In Vitro and In Vivo Bioreactor Strategies for Bone Defect Repair In Vivo Bioreactor Strategies for Bone Defect Repair.

Several studies have demonstrated promising when Abstract The key elements for bioartificial bone formation in three-dimensional matrices in any tissue engineering concept are a high number of osteogenic cells and supplies of oxygen and nutrition [1]. In the treatment of large bone defect, tissue engineering is challenging the problem to obtain scaffolds able to release growth and differentiation factors for mesenchymal stem cells, osteoblasts and endothelial cells in order to achieve faster mineralization and activate a stable vascularization. Bone is a highly vascular structure: like all other living tissues, it is supplied by several blood vessels; on the contrary cartilage is avascular, a lymphatic and has the lowest cellular density of any tissue in the body with less than 5% cells by volume. Despite important progress in engineered scaffolds for cartilage lesions, bone tissue scaffolds are still facing diffusion issues, specially a lack of functional network of blood vessels. Three-dimensional scaffolds used in bone tissue engineering, increase the complexity of the culture because of the difficulty in supplying oxygen and nutrients to the cells. Researchers are developing in vitro and in vivo bioreactor based strategies for solving these functional problems. The concept of bioreactor for growing “functional engineered tissues” has not only an in vitro application but can be extended to an in vivo approach. The aim of this mini review is to analyze current trends in applying the concept of bioreactor to bone tissue engineering, comparing in vivo and in vitro methods currently proposed


Introduction
The ideal bone graft has several features that can be achieve in different ways combining in vitro and in vivo approaches. Bone is a dynamic, highly vascularized tissue with the unique capacity to heal and remodel without leaving a scar [2]. An ideal functional bone graft should have some important characteristics like biological safety, osteoinductive and angiogenic potentials, low donor site morbidity, no size restrictions, rapid accessibility to surgeons, long shelf life and reasonable cost. Even if for bone defect reconstruction several strategies have been already applied, none of currently available bone substitutes own all the above-mentioned properties.
The present mini review focuses on the different solutions adopted to create bone scaffolds using bioreactors starting from the clinician "in vivo" and scientist "in vitro" point of view. Bioreactors is an apparatus for growing an organism or cells in the context of cell culture. The term bioreactor it may refer to any manufactured or engineered device or system capable to support a biologically active environment. Bioreactors are classically used in industrial fermentation processing, wastewater treatment, food processing and production of pharmaceuticals and recombinant proteins (e.g. antibodies, growth factors, vaccines, and antibiotics).
Bioreactors in the field of bone tissue engineering they are important because are able to provide an in vitro environment mimicking the in vivo conditions; for example mechanical compression and hydrostatic fluid pressure are important regulating factors for cell physiology in bone and can facilitate tissue formation [3,4]. Several studies have demonstrated promising results when in vitro dynamic conditions were applied before implantation, describing the effect of many variable and how those can influence cell metabolisms [5][6][7]. Furthermore, bioreactors provide more standard culture conditions than in vivo tissue regeneration, thus it is useful for systematic, controlled studies of cellular differentiation and tissue development in response to biochemical and mechanical cues. Regenerative medicine takes advantage of the body natural capacity to regenerate, on the contrary in tissue engineering bone substitute are manufactured in an industrial setting despite the need for adaptation in the human body. The borders between regenerative medicine and tissue engineering are today narrower, often merging in a bigger topic (TERM), determining the foundation and growth of an international scientific society has been founded with the mission of bring together the international community engaged or interested in both fields (TERMIS). All engineered tissues need adaptation in human body and artificial bone tissue had been experienced vascularity issues and creeping substitution when implanted in clinical setting. That's the main reason why in vivo bioreactor concept has been proposed: microsurgery techniques and tissue engineering are ideally mixed in order to increase the quality of artificial bone substitute.

In Vitro Bioreactors
Tissue engineering aims to realize biological functional substitutes to be used to repair or regenerate damaged tissues.
Tridimensional scaffolds are necessary to sustain living and functional constructs. They are designed to be biodegradable and bioresorbable, made of synthetic or natural origin materials, to have specific chemical properties and to exhibit the proper geometrical dimensions, structure and chemistry as close as possible to the original tissue. To achieve tissue remodeling, cell culture must tangibly differ from the traditional static 2D culture.
In fact, traditional cell cultures are usually carried out on multi-well polystyrene plate, containing a specific treated surface to promote cell adhesion. Cells are seeded with medium and then plates are placed in an humified incubator where the environmental conditions of 37°C and 5% CO 2 are maintained. The temperature is, obviously, physiological, while the CO 2 is necessary to stabilize pH in the culture. As previously mentioned, a bioreactor can be defined as any apparatus that attempts to mimic physiological conditions in order to maintain and encourage tissue regeneration, simulating the living organism. In a bioreactor, tissue culture is a non-steady state process in which all parameters can be measured and controlled.
Precisely, temperature, pH of the medium, gas exchange, O 2 and CO 2 level, humidity, nutrient flow and waste removal. Moreover, mechanical-biochemical stimuli can also be tuned.
Temperature must be constant around 37°C, pH value should remain between 7.2 and 7.4 acting on CO 2 level. Humidity should be enough (generally closed to condensation) to avoid medium evaporation, while dissolved O 2 level and nutrient concentrations must be enough to guarantee cell functions. The fundamental part of a bioreactor is the culture chamber: a sterile environment where the cellular constructs are housed. The seeded scaffold can be confined, that means laterally constrained, or unconfined, in which the lateral side is free to move. Growing medium with the necessary nutrients can flow inside the chamber thanks to a predisposed system. The culture medium can diffuse inside the substrate with a proper flow regime, allowing nutrients transport and waste removal. The culture chamber must also present some general characteristics: each component must be sterilized and manufactured from nontoxic materials and should be easy to assemble, still allowing Considering these factors is crucial to obtain an engineered construct with adequate properties. Therefore, it is essential to evaluate the normal living tissue conditions to develop a suitable device for driving cell functions and biosynthesis. In fact, an ideal bioreactor design, which covers different tissue engineering applications, does not exist, but can be tailored for a specific individual aim. A bioreactor for tissue engineering applications should, at some level (i) Facilitate uniform cell distribution; (ii) Provide and maintain the physiological requirements of the cell (e.g., nutrients, oxygen, growth factors); (iii) Increase mass transport both by diffusion and convection using mixing systems of culture medium; (iv) Expose cells to physical stimuli; and (v) Enable reproducibility, control, monitoring and automation [8].  to cells [10,11]. The introduction of perfusion allows nutrients transport and waste removal directly through the scaffold bulk, to overcome the limitations of diffusion. In perfusion bioreactors, porous scaffolds are usually housed on an impermeable chamber so that the culture medium can be forced to pass through (Figure1c).
The perfusion influences osteocytes/chondrocytes characteristics, such as matrix synthesis gene expression and generally stimulates cell proliferation and matrix synthesis compared to the traditional static cultures [9,[12][13][14]. While perfusion allows improving gas and medium exchange, it is also capable to activate gene expression, upregulating alkaline phosphatase (ALP) and calcium deposition [15,16]. Similar results were reported in the case of bioreactors capable to apply direct hydrostatic pressure. In general, a hydrostatic compression bioreactor can apply either intermittent (ICP) or cyclical (CCP) pressure, imposing to the liquid or the gas a square wave pulse, or a sinusoidal pressure profile [17,18].  and calibrate the effective stress/strain applied to the scaffolds. By modifying the geometry, the system also permits the application (and measurement) of a hydrostatic pressure. The bioreactor can be programmed to apply different stimulation cycles, with user programmable shape (sinusoidal, triangular, sawtooth, trapezoid shape), intensity, duration and number. Contemporary, during the standard operation, the bioreactor is designed to monitor also temperature and CO 2 levels, and to control the medium exchange.
Bioreactors from other brands follow similar approaches, with slightly different design. Despite the high technical complexity of the current bioreactor design, still the problem of proper scaffolds feeding with nutrients is not completely resolved and, while they allow better proliferation of cells in the scaffolds, they start manifesting some limitations when their size is bigger than few millimeters.

In Vivo Bioreactors
The concept of "in vivo" bioreactors is inherent in the body self-  [24] and that describes the implantation of a vascular pedicle into a new territory, followed by a neovascularization period and subsequent tissue transfer based on its implanted pedicle.
Tissue prefabrication is commonly realized in two steps. Initially, the selected tissue, is designed into the required configuration and

Muscular Flap or Pouch
A perfused muscle is a poor bed for cancellous bone graft but local condition of good perfusion which are likely to be obtained with fascial or muscular flap wrapped around an autologous bone graft could promote bone tissue regeneration (Figure 3) [26].
Vascularization induced by this method is called "inosculation", in which blood vessel already present in the graft rapidly connect with the vascularized muscular flap. The preformed micro-vessels simply must develop interconnections to the host microvasculature to get fully blood perfused within a short period of time. Although inosculation of preformed micro-vessels is a very promising strategy in tissue engineering, some studies have shown that adequate blood perfusion of artificial bone graft is not guaranteed during the very first days after implantation of the tissue construct [27,28] and much of the inosculation process has to rely on favorable local conditions. Bone formation beneath "standard muscular flaps" has been successfully induced using bioceramics scaffolds seeded with autologous bone marrow stromal cells [29,30]. Prefabricated vascularized bone grafts have also been tested for jaw reconstruction with a thorough in vivo evaluation in a pig model [31,32]. In the clinical setting, a large mandibular defect was successfully reconstructed using custom made titanium cage, realized according to CT scan followed by 3D reconstruction and filled with bone marrow aspirate, xenogeneic bone minerals and OP-1, after a prefabrication period of 7 weeks. The patient obtained both a functional and an esthetic mandibular reconstruction. This landmark case has offered a novel method for realizing custom bone grafts, meanwhile avoiding the development of secondary bone defects. Taking inspiration from these promising results, researchers continue to apply, trying to improve the muscle strategy for applications in clinical cases [33]. There is the need of new "in vitro" techniques to improve the inosculation process "in vivo", some recent studies have demonstrated the effectiveness of combining these two approaches to improve bone graft vascularity [34]. The use of a muscular pouch for improving blood vessel network formation in artificial bone grafts is, by now, the most used strategy within the concept of "in vivo bioreactors" and is reported in several studies both on small and large animal models. As for now, it is the only in vivo strategy that has been applied in a clinical setting and whose potentialities were demonstrated on humans.

Periosteal Flap
In the periosteal flap strategy, a periosteal flap is used to wrap the tissue-engineered construct or to cover the chamber containing the tissue-engineered construct. Therefore, the periosteum- procedure. The osteoperiosteal decortication procedure described by Judet [35] consist in the dissection of the bone periosteum membrane from the cortex, using a sharp chisel, so that small bone pieces remain attached to periosteal tissue; bone chips 1 to 3mm thick are elevated for 5-10cm proximal and distal to the fracture site and for 60-75% bone circumference. Osteoperiosteal decortication is a reliable technique that leads to predictable, satisfactory results.
Although this technique has been in use for a long time, it continues to be effective for treating diaphyseal nonunion [36].
The Masquelet technique for bone gap is based on the application of a cement spacer within a fixed bone defect, whose role is to provide mechanical stability and to act as a foreign body that allows the propagation of periosteal-like membrane on its surface [37]. In orthopedic surgery the use of periosteal flap is widespread. The medial femoral condyle as a free cortico periosteal flap has demonstrated its potential when applied to upper limb defects [38]. Some studies have demonstrated a better new tissue formation when comparing with bone graft implanted against muscle fascia [39,40]. The use of periosteal flap to wrap tissue engineered construct in the rat femur was reported by Vögelin E. to significantly increased bone formation and prevented heterotopic ossification [41,42]

Arteriovenous loop (AV loop)
Erol and Spira in 1980 developed in a rat an arterio-venous loop model interposing a venous graft between the femoral artery and vein in the thigh, to create a prefabricated full thickness skin graft. with this they also described the first AV-loop model [47].
The creation of a vascular axis using vein grafts holds promise for generation of vascularized bone units relatively independent of anatomical limitations and without the creation of a significant donor site defect [47][48][49][50]. Inadequate adjacent arterial supply and venous outflow either due to the injury or to preexisting  a) The in vitro approach allows the realization of ad-hoc scaffolds to cover bone gaps with precision after 3D imaging reconstruction (CT or MRI); it utilizes just artificial resources without any donor site morbidity; it uses standardized tissue manufactured in controlled close system, with consequent reduction of costs.
b) The in vivo approach permits to implant a tissue ready to his function; provides a tissue with a vascular network through microsurgery techniques; allows to implant a bone graft that doesn't undergo creeping substitution; permits the reconstructing of tissue loss in one procedure.
In the present literature, it was hard to find a single winning approach to bone reconstruction: in most cases different strategies were applied together to create engineered bone tissue, as evidenced both in small and large animal studies. Even if the body osteogenicity capacity, emblematic in pathologic heterotopic bone formation, it is well known; the new bone formation needs artificial scaffolds and growth factors in the case of critical size defects.
Considering that neither scientists neither the microsurgeons could solve alone the problem of critical size bone reconstruction in trauma, the most promising approach is probably integration of the different approaches. The route to a vascularized bone graft could, as a principle, starts from the "in lab" realization of a cell constructs with osteocyte phenotype expressed and bone synthesis genes activated taking full advantage of the in vitro bioreactor approach.
Only at this time, the alive, but non-vascularized cell construct could undergo to "in vivo" bioreactor maturation for vascularization development. Such a multidisciplinary approach would inevitably imply the integration of knowledge and side-by-side collaboration of clinicians, engineers, biologists and physicists, the only key for a successful ending.