Integration of clinical perspective into biomimetic bioreactor design for orthopedics

Abstract The challenges to accommodate multiple tissue formation metrics in conventional bioreactors have resulted in an increased interest to explore novel bioreactor designs. Bioreactors allow researchers to isolate variables in controlled environments to quantify cell response. While current bioreactor designs can effectively provide either mechanical, electrical, or chemical stimuli to the controlled environment, these systems lack the ability to combine all these stimuli simultaneously to better recapitulate the physiological environment. Introducing a dynamic and systematic combination of biomimetic stimuli bioreactor systems could tremendously enhance its clinical relevance in research. Thus, cues from different tissue responses should be studied collectively and included in the design of a biomimetic bioreactor platform. This review begins by providing a summary on the progression of bioreactors from simple to complex designs, focusing on the major advances in bioreactor technology and the approaches employed to better simulate in vivo conditions. The current state of bioreactors in terms of their clinical relevance is also analyzed. Finally, this review provides a comprehensive overview of individual biophysical stimuli and their role in establishing a biomimetic microenvironment for tissue engineering. To date, the most advanced bioreactor designs only incorporate one or two stimuli. Thus, the cell response measured is likely unrelated to the actual clinical performance. Integrating clinically relevant stimuli in bioreactor designs to study cell response can further advance the understanding of physical phenomenon naturally occurring in the body. In the future, the clinically informed biomimetic bioreactor could yield more efficiently translatable results for improved patient care.

electrical, or chemical stimuli to the controlled environment, these systems lack the ability to combine all these stimuli simultaneously to better recapitulate the physiological environment. Introducing a dynamic and systematic combination of biomimetic stimuli bioreactor systems could tremendously enhance its clinical relevance in research. Thus, cues from different tissue responses should be studied collectively and included in the design of a biomimetic bioreactor platform. This review begins by providing a summary on the progression of bioreactors from simple to complex designs, focusing on the major advances in bioreactor technology and the approaches employed to better simulate in vivo conditions. The current state of bioreactors in terms of their clinical relevance is also analyzed. Finally, this review provides a comprehensive overview of individual biophysical stimuli and their role in establishing a biomimetic microenvironment for tissue engineering. To date, the most advanced bioreactor designs only incorporate one or two stimuli.
Thus, the cell response measured is likely unrelated to the actual clinical performance.
Integrating clinically relevant stimuli in bioreactor designs to study cell response can further advance the understanding of physical phenomenon naturally occurring in the body.
In the future, the clinically informed biomimetic bioreactor could yield more efficiently translatable results for improved patient care.

| INTRODUCTION TO CLINICALLY RELEVANT BIOREACTORS
A bioreactor, put simply, is a vessel that maintains a specific microenvironment and allows biochemical reactions to occur. 1 In tissue engineering (TE) applications, the microenvironment must be closely monitored and tightly controlled to ensure a high degree of accuracy and reproducibility amongst biological constructs. 2,3 These properties have made bioreactors an indispensable component of any bioprocess, irrespective of the end product, which can take the form of chemicals, pharmaceuticals, cells, tissues, or organs. Early bioreactors focused mainly on controlling purely environmental factors, such as temperature, pH, aeration, agitation, pressure, nutrient concentration, and waste removal. 2,4,5 However, in certain fields, such as TE, a new era of bioreactors has arisen to incorporate additional physiologically-derived factors, such as mechanical, electrical, and chemical cues. 1 Despite these major changes in design, the common thread connecting all bioreactors is the ability to provide a controlled microenvironment for a product of interest. In TE and other research applications, the purpose of controllability is to evoke the scientific method; to be able to isolate variables and measure changes in response to variation, allowing for easier automation and reproducibility of experiments, which is essential for any study design.
Classical TE techniques typically involve the seeding of cells onto a supporting matrix, or scaffold, and supplying additional growth factors to promote cell adhesion, alignment, migration, proliferation, differentiation, and new tissue production. 6 This combination of cells, scaffolding, and growth factors is known as the TE paradigm. [6][7][8] Bioreactors are often added to the triad to supply biophysical stimulation to the cell scaffold and improve tissue formation metrics. Bioreactors in TE have three main uses: to maintain a specific cellular microenvironment, whether that be physiological or pathological, in order to better understand cell and molecular physiology/pathophysiology; to expand cell lines for gene/cell therapies or grow functioning tissue in vitro for clinical applications; and to test potential treatments for new therapeutic targets. 7,9 Bioreactors also have other clinical uses, such as testing biomedical implants and facilitating cell seeding onto scaffolds ( Figure 1). For many pharmaceuticals and implants, pre-clinical animal models are required by the FDA for testing. Bioreactors have the potential to replace pre-clinical animal testing models, saving labor, time, and money. 10 Research oriented bioreactors have had variable success over the years in the cultivation of engineered tissues, partly due to the lack of standardized environmental parameters and loading regimes. 11 Yet despite the research success seen for specific bioreactor configurations, translation to the clinical setting has been limited. Classical TE methods have had fairly limited success for growing tissues in vitro and are only consistently used for thin or avascular tissues, such as skin, due to the diffusion limit of oxygen (100-200 μm). 10,12 As for larger, vascular tissues, like those needed to treat large bone defects, the speed of vascularization after implantation is often too slow to ensure adequate nutrient transport to the core. 12,13 Thus, in vitro strategies for enhancing vascularization before implantation are being investigated. Biomimetic bioreactors are being explored as one of these potential strategies, since research has implied that a wellestablished, physiologically relevant microenvironment helps promote tissue growth and vascularization in vitro. 10,[13][14][15] If these barriers can be addressed, the gap between research and clinical bioreactors will narrow and clinical translation may be feasible for improved patient care.
To better recapitulate the in vivo microenvironment, modern bioreactor designs for TE applications have shifted away from classical systems, which only focus on environmental cues and nutrient transfer, toward more specialized systems that incorporate additional biophysical stimuli, such as compressive loading 16,17 or electrical polarization, 18,19 to facilitate specific cell behaviors. These physiological stimuli are especially relevant for bone tissue engineering (BTE) applications where mechanical and electrical loads play a key role in cell signaling and long-term tissue functionality. 3 Several groups have designed modern bioreactors that strive toward enabling long-term in vitro tissue functionality. However, these designs typically involve only a single biophysical stimulation type. For example, some bioreactors use purely mechanotransduction principles for musculoskeletal tissues, 11,16,17,[20][21][22][23][24][25]  Despite the individual successes found in these studies, emerging evidence from research into the various cellular signal transduction pathways suggests that multiple integrated stimuli could further enhance tissue growth and functionality. [43][44][45] In a pilot ovine study conducted by Friis and Arnold, a novel piezoelectric interbody implant was placed in the lumbar segment of the spine to assess fusion progression and bone growth, while two other levels of the spine served as controls. 46 The circuitry accompanying the piezoelectric materials inside the implant produced a capped negative current on the electrode of the implant surface in sync with sufficient mechanical loading. 46 This form of mechanically synced electrical stimulation (MSES) is inherently F I G U R E 1 Various applications for TE bioreactors distinct from the presently available battery-operated implantable electrical stimulation devices that produce constant direct current irrespective of the mechanical load. 46 The ovine study included radiographic fusion assessment at 6 weeks and 4 months, biomechanical testing, and histological analysis at 4 months, to assess fusion mass and time-to-fusion ( Figure 2). 46 The results of this pilot ovine study showed that spine segments treated by MSES with the piezoelectric device had an earlier and more robust fusion, as measured radiographically, biomechanically, and histologically. 46 Additionally, no pathologic bone formation or adverse bone growth were detected in the spinal canal. 46 The piezoelectric implant supplied a coupled mechanical and electrical load to the bone resulting in faster fusion, which supports the theory that multiple integrated stimuli could enhance bone tissue formation. However, the cellular mechanism for this enhanced bone growth remains unknown, and thus exemplifies a need for a biomimetic bioreactor that utilizes multiple stimuli to mimic the in vivo environment and provide quantitative analysis.
This review article stresses the need for clinically informed, multiple stimuli, TE bioreactors by first providing a brief historical summary on the progression of bioreactors, from classical to modern, describing the major advances in design with respect to clinical significance. Next, a comprehensive overview of the individual biophysical stimuli and their role in establishing a biomimetic microenvironment for TE, with a specific focus on the cellular response to specific loading regimes is provided. Finally, a model for future TE bioreactors that simultaneously incorporates several of these biophysical stimuli to create a more physiologically relevant microenvironment for specific cell types is also presented.
Even the most advanced bioreactor models cannot recapitulate a physiological environment accurately. Overall, the need to integrate diverse stimuli simultaneously to drive cells toward a desired phenotype and offer a clinically informed bioreactor design for a patient-first mindset for diagnosis and treatment is highlighted.

| EVOLUTION OF TISSUE ENGINEERING BIOREACTORS
One of the earliest instances of industrial fermenters, before the term bioreactor was even coined, was developed by Chaim Weizmann in the early twentieth century to produce butanol and acetone on a large-scale for the war effort. 5,47 Since then, bioreactors have been constantly adapting to meet the needs of the end product ( Figure 3). With the increasing demand for mammalian cell culture production came the need for two more distinctive bioreactor applications: stimulating cell expansion/ aggregation and promoting tissue formation. However, when culturing cells, static culture vessels, often used in early fermenters, do not provide adequate homogenous nutrient supplies. 48 Different modes of operation have been employed in TE bioreactors with varying success and are classified by the nutrient input, waste output, and cell harvesting techniques. 49 The mode of operation forms the core for each bioreactor design, since many engineering parameters are dictated by the mass transfer, aeration, and nutritional needs.   Figure 5). [54][55][56] One of the most conventional bioreactors in chemical engineering and biopharmaceuticals is the stirred-tank bioreactor. 54 The main characteristics of stirred-tank bioreactors are the free-floating cells in media and the agitator arm (or impeller). The impeller performs a wide array of functions, including heat and mass transfer, aeration, and mixing. 54 The geometric parameters of the impeller and tank, such as the off-bottom clearance, the impeller size, and the ratio of liquid height to tank diameter, can affect the performance of the stirredtank bioreactor. 54 Typically, stirred-tank bioreactors produce high shear stresses, which are not ideal for fragile mammalian cell cultures.
Thus, for TE applications, stirred-tank bioreactor designs are modified to reduce damage to cells by minimizing hydrodynamic shear forces induced by agitation and air bubbles. 57,58 Spinner flasks are very similar to stirred-tank bioreactors, except the cells are seeded on either scaffolds, that are fixed to needles protruding from the vessel, or onto microcarriers. 55,59 The stirring element also differs from the typical stirred-tank bioreactor. Frith et al. describe spinner flasks as comprised of a magnetic stirring arm with two side arms that allow for the addition or subtraction of substance and aeration to the contents of the flask. 60 The side-arms, also referred to as the inlet and the outlet to the confined space, contain filters that help limit contamination while also ensuring oxygenation.
Spinner flasks are typically used to expand embryonic and adult stem cells (e.g., human hematopoietic stem and progenitor cells, human embryonic stem cells, and mouse embryonic stem cells) for both research and stem cell therapy purposes. 48 Spinner flasks are also routinely used for dynamic cell seeding 61 and inducing specific geometries for cell clusters. 62 Despite the success and versatility of spinner flasks, some drawbacks include the possible formation of a dense superficial cell layer, which could block oxygen and nutrient supplies to the construct's core, and the formation of shear stress gradients from nonhomogeneous forces. 48,55 Spinner flasks are also routinely used for dynamic cell seeding 61 and inducing specific geometries for cell clusters. 62 Rotating wall vessels (RWV), or rotary cell culture systems (RCCS), were first used to simulate microgravity conditions, but have since been adapted for use in dynamic three-dimensional (3D) culture systems. 55 Frith et al. describes rotating wall vessels as cylindrical chambers with internal, membrane-covered cylinders that aid in drawing oxygen into the space while rotating the vessel. 60 The rotating wall helps to diffuse nutrients in the media by generating high mass transfer rates and small levels of shear stress. The cells can be rigidly attached to the wall via microcarrier scaffolds, rigidly attached to the core via rotating beds, or free-falling. 55 For the free-falling environment, the cells are supported by balancing the forces acting on the construct. The applied forces are the drag force (F d ), the centrifugal force (F c ), and the gravitational force (F g ). 59 The rotation of the wall around the scaffold creates an equilibrium of forces that allows for the scaffold to be held up in the medium without colliding with the vessel. Successful growth of human hematopoietic stem and progenitor cells, 63 human mesenchymal stem cells (hMSCs), 64 and mouse embryonic stem cells have been shown in RCCS. 65 Changes in cell behavior have also been identified between cells cultured in RWVs and spinner flasks, suggesting that the dynamic 3D environment influences MSC properties. 60 Although RWVs have been demonstrated to promote differentiation, some controversy exists on whether the dynamic environment promotes or inhibits osteogenesis. For example, some studies have shown that RWVs increase adipogenesis but decrease osteogenesis, 66 while others present evidence that RWVs increase both. 60 Perfusion flow bioreactors are the most commonly used bioreactor for TE applications involving three-dimensional bone stimulation due to overcoming the diffusional limitations of rotation-based and stirring bioreactors. 50 Perfusion flow reactors are commonly used for seeding scaffolds and culturing the subsequent construct. Scaffold seeding with perfusion bioreactors produces a more uniform cell distribution compared with other agitation methods. 2 In perfusion bioreactors, solutions under continuous or pulsatile laminar flow are pumped through the entire scaffold, enabling mass transport of nutrients and oxygen. 55 The fluid flow produced by perfusion bioreactors also exhibit flow patterns most similar to those experienced in native tissues, such as bone and blood vessels. Perfusion bioreactors can be further divided into systems using direct or indirect media flow. In direct perfusion, the construct is tightly sealed, forcing the flow to travel through the construct's pores. However, in indirect perfusion there is only a loose seal, allowing for the flow to take the path of least resistance. 55 Since direct perfusion allows for easier control of flow-induced stresses on the construct, it is often preferred to indirect perfusion. MSCs are one of the most common cell types used in perfusion bioreactors. 48 There have been reports that lower perfusion rates increase MSC expansion, while higher perfusion rates decrease expansion due to the effects of high hydrodynamic stress. 48 Thus, careful design considerations must be given to ensure the hydrodynamic stresses produced by the fluid flow do not damage the cells. In one study, a perfusion-based microbioreactor for rapid cellular disease diagnosis was designed with piezoelectric transducer integration in order to closely measure the shear stresses being applied to the cells. 67 The microbioreactor design was modified in order to maintain shear stresses in the milli-Pascal range while still applying fluid volume flow rates between 0.03 and 3 μL/min. 67 While all the classical bioreactors mentioned in this section are still used in the field of TE today, there has been a shift toward perfusion bioreactors due to the higher control of shear stresses and uniform flow. It can be argued that a perfusion flow system coupled with a continuous batch mode creates an environment that best recapitulates the physiological environment for most biological tissues, and thus allows for better translation into the clinical realm. The driving factor for the shift from classical to modern bioreactors, and the driving factor behind all TE bioreactor designs, is the desire to improve tissue formation metrics by simulating the in vivo microenvironment.
Future bioreactors should also be designed with these goals in mind.

| BIOREACTOR ENVIRONMENTAL REQUIREMENTS
Recall that a TE bioreactor's main purpose is to create and maintain a specific microenvironment for biological constructs. This microenvironment is designed to elicit specific outcomes, such as a change in cell phenotype, maintaining cellular metabolism, or promoting cellular proliferation. It is important to note that the cellular outcome is not only dependent on the microenvironment but also the types of cells used in the biological construct. For example, a specific microenvironment might promote cellular maturation in one cell type but hinder the expression of another cell type's desired phenotype. Thus, it is important to choose and optimize the microenvironment toward a specific cell type and desired outcomes. Thus, when designing a bioreactor, understanding the factors that promote or inhibit specific cell functions is important to ensure that the appropriate microenvironment is being created. 1 The microenvironment can be broken up into two types: the physiochemical environment and the physiological environment.
The physicochemical environment embodies the abiotic factors of the

| Mechanical stimulation
Bone response to mechanical stimulation has long been a topic of interest. According to Wolff's law, bone is an adaptive tissue that changes its geometric and structural properties in order to meet the demands of its physical environment. 53,73 In other words, the mechanical loads applied to bone play an important role in determining its mechanical properties, such as the elastic modulus, ultimate As with all biophysical stimuli, the loading regime plays a vital role on tissue and cell response, and since these regimes are not standardized, the success of mechanical bioreactors has been limited (  In the field of orthopedics, tension bioreactors are typically used to facilitate the growth of muscles and tendons in vitro. [82][83][84] However, they can also be used to grow some hard tissues, such as fibrocartilage, or more specifically the meniscus. The meniscus is subjected to both compressive and tensile forces in vivo. Thus, dynamic compressive and tensile forces have been applied to meniscus constructs in tandem with an observed increase in the following: proliferation, collagen, osteocalcin, and stiffness. 85,86 Unlike compressive and tensile forces, which are typically applied directly to the substrate, shear loading is applied to cells within biore- Most studies agree that pulsatile fluid flow is a much more potent stimulator of bone and cartilage cells compared with the other dynamic patterns. 3 It is also important to note that if the rate of shear flow exceeds a certain threshold seeded cells may detach from the matrix. Thus, the optimal flow rate for each bioreactor setup should be determined based off the combination of cell types and scaffold material. 55 Although the most common types of mechanical stimulation applied to bone and cartilage in vitro are compression, tension, and shear, recent literature has suggested that torsion also plays an important role in the mechanotransduction process, specifically for long bones. 87 Torsion has been shown to promote the shaping of tubular structures in bone, which is essential for the development of shafts in long bones and the femoral neck. 88 Thus far, torsion bioreactors have shown promising results, upregulating the gene expression of collagen type 1, tenascin C, and collagen type 3. 89 Additional research is needed to determine the optimal loads and angles for torsional stimulation. In addition to long bones, torsion bioreactors are also used to culture intervertebral discs, since the spine is routinely subjected to compression and torsion. 87,90 With this dual loading in mind, some bioreactors are being designed with multiple levels of mechanical stimulation to better mimic in vivo conditions.
In the past few decades, ultrasound stimulation has been studied as a form of mechanical loading in bone and cartilage TE applications.
Of particular interest is the effects of low-intensity pulsed ultrasound (LIPUS) on bone healing properties. [91][92][93] LIPUS is a gentle form of mechanical energy that is transmitted through tissue and is a widely used tool for therapy and diagnosis. 93 LIPUS has been shown to affect the differentiation of osteoblasts and upregulates prostaglandin E 2 and cyclooxygenase 2 for accelerated bone regeneration. [91][92][93] Ultrasound has also been used in tandem with dynamic compressive loading, resulting in increased osteogenic gene expression compared with the individual stimuli. 20 In summary, mechanotransduction can be triggered by any of the listed forms of mechanical stimulation. However, in order to push for clinical relevance, the loading types and regimes should be chosen to reflect the in vivo microenvironment of the specific tissue type. As such, the best in vitro microenvironment created by a bioreactor is likely one that incorporates several mechanical, as well as chemical and electrical, stimuli.

| Electrical stimulation
Electrical stimulation has been studied as a method for enhancing bone healing for several decades. In the 1950s, Fukada and Yasuda discovered that the collagen molecules in bone exhibit piezoelectric properties, allowing the tissue to create a dipole moment when under strain. 52 It was later discovered that streaming potentials, or electric fields generated by stress-generated flow of ionic fluids, also exists in bone. 50 Furthermore, when a bone is fractured, the gap within the fracture site becomes negatively charged, likely playing a role in attracting inflammatory and reparative cells. 94  Whereas a transcutaneous bone stimulator supplies direct current (DC) directly to the defect. Each of these methods can be adapted to be used in TE bioreactors to grow viable tissue constructs before being implanted into defects.
Studies have shown that electrical stimulation heavily influences cellular behavior, such as adhesion, migration, proliferation, differentiation, mineralization, extracellular matrix deposition, and vascularization, all of which are essential in BTE treatments (Table 3). 15,18,95 However, as with mechanical stimulation bioreactors, the specific loading regime plays a T A B L E 3 Summary of cellular behaviors in response to electrical loading for in vitro and animal studies  Gittens et al.
T A B L E 4 Summary of cellular behaviors in response to a combination of chemical and mechanical or electrical loading for in vitro and animal studies Note: Upregulation ("), downregulation (#), and no significant changes (À). vital role on the success of the treatment. For the percutaneous devices, the electrical stimulation is typically characterized by the electric field magnitude that permeates through the skin and soft tissue to interact with the bone defect site. 35,[96][97][98][99] On the other hand, transcutaneous DC devices report the electrical stimulation as a current, current density, charge, electric field, potential, or a combination of the previously mentioned. 18,38,97,[100][101][102][103] Recently, it has been speculated that the current density and electric field play a more important role in cell behavior mainly due to heating effects, which may explain the inconsistent results of studies that only reported on the current magnitude. 100 Generally speaking, electric fields should remain below 10 V/cm and current density below 1-2 mA/cm 2 to avoid damaging nearby tissue. 100 Although electrical stimulation in its various forms has been extensively used in clinical and BTE applications, the specific mechanisms of action are not fully understood. However, a few hypothetical mechanisms have been proposed and are actively being studied. 43,44 For example, sclerostin is known to play a Mothers Against Decapentaplegic (SMAD), 114 and vascular endothelial growth factor (VEGF). 115,116 Additionally, hormones associated with orthopedics like estrogen, 117,118 androgen, 119,120 and parathyroid hormone (PTH) [121][122][123] have been studied immensely. Other important molecules found in orthopedics are calcium, 124 vitamins (specifically vitamin D 124,125 ), and nitric oxide (NO). [126][127][128] The small molecules involved in orthopedics make up key aspects of the bone regeneration pathways. 43  The combination of chemical and mechanical factors in a contained environment has been shown to aid in bone healing. For example, the synergistic effects of PTH with compression was exemplified in a study by Kim et al. 71 The results of this study showed that the combination of both stimuli significantly increased the bone formation rate compared with the diminishing effects of only one of the two stimuli. 71 Single stimuli showed initial positive results, however, after 4 weeks the bone response diminished to the level of baseline control animals. 71 Another group analyzed the phenotypic differences in calvarial and femoral osteoblastic responses to several chemical and mechanical factors, including the induction of osteogenesis through compressive loading, estrogen, growth factors, and cytokine stimulation. 153 The goal of this study was to investigate if there was a difference response in these two types of bone cells since skull/calvarial bones tend to have osteoporosis-resistant nature. 153 The mechanical loading results of this study showed that there was an induction of two early response genes expression in femoral osteoblasts but remained unchanged in the calvarial osteoblasts. 153 Additionally, the estrogen receptor beta (ERβ) expression was upregulated in calvarial osteoblasts, and the estrogen responsive transcriptional repressor (RERG) was expressed 1,000-fold greater levels in calvarial osteoblasts compared with femoral osteoblasts. 153 Ultimately, the results of the study showed how there are functional differences between calvarial and femoral osteoblasts in vivo and a better understanding might lead to a better therapeutic prospect. 153 The pilot ovine spinal interbody device study conducted by Friis and Arnold involved a mixed mode of mechanical and electrical stimulation. 46 To investigate the pathway triggered in response to the dual modes of stimuli there is a need for a biomimetic bioreactor to quantify on a cellular level the physiologic mechanism of this increased bone growth. Coined mechanically synced electrical stimulation (MSES), the synergistic effects of physiological loading of the sheep to initiate the piezoelectric circuit in the interbody device showed better bone healing in less time than the controls. 46 Knowing what pathway is utilized due to the introduction of the interbody device and physiological mechanical loads would allow for clinically applicable and translatable information.
Progress toward clinically relevant bioreactors has been made, but a true physiological representation of cell microenvironments has yet to be realized. However, recent studies are reaffirming the notion that physiological loads are critical for enhancing cellular behaviors.
One approach could be mimicking the physiological responses, with all forms of stimuli, such that the environment promotes innate cell and tissue repair processes during disease or injury. It would be interesting, and of clinical relevance, to design a system where such responses could be controlled by variable inputs integrated within a reactor system (Figure 7).

| FUTURE DIRECTIONS
Diversification of bioreactors enables researchers to better understand how various changes in physiologic conditions affect the human body.
The human body is an extremely complicated machine that makes the translation between benchtop to bedside particularly difficult. Designing a clinically relevant biomimetic bioreactor that simultaneously affects mechanical, electrical, and chemical stimuli would further our understanding and reduce time on a design that would not work in clinical practice. While there are a few ways to interact with a bioreactor system, batch, fed-batch, and continuous batch systems, we believe that continuous batch systems will better recapitulate and mimic the physiological environment. Additionally, it will take a bioreactor that can provide mechanical stimulation, apply electrical loads, and incorporate chemical factors to fully see the multiple stimulation machine come together.
Studying how each one of these cues and factors are incorporated into the system would lead to better engineered orthopedic devices.