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Fig. 3 Rotating
bioreactors; (a) preliminary
rotating bioreactor; (b)
disc-xated model rotating
bioreactor; (c) rotating bed
bioreactor (RBB)
a
b
c
walls. This led to construct destruction and disruption of cellular attachment.
Aiming for improving different aspects of rotational bioreactors, different modalities have been introduced. One is named rotating bed bioreactor (RBB). RBB design
includes tight xation of seeded scaffolds onto the rotating axis of a bed [32], which
has successfully prevented the construct-wall collision. Comparing to spinner
asks, the rotating bioreactor systems are observed to provide higher rates of cellular proliferation and differentiation [7, 8, 29] and complied with GMP standards
[29, 34]. However, medium inltration, mineralization, and culturing benets are
still conned to the outer proportion of the structure, meaning that rotational forces,
being used with these techniques, are still incapable of piercing through the miniature pores of scaffolds and the internal nutrient transport is still restricted [35]
(Fig.3).
2.1.3 Perfusion-Based Bioreactors
Perfusion-based bioreactors have been designed and manufactured aiming for elimination of persistent restrictions of mass transport [8, 11, 29]. Comparing to other
cultural settings, perfusion-based bioreactors have shown the best results in terms of
cellular proliferation, differentiation, and viability of BTE [36].
A basic perfusion-based setup consists of a pump, a tubing circuit, a reservoir,
and a perfusion cartridge. Containers, or cartridge components, are designed for
harboring of the constructs [9]. With perfusion-based systems, tubes with a
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peristaltic roller pump are utilized to pump the media into the chamber; creating a
laminar uid ow within the chamber [9]. The media is predicted to ow in a closed
loop or be discarded from the main chamber by associated vessels. Various modalities have been introduced and have proved successful even media delivery through
construct pores. Of note, gas-permeable silicon tubes and oxygenator devices can be
merged into the system for medium oxygenation [9, 11, 26, 29]. The system’s osteo-
genic stimulation ability is dictated by performed uid ow modes. Literature states
that the perfusion-based bioreactors are the most procient shear-loading bioreactors for BTE purposes. According to Nokhbatolfoghahaei etal. [8], 73% of studies
have utilized perfusion-based bioreactors only for BTE, highlighting the point that
perfusion- based bioreactors can potentially diffuse media through the construct
pores and provide better nutrient and substance transfer into the construct core.
Ideal perfusion ow rates have been calculated to be within the range of 1–600mL/h.
Flow rates beyond this maximum can impose cells with excessive shear stress, leading to their ultimate washout. Given that bioreactors are now a part of the Tissue
Engineering Quadriad, apt clinical application of BTE necessitates optimization of
bioreactor-associated parameters, including media ow rate, as well [8].
Based on their pumping method, perfusion-based bioreactors are categorized as
indirect and direct perfusion systems [7, 11, 29]. With indirect perfusion bioreactors, constructs are xated onto a loosely sealed cassette, allowing ow of media to
entail the space within and around the scaffold through the path of least resistance.
Flow-derived shear stresses cannot affect cells placed at the core of construct. Given
this, production of heterogeneous ow prole may occur. Conversely, direct
perfusion- based bioreactors have been developed to address the limited internal
mass transfer. Their application can exert biophysical forces onto the internally cultivated cells, through media ow. Their design entails press-tting of scaffolds onto
a cassette while perfusion cartridges are sealed onto them. This has been developed
to avert leaking of media around constructs and the media ends up being perfused
directly through the scaffold pores. Direct perfusion-based bioreactors are often
onerously manufactured due to scaffold customization and the strict prerequisites
surrounding the scaffold design with regard to its pore size and porosity [7, 29, 37,
38] (Fig.4).
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2.2 Compression Bioreactors (Direct Mechanical Stress)
Physiologically, bone tissue must be subject to at least 500 μStrain to prevent gradual bone loss over time. Beyond 1000 μStrain loads can result in preservation of
bone’s basic geometry, and new bone formation occurs following the application of
loads within the range of 1000–4000 μStrain [30, 39]. Various investigations have
conrmed the mechanical conditioning phenomena that take place once direct
mechanical strains are applied on tissue in forms of bending, stretching, contraction, and compression. Direct mechanical stresses are applied using three strategies
[30, 39]: (a) the 4-point bending, (b) the uniaxial cyclic stretching, and (c) the uniaxial mechanical force application. The rst uses two bolts and can progress
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H. S. H. Boroojeni and H. Nokhbatolfoghahaei
Fig. 4 (a) Direct perfusion-based bioreactor; (b) indirect perfusion-based bioreactor
passively or with loading, the second is based on alternative application of uniaxial
forces on silicon plates, and the last one involves pressure application via pistons.
Direct mechanical stimulation using bioreactors has showed elevated rates of proliferation, differentiation toward osteogenic lineage, and matrix formation. However,
several studies have reported cellular stimulation on matrices composed of biomechanically instable Col1 gels, originated from mechanical stress [29]. Given that a
minimal mechanical stability is essential for constructs to be competently implied in
bone regeneration, instable gels bear drawbacks. Cultivating large constructs in
mechanical load-based bioreactors has been imposed with hurdles about media diffusion, resulting in the need for additional compromising strategies (Fig.5).
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Fig. 5 Direct mechanical
strain bioreactors
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2.3 EMF-Based Bioreactors
EMF-based bioreactors have a chamber having two Helmholtz coils, powered by an
electromagnetic eld. The BTE constructs are harbored within the powered coils
with tunable amplitude, frequency, and intensity that allow controllable stimulation
of cultivated cells. It has been showed that bony cells are strongly frequency selective. They are most thrivingly proliferated and differentiated osteogenically, at
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Fig. 6 EMF-based
bioreactors
H. S. H. Boroojeni and H. Nokhbatolfoghahaei
15Hz EMF [8, 29, 40]. In terms of BTE, comparing to static systems, EMF-based
bioreactors have indicated a promoted osteogenic cellular differentiation.
Pulsed electromagnetic elds (PEMF) can stimulate proliferation of osteoprogenitor cells while being noninvasive and following GMP standards. Nonetheless,
they need high acquisition costs [8, 29, 40] (Fig.6).
2.4 Combined Bioreactors
Combined bioreactors have been introduced to enhance mimicking of physiological
conditions through conating various design systems [7–9, 24, 41–45].
2.4.1 Applications andAdvancements
Using computational tools, variables (i.e., ow elds, shear stresses, mass transport, etc.) can be calculated. This provides researchers with possible determination of mass transport rates and cell viability relations. In addition, theoretical
modelling has enabled assessment of the relations between cell viability and density, and diffusion radius in BTE constructs, under sustained nutrient supply [9].
With regard to this, Muschler etal. [46] came up with a differential equation for
calculating the relation between cell viability and density, and diffusion radius in
materials, aiming to determine the stage wherein hypoxic conditions appear.
Mathematical modelling has been used to illustrate links among theoretical versus
experimental data of nutrient and oxygen supply and metabolic phenomena.
Moreover, the impact of micromechanical stimuli of cell-surrounding environments has been studied with mathematical modelling. Using nite element analysis (FEA), dened stress and strain elds are calculated for further implementation
through bioreactor systems, developing simplied databases into systems reective of real deformations in tissue chambers. Despite their still-vague practicality
in some elds, the biologically relevant calculated magnitude has, at least, enabled
discrimination of hypophysiologic, physiologic, and hyperphysiologic load applications [4].
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Accumulating data has demonstrated BTE-specic bioreactors to be critical factors and crucial methodological steps for orchestrating efcient bone regeneration.
However, invitro characterization of cells, via mineralization assay, gene expression, etc., after being seeded upon scaffolds, must be an integrated step of evaluative
measures during construct development. This is to avoid concerns of osteogenic
activity, immunological matters and rejection risks, prior to invivo application [47].
Technically, invitro bioreactors can be classied as “closed systems” for those
that allow gas exchange through ports and lters and “open systems” for those consisted of conventional reservoirs. Sterility and kept cell and tissue viability of closed
bioreactor systems highlight their application, whereas open systems need regular
manual handling in terms of media exchange, cell seeding, etc., limiting their application with high manufacturing standards and, in particular, clinical application. In
addition to mechanical variables, implementation of supplementary systems for
monitoring and tuning of physicochemical stimuli is of note. Moreover, tunable
culture conditions, including temperature, pH, nutrients, and oxygen supplies, could
benet the system in terms of reproducibility and standardization. In light of this,
implementation of devices for real-time evaluation of cells and analysis tools for
monitoring of material, biological, and metabolic variables (i.e., e-modulus, degradation, cell count, cellular differentiation, pH, oxygen and nutrient concentration,
etc.) could further benet consecutive yet different stages of cell and tissue maturation. Hence, using micro computerized tomographies, substance-delivery monitoring, ow determination, and uorescence microscopy, predictive measures in terms
of construct development and maturation have been introduced [4, 48].
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2.5 In Vivo Bioreactors
Albeit there are numerous endeavors in terms of BTE, clinical translational solutions are yet to be advanced. Developing bioreactors with optimized dynamic forces
to mimic the physiological conditions of the body, cellular differentiation and tissue
remodeling phenomena are still restricted in terms of physiological stimulation.
Lack of mature and functional vascularity within larger construct has also been a
key challenge. A complex network of interacting factors (i.e., spatiotemporal control, intracellular and cell-ECM interactions, hypoxic conditions, ow, and circadian clock) regulates the angiogenic phenomena. However, such complexity has
been rudimentarily simulated invitro, even using our most currently advanced technologies [49, 50]. Additionally, concerns exist about cellular karyotype alternations
that occur during invitro manipulation, putting emphasis upon the necessity of cell
karyotyping before clinical translation. Moreover, working with invitro bioreactors
necessitates not only expensive technical equipment with rather complex assembly
but also presence of specially trained technicians [29, 51].
With regard to the mentioned constraints, one clinically promising approach for
translational BTE application can be harnessing a living body to generate customized pre-vascularized autologous tissue for reconstructive purposes, bridging the
conventional reconstructive medicine and BTE [51–53]. In the nineteenth century,
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consequent to rhBMP2 administration, intramuscular ectopic bone formation was
seen, leading to the development of the initial ideas of invivo bioreactors (IVB).
This approach lies in the idea of using living creatures as living bioreactors while
leveraging their inherent self-regenerative aptitudes. This manipulation is conducted
through providing the tissue with a consecutive stream of cells (i.e., immune cells
and osteogenic and angiogenic cells), nutrients, oxygen, and native signals [54].
IVB have been developed to serve cells in the living models. With IVB, autogenous
skeletal muscles serve as niches for in situ fabrication of pre-vascularized constructs. Upon the inspection of biological advancements, the constructs translated
into defect sites as bone grafts [53, 55]. Of note, the body provides the BTE constructs not only with procient stimuli but also with supply of necessary growth
factors and biotic substances of host. Various IVB systems, developed in mice, rats,
rabbits, miniature pigs, etc., have been investigated while aiming for optimal means
of producing pre-vascularized constructs capable of forming new bone tissue [51,
55, 56]. Their application has evaded the excessive stages of exvivo manipulations
over cell and growth factors. Therefore, the functional inherent properties of cells
are more likely to be preserved.
Although preclinical and clinical studies on the efcacy of invivo bioreactors
have used diverse set of models and study designs, their principles have been parallel. They have all aimed for anatomical site of implantation that acts as a regenerative niche while also developing optimal BTE constructs for implantation. Their
results demonstrate the potential of in vivo prefabricated bone grafts in criticalsized bone defects [53]. However, it is crucial to discriminate different IVB techniques in regard to their regenerative potential, in clinically relevant models in the
absence or minimal presence of exogenous factors or transplanted cells [51, 53, 57].
2.5.1 Surgical Strategies
Since tissue fragments are developed in invitro settings, and almost always lack
neurovascular networks, their internally located cells may experience hypoxic conditions. In addition, there is a lack of efcient transferal of waste and nutrients from
and toward cells. To address this, many surgical strategies have been designed for
immediate vascularization of transplanted grafts. The most predominant strategy for
invivo bone healing is currently co-translation of both bone- and vessel-forming
cells into the defect region [58, 59].
Prefabrication is a surgical term referring to vascularization of an avascular territory from an implanted vascular pedicle. It was rst dened by Shen as vascular
pedicle transplantation into another territory with later neovascularization. This
involved transplantation of a dened tissue fragment to another site to allow formation of neovascular networks [60]. Additionally, in terms of BTE, prefabrication
discusses the development of substitutive constructs being implanted within a vascularized recipient site, aiming for production of a prefabricated bone graft [53],
and upon vascularization, a bone ap is elevated based on the anticipated size and
geometry of initial defect [61, 62]. Flap prefabrication technique has been a prime
choice of intervention to bridge BTE and oral and maxillofacial reconstructive
surgeries.
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Through combination of invivo bioreactor concept and prefabrication surgical
techniques for vascular translation of tissue fragments, a regenerative strategy has
appeared. It includes both transferal of a regenerating niche for best reconstruction
and addressing the shortcomings of in vitro bioreactors for BTE. This strategy
allows chemotaxis of undifferentiated cells from neighboring sites and the circulating bloodstream, delivery of growth factors and signaling molecules, and imposition of other stimuli.
The recipient bed tissue plays a signicant role in terms of guring out which
sort of interactions are to be received by the construct. The recruitment of endogenous cells, establishment of neurovascular networks, and the overall bone graft
prefabrication results are highly dependent upon the recipient tissue type.
Orthotopic, namely, the direct reconstruction of defect using constructs and no
further need for secondary surgery, or ectopic prefabrication techniques are both
employed. Since orthotopic prefabrication can lack ability due to the inadequate
regenerative potency of defect site microenvironment following chronic infection,
degenerative conditions, radiotherapy, or substantial degeneration rates, the ectopic prefabrication technique can also be alternatively chosen. Selection of the ectopic site must be in accordance with the adequate circumstances that the recipient
site offers in terms of cell colonization, vascularization capacity, physical stimulation, and facile association during operation. With regard to in vivo bioreactor
approaches, two sets of techniques are present: the ap and the vascular techniques.
The former includes subcutaneous pocket, muscular pouch or ap, and periosteal
ap, and the latter includes axial vascular bundle (AVB) and arteriovenous
loop (AVL).
Flap Techniques
The BTE constructs are implanted at a corresponding chamber, remote to the defect
site. Once the prelamination, namely, the vascularization of an avascular construct
from a vascular host territory, phase has passed, a ap containing the construct is
elevated and transferred to the defect site. A vascular pedicle of the ap is microsurgically anastomosed to the vasculature of the bed, enabling immediate perfusion of
the construct at a physiologic rate. Although employing this approach can evade
bone harvesting, elevation of a muscular ap and its donor site morbidity can still
be considerably challenging. Moreover, the necessity of performing at least two
major surgeries that dictate a post-operation treatment period of few months must
also be considered.
Subcutaneous Pocket
The simplest invivo bioreactor approach for prefabrication of BTE constructs is the
“subcutaneous pocket.” It involves establishment of an articial space between the
supercial and the deep subcutaneous fascia, supplying apt regenerative requisites.
Prefabricated bone grafts with complex phenotypes and custom-made geometries
have been made accordingly. This approach offers a exible selection of anatomical
locations with facile transplantation of grafts while risks of donor site morbidity and
other complications remaining minimal. However, in a surgical context, the
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subcutaneous implantation sites are relatively avascular, lacking an abundant blood
supply and the requisite stem cell, growth factor, and hormonal stream that are necessary for bone regeneration, when compared to other in vivo bioreactor-based
approaches. On the other hand, muscle tissue offers rich resources of neurovascular
networks [63–65].
Muscular Pouch or Flap
The muscular pouch or ap strategy is based on the idea of inducing ectopic neovascularization and bone regeneration, via enveloping the BTE constructs as an
intramuscular pouch or pedicled ap. Muscles have copious amounts of native
skeletal progenitor cells, capable of differentiating into bone cells. This obviates
the need for transplantation of exogenous osteoprogenitor or stem cells to some
extent. Inherently present molecules of osteogenic pathways, such as BMPs,
TGF-beta1, and IGF-1, are present in sites of muscular injury, capable of diffusing into the BTE construct and naturally upregulating the activation of osteogenic
signals. Experimental and clinical studies have used muscular pouch or ap to
generate prefabricated bone grafts. According to Warnke etal. [66], a subtotal
construct composed of a titanium mesh cage and bone mineral blocks with BMP-7
and bone marrow aspirate containing undifferentiated precursors has indicated
growth within the latissimus dorsi muscle. Following the microsurgical transferal
of the surrounding muscle and construct unit to the mandibular defect site at week
7, apt integration and aesthetic and functional outcome were obtained. Mesimäki
et al. [67] have reported successful maxillary reconstruction with IVB-based
microvascular BTE prefabrication ap. They used a rectus abdominis muscular
pouch and MSCs, BMP-2, and βTCP scaffolds in a titanium cage. Moreover, several studies have aimed for clinical application of IVB-based BTE constructs,
either holding harvested cells and growth factors or not, reporting inconsistent
results [57, 68–74].
Periosteal Flap
The periosteum is also a highly vascular bi-layered tissue. It is made up of a
brous outer layer with spread broblasts and collagenous bers and a cambium
inner layer containing skeletal progenitor cells and osteoblasts. The former provides structural durability of periosteum, and the latter offers appositional bone
growth, cortical bone formation, and remodeling capacities [75]. Given that the
periosteum has these inherent features, the periosteal ap for envelopment of BTE
constructs or a containing chamber of which, as an invivo bioreactor, can provide
the potential of proper clinical results. It supplies a broad spectrum of growth factors. In addition, its cambium layer acts as a rich reservoir of periosteum MSCs.
It has a rich neurovascular environment with copious neurovascular-borne nutrients, capable of enhancing the prefabrication phase. The periosteal ap also follows the principles of guided bone regeneration (GBR) concept, making a
relatively safe and competent choice of ap. However, their donor sites are relatively limited and avert implication of periosteal aps for large and/or geometrically complex or customized BTE constructs [76, 77]. Abu-Shahba et al. [78]
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investigated the signicance of periosteal vascular supply for prefabricating BTE
aps to reconstruct mandibular defects in sheep models. Their computed tomography (CT) and micro-CT analyses revealed an improved new bone formation
rate, while the volume of remnant biomaterial had diminished. According to their
histological analysis, majority of newly generated bone tissue had appeared from
defect edges. On the other hand, new bone islands in their periosteal ap-associated group were larger, coupled with enhanced vascularization and remodeling,
conclusively showing that periosteal aps had contributed to the promoted reconstructive potential of BTE prefabricating aps and their regenerative capacity
once transplanted into the defect site.
Vascular Techniques
Axial Vascular Bundle (AVB) andArteriovenous Loop (AV loop)
The present regenerative approaches are chiey rooted in obtaining vascularization
extrinsically through neovaculatures sprouting from recipient site. However, the
recipient site may not always provide requisites of neovascularization. For instance,
irradiated, chronically infected, or excessively degenerated tissues cannot supply
the desired neovascularization specics and minimums. Therefore, in lieu of ap
elevation and extrinsic vascularization of BTE constructs, intrinsic axial vascularization strategies, such as the axial vascular bundle (AVB) and arteriovenous loop
(AV loop) technique, can be employed. Intrinsic osteogenesis and vascularization
for prefabrication purposes take place accordingly.
In the AVB model, a central artery and vein within the construct provide a stream
of cells and signaling and nutrient molecules. It, concomitantly, disposes cellular
waste and metabolic by-products. The AVB model has shown apt results in terms of
vascularization and osteogenesis in BTE constructs. Prefabricating with the use of
AVB models contributes to formation of axial pattern bone grafts, translated in the
form of pedicled or free aps. The potential for clinical application of AVB models
has been proven using the supercial inferior epigastric vessels, the saphenous vascular bundles, the femoral vascular bundles, and the perforating vessels from the
thoracodorsal trunk, as parts of invivo bioreactors.
When performing the AVB strategy, selection of long and supercially located
artery and vein, for later transferal of pedicled ap to a remote site, is essential.
This restricts the amount of proper donor AVBs. Furthermore, a limited contact
surface area is present between the AVB and the BTE construct. This hampers the
prefabrication of an ideally ossied and vascularized bone graft over conventional
periods of time. Combined use of AVB and tissue aps can compensate for this
drawback. Also, trauma, injury, or presence of established vascular disorders can
inhibit application of this strategy [79, 80]. In such cases, AV loop models can be
alternatively harbored within the containing chambers. AV loops are iatrogenic
arteriovenous stulas, generated via placement of a venous graft between the opted
artery and vein, or microsurgical anastomosis of the two without any graft mediation. This “neopedicle” is a supportive provisional sprouting matrix that buds capillary systems into the adjacent sites while being perfused. The remodeling of these
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