][51][52]. Q. Zhang et al. [81][49] investigated the effects of growing chondrocytes on a range of polydimethylsiloxane (PDMS) membranes stiffnesses: soft to stiff (stiff being similar to commercial petri dish). They showed that 78% of the cells grown on soft substrates exhibited and maintained a round chondrocyte morphology, while on stiffer substrates only 41% of the cells presented with a spherical morphology, with 59% having a stretched fibroblastic morphology [81][49]. E. Schuh et al. found that stiffer substrates lead to higher proliferation rates but that stiff substrates also led to phenotypic changes associated with low collagen type II and aggrecan expression, and high collagen type I expression [82][50]. By contrast, softer substrates promoted the maintenance of the chondrogenic phenotype with high collagen type II and aggrecan expression, and lower collagen type I expression [82][50]. Chondrocytes grown on different substrate rigidities also showed apparent differences in F-actin distributions [83][51] and actin depolarization has been shown to enhance the chondrogenic potential [85[53][54],86], while the loss of chondrocyte phenotype correlates with increased RhoA signaling and the presence of stress fibers [83,84][51][52]. On 54–135 kPa substrates, chondrocytes presented highly organized parallel stress fibers, with a wide spread polygonal morphology [83][51]. By contrast, on 1.4–6 kPa substrates chondrocytes had a much smaller and rounded morphology with actin filament extensions found only in few cells [83][51]. Because actin filaments are able to transmit force to the nucleus, substrate stiffness has been shown to contribute to lineage determination, and affect expression of NE proteins, including Lamin A/C [87,88][55][56].

5.3. Nuclear Lamins, Hetrochromatin and Euchromatin

Lamins are intermediate filament proteins that reside primarily within the internal periphery of the nucleus. Lamins are encoded by three genes: lamins A and C are alternative splice products of the LMNA gene, lamin B1 and lamin B2 are encoded by the LMNB1 and LMNB2 genes respectively [89][57]. Lamins are necessary to maintain nuclear structure and mechanical properties [90,91][58][59]. Lamins A/C primarily contribute to nuclear rigidity, while B-type lamins provide the nucleus with elastic properties [89,92][57][60]. Lamins have been shown to protect nuclear DNA against mechanical forces [93][61]. DNA in cells is generally found in one of two states. Heterochromatin is densely packed chromatin located at the periphery of the nucleus and is typically transcriptionally inactive [94][62]. Euchromatin on the other hand is gene rich with higher transcriptional activity and is located centrally with open structures [94][62]. The organization of chromatin is key to gene regulation and cell-fate determination [95][63]. Advances in microscopic imaging and molecular approaches have provided important insights into DNA localization and folding in normal versus disease states. The genome organization is an important player in regulating gene activity [96,97,98][64][65][66]. Lamins play an important role in chromatin organization [99][67], interacting with chromatin via lamina-associated domains (LADs) found mostly in heterochromatin. LADs are found in chromatin regions that contain mostly silent or weakly-expressed genes [100][68] and is enriched with repressive histone modifiers: H3K9me2, H3K9me3, and H3K27me3 [63][69]. Thus, the nuclear lamina helps to establish a repressive nuclear compartment at the nuclear periphery. In the process of chondrocytes expansion for ACI, passage 0 (P0) chondrocytes have a rounded nucleus that is located in the center of the cell and expresses chondrocyte markers COL2A and SOX9. Using high resolution strain analysis to map mechanical strain on these chondrocytes, strain localization was distributed equally to heterochromatin and euchromatin at P0. At later passages, chondrocytes had a much flatter nucleus that was no longer centrally located in the cell. These later passage chondrocytes also had a higher strain in the heterochromatin and a higher expression of COL1A1 [101][70]. Interestingly, late passage chondrocytes maintained LMNB1 and LMNB2 gene levels but had a significantly lower expression of lamin A/C, suggesting that a loss of nuclear structural integrity contributes to the expression of repressed genes. It is important to note, the loss of lamin A/C is also indicative of loss of resistive force around the nucleus periphery [92][60]. Similarly, Nava et al. showed the application of stain on progenitor cells leads to a decrease in nuclear envelope tension to prevent DNA damage. The reduction in tension is mediated by the reduction in H3K9me3 lamina-associated heterochromatin [63][69].

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