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Recent Advances in Bioreactors in Tissue Engineering and Regenerative Medicine

JP Ruiz*1, N Ecker1, D Pawley1 and HS Cheung1, 2

1Department of Biomedical Engineering. College of Engineering. University of Miami. Coral Gables, Florida, USA; 2Geriatric Research, Education and Clinical Center. Miami Veteran’s Affairs Healthcare System. Miami, Florida USA

Abstract: The goal of the field of tissue engineering has always been to provide constructs that can repair, replace, or aid in the healing of damaged organs and tissues. In order to do this successfully however, a close understanding of the tissue to be replaced is required, from the molecular to the systemic level. In recent years, it has been understood that the physio-logical environment of many tissues is modulated and maintained by a plethora of subtle mechanical, chemical, and elec-trical cues, all working in synch. Bioreactors serve the purpose of mimicking this physiological environment at the bench top, in an effort to precondition tissue constructs to develop better differentiation, integration and mechanical compatibil-ity to avoid complications post transplant. However, exactly mimicking every single subtle cue that takes a part in the de-velopment of certain tissues is currently beyond our reach, as there is still a lack of understanding of said cues and interac-tions. Despite this, the advances in the design of bioreactors have been tremendous, from the first simple incubator to bioreactors that now incorporate a vast amount of forces and stimulation to different tissues. This review paper takes a look at the advances and development of bioreactors in the past five to ten years, as well as assesses those currently being used in the field and their efficacy in preconditioning functional tissue constructs.

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INTRODUCTION

Since its beginning stages, the field of Tissue Engineer-ing (TE) has had its share of obstacles to overcome. And while the years of research in the field—20 years to be ex-act—have overcome many of these obstacles, host integra-tion, immune response, mechanical mismatch, and upscaling remain critical issues that the field needs to address. Tissue Engineering was originally defined by two fathers of the field, Langer and Vacanti, as “an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function” [1]. To this day, this remains the most concise definition which most clearly de-fines the field and its goals. In order to successfully restore, maintain, and improve tissue function however, a clear un-derstanding of the tissue in question is required. From before the inception of tissue engineering as a field and more so during recent years, it has been understood that a tissue’s environment is maintained and developed by many mechani-cal, chemical, and electrical cues. Mechanotransduction, for example, is a cell or tissue’s ability to receive and interpret mechanical cues and translate those cues into chemical intra-cellular cues that affect a cell’s phenotypic expression [2]. The complexity of the cues is addressed in tissue engi-neering from many angles, from scaffold and cell type, to pre-transplantation conditioning [3]. While we are nowhere

*Address correspondence to this author at the Department of Biomedical Engineering. College of Engineering. University of Miami. Coral Gables, Florida, USA; Tel/Fax: ?????????????; E-mail: ???????????????

close to deciphering and understanding exactly every single one of these minute cues that controls the development and functions of tissues, we have been able to mimic some of these cues in order to address some of the issues associated with tissue engineering. Bioreactors are “tissue-culture de-vices that provide a controllable, mechanically active envi-ronment that can be used to study and potentially improve engineered tissue structure, properties, and integration” [4]. From the simple lab bench incubator to the most complex of bioreactors, the goal remains the same: controlling one or multiple aspects of a cell’s environment in order to aid in the development of the tissue. Taking advantage of mecha-notransduction for example, many bioreactors apply different types of mechanical forces to stimulate the cells and devel-oping tissues [5]. This kind of bioreactor has been employed as early as the 80’s for myofibroblast research [6], until to-day, with a recent study using mechanical stretching to stimulate corneal constructs [7]. The field of Tissue Engi-neering has tried to design replacement tissue for almost every tissue for which there is a major disease paradigm. Because of this, the applications of bioreactors are almost limitless. This review however, focuses on the design and development of bioreactors during the past five to ten years, in fields which bioreactor application has had a large impact: skeletal muscle, cardiovascular tissues, bone, cartilage, and neural tissues.

SKELETAL MUSCLE BIOREACTORS

The field of skeletal muscle engineering differs slightly from those of the other muscle tissue counterparts. Unlike smooth muscle, skeletal muscle needs to react to and pro-

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duce higher levels of force. And although it is similar to cardiac muscle in that it can be stimulated through electri-cal and mechanical means, the paradigms for producing functional skeletal muscle constructs are much better un-derstood than those needed to produce functional cardiac constructs. One of the reasons for this is that, unlike cardiac muscle, skeletal muscle has the physiological means for regeneration and healing after injury through progenitor and satellite cells [8]. The idea of using an outside stimulus to enervate or stimulate muscle, whether in in vivo or in vitro has been ex-plored since the beginning of the twentieth century [9]. Be-cause of its ability to become stimulated by electrical [10, 11] and mechanical cues however, bioreactors which stimu-late myocytes through electrical or mechanical means have been the most extensively explored as viable means of pre-conditioning tissue engineered muscle constructs [9]. Mechanical stimulation of myocytes in vitro has been explored since the late eighties in early studies that studied the physiology of cultured myocytes after dynamic me-chanical stimulation. These studies saw an alignment of the majority of the culture to the uni-axial direction of strain [12]. Many more studies have been done since then. The most recent of these studies have found that stretching skeletal muscle constructs seeded with muscle precursors (typically C2C12 cells) leads to an increase in skeletal muscle phenotype as well as return of functionality when implanted in vivo. One of these studies showed that me-chanical preloading of constructs leads on an increase in Myosin Heavy Chain (MHC) protein accumulation in the engineered constructs [13]. This is a protein that plays a critical role in muscle contraction, and one of the markers used for determining differentiation into muscle cell lines. Other studies have shown that uniaxial and multiaxial stress to C2C12 myocytes produces activation of similar pathways. However, multiaxial strain causes an increase in the phosphorylation of the p70Sk6 pathway where uniaxial strain does not. They also found that this particular path-way is dependent on the integrity of the myocytes’ cy-toskeleton, suggesting that multiaxial stress causes cy-toskeletal mechanotransduction where uniaxial stress does so through other means [14]. Another group has focused largely on seeding acellular porcine bladder scaffolds with C2C12 murine myocyte pro-genitors and studying the effects that dynamic stretching has on these scaffolds as treatment for Volumetric Muscle Loss. Their studies have shown that cyclic stretching in a bioreac-tor does in fact increase the healing rates and restores func-tionality close to 75% of that of the uninjured muscle [15, 16]. This same group has also done studies that showed seed-ing a scaffold prior to mechanical stimulation as well as fol-lowing stimulation produce the fastest heal time, as well as a long-time healing paradigm [17]. This suggests that in order to produce the best healing and times and muscle repair, a mix of myocyte progenitors as well as progenitors committed to the myocyte differentiation pathway are required. Other studies that mechanically stimulate skeletal muscle tissue con-structs have also been reviewed in greater detail [18]. A very recent study has shown the electrical excitability of different muscle cells, from adult and neonatal, to primary

and cultured cell lines as well as adult denervated cells. These studies have shown that adult myocytes are the most exciteable, but that denervated cells, while originally having a level of exciteability close to that of 4 day neonatal myo-cytes, regain excitability after electrical stimulation [19]. This shows the promise of electrical stimulation as a way of preloading tissue engineered muscle constructs prior to im-plantation in order to increase the functionality of the im-planted construct. In vitro, electrical stimulation of C2C12 myocytes has been shown to accelerate sarcomere assembly through a de novo pathway [20], as well as to modulate IGF binding pro-tein transcript levels, both critical in myogenic differentia-tion [21]. One group designed and developed a very afford-able bioreactor system that allows for electrical stimulation of 2D and 3D myocyte cultures. Electrical stimulation with their bioreactor led to an increase in protein production in the 2D constructs and to an increase in force production and excitability in the 3D constructs [22]. Other bioreactor types, such as the perfusion bioreactor, have been tested on engineered muscle constructs, though few have shown as promising results as the electrical and mechanical stimulation bioreactors. Two recent studies have been done on engineered muscle constructs. These two stud-ies saw an increase in the cell distribution throughout the scaffold as well as cell viability, and one study found an in-crease in the fraction of proliferating cells [23, 24]. These effects however, are known effects of the perfusion bioreac-tor in general, regardless of tissue or cell type. Another study tested the effects of shear stress on myocytes and found little no to no difference in treatment and control groups [14], suggesting that a perfusion bioreactor would not be ideal for this application other than to increase cell viability and dis-tribution throughout the construct.

VASCULAR BIOREACTORS

It is estimated that in 2005, 17. 5 million deaths were due to cardiovascular disease [25]. Due to the large incidence of cardiovascular disease in the United States alone, the amount of research being employed to prevent, treat, and cure car-diovascular disease is predominantly large. This is true also of the amount of research working towards engineering con-structs to replace cardiovascular tissue. Because of this, our review breaks the cardiovascular research into two areas—cardiac and vascular—in order to review this research in more detail.

HEART VALVE

The field of vascular tissue engineering mainly involves the engineering of constructs for the replacement of heart valves or vasculature. It is estimated that every year, more than 100, 000 US patients need their dysfunctional or dis-eased valves replaced with a prosthetic or replacement valve [26]. Because of this, there is a large market for the devel-opment of suitable replacements. The development, imple-mentation, and success of various strategies for heart valve replacement have previously been reviewed in great detail [25-28]. Here however, we explore those methods that ex-plicitly involve the use of some bioreactor for the precondi-tioning or fabrication for said heart valve replacements.

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Because of the dynamic mechanical environment that heart valves are constantly exposed to physiologically, the in vitro mechanical stretching of engineered heart valves is thought to have an effect on cell phenotype and valve prop-erties. It has been shown that cyclical, circumferential stretching of porcine aortic heart valve sections leads to an increase in collagen levels as well as an increase in α-Smooth Muscle Actin (α-SMA) [29, 30]. Another study in-terestingly found that when aortic leaflet firbroblasts are seeded in 3D collagen hydrogels and stretched biaxially in an anisotropic fashion, it leads to a decrease in overall expres-sion of α-SMA as well as Vimentin. Most of the cells how-ever, manage to rearrange themselves orthogonally to the anisotropy of the stretching. Those that do, have an increase in α-SMA expression [31]. The results of other papers corre-late with the results of this previous one, suggesting that me-chanical stretching—at pathological levels of stress—leads to an decrease in α-SMA, Vimentin, and Calponin [32] as well as an increase in the expression of serotonin receptors, associated with many heart valve pathologies [33]. Because of this, as well as the fact that a heart valve’s geometry is difficult to stretch as a whole, there is only one study that we know of that has tried to stretch valves as a whole in order to precondition them prior to implantation [34]. Placing the valve over a latex tube, the tube is distended by increasing the level of fluid within the tube, thus distending the attached valve as well. This study showed that such distension led to better tensile stiffness and stiffness anisotropy. It is interest-ing to note however, that while all of these ex vivo studies together point to the fact that while mechanical stretch can have a positive effect on the phenotype of interstitial valvular cells, it can also be damaging to them at pathological levels of stress. The perfusion bioreactor is the most common bioreactor employed in the field of heart valve engineering, as it pro-vides easy loading and preconditioning of the valve in physiological conditions. It also allows the researchers to test the valve’s efficacy under a variety of conditions, such as hypo and hypertension, prior to implantation. To date, many different types of perfusion bioreactors have been designed for this purpose. While most of the studies claim these biore-actors to be “special” or “novel”, all are slightly different designs to the classic perfusion pump bioreactor. One of these bioreactors, for example, was designed for real time measuring of the heart valve’s compliance during perfusion [35]. The values estimated by the software for compliance during systolic and diastolic pressure very closely resembled the values measured by traditional methods. In order to most closely resemble the systole and diastole of a beating heart, many bioreactors employ a wave generator which pumps a fluid, normally air or water, into a chamber. This chamber is separated from the main chamber (containing media) by a diaphragm, normally rubber, which distends, pushing media through the valve. The media then recycles through another chamber where it can be changed for fresh media or replen-ished with treatments (growth factors, stem cells, more nutri-ents) and then returns through another valve, which prevents flow through the return circuit during systole [35-43]. The flexibility of this kind of bioreactor lies in its ability to be loaded with most type of heart valve grafts, from those made purely of biomaterials [41-43] to ex-vivo porcine or ovine

grafts [35, 37-40]. Most of these studies have used this bioreactor to perfuse and seed Endothelial Cells [37, 39, 43] and other valve leaflet cells onto the constructs [41, 42] or to decellularize ex vivo xenografts [40]. These studies seem to agree that preconditioning heart valve grafts in this kind of bioreactor leads to a graft better suited for implantation, and have shown increases in anti-thrombotic behavior [39], in-creased cell attachment, alignment and expression of α-SMA [41], as well as collagen deposition [41, 43] and better me-chanical properties [42]. Due to its versatility and ability to closely mimic physiological conditions in a controlled set-ting, this is probably the better bioreactor in the field of valve tissue engineering.

VASCULATURE

Due to the almost identical physiological conditions that both vasculature and heart valves are exposed to, research pertaining to these two fields is somewhat similar. Like re-placement heart valve constructs, replacement vascular con-structs need to bypass the issues of thrombosis and intimal hyperplasia, as well as have appropriate mechanical proper-ties to avoid rupture, failure, and mechanical mismatch at the site of implantation [44]. Because of these similarities, the research that has arisen involving bioreactors for the precon-ditioning of vascular grafts mainly involves either mechani-cal stretching bioreactors and perfusion bioreactors, as with heart valve research. Unlike those bioreactors employed for heart valve re-search however, much of the bioreactors employed for me-chanical stretching of tissue engineered vasculature include some sort of perfusion mechanism which in turn distends the constructs, providing the mechanical stretching as well as all of the advantages of regular perfusion bioreactors [45-50]. Some of these studies designed their bioreactors to cater to the perfusion of small vessel constructs [47-49]. Of these, one study was equipped to provide real-time data on the distension of the vessel constructs using a built-in LED system [47], while another seeded Endothelial Cells through perfusion, but only 13 days after the construct had been seeded with Smooth Muscle Cells, providing a lay-ered construct similar to blood vessels [48]. Of the bioreac-tors designed for the larger constructs, one is worth noting in that it also incorporates rotation to minimize the effect of gravity on the cell growth and matrix deposition of the con-struct. This additional feature ensured a more uniform dis-tribution of cells and matrix throughout the construct [45]. Together, these studies show that this bioreactor style for preconditioning of vascular constructs decreases thrombo-sis [50], increases collagen fiber anisotropy and alignment [46] increases α-SMA expression [49, 50] and compliance of the constructs [47]. Only one of the studies reviewed showed a negative effect of mechanical stretching on the mechanical properties of vascular tissue [51]. However, this was a study that used a vacuum pump to provide the levels of mechanical loading instead of a perfusion-distention bioreactor. One interesting area in which the field is headed is that of perfusing the decellularized vascu-lature of whole tissue constructs in order to maintain the vascular tree architecture but replace the xenogeneic or allogeneic cells of the original donor organ [52].

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CARDIAC BIOREACTORS

The field of cardiac tissue engineering is one of the faster-growing fields in tissue engineering due to the preva-lence of cardiovascular disease around the world. This exten-sive field and the strides that have been made in it have been previously reviewed [53]. Due the abilities of cardiac muscle to become excited through both electrical and mechanical stimulation, similar to skeletal muscle, the bioreactors that have arisen for the stimulation and preloading of cardiac constructs are either mechanical or electrical bioreactors. Despite this tendency in the field to build electrical and mechanically driven bioreactors, a few studies have tried to use different bioreactor modalities for the culture and differ-entiation of cardiomyocytes. One of these studies tested the effects of a stirred suspension bioreactor on the differentia-tion of embryonic stem cells (ESCs) into beating cardiomyo-cytes through growth factor treatment. The study encoun-tered an increase in the amount of spontaneously contracting embryoid bodies (EBs) as well as an increase in the total number of cardiomyocytes extracted [54]. There was also an increase in the amount of GATA-4, Myosin Heavy Chain and Myosin Light Chain expression in the cultures. Very interestingly, another study which replicated these same conditions, found that differentiating ESCs into cardiomyo-cytes in stirred suspension leads to cultures which, despite differentiation into spontaneously beating cardiomyocytes, retain the ability to express ESC makers, form ESC-like colonies, as well as generate teratomas [55]. Future work will need to be done, but these two studies raise questions pertaining to the role that bioreactors will play in the devel-opment of safe alternatives for the differentiation of cardio-myocytes from ESCs. As described previously, the perfusion bioreactor has been used for a variety of tissue engineering applications to uniformly seed cells onto a scaffold, as well as to provide a more homogenous level of nutrients to cells seeded throughout the scaffold. Because of these clear ad-vantages, perfusion bioreactors have also been tested on tis-sue engineered cardiac constructs. Studies have, for the most part, corroborated the results of analogous studies in other fields. The perfusion bioreactor leads to an increase in viabil-

ity of cells [56], total protein expression [57, 58] and prolif-eration of cells [58]. Mechanical stretching has risen to the forefront of the field as a way of preconditioning constructs in order to make their physical properties match those of the myocardium or ventricular wall, depending on the application. In data that is currently under review for publication, our group has shown that multipotent periodontal ligament-derived stem cells (PDLs), can be driven down a cardiac differentiation path-way through the sole use of cyclic mechanical stretching on a custom built bioreactor. (Fig. 1) By stretching these cells on silicon scaffolds, we were able to increase the expression of cardiac markers such as cardiac troponin-t and myosin heavy chain, among others. And while another study has also found that seeding rat neonatal cardiac myocytes in a 3D fibrin gel, and subjecting them to cyclic stretch does not have an effect on the actual physical properties of the fibrin gel, no tests were done on the phenotypic expression of the car-diomyocytes seeded on the gel [59]. These and other studies however, have paved the way for a new generation of biore-actors that now incorporate mechanical stretching to other bioreactor modalities and so create a synergistic effect with electrical stimulation. Such a mechanical stretching/electrical stimulation bioreactor is currently being designed and devel-oped by our group. A variety of bioreactors have been designed to provide electrical stimulation to cardiac replacement constructs. Studies have been done to test these various bioreactor de-signs, as well as to optimize said bioreactors [60, 61]. It has been shown by different studies that electrical stimulation of cardiomyocytes and stem cells has positive effects on the development and organization of said tissue. One of these studies found that the direct application of an electrical stimulus to human embryonic cells led to the spontaneous contraction of the cells, as well as and increase in sarcomeric organization and troponin-t expression, suggesting a differ-entiation towards a myogenic lineage [62]. Other studies have confirmed these effects, confirming that electrical stimulation of both stem cell and cardiomyogenic lines lead to increases in proliferation, alignment, elongation, and ex-

Fig. (1). A) Double-chamber mechanical stretch bioreactor driven by stepper motor used in the stimulation of multipotent stem cells to a cardiomyogenic lineage. B) View of a single chamber with attached silicone membranes for stretching [59].

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pression of cardiac markers and protein expression [63, 64]. One study went so far as to suggest using regular skeletal muscle myocytes for cardiac regeneration after electrical stimulation. While this study found an increase in the prolif-eration of myoblasts, they found nothing to suggest that elec-trical stimulation would drive the myocytes towards a more cardiac lineage [65]. Of the bioreactor designs to stimulate and preload car-diac constructs, perhaps the most interesting are the ones that most closely mimic the heart physiology and condi-tions. In the introduction we mention that the role of a bioreactor is to most closely mimic the various stimuli that cells receive from their environment to preload or accustom tissue engineered constructs to the in vivo environment prior to implantation. Recently, various groups have de-signed bioreactors which incorporate perfusion, mechanical stretching, and electrical stimulation all at once [66-69]. Of these, two incorporated electrical stimulation with constant perfusion [67, 69], and found a synergistic effect on elon-gation and striation as well as an increase in expression of cardiac markers such as conexin-43. Other, newer studies, incorporate all three methods: perfusion, electrical, and mechanical stimulation [66, 68]. One group has yet to do any testing of their bioreactor, as their proof-of concept merely tests for cytoxicity of the materials used and cell elongation along direction of stretch [68]. However, the other study has already shown that use of their bioreactor on rat cardiomyocytes leads to higher cardiac protein ex-pression relative to controls [66]. These bioreactors point to the direction in which the field is headed. While no major design breakthroughs have been made in new bioreactor types, the incorporation of different bioreactor types shows the synergistic effect that different modalities have when incorporated into a single bioreactor.

BONE BIOREACTORS

The field of bone tissue is similar to other fields in tissue engineering in that it involves the in vitro expansion of cells on a scaffold followed by implantation into the bone. The use of bioreactors in this field is varied. Some studies use bioreactor preconditioning in order to more closely match the construct’s mechanical properties to those of the implant site. Others use a cellularized scaffold and use bioreactors to induce osteogenic differentiation. A variety of bioreactor systems including the spinner flask, perfusion, compression and rotating wall bioreactors have been implemented for the purpose of improving in vitro culturing of tissue engineered bone constructs. As described in previous sections, a spinner flask biore-actor may be used to overcome problems of uneven cellular distribution within a scaffold. It has the added benefit of pro-viding shear stress to the cells, which has shown to have positive effects on cell proliferation and differentiation [70]. These studies on spinner flask bioreactors both seed human Mesenchymal Stem Cells (hMSCs) onto porous scaffolds. One study used a larger aqueous-derived porous silk scaf-fold, with enlarged pore sizes. Results saw enhanced cell proliferation, and histological analysis showed bone-like structures after 56 days of culture [71]. A similar study used a coralline hydroxyapatite scaffold with different pore sizes

of 220 and 500 mm for expansion. Results showed that the 200 mm pore scaffold exhibited faster osteogenic differentia-tion, though the 500 mm scaffold had an increased prolifera-tion rate and a greater number of cells [70]. Both studies conclude spinner flask bioreactors are an efficient and inex-pensive method for cultivating hMSCs in 3D scaffolds. Fur-thermore, these studies show that pore size is an important factor in controlling bone formation during dynamic cultiva-tion [71]. This is important as larger pore sizes most likely lead to an increase in the convection and diffusion of nutri-ents throughout the scaffold. Besides the evident benefits of the spinner flask, one drawback is that it can be a turbulent environment for seeded cells near the periphery of the scaf-fold due to high shear forces. Earlier in the paper we described the perfusion bioreac-tor, and how it is used to maintain the scaffold’s environment of cell culture conditions, obtain a more even cell distribu-tion through a uniform rate of perfusion, and increase nutri-ent delivery to the whole scaffold. Currently, many studies involve seeding hMSCs on various types of scaffolds. Two studies focused on seeding hMSCs on decellularized bone. One study conducted on decellularized bovine trabecular bone showed that the rate of perfusion during cultivation increases the amount of cells, cell distribution throughout the constructs as well as increases in osteopontin and collagen expression [72]. Another study implanted a fully decellular-ized scaffold in the temporomandibular joint of the condylar bone as the tissue model and showed tissue growth by the formation of confluent layers of lamellar bone and formation of osteoids. Cells were fully viable at a physiologic density [73]. Another technique develops a method for an aggregated cell-containing construct in a perfusion bioreactor system. HMSCs are encapsulated in tightly packed alginate beads. These form a single construct or tubular growth chamber where nutrient transfer is enhanced and the cells are exposed to shear stress [74, 75]. Results show that the cells in the perfusion bioreactor indicated early osteoblastic differentia-tion, as well as a significant increase in gene expression lev-els of osteocalcin, osteopontin, and bone morphogenetic pro-tein 2 (BMP-2) in the culture, which increased with in-creased perfusion rates [75]. In the perfusion culture, fluid flow can exert shear stress on the cells seeded on the scaf-fold, improving the mass transport of the cells [76]. One study showed that increasing flow shear stress accelerated osteogenic differentiation and improved the mineralization of the extracellular matrix (ECM) [76]. A similar study showed that bioreactor-cultured scaffold reached cellular confluence earlier, with greater cellularity, and maintained high cellular viability in the core. Additionally, bioreactor culture was associated with greater osteogenic induction, alkaline phosphatase expression, and bony nodule formation, and in vivo ectopic bone formation in immunodeficient mice, compared with the static-cultured scaffold [77]. A recent study utilized two perfusion flow conditions (parallel flow [PF] and transverse flow [TF]) to understand the impact of flow configuration of cellular construct development during both preculture and in growth media, and how it affects os-teogenic induction. The TF reduced cell proliferation and osteogenic induction in the preculture. In contrast, PF main-tained cell proliferation under the osteogenic induction but

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resulted in localized cell aggregation, influencing osteogenic differentiation [78]. This is most likely due to the fact that the forces experienced by the cells with TF are damaging to the cells, while PF produces forces that do not damage the cells, but allow nutrients to be more evenly distributed throughout the construct. Most studies conducted with perfusion bioreactors are in vitro studies, though in vivo studies provide critical informa-tion for clinical applications. In one study, a perfusion cul-tured scaffold made of natural coral was transplanted subcu-taneously in sheep; the bone constructs were shown to be osteogenic [79]. A similar study using a sheep model evalu-ated the effect of a large perfusion bioreactor on the postero-lateral spine. The study saw a significant increase in bone density and trabecular thickness than non-implanted bone substitutes [80]. Perfusion bioreactors serve as a promising method for custom bone constructs, spine fusion procedures, bone grafts, craniofacial and orthopedic applications. Studies have shown that compression bioreactors can also be used for the purpose of cell proliferation for os-teogenic differentiation. Our group used a scaffold subjected to dynamic mechanical compression previously demon-strated to induce chondrogenic and osteogenic differentia-tion. Results showed that inhibiting ERK1/2 augments os-teogenic cell response and significantly increases their ex-pression of alklaline phosphatase, collagen type I, and osteo-calcin confirmed by histochemical staining [81]. In a com-parative study of a perfusion and compression bioreactor, results indicate that the perfusion and on-off cyclic mechani-cal stimulation maintain cell viability and promote prolifera-tion, though the perfusion group had the highest tensile modulus, higher cell density, and higher degree of cell pro-liferation [82], reaffirming the promising results of the perfu-sion bioreactor. Rotating-wall vessel (RWV) bioreactors are also prom-ising in the field of osteogenic differentiation because these bioreactors have high rates of mass transfer and small shear loads. A very recent study saw bone tissue containing vas-cular-like structures was generated, providing cells with an advantage in the construction of 3D bone tissue with blood vessels [83]. Another group showed the number of cells cultured in the bioreactor was five times higher than those cultured in a T-flask. The tissue-engineered bone grew very well in the bioreactor [84]. A unique study used a NASA-approved RWV to develop osteoblast-like cell cultures un-der microgravity and evaluated osteoblast phenotype and cell function. The rat osteoblast-like cell line was grown in the RWV at gravity of 0. 008g. Results showed that cells under microgravity experienced reduced osteoblast life span which may result in inefficient osteoblast and in-creased osteoclast activity. This is potentially what contrib-utes to bone loss in individuals subjected to long periods of weightlessness [85]. The study shows that inhibiting the force of gravity on the bone inhibits the differentiation of bone cells and the strength of the bone construct. Due to these conflicting studies, it is hard to determine the true potential of the rotating wall vessel bioreactor, for the growth of TE bone constructs.

CARTILAGE BIOREACTORS

Adult cartilage has a limited healing capacity. Damages resulting from disease or injury increase over time and cause severe pain [86] Currently there is no surgical procedure available to treat large and deep cartilage defects associated with advanced diseases such as osteoarthritis [87]. For this reason cartilage tissue bioreactors serve as a promising means for the successful regeneration of damaged or dis-eased cartilage. Different methodologies for tissue engineer-ing of cartilage have been previously and extensively re-viewed in detail [88]. In this review paper however, we focus on those applications which pertain directly to the use of bioreactors for the field of cartilage engineering. A variety of bioreactor systems including the perfusion, cyclic compres-sion, dynamic compression, hydrostatic pressure, and hydro-dynamic pressure bioreactors have been implemented to in-duce chondrogenic differentiation. A perfusion bioreactor is used in the field of cartilage tissue engineering as a design mechanism to perfuse a cell suspension directly through the pore of a three-dimensional scaffold as well as perfuse culture media to maintain cell viability within the seeded construct [87]. One study seeded human chondrocytes through a 50 mm diameter scaffold. Results showed that these tissues grown in the bioreactor were viable and homogenously cartilaginous, with biome-chanical properties such as those of native cartilage. Con-trastingly, the tissues generated by conventional manual pro-duction in this study were inhomogeneous and contained large necrotic regions [87]. A similar study used a recirculat-ing flow-perfusion bioreactor was used to stimulate the mo-tion of a native diarthrodial joint by offering shear stress and hydrodynamic pressure simultaneously [89]. They found that cells in the perfusion bioreactor showed significantly better cell morphology and zonal organization, as well as presented more characteristics of native articular cartilage [89]. In yet another study, chondrocytes and osteoblasts composites were co-cultured using a perfusion bioreactor. Results showed that the chondrocytes and osteoblasts showed fine adhesive pro-gression and proliferation in a β-tricalcium phosphate (β-TCP) scaffold [90]. Therefore the perfusion bioreactor is deemed beneficial because it provides sustained nutrient supply and gas exchange into the center of a large scaffold, allowing the chondrocytes and osteoblasts to survive and proliferate. Two studies seeded human chondrocytes on po-lyglycolic acid (PGA) scaffold and cultured the cells in per-fusion bioreactors. One study showed that gradually increas-ing the flow rate resulted in larger constructs, an increase in glycosaminoglycan (GAG) retained in the extracellular ma-trix, and an increase in GAG concentration in the tissues compared to the control [91]. The other study pre-cultured the cells in shaking T-flasks and perfusion bioreactors, show-ing that mechanical treatment improved the amount and quality of cartilage produced in comparison of both culture methods. Results also showed that the perfusion bioreactor proved more beneficial in producing collagen type II [92]. Recent studies have set out to compare the synergistic effects of a perfusion bioreactor and mechanical stimulation on the properties of TE cartilage. One group centrifuged high-density porcine chondrocytes onto an agarose layer. The results showed that the constructs cultured in the biore-

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actor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus in comparison to the static constructs [93]. A different study tested the con-fluence of perfusion and mechanical stimulation on bone marrow stromal cells seeded on a collagen meniscus implant. Results showed proliferation using continuous perfusion and differentiation if also fostered by mechanical stimulation [94]. Studies on cyclic compression bioreactors have shown that they can be used for the purpose of cell proliferation for chondrogenic differentiation. Our group studied the effects of cyclic compression on these very properties. (Fig 2) Hu-man Mesenchymal Stem Cells (HMSCs) were seeded in the scaffold and subject to cyclic compression. Three concentra-tions of fibrin gel and three different stimulus frequencies were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Results show that cyclic compression at frequencies >0. 5 Hz and gel concentration of 40 mg/mL fibrinogen ap-pears to maintain cellular viability within scaffolds [95, 96].

Another study embedded hMSCs in a fibrin/polyurethane scaffold and applied compression, shear, or a combination of both stimuli. Both compression and shear alone proved in-sufficient for chondrogenic induction of hMSCs, though the application of shear superimposed upon dynamic compres-sion lead to significant increases in chondrogenic gene ex-pression. Histological analysis detected sulphated glycosa-minoglycan and collagen II only in the combined compres-sion and shear group [97]. Stimulating scaffolds with a bioreactor which simulates natural joint movements holds great potential to produce cartilage. This is the reason dynamic compression bioreac-tors prove promising in the field of cartilage tissue engineer-ing. One study used two methods based on atomic force mi-croscopy (AFM) to obtain information about the quality of the engineered cartilage surface [98]. Bovine chondrocytes were seeded on polyurethane scaffolds and subjected to dy-namic compression applied by a ceramic ball in loading group 1 (LG1), and dynamic compression applied with a ball oscillating over the scaffold generating a sliding motion in

Fig. (2). A) Schematics for sinusoidal compression bioreactor used in the stimulation of multipotent stem cells to a chondrogenic and os-teogenic lineage. B) Loading configuration for compression of constructs [95].

8 Current Tissue Engineering, 2013, Vol. 2, No. 2 Ruiz et al.

loading group 2 (LG2). Results showed the presence of col-lagen type II and aggrecan staining in all constructs, and appeared to be deeper area in loaded constructs versus the unloaded scaffolds. Sliding-type biomechanical movement favors regeneration and maintenance of functional articular surfaces and supports the development of mechanically com-petent engineered cartilage [98]. Another study seeded porcine chondrocytes in hydroxyapatite carriers, resulting in scaffold-free cartilage-carrier constructs which were placed in a custom-made bioreactor. Results showed that the highest compression amplitude of 20% had the strongest positive effect on the mechanical properties of the TE cartilage com-pared to the unloaded control [86]. The data suggests that preconditioning with higher load amplitudes might be a promising method of generating stiffer tissue and may assist in accelerating the cultivation of mechanically competent tissue engineered cartilage. Different methodologies for tissue engineering of carti-lage using hydrostatic pressure (HP) bioreactors have been previously and extensively reviewed in detail involving the chondroprotective effects of HP, the use of HP for chondro-genic differentiation, and HP mechanotrasnduction [99]. One group seeded MSCs in type I collagen sponges and exposed them to HP. Results showed that with HP loading, proteo-glycan staining increased markedly. Also results showed that genes associated with chondrogenic differentiation, includ-ing aggrecan, type II collagen, and Sox 9 increased signifi-cantly [100]. A similar study was conducted on an alginate hydrogel scaffold seeded with bovine articular chondrocytes. Results show that the Ca2+ signaling response to direct per-fusion of chondrocytes-seeding scaffolds increased with flow rate and was found more directly dependent on fluid velocity rather than shear stress or hydrostatic pressure [101]. The data suggests that flow-induced Ca2+ signaling response of chondrocytes may be due to mechanical signaling pathways, influenced by the 3D nature of cell shape. A different group used ovine bone marrow mesenchymal cells (BMMCs) and seeded them in non-woven filamentous plasma treated poly-ester scaffolds and applied pulsatile HP [102]. Results showed that the glycosaminoglycan (GAG) content, the de-oxyribonucleic acid (DNA) content, and the collagen content increased after the application of pulsatile HP. The data sug-gests that a light pulsatile HP applied at a low frequency has a cumulative stimulatory effect on the BMMCs metabolic activities including cell proliferation and synthesis of the extracellular matrix. Hydrodynamic conditions mimic the motion-induced flow fields between the articular surfaces in the synovial joint and induce formation of a distinct superficial layer in tissue engineered cartilage hydrogels with enhanced produc-tion of cartilage matrix proteoglycan and type II collagen. One study used chondrocytes-seeded agarose hydrogels and cultured them in a hydrodynamic pressure bioreactor. Re-sults showed enhanced metabolism of type II collagen and proteoglycans, including aggrecan and a surface protein Pro-teoglycan 4, and induces a distinct superficial layer with en-hanced collagen alignment [103]. A similar study engineered cartilage constructs and cultivated them in four well-defined hydrodynamic environments within a bioreactor [104]. The results suggested that even for similar composition, con-structs could exhibit different mechanical properties due to

differences in their ultrastructure that can be modulated by hydrodynamic parameters. Improved mechanical properties were observed in constructs placed in the hydrodynamic pressure bioreactor. A recently developed bioreactor is capa-ble of conducting dynamic stimulation and mechanical evaluation into a single batch-testing research platform [105]. This mechanoactive transduction and evaluation bioreactor (MATE) provides accurately applied static and dynamic loading of six specimens with minimal hardware. Though not much testing has been conducted with MATE, this bioreactor proves promising in the field of cartilage en-gineering because it can efficiently map the biomechanical development of tissue engineered constructs during long-term culture.

NEURAL BIOREACTORS

The field of Neural TE is different to that of other TE fields in that the complexity and delicacy of the neural con-nections of the central and peripheral nervous systems (CNS, PNS) make constructs and transplant of those constructs a bigger challenge. The application of bioreactors to this field reflects this as well. While bioreactors in other TE fields stimulate and preload constructs, neural TE bioreactors focus mainly on the culturing and proliferation of Neural Stem Cells (NSCs) and Neural Progentiors (NPs) to produce enough cells to be clinically applicable and relevant. The culture of NSCs and NPs has been developed for quite some time, and it is now known that they grow best in 3D cultures [106]. When grown in suspension, both cell types have a tendency to aggregate into large cell bodies known as neural spheres, or neurospheres. Bioreactors in the field are used to control the growth, proliferation, size, and phenotype of these neurospheres, and are mostly either spinner-flask bioreactors—also known as suspension bioreactors in this field—or rotating wall vessel (RWV) bioreactors. As previously mentioned in other sections, the role of RWV bioreactors, which cancel out most forces acting on constructs and simulate microgravity, is somewhat debated within the literature. Because of mechanotransduction and its effect on cell development, studies argue that this bioreactor is detrimental to cellular proliferation and differentiation, particularly in the area of bone growth. However, many stud-ies have shown that this bioreactor actually increases cell proliferation and alters phenotype positively. This is the case in Neural TE. One study, for example, found that if grown on traditional tubular or cylindrical scaffolds, culture in the RWV led to a decrease in proliferation of the cells compared to static cultures. A special spiral scaffold developed by the group however, showed increased cell proliferation in the static culture and an even greater proliferation when cultured in RVW [107]. One other study suggests that a microgravity environment stimulates mitochondrial activity in NSCs, as evidenced by an increase in mitochondrial gene expression [108]. Yet another study found that culturing NPs in an RWV bioreactor led to formation of larger neurospheres and higher DNA content that in static culture [109]. It is interest-ing to also note that these neurospheres did not form tumors when implanted into immunodeficient mice, as some neuro-spheres with stem cell or stem cell-like precursor cells tend to do. Regarding cell phenotype, one study found that grow-ing NSCs in a RWV bioreactor led to a ten-fold greater

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number of cells which were positive for Nestin, TuJ1, and GFAP, all critical neural factors. Those cells expressing TuJ1 had extensive neurite outgrowth, suggesting the strong role that this kind of bioreactor can have in the development of neural-like constructs [110]. This study also showed that at the beginning, cultures grown in this type of bioreactor exhibited a lag in cellular differentiation, but caught up and surpassed static cultures at around week 3. This suggests that in order to harness the full power of such a bioreactor, a bet-ter understanding of the mechanisms and time points of this bioreactor on the development of neural constructs is re-quired. It has been known for some time that spinner flask (sus-pension) bioreactors can be used to increase the proliferation rate of neural stem cells and neural progenitors, as well as control the size of neurospheres. The ideal media and pas-saging conditions for culturing such constructs in suspension bioreactors have also been previously researched and devel-oped [111, 112]. The role of suspension bioreactors on the culture and scale-up of NSCs and NPs has also been previ-ously reviewed, though not recently [113]. One of the big-gest advantages to growing neurospheres in spinning flask bioreactors is the ability to control the size of the neuro-spheres. Studies have found that the shear forces experienced by the neurospheres in these bioreactors limits the size of to which these neurospheres grow [114]. This study also showed that an increase in agitation rate leads to the decrease of neurosphere diameter. This is critical as larger neuro-spheres develop necrotic cores, due to a decrease in the dif-fusion of nutrients to the center of the sphere. Another clear advantage to this kind of 3D culture is the ability to upscale suspension bioreactors [115, 116]. The same group that studied upscalability in this bioreactor type found that the ideal stirring rate is that of 100 rpms, at which point cultured cells maintain Nestin expression after culture and expansion and can still differentiate in glial and neural phenotypes. While these two bioreactors dominate the field and are used most extensively, there have been studies using other kinds of bioreactors. Studies have used a perfusion bioreac-tor to constantly perfuse cells [117, 118]. Both studies found an increase in cell viability and proliferation due to medium perfusion. One study also discovered that shear rates that were too high decreased viability, suggesting that while fresh media perfusion increases nutrient delivery and waste re-moval, high shear stresses are detrimental to the cells [118]. Another study is the only study that we are aware of that uses mechanical stimulation in this field. Cells were seeded on the edge of a surface and allowed to attach and grow. The axons of said cells, when attached to the stationary bottom edge, were then stretched by the very small stretching of the top layer on which the cell bodies were seeded. The publica-tion is a video publication, and the design of the bioreactor, as well as accelerated video of the stretching axons, is avail-able online, and presents the interesting, and previously un-explored area of mechanical stimulation of neurons [119]. Another study looked at the effect of hydrostatic pressure on myelination. This however, was done not with a TE goal in mind, but to better understand myelination and demyelina-tion in injury models. They found that this kind of biophysi-cal stimulation induces demyelination and Schwann cell pro-liferation [120].

CONCLUSION

In the past ten years, little has changed in the design of the major bioreactor modalities. Perfusion, mechanical stretching, electrical stimulation, rotating wall vessel, and spinner flask bioreactors all remain the same in basic design and concept. While recent studies have claimed to develop novel or new bioreactors, the truth remains that no ground-breaking bioreactor designs have been made recently. What has evolved however, is our understanding of the value that each of these bioreactor modalities has for different tissue engineering applications. The perfusion bioreactor, for ex-ample, has the widest range of applications, as it provides homogenous nutrient transport and cell seeding throughout the construct, regardless of cell type or scaffold material. Others, such as the electrical stimulation bioreactor, are more restricted to the fields of cardiac and muscular tissue engi-neering. What has also advanced is the ways in which biore-actors are applied and used. As our understanding of the dif-ferent forces and signals which play a role in tissue devel-opment increases, so does our understanding of the ways in which we can apply the bioreactor designs available for op-timal tissue development and integration. The biggest ad-vances in bioreactor use and development have perhaps then, been in those areas in which multimodal bioreactors have been developed. The most novel bioreactor designs are those which incorporate multiple forms of stimulation in synch, such as mechanical and electrical stimulation for muscle development. These are the bioreactors which point to the future of the field. In a few years, the perfusion bioreactor will probably disappear as a stand-alone bioreactor, and rather become a feature that all new bioreactor designs in-corporate, as was seen in some of the multimodal cardiac bioreactors. Perhaps we might even see the development of a new tissue culture incubator with easy access perfusion as an option for all culture types. Bioreactors which more closely monitor multiple stimuli will usher in the next generation of bioreactors that will aid in the development of replacement tissues that will have better incorporation and function once implanted into sites of disease and injury.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

Declared none.

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Received: ??????? ??, 201? Revised: ????? ??, 201? Accepted: ????? ??, 201?