Current Stem Cell Research & Therapy, 2012, 7, 00-00
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The Role of Bioreactors in Cartilage Tissue Engineering Nigel Mabvuure1, Sandip Hindocha*,2, Wasim Khan3 1
Brighton and Sussex Medical School, Audrey Emerton Building, Eastern road, Brighton, BN2 5BE; 2Department of Plastic Surgery, Whistin Teaching Hospital, Warrington Road, Liverpool. L355DR; 3University College London Institute of Orthopaedics and Musculoskeletal Sciences, Royal National Orthopaedic Hospital, Stanmore, Middlesex, HA7 4LP, UK Abstract: Cartilage tissue engineering is concerned with developing in vitro cartilage implants that closely match the properties of native cartilage, for eventual implantation to replace damaged cartilage. The three components to cartilage tissue engineering are cell source, such as in vitro expanded autologous chondrocytes or mesenchymal progenitor cells, a scaffold onto which the cells are seeded and a bioreactor which attempts to recreate the in vivo physicochemical conditions in which cartilage develops. Although much progress has been made towards the goal of developing clinically useful cartilage constructs, current constructs have inferior physicochemical properties than native cartilage. One of the reasons for this is the neglect of mechanical forces in cartilage culture. Bioreactors have been defined as devices in which biological or biochemical processes can be re-enacted under controlled conditions e.g. pH, temperature, nutrient supply, O2 tension and waste removal. The purpose of this review is to detail the role of bioreactors in the engineering of cartilage, including a discussion of bioreactor designs, current state of the art and future perspectives.
Keywords: Bioreactor, cartilage, cartilage engineering, chondrogenesis, design, scaffold. INTRODUCTION
THE IN VIVO CONTEXT OF CARTILAGE
Conditions resulting from damage and degeneration to articular cartilage present a significant burden to individuals and their respective public health systems. Osteoarthritis (OA), a chondrodegenerative disorder whose prevalence increases with age, is a major cause of disability. Progressive osteoarthritis can lead to joint replacement, a procedure fraught with the risks of both the surgery and the anaesthetic used. With an aging population, there is clearly an increased impetus to develop therapies that will benefit the increased number of people to be afflicted by chondrodegenerative diseases at a minimal cost and maximal efficacy. It is no surprise, therefore, that a great deal of efforts have been invested in developing effective therapies for this and related conditions, for decades. The field of cartilage tissue engineering is concerned with developing in vitro cartilage implants that closely match the properties of native cartilage, for eventual implantation to replace damaged cartilage. The three components to cartilage tissue engineering are cell source, such as in vitro expanded autologous chondrocytes or mesenchymal progenitor cells, a scaffold onto which the cells are seeded and a bioreactor which attempts to recreate the in vivo physicochemical conditions in which cartilage develops [1].
There are different types of cartilage depending on which part of the body they are located in and the function they undertake, namely fibrocartilage, elastic cartilage and hyaline cartilage. Hyaline cartilage forms the articular cartilage that lines joint surfaces and is destructed in OA. Understanding the biomolecular structure of articular cartilage and the forces that act on cartilage naturally in joints provides measurable goals on which we can compare the quality of engineered cartilage. It is not sufficient to understand just the composition of cartilage, but the relative concentrations of the constituents in mature, functioning articular cartilage also need to be understood.
The purpose of this review is to detail the role of bioreactors in the engineering of cartilage, including discussions of bioreactor designs, current state of the art and future perspectives.
*Address correspondence to this author at the Department of Plastic Surgery, Whistin Teaching Hospital, Warrington Road, Liverpool. L355DR, UK; E-mail:
[email protected] 1574-888X/12 $58.00+.00
Current tissue engineering paradigms have not been able to produce clinically useful cartilage as they have resulted in cartilage with inferior physicochemical properties (Table 1) [2]. One of the reasons progress towards clinically useful cartilage has been slow has been the neglect of the role of mechanical forces in chondrocyte culture. Bioreactors have been defined as devices in which biological or biochemical processes can be re-enacted under controlled conditions e.g. pH, temperature, nutrient supply, O2 tension and waste removal [1, 3, 4]. It is hoped that their use in cartilage engineering will lead to the development of reproducible protocols for the development of clinically useful cartilage constructs. CONSIDERATIONS IN BIOREACTOR DESIGN Bioreactors vary widely in their complexity and capability. The common laboratory culture dish is the simplest and most widely used bioreactor [4, 5]. They are relatively cheap to purchase and are widely available in most laboratories.
© 2012 Bentham Science Publishers
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Table 1.
Mabvuure et al.
Properties of Tissue-Engineered Cartilage Constructs and Human Foetal and Adult Articular Cartilage.*[2] Reproduced with Permission.
Culture system or cartilage source
Tissue property Cell concentration (cells g–1 dw x 10–6) 340±13
Water content (% w/w)
GAG concentration (% dw)
Total collagen concentration (% dw)
Collagen type II concentration (% dw)
Collagen type II as a percentage of total collagen (%)
90±0.3
26±2.1
11±3.4
8.5±1.8
88±11
Tissue-engineered cartilage using human adipose-derived stem cells after 5 weeks of culture
340±24
84±1
2.5±0.076
14±2.0
0.22±0.054
1.6±0.15
Human foetal articular cartilage
1350±43
89±0.4
42±4.1
19±1.5
17±1.6
77±8.4
Human adult articular cartilage
40±10
79±0.3
17±0.51
54±0.39
47±6.3
86±11
Tissue-engineered cartilage using human chondrocytes after 5 weeks of culture
*Results are mean±standard error for triplicate bioreactor cultures or ex vivo cartilage samples. dw, dry weight.
They however are not suitable for the culture of cartilage for clinical applications due to several limiting factors. Their small size is not conducive to the culture of three dimensional cartilage constructs for eventual in vivo transplantation. Such three dimensional (3D) cartilage structures would require a bioreactor system which allows the reach of nutrients and oxygen to the core of the construct, and the complementary removal of waste. This has been the major consideration is designing the various types of bioreactors which aim to provide nutrition in different ways. Other considerations include the repeatability of the process. Clinical grade cartilage constructs will need to have predictable batch-afterbatch properties and this can only ben managed in bioreactors. Asepsis is mandatory to ensure no infirmity is transferred to the patient and hence, closed system bioreactors have been developed with this consideration in mind. TYPES OF BIOREACTORS AVAILABLE FOR CARTILAGE ENGINEERING As mentioned above, the common laboratory culture dish is the simplest bioreactor. Because this culture system provides static conditions, it often results in a non-homogenous chondrocyte distribution within the scaffold [4]. Furthermore, whilst static cultures do result in glycosaminoglycan (GAG) expression, a great deal of these macromolecules are not retained in the scaffold but released into tissue culture medium and an outer fibrous capsule forms around the scaffold [6]. This capsule limits the mass transport of nutrients into the core of large scaffolds and hence affects the extent to which such constructs can mimic native cartilage. Due to these limitations, more complex bioreactors have been designed. 1. Rotating Wall Vessel (RWV) Bioreactors The RWV was designed specifically by the National Aeronautics and Space Administration (NASA) to allow the culture of shear-sensitive mammalian cells in a microgravity environment (Fig. 1b) [7]. This bioreactor subjects the scaf-
fold to dynamic laminar flow and enhances nutrient flow to the core of the scaffold. The improved nutrient supply, when bone-marrow derived mesenchymal stem cells (bMSCs) are cultured in a scaffold, results in a statistically significant higher concentration of GAGs than a pellet culture control [8]. The GAG concentration in cartilage cultured in RWV bioreactors more closely resembled hyaline cartilage than the controls. Further supporting evidence was provided by Yoshioka et al. [9]. They transplanted RWV bioreactorderived three-dimensional cylindrical allogeneic cartilage aggregates into osteochondral defects of Japanese white rabbits. This group developed reparative tissues resembled hyaline cartilage and importantly, did not develop fibrocartilage. The mean histological score of these reparative tissues was significantly higher than the control defects in which no cartilage constructs were transplanted. When dedifferentiated chondrocytes were cultured in RWV bioreactors, collagen II expression was detected, signalling that these chondrocytes redifferentiate and recommence the production of cartilagespecific extracellular matrix (ECM) [10]. The RWV bioreactor optimises nutrient reach throughout the cartilage construct and therefore is preferred for cartilage engineering as it promotes cartilage growth and differentiation more than other culturing techniques [11]. 2. Spinner Flask In spinner flask bioreactors, cell-seeded scaffolds are attached to needles and are suspended from the top cover of a flask, bathed in culture medium which is mixed with a magnetic stirrer placed at the bottom of the flask (Fig. 1a) [12]. This creates convection currents which stop the formation of nutrient concentration gradients at the border of the scaffold. Cell seeding in spinner flasks improves the cellularity of polymer constructs compared with three other seeding techniques; custom vacuum system combined with a perfused bioreactor or with an orbital shaker, and orbital shaker [13]. The importance of this is not to be underestimated since studies have shown that when scaffolds are seeded at high densities, chondrogenesis is enhanced in 3D constructs
Bioreactors in Cartilage Bioengineering
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fusion flow bioreactors are also useful in increasing ECM retention in scaffolds during culture. Shahin et al. found that applying low and gradually increasing medium flow rates to human chondrocytes in polyglycolic acid scaffolds in perfusion flow bioreactors resulted in larger constructs, a 4.0-4.4fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with a flow rate of 0.2 mL/min [17].
Fig. (1). Representative bioreactors for tissue engineering applications. (A) Spinner flask (B) Rotating wall vessel (C) Direct perfusion.[5] Adapted with permission.
[14]. Spinner flasks also allow the introduction of turbulence into the culture system, presumably increasing the flow of nutrients around the construct. Evidence shows that cartilage constructs derived from mixed cultures, such as stirredflasks, have more regularly shaped chondrocytes and contain up to 70% more cells, 60% more GAGs and 125% more total collagen when compared to constructs from static cultures [15]. Stirred-flask bioreactors, though simple, meet the basic design consideration of facilitating nutrient and oxygen transport throughout the medium and reduce the concentration boundary layer at the construct surface [5]. However, even with these bioreactor designs, it is still a significant challenge to ensure nutrient reach to the core of 3D cartilage constructs. Although the spinner flask bioreactor removes external diffusional limitations, the inability of medium and other nutrients to penetrate within the porous network of the scaffold remain [12]. There was a need, therefore, to develop a new type of bioreactor to attempt to address this issue otherwise the in vitro engineering of cartilage constructs large enough for clinical utility would remain an elusive target. 3. Flow Perfusion Bioreactors In contrast, dynamic laminar flow in rotating bioreactors provided efficient oxygen supply and resulted in the retention of newly synthesized macromolecules, the maintenance of cartilaginous tissue morphology, and the best overall tissue structure and composition for both engineered and natural cartilage (Fig. 1d) [6]. Da Silva et al. compared the chondrogenic potential of human bMSCs cultured on fibre mesh scaffolds in a flow perfusion bioreactor compared to static culture [16]. They found that constructs which were cultured in the flow-perfusion bioreactors had higher ECM and collagen type II production and took shorter to differentiate. Per-
Recent studies have begun to focus on developing bioreactors in which cartilage of clinically-useful sizes can be engineered. Santoro et al. used computational fluid dynamics models to optimize the flow profile in their new flow perfusion bioreactor [18]. Using their system, they seeded human chondrocytes uniformly into a 5 cm diameter, 3mm depth scaffold, achieving a uniform distribution. Bioreactor-grown constructs were homogeneously cartilaginous and had biomechanical properties approaching those of native cartilage as opposed to constructs generated by conventional manual methods. There has been a steady accumulation of data supporting the suitability of perfusion flow bioreactors for cartilage engineering applications. These bioreactors have the main advantage of facilitating the transport of nutrients into the inner core of the cartilage constructs through the connected pores o f the scaffolds. If we are to realise the goal of developing large constructs for clinical application, perfusion flow bioreactors seem to provide reasonable hope, as shown by Santoro et al.’s study. EXPERIMENTAL DATA ON THE ROLE OF BIOREACTORS IN CARTILAGE ENGINEERING A great deal of progress in cartilage engineering was made whilst ignoring the physical environment in which cartilage develops and is maintained. However, as already discussed, a plateau in the quality of derived cartilage was reached without achieving constructs of native cartilage qualities. This led to a paradigm shift to more “complete” protocols which take into account the biochemical as well as the physical environment. The physical factors most studied are oxygen tension, hydrostatic force, compression, low shear and high shear forces. The controlled introduction of these loads to culture systems requires the use of automated bioreactors. Extensive reviews of how these forces affect chondrogenesis have been detailed elsewhere. Here, the latest studies are summarised. 1. Oxygen Tension In the embryo, the first stage of chondrogenesis begins when uncommitted mesenchymal chondroprogenitors condense to form a cartilaginous anlage. This anlage is the template on which endochondral ossification subsequently occurs. This anlage is in relatively hypoxic environments and therefore, the role of oxygen in chondrogenesis is of current intense research interest. Several studies have demonstrated that the biochemical composition of in vitro cartilage more closely mimics native tissues when cultured in hypoxic conditions [19-21]. This importance of a hypoxic microenvironment is further supported by studies showing that bovine articular chondrocytes dedifferentiate in normoxic conditions, but redifferentiate under low oxygen tension conditions [22]. Others have suggested that low oxygen tension is
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a more potent promoter of chondrogenic differentiation than dynamic compression in porcine MSCs [23]. However, the effect of oxygen is not uncontroversial as some studies have shown seemingly conflicting results to studies suggesting a stimulatory effect of low oxygen tension on chondrogenesis. An example is Malladi et al. who found that the differentiation of adipose-derived mesenchymal cells into chondrocytes was inhibited in a 2% oxygen environment [24]. Other studies of chondrocytes embedded in a scaffold have demonstrated an enhanced ECM formation under more aerobic conditions [25, 26]. Despite the ongoing controversy regarding the role of oxygen, [27] it is not controversial to note that oxygen plays a significant role in chondrogenesis, although the role has not yet been specifically delineated. Bioreactors have been instrumental in the gathering of current research evidence in attempts to understand the role of oxygen. When that role becomes understood, bioreactors will also have a role in standardising optimum oxygen conditions for in vitro cartilage growth. 2. Hydrostatic Pressure A prevailing hypothesis is that since cartilage is exposed to intermittent hydrostatic pressure (HP) due to everyday activities, introducing this load, in combination with other mechanical loads, to chondrocyte culture might increase matrix synthesis of in vitro-engineered cartilage [28, 29]. Indirect application of HP is the most important load occurring in joints [30]. Ogawa et al. compared the histological, immunohistochemical, and gene expression characteristics of cartilage constructs cultured under two different conditions; HP at 0-0.5 MPa and 0.5 Hz and at atmospheric pressure [31]. The HP constructs accumulated ECM faster, maintained cell number for longer and had increased chondrocyte-specific gene expression especially after 2 weeks. They concluded that cyclic HP was effective in enhancing ECM accumulation and chondrocyte-specific gene expression. Others have shown that intermittent HP loading during redifferentiation of chondrocytes in alginate beads and chondrogenesis resulted in a statistically significant increase in GAG and collagen type II synthesis compared to controls without loading [29]. The application of intermittent HP at levels between 7-10 MPa is regarded ideal for cartilage tissue engineering [28, 32]. Another important reason for the tight control of HP levels within this range is because HP impacts the solubility of oxygen and may therefore affect oxygen tension [28]. To produce these conditions reproducibly on an industrial scale requires the use of bioreactors. However, the precise effect on HP is not yet agreed upon as some authors have found results conflicting with the general trend. Wenger et al. found that applying cyclic HP for 4 hours at 5 MPa using a 1 Hertz sinusoidal frequency significantly increased the proportion of apoptotic cells in the cartilage-constructs [33]. Hence, the role of bioreactors will not only be limited to the study of appropriate levels of HP, but also to the further understanding of the effects HP has on chondrogenesis and cartilage. Further specific review of the role of hydrostatic forces in chondrogenesis is provided by Elder et al. [32].
Mabvuure et al.
3. Compressional Forces The vast majority of studies on the effects of mechanical loading have investigated compressional forces. This is perhaps unsurprising as the shock-absorber function of articular cartilage is perhaps the most intuitive. Bioreactors have provided the controlled environmental conditions in which studies assessing the effects of compressional forces of cartilage have been conducted. Although static compression has increased chondrogenic gene expression in some studies, [34] many groups have shown that dynamic compression promotes chondrogenic more than static compression does [3537]. Several studies on MSCs have shown an increase in markers of chondrogenic differentiation when dynamic compression is applied as opposed to static compression [38-40]. This is in keeping with the everyday function of articular cartilage which experiences dynamic compression during arm and leg movement. Hoenig et al. assessed the short-term influence of variable dynamic compressive strain amplitudes (5%, 10%, and 20% 3000 cycles/day, 1Hz) on mechanical and biochemical properties of scaffold-free cartilage constructs seeded with primary porcine chondrocytes [41]. Although no difference in GAG or collagen type II content between the loaded and the control groups was found, compression amplitude was positively correlated with strain amplitude with 20% having the strongest positive effect on the mechanical properties of the cartilage constructs. This suggests that preconditioning engineered cartilage with higher load amplitudes may generate stiffer, more clinically relevant tissue [41]. In the long term, dynamic loading also improves the mechanical properties of chondrogenic MSC-seeded cartilage constructs [42]. As with other aspects of chondrogenesis and cartilage development, a consensus is yet to be reached on the precise role of dynamic compressive loads. Thorpe et al. investigated the influence of dynamic compressive loading on chondrogenesis of porcine MSCs in the presence of tumour growth factor (TGF)-3 [43]. They found that after 42 days in culture, the free-swelling specimens had increased expression of proteoglycans, GAGs and collagen type II than the dynamically compressed constructs. Once again, newer bioreactor designs and protocols will need to be designed to provide definitive evidence. 4. Low and High Shear Loads There is a distinction between bioreactors applying high and low shear forces. Stirred systems such as spinner flasks and direct perfusion systems apply high shear forces whereas systems such as the RWV apply low shear forces [28]. It is important to understand the effect of different types of shear forces as they can significantly affect cartilage homeostasis. For example, high fluid shear forces applied to chondrocytes can cause the recapitulates of the earmarks of OA such as the induction of cyclooxygenase-2, prostaglandins (PGs), and interleukin-6 (IL-6) [44, 45]._ENREF_40 Other studies have shown have shown that high mechanical shear stress can induce chondrocyte death via apoptosis [46]. Other studies have shown conflicting results. When 1 dyne/cm2 fluid shear was applied continuously for 3 days, primary bovine articular chondrocytes produced constructs with significantly
Bioreactors in Cartilage Bioengineering
higher amounts of total collagen type II compared to static controls [47].
Current Stem Cell Research & Therapy, 2012, Vol. 7, No. 4
[2]
AUTOLOGOUS ENGINEERING OF CARTILAGE The relatively new concept of in vivo bioreactors is based on the appreciation that although much progress has been made in attempting to mimic in vivo conditions, the complexity of the processes is such that there is still a way to go before bodily conditions can be matched. It therefore seems logical to introduce systems where the cartilage construct is grown in the body, using the body as the bioreactor. Stevens et al. called this space created in the body, where cartilage cells can then be grown, an “in vivo bioreactor (IVB)” [48]. In New Zealand White rabbits, they created in vivo bioreactors between the surfaces of the tibia and the periosteum, which is rich in pluripotent cells. A biocompatible calciumalginate gel that crosslinked in situ was injected into the bioreactor space. After 12 weeks, the bioreactor space had been reconstituted by functional living bone without the addition of any exogenous growth factor administration. A recent study by Emans et al., they hypothesised that if a gel had properties which limit vascularization, it would favour a hypoxic microenvironment which promotes chondrogenesis. They created IVBs in New Zealand white rabbits and injected them with a hypoxia-promoting agarose gel. The IVBs filled with collagen type-II and proteoglycan positive neocartilage which was hypercellular and capable of remodelling within a full-thickness osteochondral defect with complete integration. A significant finding was that this cartilage did not undergo calcification even after 9 months and is an encouraging finding for the clinical use of such cartilage. This is a promising area of cartilage engineering in which more studies are required to investigate its potential. CONCLUSIONS The potential of bioreactors in improving cartilage engineering has been realised for some time. Their use stemmed from the realisation that traditional bioengineering methods were failing to produce neocartilage of a quality sufficient enough justify clinical utility. Bioreactors have been used to introduce various mechanical loads, normally present in vivo, to cartilage culture protocols. They are also crucial in providing standardised conditions such as pH and oxygen tension which is crucial for batch reproducibility. However, many controversies still remain with regard to the specific actions of various mechanical stimuli. Bioreactors will also be instrumental in providing the conditions necessary to further study these stimuli. There are also now new ideas in bioreactor use such as the in vivo bioreactor, which provides hope for an avenue that might provide in vivo conditions to cartilage engineering. CONFLICT OF INTEREST
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Received: February 24, 2012
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Revised: March 01, 2012
Accepted: April 29, 2012
