Chapter 8. Comparison of static vs dynamic culture

Transcription

1 Chapter 8 Comparison of static vs dynamic culture 8.1. Literature Review Articular cartilage is a load-bearing connective tissue in which its functions not only transmitting the compressive joint loads to the underlying subchondral bone, but also providing a low-friction interface between the contacting cartilage surfaces of the joint. Thus, in its native physiological conditions, it experiences complex loading, which includes compressive and shear forces. These forces had been shown to play important roles on regulation, development and maintenance of articular cartilage in vivo [Grodzinsky et al. 2000]. Cartilage grafts developed under static environment often curtailed by cells death, limited production of extracellular matrix, which led to inferior mechanical properties and ultimately failure of the grafts. Relating to the in vivo physiological conditions, these observations may be due to the lacks of mechanical stimuli experienced by the isolated cells in the grafts. In vitro study conducted by Waldman et al. exhibited that intermittent mechanical stimulations, either compressive or shear loading resulted in enhance cartilaginous tissue formation of cultured bovine chondrocytes [Waldman et al. 2003]. Culturing cartilage grafts in dynamic conditions, i.e. in bioreactor, is hence seen as one of the alternatives to provide mechanical stimulation mimicking in vivo settings. Several groups investigated the use of bioreactor for cartilage tissue engineering, which included spinner flask system [Vunjak-Novakovic et al. 1996, Freed et al. 1994], rotating wall vessel (RWV) [Freed et al. 1997, Freed et al. 1998], perfusion flows reactor [Dunkelman et al. 1995], and concentric cylinder bioreactor [Saini & Wick 2003]. 92

2 These studies reported less cell deaths, more homogenous cell distribution throughout the construct, illustrating sufficient mass transfer. The advantage would be thicker constructs can be produced as opposed to static that limited to only a few mm thickness. Enhanced proteoglycans production and collagen type II expression with homogenous extracellular matrix expression through out the constructs illustrating better maturation, cartilaginous tissue formation and cartilage graft function. Based on this, utilizing bioreactor is a promising approach to achieve functional cartilage tissue engineering Materials and Methods Setting up spinner flask system. Comparing the effect of culturing environment to the constructs was done in a simple system, i.e. spinner flask (Figure 8.1a and b). When scaffolds were tried in the first system (Figure 8.1a), where scaffolds were stuck in to a stainless steel rod and suspended in the medium, due to the characteristics of the hydrogels that were slippery and the size of the pores that were considered big, the scaffolds tended to slide off from the rod. Instead, scaffolds were then cultured using the second system (Figure 8.1b). In the second system, the wire was shaped resembling a nest-like structure. a Figure 8.1. (a) Schematic diagram of spinner flask with scaffolds stuck into a stainless steel rod and suspended in the medium. (b) picture of nest-like wire system to suspend scaffolds in the medium. b 93

3 Constructs were seeded with chondrocytes using alginate as the matrix as previously described at section 6.2. The constructs were then cultured for two weeks in static environment, i.e. the 24-well plate before transferred to a spinner flask system. In the spinner flask system, constructs were cultured for another 4 weeks, changing medium was done once every two weeks, and harvested for various assays Assessments. Assays for constructs were for cell morphology, with light microscopy, cell metabolic activity using AlamarBlue assay, cell proliferation with dsdna quantitation assay, PicoGreen, cell viability using confocal laser microscopy with FDA/PI staining, and extracellular matrix assay by alcian blue and silver staining. Methods have been described in the previous sections Results and discussion Light microscopy and confocal laser microscopy. At the end of 6 weeks a lot of chondrocytes including a number of cell balls were observed in both of the samples namely in static culture and in spinner flask culture. But the cells grown in the spinner flask were observed to have intense dark appearance and higher cell numbers (Figure 8.2a). The morphology was maintained in round shape similar to that of native cartilage. It was corroborated in confocal microscopic pictures that cells adopted round morphology after 6 weeks culturing with more viable cells in spinner flask culture than in static culture as shown in Figure 8.2b and c. 94

4 The maintenance of chondrocytes in round shape is desirable for maintaining production of extracellular matrix distinctive of cartilage. In other study, chondrocytes often adopted fibrobastic and more elongated morphology, especially in the outer layer due to the shear stress experienced by the chondrocytes [Akmal et al. 2006]. This in turn would produce more of collagen type I instead of collagen type II. a b Static c Figure 8.2. (a) Light microscopy picture after culturing for 42 days, 4 weeks in spinner flask. (b) and (c) are the confocal laser microscopy pictures for comparison of cell viability in static and dynamic culture. As for cellular viability, the higher number of viable cells in dynamic cultured was expected as dynamic culture should enhance the mass and nutrient transfer. Constructs produced by static culture larger than 1 mm had been found to contain hypoxic, necrotic centre with viable cells at the rim. Penetration of mass and nutrient may achieve no further than 240 µm from the surface. It represents that the limitation of mass transfer is a great challenge to be addressed. Spinner flask External mass-transfer limitation may be alleviated by the spinner flask system. Continuous stirring induces mixing of oxygen and nutrient through out the medium 95

5 hence reducing the concentration boundary layers at the construct surface [Martin et al. 2004]. The rotating fluid environment generated dynamic laminar flow efficiently which reduced diffusional limitation of nutrients and wastes while producing low level of shear Metabolic activity and cell number. It was evident by the AlamarBlue TM test results that at the end of 6 weeks, the spinner flask cultured cells showed higher metabolism compared to static culture system (Figure 8.3a). As per the PicoGreen assay, the dsdna values were observed to be 2 times for the spinner flask culture than the static culture system (Figure 8.3b). a b 1 STATIC 2 SPINNER FLASK ds DNA values( n g /m l) CULUTURE METHODS Figure 8.3. (a) Metabolic activity assay by AlamarBlue TM assay showed relatively higher % reduction indicating higher activity in spinner flask compared to static. (b) ds-dna quantitative assay by PicoGreen assay also indicating higher value in spinner flask culture. The higher degree of metabolism as shown for the spinner flask cultured constructs may be due to the true higher degree of metabolism or may also be due to the higher cell number in the constructs. It has been explained before that % of reduction measured by AlamarBlue TM correlated with the cell number [Ng et al. 2005]. Higher degree of metabolism was also suspected as hydrodynamic environment may stimulate cell to 96

6 proliferate more and produce more extracellular matrix [Martin et al. 2004, Bueno et al. 2005]. However, if we take a look at the PicoGreen measurement result, it was obvious that the cell number was significantly higher at the final time point of culturing. Hence it can be mentioned that corroborated with other studies, culturing in dynamic environment increased mostly the cell proliferation. Increase in metabolic activity can also be suggested as metabolism is increased when cell proliferates. As for the increase metabolism due to the production of extracellular matrix, it is further explained in the section below Alcian blue and silver staining Production of extracellular matrix distinctive of cartilage tissue was done by assessing proteoglycans production by Alcian Blue and assessing collagen type II production by SDS-PAGE and stained with silver staining. The presence of proteoglycans was shown in intense alcian blue stain in Figure 8.4. Comparing static and dynamic culture we can see that more intense color was exhibited at the rim of spinner flask construct sample. Although at the rim of static cultured constructs we observed more intense stain compared to the inner area, but dynamically cultured showed the most intense colour. This indicated proteoglycans strongest presence was at the peripheral of dynamically cultured constructs. 97

7 a b Figure 8.4. Alcian blue staining revealing proteoglycan presence of (a) static cultured and (b) dynamic cultured constructs. The presence of collagen-ii was found in silver staining indicating the synthesis of extracellular matrix (Figure 8.5). The media containing the cell-scaffold construct has significantly more collagen-ii content than the scaffold itself indicating the secretion of extracellular matrix proteins to the surrounding space in the form of soluble collagen M C1 C ST SF CELLSHEET STM SFM Figure 8.5. Silver staining results comparing collagen production in statically and dynamically cultured construcs and medium. Intense band were seen in medium used to culture the constructs (dashed box) but not in the constructs lane (dotted box). 98

8 . It is important to note as well, that more intense band was observed for medium of spinner flask culture. It illustrated that dynamic cultured in fact, stimulated more production of extracellular matrix, in particular collagen type II. More collagen type II found in the medium compared to the scaffold construct itself, showed that deposition of collagen type II into the constructs were not achieved as desired. It is in contrast with other studies that claimed increased deposition of extracellular matrix in the constructs exposed to hydrodynamic forces [Bueno et al. 2005, Mauck et al. 2003, Hung et al. 2004, Vunjak-Novakovic et al. 1996]. Nevertheless, it was in agreement with another study using rotating wall bioreactors which showed no significant increase of deposition of extracellular matrix into the constructs compared to static culture [Aufderheide et al. 2005] Conclusion Investigation of exposing dynamic environment to the novel soft- PCL/matrix/chondrocyte constructs led us to conclude several facts. Spinner flask method clearly exhibits its superiority over the static method with the obvious differences in higher cell number, higher metabolism and higher cellular viability for the spinner flask culture. This implied better nutrient and mass transfer had been achieved. Hydrodynamic forces had also increased the production of extracellular matrix distinctive of articular cartilage such as collagen type II. However, it was also concluded that the extracellular matrix produced, most were soluble and released to the medium instead of being deposited to the constructs. Shear forces generated by the rotating fluid may be enough for increasing cell number and 99

9 stimulate production, but deposition of the matrix to the constructs may have to be achieved with different approaches. Spinner flask being the simplest bioreactor may not provide the correct cues for deposition of matrix. Perhaps, mechanical stimulation more similar to the native environment such as deformation or intermittent flow bioreactors will enhance matrix deposition and produce better cartilaginous tissue. This is one of the interesting issues to be addressed in the future works. 100