**4. Systems for 3D cultivation of bone tissue**

*Ex vivo* tissue-engineering (TE) strategies for *de novo* generation of bone tissue enclose the combined use of autologous bone-forming cells and three-dimensional scaffold materials serving as structural support for the cells [118]. Bioreactors are used as a tool for studying and mimicking *in vivo* conditions in an *in vitro* environment for the growth of tissue substitutes and represent the device used to develop biological processes by closely monitoring and controlling the environment [119]. Parameters that must be controlled and appropriately adjusted in order to perform controlled and successful experiments are:


always the risk of rejection due to reduced bioactivity. Degradation of synthetic polymers becomes the major issue because most of them are degraded by hydrolysis, causing lower local

Since ceramics have excellent osteoinductive properties but low mechanical strength, and synthetic polymers exhibit poor osteoinductivity but better mechanical strength and degrad‐ ability, in the past decade researches have been trying to develop the scaffolds made of ceramic and polymer composites. Most commonly used 3D composites are made of synthetic polymers such as poly(lactic) acid (PLA), PGA, poly(ε-caprolactone) (PCL), poly(lactic-co-glycolide) (PLGA), poly(propylene fumarate) (PPF) and natural polymers such as collagen type I and chitosan. These composites have rigid sponge-like structures often containing HA (133-138 from three-dimensional alexander). Hydroxyapatite increases attachment of mesenchymal

stem cells, differentiation to osteoprogenitors and promotes cell survival [109, 110].

The ideal scaffold is difficult to obtain and should be biocompatible, bioresorbable, osteocon‐ ductive (must allow bone cells to adhere, proliferate and secrete extracellular matrix), ostein‐ ductive (with the ability to induce new bone formation), osteogenic (should act as MSCs and osteoblasts reservoir), structurally similar to bone enabling formation of strong bonds with surrounding bone tissue, as well as it should be easy to use and cost-effective. New approach includes development of methods to isolate and transplant bone tissue-forming cells, bioactive matrix materials that act as tissue scaffolds mimicking what happens in nature, and delivery of bioactive molecules within scaffolds. In the past two decades, many 3D systems have been

**Figure 2.** Bone graft grown on scaffold made of chitosan and hyaluronic acid. Section of graft stained with hemalaun/

eosin staining shows equal cell distribution, formation of extracellular matrix and scaffold residues.

**3.3. The ideal scaffold for bone tissue engineering**

pH and cell necrosis [108].

234 Advanced Techniques in Bone Regeneration


Tissue-engineering bioreactors can be used to aid the *in vitro* development of new tissue by providing biochemical and physical regulatory signals to cells, encouraging them to undergo differentiation and produce extracellular matrix prior to *in vivo* implantation [120].

This 3D cell expansion on a scaffold poses several challenges. The first challenge is the **transport of nutrients** to cells and **removal of waste** metabolites from the interior of the scaffold. In 2D cell culture diffusion provides nutrients and oxygen to all cells as well as waste removal, but in 3D constructs diffusion is insufficient [121]. That represents an important issue in tissue engineering, limiting the tissue growth due to insufficient nutrient transport [122]. To overcome this problem, scientists developed more complex bioreactor systems 3D tissue culture to improve the media flow and transport of nutrients to cells which contribute to balanced development of tissue [118]. Dynamic bioreactor culture systems are essential for *in* *vitro* cultivation and maturation of tissue-engineering bone grafts, in particular for larger bone grafts where the core of the scaffold is more than 200 μm from the surface. Bioreactors improve the mass transport of nutrients and overcome the diffusion limitation of traditional static culture [123]. Bioreactors bring several advantages into the culture of functional tissues. They do not only increase mass transport inside three-dimensional structures but also reduce the handling steps, hence reducing contamination potential.

Fluid shear stress caused by mixing or perfusion the medium is also very important for bone tissue engineering because it exposes the cells to mechanical stimulation. *In vivo*, mechanical loading increase production of prostaglandins, alkaline phosphatase, collagen type I, along with osteoblast proliferation and mineralization [124]. Mechanical loading of the skeleton causes interstitial fluid flow through lacunar and canalicular space of bones. The cells lining these spaces are then influenced by the mechanical stimulation provided by the fluid flow, differentiating or proliferating accordingly [125, 126]. Based on this knowledge, it is clear that the recapitulation of these mechanisms *in vitro* is essential for the growth and the regenerative properties of human osteoprogenitor cells seeded onto scaffolds [127]. *In vitro*, mechanical stimulation can encourage cells to produce extracellular matrix (ECM) in a shorter time period and in a more homogeneous manner than in static culture [128]. A benefit of ECM production is the increase in mechanical steadiness of the scaffold and tissue graft. Another important advantage of bioreactors is **induced cellular differentiation**. Mechanical stimuli can be used to encourage stem cells down a particular path and hence provide the cell phenotype required [129].

As well as providing mechanical stimulation, bioreactors can also be used to improve **cellular spatial distribution**. A heterogeneous cell distribution is a major problem in developing threedimensional tissue or organ *in vitro* [130]. Scaffolds in larger size range are easily fabricated, but problems arise with culturing cells on these scaffolds. As the size of the scaffold increases, diffusion of cells to the center becomes more difficult. Static culture conditions result in scaffolds with few cells in the center [131]. Thus, bioreactors can be used in tissue-engineering applications to overcome problems associated with traditional static culture conditions, improve cellular distribution and accelerate construct maturation [132] while applying biophysical signals to constructs to improve tissue formation *in vitro* prior to *in vivo* implan‐ tation [120].

The ultimate design of a tissue engineering bioreactor system must: (i) ensure a controlled and rapid cell growth; (ii) facilitate uniform cell distribution; (iii) provide and maintain the physiological requirements of the cell (nutrients, oxygen, growth factors); (iv) increase mass transport both by diffusion and convection using mixing medium systems (v) expose cells to physical stimuli; and (vi) enable reproducibility, control, monitoring and automation. For this purpose, different dynamic culture systems have been developed. These systems improve nutrient delivery to the cells and generate shear stress that promotes cell differentiation into osteoblastic phenotype. Bioreactors for bone engineering applications are broadly classified into few main categories, including rotating wall vessel, spinner flask, perfusion bioreactor and compression systems. In addition to these, combinations of different types of bioreactors have been explored in order to better mimic the bone physiological environment *in vitro* and all these systems for tissue culture are used to achieve a homogeneous cell growth within the scaffold [120].

#### **4.1. Rotating wall vessel bioreactor**

*vitro* cultivation and maturation of tissue-engineering bone grafts, in particular for larger bone grafts where the core of the scaffold is more than 200 μm from the surface. Bioreactors improve the mass transport of nutrients and overcome the diffusion limitation of traditional static culture [123]. Bioreactors bring several advantages into the culture of functional tissues. They do not only increase mass transport inside three-dimensional structures but also reduce the

Fluid shear stress caused by mixing or perfusion the medium is also very important for bone tissue engineering because it exposes the cells to mechanical stimulation. *In vivo*, mechanical loading increase production of prostaglandins, alkaline phosphatase, collagen type I, along with osteoblast proliferation and mineralization [124]. Mechanical loading of the skeleton causes interstitial fluid flow through lacunar and canalicular space of bones. The cells lining these spaces are then influenced by the mechanical stimulation provided by the fluid flow, differentiating or proliferating accordingly [125, 126]. Based on this knowledge, it is clear that the recapitulation of these mechanisms *in vitro* is essential for the growth and the regenerative properties of human osteoprogenitor cells seeded onto scaffolds [127]. *In vitro*, mechanical stimulation can encourage cells to produce extracellular matrix (ECM) in a shorter time period and in a more homogeneous manner than in static culture [128]. A benefit of ECM production is the increase in mechanical steadiness of the scaffold and tissue graft. Another important advantage of bioreactors is **induced cellular differentiation**. Mechanical stimuli can be used to encourage stem cells down a particular path and hence provide the cell phenotype required

As well as providing mechanical stimulation, bioreactors can also be used to improve **cellular spatial distribution**. A heterogeneous cell distribution is a major problem in developing threedimensional tissue or organ *in vitro* [130]. Scaffolds in larger size range are easily fabricated, but problems arise with culturing cells on these scaffolds. As the size of the scaffold increases, diffusion of cells to the center becomes more difficult. Static culture conditions result in scaffolds with few cells in the center [131]. Thus, bioreactors can be used in tissue-engineering applications to overcome problems associated with traditional static culture conditions, improve cellular distribution and accelerate construct maturation [132] while applying biophysical signals to constructs to improve tissue formation *in vitro* prior to *in vivo* implan‐

The ultimate design of a tissue engineering bioreactor system must: (i) ensure a controlled and rapid cell growth; (ii) facilitate uniform cell distribution; (iii) provide and maintain the physiological requirements of the cell (nutrients, oxygen, growth factors); (iv) increase mass transport both by diffusion and convection using mixing medium systems (v) expose cells to physical stimuli; and (vi) enable reproducibility, control, monitoring and automation. For this purpose, different dynamic culture systems have been developed. These systems improve nutrient delivery to the cells and generate shear stress that promotes cell differentiation into osteoblastic phenotype. Bioreactors for bone engineering applications are broadly classified into few main categories, including rotating wall vessel, spinner flask, perfusion bioreactor and compression systems. In addition to these, combinations of different types of bioreactors have been explored in order to better mimic the bone physiological environment *in vitro* and

handling steps, hence reducing contamination potential.

236 Advanced Techniques in Bone Regeneration

[129].

tation [120].

Cells that grow *in vitro*—outside the body in 2D layers do not behave in the same way as cells grown *in vivo*—inside the body. *In vivo* cells grow three-dimensionally and form tissues that have modified their structure to perform a specific function and secrete extracellular matrix. Two-dimensional growth represented a limit to the scientists who wanted to understand mechanisms that govern cell behavior and tissue formation. In the 1970s, a small NASA group of scientists began to think about space as a possible answer. The group believed if cells could be grown without the Earth's gravity influence, they would not settle to the bottom of the culturing container, instead they would be suspended in the medium and therefore might compound and form tissue that more closely resembles the tissue in the body [133]. The rotating-wall vessel (RWV), developed by NASA, was originally designed to protect cell cultures from high shear forces generated during the launch and landing of the space shuttle. When the device was tested on the Earth for cells in suspension, cells aggregated and formed structures similar to tissues. These observations led to the possibility that the bioreactors might be used to study co-cultures of multiple cell types and their association, proliferation and differentiation during the early steps of tissue formation [134].

The RWV bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organi‐ zation and specialization, samples constructed in this bioreactor more closely resembled the original tumor or tissue found in the body. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues cultured in rotating bioreactors [133].

The RWV bioreactor (**Figure 3A**) consists of a cylindrical growth chamber with a gas exchange membrane. The solid-body rotation is accomplished by a vessel rotating horizontally around its axis, randomizing the gravitational forces acting on the cell surface. The culture chamber is completely filled with medium and is oxygenated through a silicone rubber membrane by an air pump that draws incubator air through the filter. As the vessel rotates, the liquid inside accelerates until the entire fluid mass is rotating at the same angular rate as the wall. Thus, this environment eliminates most of the disruptive shear forces associated with a conventional bioreactor, scaffolds and cells obey simple kinematics and are uniformly suspended in the culture medium with minimum shear forces. In this environment, cells aggregate and undergo three-dimensional growth to form tissue-like structures. As aggregates grow during culture, the speed of vessel rotation is increased to contrary gravitational sedimentation [134].

Cultures using an RWV bioreactor proved useful for growing tissues, such as bone. Many studies showed enhanced proliferation, distribution and differentiation of osteoprogenitor cells on scaffolds when cultured in a free fall manner in RWV-based bioreactor systems [135]. Until today, many designs of rotating bioreactor systems have been developed for dynamic 3D bone tissue engineering. One of them is RWV bioreactor with the scaffolds attached to the external wall by use of stainless steel clamps. External and internal cylinders were driven by

**Figure 3.** Design of rotating vessel (A) and spinner flask (B) bioreactor systems.

step motors and compared to the cultivation in static culture. The use of RWV resulted in better cell proliferation and differentiation. The second one is a rotating bed bioreactor (RBB). In an RBB, constructs are attached directly on the axis and moved between gas and liquid phases in an alternating manner. One major benefit of the system besides the positive effect in terms of proliferation and differentiation is the compatibility with good manufacturing practices (GMP) standards. Disadvantage of RWV system is the collision of scaffolds with the bioreactor wall, which may damage scaffolds and disrupt seeded cells. This can be omitted by the use of the RBB concept. Another major disadvantage of the rotating system is that mineralization is limited to the outer part of the scaffold. Internal nutrient transport limitations could not be eliminated by rotation-based bioreactor systems [118]. Rotating wall vessels are limited to the small-sized constructs due to insufficient transport inside the scaffold. Additionally, because of the low range of values of shear stress, these systems may not be efficient in promoting robust osteogenic differentiation. On the other hand, rotating wall vessels allow the accom‐ panying culture of several cell/scaffold constructs. These systems could be adopted to engineer thin bone substitutes for the reconstruction of flat bones or as bone patches for restorative applications of the skeletal system [3].

#### **4.2. Spinner flask bioreactor**

A simple bioreactor system based on media mixing is the spinner flask (**Figure 3B**). Spinner flasks are composed of a glass media reservoir with side arms that can be opened to remove scaffolds and media and also to allow gas exchange. The flask has a stir bar mechanism that stirs the media in the flask. They are often used in bone tissue engineering because of the ability to increase expression of early osteoblastic marker alkaline phosphatase, late osteoblastic marker osteocalcin and calcium deposition as compared to static culture and rotating wall bioreactors. This effect is the result of convective transport of nutrients to the surface of the scaffold in contrast to the purely diffusional transport in static culture. It also increases the concentration of oxygen throughout the scaffold [136].

Scaffolds are hanging attached to vertical needles from the top of the vessel and are submerged in the medium. The top of the vessel is usually used for gas exchange and medium oxygenation. Mixing of the medium is maintained by stir bar mechanism at the bottom of the vessel. The convective forces generated during stirring moderate the nutrient concentration gradients at the surface of the scaffold and produce turbulences that enhance mass transport according to the center of the samples [3].

Commonly, spinner flasks are around 120 ml in volume (up to 8 liters), are run at 50–80 rpm and 50% of the medium used in them is replaced every 2 days [137]. An important advantage of the spinner flask design is its maintenance of well-mixed environment within the flask. However, spinner flasks are not always an ideal solution, since the constant mixing motion causes turbulent flow within the capsule and the associated high shear stress. Spinner flasks have been modified form their original design to reduce the turbulent flow. Current designs induce small waves during mixing instead of the rough, turbulent flow induced from tradi‐ tional spinner flasks. Spinner flasks are intended for small-scale production and do not appear to be used as much as other types. They are primarily used for the seeding of cells in 3D scaffolds until they are ready for more large-scale cell culture procedures [119].

#### **4.3. Perfusion bioreactor**

step motors and compared to the cultivation in static culture. The use of RWV resulted in better cell proliferation and differentiation. The second one is a rotating bed bioreactor (RBB). In an RBB, constructs are attached directly on the axis and moved between gas and liquid phases in an alternating manner. One major benefit of the system besides the positive effect in terms of proliferation and differentiation is the compatibility with good manufacturing practices (GMP) standards. Disadvantage of RWV system is the collision of scaffolds with the bioreactor wall, which may damage scaffolds and disrupt seeded cells. This can be omitted by the use of the RBB concept. Another major disadvantage of the rotating system is that mineralization is limited to the outer part of the scaffold. Internal nutrient transport limitations could not be eliminated by rotation-based bioreactor systems [118]. Rotating wall vessels are limited to the small-sized constructs due to insufficient transport inside the scaffold. Additionally, because of the low range of values of shear stress, these systems may not be efficient in promoting robust osteogenic differentiation. On the other hand, rotating wall vessels allow the accom‐ panying culture of several cell/scaffold constructs. These systems could be adopted to engineer thin bone substitutes for the reconstruction of flat bones or as bone patches for restorative

**Figure 3.** Design of rotating vessel (A) and spinner flask (B) bioreactor systems.

A simple bioreactor system based on media mixing is the spinner flask (**Figure 3B**). Spinner flasks are composed of a glass media reservoir with side arms that can be opened to remove scaffolds and media and also to allow gas exchange. The flask has a stir bar mechanism that stirs the media in the flask. They are often used in bone tissue engineering because of the ability to increase expression of early osteoblastic marker alkaline phosphatase, late osteoblastic marker osteocalcin and calcium deposition as compared to static culture and rotating wall

applications of the skeletal system [3].

**4.2. Spinner flask bioreactor**

238 Advanced Techniques in Bone Regeneration

Spinner flasks and rotating wall bioreactors do not effectively perfuse media through the center of the scaffold. Bioreactors that use a pump system to perfuse media directly through a scaffold are known as perfusion bioreactors [136]. In perfusion bioreactors, scaffolds are placed in the perfusion chamber (**Figure 4A**) in a press-fit manner so that the medium is forced to pass through the center of the samples [3]. Flow perfusion bioreactors have been shown to provide more homogeneous cell distribution throughout scaffolds. This has resulted in greater cellularity throughout the scaffold in comparison to static controls, suggesting the better nutrient exchange [120].

These bioreactors have an advantage over the others because they provide a uniform mixing of the media, enabling better control of the environment and the physical stimulation of the cells in the bone tissue [121]. They are very effective for the culture of mesenchymal stem cells and have been shown to induce osteogenesis. This is attributed to the ability of the perfusion system to increase the transport of oxygen and nutrient through the scaffold and expose the cells to the mechanical stimulation [137]. The optimization of the perfusion bioreactor protocols for tissue engineering must ensure balance between the transport of substances and waste metabolites and hold newly synthesized tissue within the scaffold, taking care of the fluid flow rate which goes through the pores [120]. Many different perfusion bioreactor systems have been developed, but most systems are based on the similar basic design—media reservoir, pump, tubing circuit and scaffold chamber. The scaffold is sealed within the chamber so media cannot flow around it. Thus, media flows directly through the pores of the scaffold [136]. Scaffolds should have interconnected pores and should have between 70 and 99% porosity in

**Figure 4.** Design of the scaffold chambers in perfusion (A) and compression (B) bioreactor systems.

order to facilitate direct perfusion. In most cases, the major difference between the systems is the design of the perfusion chamber because it is the key element to ensure deep perfusion of the scaffold center [121]. Despite these difficulties, many perfusion bioreactor systems have been developed and tested for bone tissue-engineering purposes [136]. The pump produces a force that travels through the tubing circuit and perfuse the media through the scaffold pores in a continuous or noncontiguous way [120]. This force represents the perfusion flow rate applying mechanical stimulation in the form of shear stress to cells in the scaffold. This mechanical stimulation proved to be a powerful tool to stimulate osteogenic differentiation, and data show that cell-matrix and cell-cell junction molecules are capable of converting mechanical stimuli into biochemical signals.

One of the most important parameters when optimizing a bioreactor is the flow rate. It depends on the composition, porosity and geometry of the scaffold. The pump is capable of precisely and consistently pumping flow rates from 0.01 to 6.0 ml/min through each chamber [138]. Still, there is a big variation of values and there are not many studies that compare a significant range of flow rates. It appears that the increase in flow rate leads to an increase in the deposition of mineralized matrix. Very low flow rates such as 0.01 ml/min have been reported to lead to higher cell viability, but this does not seem an optimal flow rate for bone tissue engineering as it might be too low to actually accomplish an adequate distribution of nutrients, oxygen and removal of waste products. It is also necessary to bear in mind that lower values of flow rate will provide lower values of shear stress, which might facilitate cell attachment and spreading, hence leading to higher values of cell viability. Despite the wide variation of the flow rates tested, it seems that the optimal values would range from 0.2 to 1 ml/min, depending obviously on the system being used. This is the range of values that seems to have a more positive effect on osteoblastic differentiation, ECM deposition and distribution [121]. Perfusion bioreactor is so far the only system that produces such a force, making it ideal for growing large bone grafts *ex vivo* [139].

#### **4.4. Compression bioreactors and combined systems**

Compression bioreactors (**Figure 4B**) were intended to mimic the bone physiological *in vitro* environment, characterized by repeated mechanical stimulation required for functional bone regeneration. Many studies provide evidence that mechanical loading, when combined with flow perfusion, can play a main role in promoting survival and functional osteogenic differ‐ entiation of the cells within the scaffold. Short-term mechanical stimulation enhanced the expression of several genes encoding for factors involved in osteogenesis, including *RUNX2*, *osteopontin*, *integrin*-*β1*, *TGFβR1*, *SMAD5*, *annexin*-*V* and *PDGFα* [3]. These experiments demonstrate that even short mechanical stimuli can be sufficient to activate the osteogenic differentiation pathways in human mesenchymal stem cells. Compression bioreactors systems consist of a motor, a system providing linear motion and a compression chamber in which one or more clips apply static or dynamic compressive loads directly to the scaffold [3]. The bioreactor chamber holds the scaffold in place and ensures hermetic sealing as well as force transmission onto the cell-seeded scaffold. It consists of medium flow distributors, a flexible force transmitting disk and the intended space for scaffold placement. The power transmission rack includes a plunger, a pre-load screw and a cam-shaft. The chamber is placed on the clip and fixed via tightening of the pre-load screw. The camshaft moves the clip in order to apply a sinusoidal compression pattern onto the bioreactor chamber [140]. The system can be controlled by a signal generator, and load response can be measured by using linear variable differential transformers and load cell, respectively. In contrast to static culture, mass transfer is considerably improved in compression bioreactor culture since compression leads to the fluid flow through the scaffold [141]. The compression bioreactors provide a promising tool for bone fracture research and for *in vitro* estimate of alternative fracture treatments based on engineered tissue grafts, allowing the reduction of animal experiments.

### **5. Conclusion**

order to facilitate direct perfusion. In most cases, the major difference between the systems is the design of the perfusion chamber because it is the key element to ensure deep perfusion of the scaffold center [121]. Despite these difficulties, many perfusion bioreactor systems have been developed and tested for bone tissue-engineering purposes [136]. The pump produces a force that travels through the tubing circuit and perfuse the media through the scaffold pores in a continuous or noncontiguous way [120]. This force represents the perfusion flow rate applying mechanical stimulation in the form of shear stress to cells in the scaffold. This mechanical stimulation proved to be a powerful tool to stimulate osteogenic differentiation, and data show that cell-matrix and cell-cell junction molecules are capable of converting

**Figure 4.** Design of the scaffold chambers in perfusion (A) and compression (B) bioreactor systems.

One of the most important parameters when optimizing a bioreactor is the flow rate. It depends on the composition, porosity and geometry of the scaffold. The pump is capable of precisely and consistently pumping flow rates from 0.01 to 6.0 ml/min through each chamber [138]. Still, there is a big variation of values and there are not many studies that compare a significant range of flow rates. It appears that the increase in flow rate leads to an increase in the deposition of mineralized matrix. Very low flow rates such as 0.01 ml/min have been reported to lead to higher cell viability, but this does not seem an optimal flow rate for bone tissue engineering as it might be too low to actually accomplish an adequate distribution of nutrients, oxygen and removal of waste products. It is also necessary to bear in mind that lower values of flow rate will provide lower values of shear stress, which might facilitate cell attachment and spreading,

mechanical stimuli into biochemical signals.

240 Advanced Techniques in Bone Regeneration

Bone defects that are due to trauma or pathological and physiological bone resorption represent a global health problem. The need for bone regeneration is one of the central issues in regenerative medicine. Tissue engineering is becoming a useful addition to medical therapies for repairing and restoring the function of bone tissue. Bone constructs elaborated with tissue-engineering principles are a promising substitute for autologous bone graft and have long been considered the golden standard for repair of large bone defects. Mesenchymal stem cells from adult tissues are the most suitable cell source for bone tissue engineering. Although the application of MSCs as cellular material facilitates the construct fabrication, more work needs to be done to fully determine the clinical potential, efficacy and safety of stem cellbased treatments. There is a constant need in the development of new scaffolds that have optimal characteristics, and are affordable as well as easy for manipulation. Bioreactor dynamic setting enables better culture conditions and mechanical stimuli for improved bone tissue growth. In spite of the existing problems, advances in the field are enormous and therapy using scaffolds, healing signals and stem cells together should be able to solve the current limitations in managing bone injuries.
