Shear bioreactors stimulating chondrocyte regeneration, a systematic review

It is commonly accepted that the mechanical stimuli are important factors in the maintenance of normal structure and function of the articular cartilage. Despite extensive efforts, the cellular mechanisms underlying the responses of articular chondrocytes to mechanical stresses are not well understood. In the present review, different types of shear bioreactor and potential mechanisms that mediate and regulate the effect of shear on chondrocyte are discussed. For this review, the search of the literature was done in the PubMed, Scopus, Web of sciences databases to identify papers reporting data about shear on chondrocyte. Keywords “shear, chondrocyte, cartilage, bioreactor” were used. Studies published until the first of March 2018 were considered in this paper. The review focused on the experimental studies conducted the effect of shear stress on cartilage tissue in vivo and in vitro. In this review, both experimental studies referring to human and animal tissues were taken into account. The following articles were excluded: reviews, meta-analysis, duplicate records, letters, and papers that did not add significant information. Mechanism of shear stress on chondrocyte, briefly can be hypothesized as (1) altered expression of aggrecan and collagen type II, (2) altered cartilage oligomeric matrix protein (COMP) serum levels, consequently, organizing the arrangement binding of glycosaminoglycans, integrins, and collagen, (3) induction of apoptosis signals, (4) altered expression of integrin.


Background
It is now commonly accepted that the mechanical stimuli are important factors in the maintenance of normal structure and function of the articular cartilage and changes its morphology in response to mechanical stimuli. Despite extensive efforts, the cellular mechanisms underlying the responses of articular chondrocytes to mechanical stresses are not well understood [1].
The mechanisms by which chondrocytes actively respond to mechanical stimuli are important for understanding the modulators and signaling pathways involved in the pathogenesis of major disabling diseases, such as rheumatoid arthritis (RA) and osteoarthritis (OA) [2]. But, due to the complexity of the signaling mechanisms, the detailed pathways remain unclear.
Different mechanical stimuli such as compressive and tensile forces modulate chondrocyte function. Articular cartilage is the highly specialized hydrated (80% water) connective tissue that experiences the solute transport in the cartilage and movement of fluid during the loading and unloading conditions (exploding water during loading, and draw back into the tissue during unloading) [3].
By the movement of fluid within cartilage, chondrocytes experience potential fluid shear stress that affects chondrocytes proliferation, apoptosis, growth and differentiation, and extracellular matrix production [4]. A number of pathways involved in transduction of the mechanical stimuli of shear stress to intracellular signaling, but despite the extensive effort, exact mechanisms remain unclear. Thus, in the present review, different types of shear bioreactor and potential mechanisms that mediate and regulate chondrocyte proliferation and matrix production are discussed.

Main text
For this review, the search of the literature was done in the PubMed, Scopus, Web of Sciences databases to identify papers reporting data about the effect of shear stress on chondrocyte. Keywords "shear, chondrocyte, cartilage, bioreactor" were used. Studies published until the first of March 2018 were considered in this paper. The review focused on the experimental studies conducted shear stress on cartilage tissue in vivo and in vitro. In this review, both experimental studies referring to human and animal tissues were taken into account. The following articles were excluded: reviews, meta-analysis, duplicate records, letters, and papers that did not add significant information (Fig. 1). Data assessment was conducted independently by 2-6 investigators using predefined terms.

Cartilage
Articular cartilage as a highly specialized avascular, aneural, and alymphatic, connective tissue is composed largely of water, collagen, proteoglycans, and cells. The primary function of articular cartilage is to provide a smooth welllubricated surface for synovial joint and to facilitate the transmission of loads. The composition and structure of articular cartilage have a direct role in its function as a lubricious, load-bearing tissue. To achieve a deep understanding of load-bearing properties, two major sets macromolecules, the proteoglycans, and collagens must first be well understood since structural interactions between these macromolecules resist compressive loads and retain water [5] (Tables 1, 2, 3, and 4).
Zonal structure of hyaline articular cartilage from the articulating surface down to the subchondral bone is shown in Table 2. Territorial structure of hyaline articular cartilage is shown in Table 3.

Chondrocyte
Within the cartilage matrix, the chondrocyte is the only responsible cell type for synthesis extracellular matrix and constitute about 2% of the total volume of articular cartilage.

Effect of mechanical stimuli on chondrocyte
Interactions between chondrocytes and the ECM, consequently, homeostasis maintenance of the articular cartilage modulated by several stimuli such as mechanical stress, soluble mediators, and matrix composition. Mechanical stimuli affecting chondrocytes are divided into four categories (dynamic compression, fluid shear, tissue shear, and hydrostatic). Here, we focused on the effect of shear stress on chondrocyte metabolism. Also, four general categories of shear bioreactors are discussed [9,10].

Effect of shear stress on chondrocyte metabolism
As mentioned above, cartilage is a highly hydrated connective tissue. Approximately, 70% of water is expelled when the tissue is loaded in compression resulting in potential fluid shear stress at or near the cellular membrane. The water was osmotically drawn back when the tissue is unloaded [11][12][13]. Therefore, chondrocyte can experience fluid shear stress when water is relocated during compression [14]. Shear stress as a mechanical stimulation has been shown to affect chondrocytes through changes in membrane potential, solute transport, or cellular deformation. It is hypothesized that articular chondrocyte metabolism is modulated by direct effects of shear forces that act on the cell through mechanotransduction processes and the properties of the cross-linked type II collagen fibrils.

Experimental studies with shear stress
Four general categories of shear bioreactors have been carried out including contact shear, fluid flow, direct fluid perfusion, and low shear "microgravity" bioreactors (Tables 5, 6, 7, and 8)

Contact shear
During the physiological situation, cartilage is rubbing against either cartilage or produce contact shear. Several studies attempted to stimulate the solid-on-solid, contact shear using bioreactors and different scaffold ( Table 5). The results of Waldman et al. study demonstrated that intermittent application of dynamic shearing forces (2% shear strain amplitude at a frequency of 1 Hz) increased both collagen and proteoglycan synthesis and improves the quality of cartilaginous tissue [15]. Also, in the study of Frank et al. through metabolic studies and application of sinusoidal macroscopic shear deformation (rotational resolution is 0.0005°), increase in the synthesis of proteoglycan and proteins was detected [16]. Several studies examined the tissue shear loading (0.01-1.0 Hz, using 1-3% sinusoidal shear strain amplitudes) on chondrocyte biosynthesis and revealed that the synthesis of protein by approximately 50% and proteoglycans by  showed that several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases increased [24]. Also, Vainieri et al. developed a model of osteochondral defect from bovine stifle joints using bioreactor that mimics the multi-axial motion of an articulating joint. Results revealed that proteoglycan 4 and cartilage oligomeric matrix protein, mRNA ratios of collagen type II to type I, and aggrecan to versican were markedly improved [25].

Fluid shear
Fluid flow bioreactor development is consistent with the hypothesis that increase in nutrient and wastes transfer lead to increase in cell metabolism. Several bioreactors including spinner flask and wavy-walled bioreactor developed for the purpose (Table 6). Gooch et al. investigated the effects of the hydrodynamic environment by using spinner flask (80 RPM) on bovine calf chondrocytes seeded on polyglycolic acid meshes. The finding of the study was higher fractions of collagen and more GAG in chondrocytes [26]. Also, Bueno et al. developed a wavy-walled bioreactor to provide high-axial mixing environment to the cultivation of cartilage constructs. Polyglycolic acid scaffolds seeded with bovine articular chondrocytes and resulted increased cell proliferation and extracellular matrix deposition [27]   Vunjak-Novakovic et al. investigated the effect of bovine articular chondrocytes seeded in fibrous polyglycolic acid in well-mixed spinner flasks. This environment resulted in the formation of 20-32-micron diameter cell aggregates that enhanced the kinetics of cell attachment [28]. In a similar study, Theodoropoulos et al. placed articular cartilage of bovine metacarpal-phalangeal joints in spinner bioreactors and maintained on a magnetic stir plate at 90 rotations per minute (RPM). The study found that there was a significant increase in collagen content, the expression of membrane type 1 matrix metalloproteinase (MT1-MMP), and aggrecan [29]. Study of Bilgen et al. that applied a wavy-walled bioreactor (WWB) demonstrated the importance of characterization of mixing and impact of changes in bioreactor geometry and operating conditions [30]. In the study of Song et al. in a spinner flask, adipose-derived stem cells (ADSCs) seeded with chitosan/gelatin hybrid hydrogel scaffolds. ADSCs differentiated into chondrocytes and expressed more proteoglycans and cell distribution [31]. In another study, Cortez et al. developed a 3D finite element model to mechanical simulate (5%, 10%, and 15% of compressive strain with frequencies of 0.5 Hz, 1 Hz, and 2 Hz) the diffusion and transport of nutrients. The findings showed that fluid shear stress improved the solute transport and chondrocyte activity [32]. Also, Chapman et al. applied a model to predict the optimal flow rate of culture medium into the fiber lumen [33]. Juhasz et al. in a study investigated the loading scheme (0.05 Hz, 600 Pa; for 30 min) on chondroprogenitor cells and showed an increase in cartilage matrix constituents of chicken embryos, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases [24]. Also, Pearson et al. applied a model of  fluid flow, nutrient transport, and cell distribution using a hollow fiber membrane bioreactor. With the model and the effect of mechanotransduction on the distribution investigated [34].

Perfusion bioreactor
Transfer nutrient through the three-dimensional biomaterial and tissue constructs is one of the serious problem and limitations of fluid shear bioreactors. Therefore, direct perfusion bioreactor with different flow rates investigated and developed to overcome the nutrient limitations (Table 7). Mizuno et al. in a study cultured bovine articular chondrocytes in 3D collagen sponges with medium perfusion (0.33 mL/min) for up to 15 days. Interestingly, the results demonstrated that these conditions that are beneficial for other cell types inhibit chondrogenesis by articular chondrocytes [35]. Pazzano et al. cultured chondrocytes seeded on PLLA/PGA under to 1 μm/s flow and demonstrated a 118% increase in DNA content, a 184% increase in GAG content, and a 155% increase in hydroxyproline content [36]. Also, culture of bovine articular chondrocytes seeded on micro-porous scaffolds under a median shear stress of 1.2 and 6.7 mPa, promoted the formation of extra-cellular matrix specific to hyaline cartilage [37]. In another study, bovine articular chondrocytes seeded on polyesterurethane foams and cultured for 2 weeks under flow rate (0.5 ml/min). The results of study indicated that mean content in DNA and GAG increased [38]. In a similar study, the culture of human chondrocytes in bioreactor applied loading (0.1 MPa for 2 h) and perfusion (2 ml) led to increase of COL2A1 expression and decrease of COL1A1 and MMP-13 expression [39]. Carmona-Moran and Wick applied perfusion bioreactor to promote chondrogenesis of human mesenchymal stem cells. Results of this culture condition showed that after day 14, collagen deposition and proteoglycan deposition increased [40]. Yu    Results indicated the number of detached cells increased [44]. Also, the finding of Tonnarelli et al. study indicated that culture of chondrocytes bioreactor culture conditions support chondrogenic differentiation [45].

Low shear bioreactor
Low shear mixing improves the growth of cells on threedimensional scaffolds and applies minimal loading to constructs. Rotating bioreactors are the most popular devices to apply low shear mixing (

Conclusion
In the field of tissue engineering, several bioreactors developed at once and at different times to apply mechanical forces to cartilage constructs. Mechanism of shear stress on chondrocyte, briefly, can be hypothesized as the following [24,29,35,41]: 1. Altered expression of aggrecan and collagen type II 2. Altered cartilage oligomeric matrix protein (COMP) serum levels, consequently, organizing the arrangement binding of glycosaminoglycans, integrins, and collagen 3. Induction of apoptosis signals 4. Altered expression of integrin.